Co-reporter:Chaitan Khosla
ACS Chemical Biology June 16, 2017 Volume 12(Issue 6) pp:1455-1455
Publication Date(Web):February 6, 2017
DOI:10.1021/acschembio.6b01155
Celiac disease is a lifelong immune disorder of the small intestine where inflammation is triggered by dietary gluten. There is an urgent need for the development of nondietary therapies for this widespread but overlooked disease. More fundamentally, a molecular understanding of gluten-induced pathogenesis in celiac disease has the potential to provide new insights into mucosal immunology. Over the past two decades, three pathogenically critical molecules—gluten, TG2, and HLA-DQ2—have served as focal points for collaborative efforts between biologists, chemists, engineers, and clinicians with an interest in celiac disease. This perspective summarizes a few examples of such multidisciplinary research directions with an emphasis on groundbreaking clinical studies that have profoundly informed the trajectory of subsequent molecular investigations. Examples of future challenges in fundamental and translational celiac disease research are also discussed.
Co-reporter:Xirui Xiao, Karthik Sankaranarayanan, Chaitan Khosla
Current Opinion in Chemical Biology 2017 Volume 40(Volume 40) pp:
Publication Date(Web):1 October 2017
DOI:10.1016/j.cbpa.2017.07.008
•The glycolipid Lipid A is the conserved, amphipathic moiety of LPS.•Lipid A binds to certain Toll like receptors and caspases with high affinity.•Bacteria alter their Lipid A structures to modulate immune activities.•Non-toxic Lipid A congeners are effective immunomodulatory agents.Lipopolysaccharide (LPS), a glycolipid found in the outer membrane of Gram-negative bacteria, is a potent elicitor of innate immune responses in mammals. A typical LPS molecule is composed of three different structural domains: a polysaccharide called the O-antigen, a core oligosaccharide, and Lipid A. Lipid A is the amphipathic glycolipid moiety of LPS. It stimulates the immune system by tightly binding to Toll-like receptor 4. More recently, Lipid A has also been shown to activate intracellular caspase-4 and caspase-5. An impressive diversity is observed in Lipid A structures from different Gram-negative bacteria, and it is well established that subtle changes in chemical structure can result in dramatically different immune activities. For example, Lipid A from Escherichia coli is highly toxic to humans, whereas a biosynthetic precursor called Lipid IVA blocks this toxic activity, and monophosphoryl Lipid A from Salmonella minnesota is a vaccine adjuvant. Thus, an understanding of structure–activity relationships in this glycolipid family could be used to design useful immunomodulatory agents. Here we review the biosynthesis, modification, and structure–activity relationships of Lipid A.
Co-reporter:Ayse Okesli, Chaitan Khosla, Michael C Bassik
Current Opinion in Biotechnology 2017 Volume 48(Volume 48) pp:
Publication Date(Web):1 December 2017
DOI:10.1016/j.copbio.2017.03.010
•Human pyrimidine nucleotide biosynthesis has been targeted for the treatment of many diseases.•Chemotherapy combining DHODH and UCK inhibitors can be a broad-spectrum antiviral.•Targeting the host cell, such an antiviral therapy could mitigate resistant viruses.The development of broad-spectrum, host-acting antiviral therapies remains an important but elusive goal in anti-infective drug discovery. To replicate efficiently, viruses not only depend on their hosts for an adequate supply of pyrimidine nucleotides, but also up-regulate pyrimidine nucleotide biosynthesis in infected cells. In this review, we outline our understanding of mammalian de novo and salvage metabolic pathways for pyrimidine nucleotide biosynthesis. The available spectrum of experimental and FDA-approved drugs that modulate individual steps in these metabolic pathways is also summarized. The logic of a host-acting combination antiviral therapy comprised of inhibitors of dihydroorotate dehydrogenase and uridine/cytidine kinase is discussed.
Co-reporter:James Kuo, Stephen R. Lynch, Corey W. Liu, Xirui Xiao, and Chaitan Khosla
ACS Chemical Biology 2016 Volume 11(Issue 9) pp:2636
Publication Date(Web):July 6, 2016
DOI:10.1021/acschembio.6b00489
Although a few well-characterized polyketide synthases (PKSs) have been functionally reconstituted in vitro from purified protein components, the use of this strategy to decode “orphan” assembly line PKSs has not been described. To begin investigating a PKS found only in Nocardia strains associated with clinical cases of nocardiosis, we reconstituted in vitro its five terminal catalytic modules. In the presence of octanoyl-CoA, malonyl-CoA, NADPH, and S-adenosyl methionine, this pentamodular PKS system yielded unprecedented octaketide and heptaketide products whose structures were partially elucidated using mass spectrometry and NMR spectroscopy. The PKS has several notable features, including a “split, stuttering” module and a terminal reductive release mechanism. Our findings pave the way for further analysis of this unusual biosynthetic gene cluster whose natural product may enhance the infectivity of its producer strains in human hosts.
Co-reporter:Thomas Robbins, Joshuah Kapilivsky, David E. Cane, and Chaitan Khosla
Biochemistry 2016 Volume 55(Issue 32) pp:4476-4484
Publication Date(Web):July 21, 2016
DOI:10.1021/acs.biochem.6b00639
Ketosynthase (KS) domains of assembly line polyketide synthases (PKSs) catalyze intermodular translocation of the growing polyketide chain as well as chain elongation via decarboxylative Claisen condensation. The mechanistic roles of ten conserved residues in the KS domain of Module 1 of the 6-deoxyerythronolide B synthase were interrogated via site-directed mutagenesis and extensive biochemical analysis. Although the C211A mutant at the KS active site exhibited no turnover activity, it was still a competent methylmalonyl-ACP decarboxylase. The H346A mutant exhibited reduced rates of both chain translocation and chain elongation, with a greater effect on the latter half-reaction. H384 contributed to methylmalonyl-ACP decarboxylation, whereas K379 promoted C–C bond formation. S315 played a role in coupling decarboxylation to C–C bond formation. These findings support a mechanism for the translocation and elongation half-reactions that provides a well-defined starting point for further analysis of the key chain-building domain in assembly line PKSs.
Co-reporter:Matthew P Ostrowski, David E Cane and Chaitan Khosla
The Journal of Antibiotics 2016 69(7) pp:507-510
Publication Date(Web):April 27, 2016
DOI:10.1038/ja.2016.41
Ketoreductases (KRs) are the most widespread tailoring domains found in individual modules of assembly line polyketide synthases (PKSs), and are responsible for controlling the configurations of both the α-methyl and β-hydroxyl stereogenic centers in the growing polyketide chain. Because they recognize substrates that are covalently bound to acyl carrier proteins (ACPs) within the same PKS module, we sought to quantify the extent to which protein–protein recognition contributes to the turnover of these oxidoreductive enzymes using stand-alone domains from the 6-deoxyerythronolide B synthase (DEBS). Reduced 2-methyl-3-hydroxyacyl-ACP substrates derived from two enantiomeric acyl chains and four distinct ACP domains were synthesized and presented to four distinct KR domains. Two KRs, from DEBS modules 2 and 5, displayed little preference for oxidation of substrates tethered to their cognate ACP domains over those attached to the other ACP domains tested. In contrast, the KR from DEBS module 1 showed an ~10–50-fold preference for substrate attached to its native ACP domain, whereas the KR from DEBS module 6 actually displayed an ~10-fold preference for the ACP from DEBS module 5. Our findings suggest that recognition of the ACP by a KR domain is unlikely to affect the rate of native assembly line polyketide biosynthesis. In some cases, however, unfavorable KR–ACP interactions may suppress the rate of substrate processing when KR domains are swapped to construct hybrid PKS modules.
Co-reporter:Brian Lowry, Xiuyuan Li, Thomas Robbins, David E. Cane, and Chaitan Khosla
ACS Central Science 2016 Volume 2(Issue 1) pp:14
Publication Date(Web):January 6, 2016
DOI:10.1021/acscentsci.5b00321
Vectorial polyketide biosynthesis on an assembly line polyketide synthase is the most distinctive property of this family of biological machines, while providing the key conceptual tool for the bioinformatic decoding of new antibiotic pathways. We now show that the action of the entire assembly line is synchronized by a previously unrecognized turnstile mechanism that prevents the ketosynthase domain of each module from being acylated by a new polyketide chain until the product of the prior catalytic cycle has been passed to the downstream module from the corresponding acyl carrier protein domain. The turnstile is closed by virtue of tight coupling to the signature decarboxylative condensation reaction catalyzed by the ketosynthase domain of each polyketide synthase module. Reopening of the turnstile is coupled to the eventual chain translocation step that vacates the module. At the maximal rate of substrate turnover, one would expect the chain release step to initiate a cascade of chain translocation events that sequentially migrate back upstream, thereby repriming each module and setting up the assembly line for the next round of polyketide chain elongation.
Co-reporter:Michael C. Yi, Brad A. Palanski, Steven A. Quintero, Nicholas M. Plugis, Chaitan Khosla
Bioorganic & Medicinal Chemistry Letters 2015 Volume 25(Issue 21) pp:4922-4926
Publication Date(Web):1 November 2015
DOI:10.1016/j.bmcl.2015.05.006
Transglutaminase 2 (TG2) is a ubiquitously expressed, Ca2+-activated extracellular enzyme in mammals that is maintained in a catalytically dormant state by multiple mechanisms. Although its precise physiological role in the extracellular matrix remains unclear, aberrantly up-regulated TG2 activity is a hallmark of several maladies, including celiac disease. Previously, we reported the discovery of a class of acylideneoxoindoles as potent, reversible inhibitors of human TG2. Detailed analysis of one of those inhibitors (CK-IV-55) led to an unprecedented and striking observation. Whereas this compound was a non-competitive inhibitor (3.3 ± 0.9 μM) of human TG2 at saturating Ca2+ concentrations, it activated TG2 in the presence of sub-saturating but physiologically relevant Ca2+ concentrations (0.5–0.7 mM). This finding was validated in a cellular model of TG2 activation and inhibition. Mutant TG2 analysis suggested that CK-IV-55 and its analogs bound to a low-affinity Ca2+ binding site on the catalytic core of TG2. A mechanistic model for the dual agonistic/antagonistic action of CK-IV-55 on TG2 is presented, and the pathophysiological implications of basal activation of intestinal TG2 by small molecules are discussed.CK-IV-55 can activate/inhibit human Transglutaminase 2.
Co-reporter:Cornelius Klöck ; Zachary Herrera ; Megan Albertelli
Journal of Medicinal Chemistry 2014 Volume 57(Issue 21) pp:9042-9064
Publication Date(Web):October 21, 2014
DOI:10.1021/jm501145a
Transglutaminase 2 (TG2) is a ubiquitously expressed enzyme that catalyzes the posttranslational modification of glutamine residues on protein or peptide substrates. A growing body of literature has implicated aberrantly regulated activity of TG2 in the pathogenesis of various human inflammatory, fibrotic, and other diseases. Taken together with the fact that TG2 knockout mice are developmentally and reproductively normal, there is growing interest in the potential use of TG2 inhibitors in the treatment of these conditions. Targeted-covalent inhibitors based on the weakly electrophilic 3-bromo-4,5-dihydroisoxazole (DHI) scaffold have been widely used to study TG2 biology and are well tolerated in vivo, but these compounds have only modest potency, and their selectivity toward other transglutaminase homologues is largely unknown. In the present work, we first profiled the selectivity of existing inhibitors against the most pertinent TG isoforms (TG1, TG3, and FXIIIa). Significant cross-reactivity of these small molecules with TG1 was observed. Structure–activity and −selectivity analyses led to the identification of modifications that improved potency and isoform selectivity. Preliminary pharmacokinetic analysis of the most promising analogues was also undertaken. Our new data provides a clear basis for the rational selection of dihydroisoxazole inhibitors as tools for in vivo biological investigation.
Co-reporter:Thomas R. DiRaimondo, Cornelius Klöck, Rod Warburton, Zachary Herrera, Krishna Penumatsa, Deniz Toksoz, Nicholas Hill, Chaitan Khosla, and Barry Fanburg
ACS Chemical Biology 2014 Volume 9(Issue 1) pp:266
Publication Date(Web):October 23, 2013
DOI:10.1021/cb4006408
Previous studies in human patients and animal models have suggested that transglutaminase 2 (TG2) is upregulated in pulmonary hypertension (PH), a phenomenon that appears to be associated with the effects of serotonin (5-hydroxytryptamine; 5-HT) in this disease. Using chemical tools to interrogate and inhibit TG2 activity in vivo, we have shown that pulmonary TG2 undergoes marked post-translational activation in a mouse model of hypoxia-induced PH. We have also identified irreversible fluorinated TG2 inhibitors that may find use as non-invasive positron emission tomography probes for diagnosis and management of this debilitating, lifelong disorder. Pharmacological inhibition of TG2 attenuated the elevated right ventricular pressure but had no effect on hypertrophy of the right ventricle of the heart. A longitudinal study of pulmonary TG2 activity in PH patients is warranted.
Co-reporter:Chaitan Khosla, Daniel Herschlag, David E. Cane, and Christopher T. Walsh
Biochemistry 2014 Volume 53(Issue 18) pp:
Publication Date(Web):April 29, 2014
DOI:10.1021/bi500290t
Two hallmarks of assembly line polyketide synthases have motivated an interest in these unusual multienzyme systems, their stereospecificity and their capacity for directional biosynthesis. In this review, we summarize the state of knowledge regarding the mechanistic origins of these two remarkable features, using the 6-deoxyerythronolide B synthase as a prototype. Of the 10 stereocenters in 6-deoxyerythronolide B, the stereochemistry of nine carbon atoms is directly set by ketoreductase domains, which catalyze epimerization and/or diastereospecific reduction reactions. The 10th stereocenter is established by the sequential action of three enzymatic domains. Thus, the problem has been reduced to a challenge in mainstream enzymology, where fundamental gaps remain in our understanding of the structural basis for this exquisite stereochemical control by relatively well-defined active sites. In contrast, testable mechanistic hypotheses for the phenomenon of vectorial biosynthesis are only just beginning to emerge. Starting from an elegant theoretical framework for understanding coupled vectorial processes in biology [Jencks, W. P. (1980) Adv. Enzymol. Relat. Areas Mol. Biol. 51, 75–106], we present a simple model that can explain assembly line polyketide biosynthesis as a coupled vectorial process. Our model, which highlights the important role of domain–domain interactions, not only is consistent with recent observations but also is amenable to further experimental verification and refinement. Ultimately, a definitive view of the coordinated motions within and between polyketide synthase modules will require a combination of structural, kinetic, spectroscopic, and computational tools and could be one of the most exciting frontiers in 21st Century enzymology.
Co-reporter:Briana J. Dunn, Katharine R. Watts, Thomas Robbins, David E. Cane, and Chaitan Khosla
Biochemistry 2014 Volume 53(Issue 23) pp:
Publication Date(Web):May 28, 2014
DOI:10.1021/bi5004316
Due to their pivotal role in extender unit selection during polyketide biosynthesis, acyltransferase (AT) domains are important engineering targets. A subset of assembly line polyketide synthases (PKSs) are serviced by discrete, trans-acting ATs. Theoretically, these trans-ATs can complement an inactivated cis-AT, promoting introduction of a noncognate extender unit. This approach requires a better understanding of the substrate specificity and catalytic mechanism of naturally occurring trans-ATs. We kinetically analyzed trans-ATs from the disorazole and kirromycin synthases and compared them to a representative cis-AT from the 6-deoxyerythronolide B synthase (DEBS). During transacylation, the disorazole AT favored malonyl-CoA over methylmalonyl-CoA by >40000-fold, whereas the kirromycin AT favored ethylmalonyl-CoA over methylmalonyl-CoA by 20-fold. Conversely, the disorazole AT had broader specificity than its kirromycin counterpart for acyl carrier protein (ACP) substrates. The presence of the ACP had little effect on the specificity (kcat/KM) of the cis-AT domain for carboxyacyl-CoA substrates but had a marked influence on the corresponding specificity parameters for the trans-ATs, suggesting that these enzymes do not act strictly by a canonical ping-pong mechanism. To investigate the relevance of the kinetic analysis of isolated ATs in the context of intact PKSs, we complemented an in vitro AT-null DEBS assembly line with either trans-AT. Whereas the disorazole AT efficiently complemented the mutant PKS at substoichiometric protein ratios, the kirromycin AT was considerably less effective. Our findings suggest that knowledge of both carboxyacyl-CoA and ACP specificity is critical to the choice of a trans-AT in combination with a mutant PKS to generate novel polyketides.
Co-reporter:Brian Lowry ; Thomas Robbins ; Chih-Hisang Weng ; Robert V. O’Brien ; David E. Cane
Journal of the American Chemical Society 2013 Volume 135(Issue 45) pp:16809-16812
Publication Date(Web):October 25, 2013
DOI:10.1021/ja409048k
Notwithstanding an extensive literature on assembly line polyketide synthases such as the 6-deoxyerythronolide B synthase (DEBS), a complete naturally occurring synthase has never been reconstituted in vitro from purified protein components. Here, we describe the fully reconstituted DEBS and quantitatively characterize some of the properties of the assembled system that have never been explored previously. The maximum turnover rate of the complete hexamodular system is 1.1 min–1, comparable to the turnover rate of a truncated trimodular derivative (2.5 min–1) but slower than that of a bimodular derivative (21 min–1). In the presence of similar concentrations of methylmalonyl- and ethylmalonyl-CoA substrates, DEBS synthesizes multiple regiospecifically modified analogues, one of which we have analyzed in detail. Our studies lay the foundation for biochemically interrogating and rationally engineering polyketide assembly lines in an unprecedented manner.
Co-reporter:Jay T. Fitzgerald ; Louise K. Charkoudian ; Katharine R. Watts
Journal of the American Chemical Society 2013 Volume 135(Issue 10) pp:3752-3755
Publication Date(Web):February 26, 2013
DOI:10.1021/ja311579s
A-74528 is a C30 polyketide natural product that functions as an inhibitor of 2′,5′-oligoadenylate phosphodiesterase (2′-PDE), a key regulatory enzyme of the interferon pathway. Modulation of 2′-PDE represents a unique therapeutic approach for regulating viral infections. The gene cluster responsible for biosynthesis of A-74528 yields minute amounts of this natural product together with considerably larger quantities of a structurally dissimilar C30 cytotoxic agent, fredericamycin. Through construction and analysis of a series of knockout mutants, we identified the genes necessary for A-74528 biosynthesis. Remarkably, the formation of six stereocenters and the regiospecific formation of six rings in A-74528 appear to be catalyzed by only two tailoring enzymes, a cyclase and an oxygenase, in addition to the core polyketide synthase. The inferred pathway was genetically refactored in a heterologous host, Streptomyces coelicolor CH999, to produce 3 mg/L A-74528 in the absence of fredericamycin.
Co-reporter:Thomas R. DiRaimondo ; Nicholas M. Plugis ; Xi Jin
Journal of Medicinal Chemistry 2013 Volume 56(Issue 3) pp:1301-1310
Publication Date(Web):January 17, 2013
DOI:10.1021/jm301775s
Whereas the role of mammalian thioredoxin (Trx) as an intracellular protein cofactor is widely appreciated, its function in the extracellular environment is not well-understood. Only few extracellular targets of Trx-mediated thiol–disulfide exchange are known. For example, Trx activates extracellular transglutaminase 2 (TG2) via reduction of an intramolecular disulfide bond. Because hyperactive TG2 is thought to play a role in various diseases, understanding the biological role of extracellular Trx may provide critical insight into the pathogenesis of these disorders. Starting from a clinical-stage asymmetric disulfide lead, we have identified analogs with >100-fold specificity for Trx. Structure–activity relationship and computational docking model analyses have provided insights into the features important for enhancing potency and specificity. The most active compound identified had an IC50 below 0.1 μM in cell culture and may be appropriate for in vivo use to interrogate the role of extracellular Trx in health and disease.
Co-reporter:James K. Chen, Justin Du Bois, Jeffrey Glenn, Daniel Herschlag, and Chaitan Khosla
ACS Chemical Biology 2013 Volume 8(Issue 9) pp:1860
Publication Date(Web):September 20, 2013
DOI:10.1021/cb400641u
Co-reporter:Briana J. Dunn, David E. Cane, and Chaitan Khosla
Biochemistry 2013 Volume 52(Issue 11) pp:
Publication Date(Web):March 1, 2013
DOI:10.1021/bi400185v
Acyltransferase (AT) domains of modular polyketide synthases exercise tight control over the choice of α-carboxyacyl-CoA substrates, but the mechanistic basis for this specificity is unknown. We show that whereas the specificity for the electrophilic malonyl or methylmalonyl component is primarily expressed in the first half-reaction (formation of the acyl–enzyme intermediate), the second half-reaction shows comparable specificity for the acyl carrier protein that carries the nucleophilic pantetheine arm. We also show that currently used approaches for engineering AT domain specificity work mainly by degrading specificity for the natural substrate rather than by enhancing specificity for alternative substrates.
Co-reporter:Xirui Xiao, Xingye Yu, and Chaitan Khosla
Biochemistry 2013 Volume 52(Issue 46) pp:
Publication Date(Web):October 22, 2013
DOI:10.1021/bi401116n
The entire fatty acid biosynthetic pathway of Escherichia coli, starting from the acetyl-CoA carboxylase, has been reconstituted in vitro from 14 purified protein components. Radiotracer analysis verified stoichiometric conversion of acetyl-CoA and NAD(P)H to the free fatty acid product, allowing implementation of a facile spectrophotometric assay for kinetic analysis of this multienzyme system. At steady state, a maximal turnover rate of 0.5 s–1 was achieved. Under optimal turnover conditions, the predominant products were C16 and C18 saturated as well as monounsaturated fatty acids. The reconstituted system allowed us to quantitatively interrogate the factors that influence metabolic flux toward unsaturated versus saturated fatty acids. In particular, the concentrations of the dehydratase FabA and the β-ketoacyl synthase FabB were found to be crucial for controlling this property. Via changes in these variables, the percentage of unsaturated fatty acid produced could be adjusted between 10 and 50% without significantly affecting the maximal turnover rate of the pathway. Our reconstituted system provides a powerful tool for understanding and engineering rate-limiting and regulatory steps in this complex and practically significant metabolic pathway.
Co-reporter:Aaron Elijah May
Israel Journal of Chemistry 2013 Volume 53( Issue 8) pp:577-587
Publication Date(Web):
DOI:10.1002/ijch.201300025
Abstract
The Type III Secretion System (TTSS) is indispensable for virulence of many Gram-negative pathogenic bacteria, including Escherichia coli, Salmonella spp., Yersinia spp., Vibrio spp., Chlamydia spp., Shigella spp., Pseudomonas spp., Xanthomonas spp., and Auromonas spp. Such pathogenic bacteria are responsible for diseases such as plague, shigellosis, chlamydia, cholera, pneumonia, and gastroenteritis. This review offers insights into the known inhibitors of the TTSS, their discovery, and their mode of action.
Co-reporter: Christopher T. Walsh;Dr. Robert V. O'Brien; Chaitan Khosla
Angewandte Chemie International Edition 2013 Volume 52( Issue 28) pp:7098-7124
Publication Date(Web):
DOI:10.1002/anie.201208344
Abstract
Freestanding nonproteinogenic amino acids have long been recognized for their antimetabolite properties and tendency to be uncovered to reactive functionalities by the catalytic action of target enzymes. By installing them regiospecifically into biogenic peptides and proteins, it may be possible to usher a new era at the interface between small molecule and large molecule medicinal chemistry. Site-selective protein functionalization offers uniquely attractive strategies for posttranslational modification of proteins. Last, but not least, many of the amino acids not selected by nature for protein incorporation offer rich architectural possibilities in the context of ribosomally derived polypeptides. This Review summarizes the biosynthetic routes to and metabolic logic for the major classes of the noncanonical amino acid building blocks that end up in both nonribosomal peptide frameworks and in hybrid nonribosomal peptide-polyketide scaffolds.
Co-reporter: Christopher T. Walsh;Dr. Robert V. O'Brien; Chaitan Khosla
Angewandte Chemie 2013 Volume 125( Issue 28) pp:7238-7265
Publication Date(Web):
DOI:10.1002/ange.201208344
Abstract
Frei vorkommende nichtproteinogene Aminosäuren sind schon lange wegen ihrer antimetabolen Eigenschaften bekannt. Entdeckt werden sie meist aufgrund der Reaktivität gegenüber der katalytischen Wirkung der Zielenzyme. Führt man sie regiospezifisch in biogene Peptide und Proteine ein, kann es möglich werden, eine neue Ära der Medizinalchemie an der Grenze zwischen kleinen und großen Wirkstoffen einzuleiten. Außerdem eröffnet die ortsspezifische Funktionalisierung von Proteinen besonders attraktive Strategien für die posttranslationale Proteinmodifizierung. Auch bieten viele der Aminosäuren, die von der Natur nicht für den Einbau in Proteine ausgewählt wurden, reiche architektonische Möglichkeiten bei Einführung in ribosomal hergestellte Polypeptide. Dieser Aufsatz fasst die Biosynthesewege zu den wichtigsten Klassen nichtkanonischer Bausteine und deren Stoffwechsellogik zusammen, die in nichtribosomalen Peptidgerüsten und nichtribosomalen Peptid-Polyketid-Hybriden resultieren.
Co-reporter:Tracy C. Holmes ; Aaron E. May ; Kathia Zaleta-Rivera ; J. Graham Ruby ; Peter Skewes-Cox ; Michael A. Fischbach ; Joseph L. DeRisi ; Masato Iwatsuki ; Satoshi O̅mura
Journal of the American Chemical Society 2012 Volume 134(Issue 42) pp:17797-17806
Publication Date(Web):October 2, 2012
DOI:10.1021/ja308622d
Guadinomines are a recently discovered family of anti-infective compounds produced by Streptomyces sp. K01-0509 with a novel mode of action. With an IC50 of 14 nM, guadinomine B is the most potent known inhibitor of the type III secretion system (TTSS) of Gram-negative bacteria. TTSS activity is required for the virulence of many pathogenic Gram-negative bacteria including Escherichia coli, Salmonella spp., Yersinia spp., Chlamydia spp., Vibrio spp., and Pseudomonas spp. The guadinomine (gdn) biosynthetic gene cluster has been cloned and sequenced and includes 26 open reading frames spanning 51.2 kb. It encodes a chimeric multimodular polyketide synthase, a nonribosomal peptide synthetase, along with enzymes responsible for the biosynthesis of the unusual aminomalonyl-acyl carrier protein extender unit and the signature carbamoylated cyclic guanidine. Its identity was established by targeted disruption of the gene cluster as well as by heterologous expression and analysis of key enzymes in the biosynthetic pathway. Identifying the guadinomine gene cluster provides critical insight into the biosynthesis of these scarce but potentially important natural products.
Co-reporter:Colin J. B. Harvey ; Joseph D. Puglisi ; Vijay S. Pande ; David E. Cane
Journal of the American Chemical Society 2012 Volume 134(Issue 29) pp:12259-12265
Publication Date(Web):June 28, 2012
DOI:10.1021/ja304682q
The macrolide antibiotic erythromycin A and its semisynthetic analogues have been among the most useful antibacterial agents for the treatment of infectious diseases. Using a recently developed chemical genetic strategy for precursor-directed biosynthesis and colony bioassay of 6-deoxyerythromycin D analogues, we identified a new class of alkynyl- and alkenyl-substituted macrolides with activities comparable to that of the natural product. Further analysis revealed a marked and unexpected dependence of antibiotic activity on the size and degree of unsaturation of the precursor. Based on these leads, we also report the precursor-directed biosynthesis of 15-propargyl erythromycin A, a novel antibiotic that not only is as potent as erythromycin A with respect to its ability to inhibit bacterial growth and cell-free ribosomal protein biosynthesis but also harbors an orthogonal functional group that is capable of facile chemical modification.
Co-reporter:Fong T Wong, Chaitan Khosla
Current Opinion in Chemical Biology 2012 Volume 16(1–2) pp:117-123
Publication Date(Web):April 2012
DOI:10.1016/j.cbpa.2012.01.018
Since their discovery, polyketide synthases have been attractive targets of biosynthetic engineering to make ‘unnatural’ natural products. Although combinatorial biosynthesis has made encouraging advances over the past two decades, the field remains in its infancy. In this enzyme-centric perspective, we discuss the scientific and technological challenges that could accelerate the adoption of combinatorial biosynthesis as a method of choice for the preparation of encoded libraries of bioactive small molecules. Borrowing a page from the protein structure prediction community, we propose a periodic challenge program to vet the most promising methods in the field, and to foster the collective development of useful tools and algorithms.Graphical abstractHighlights► New PKS gene clusters are being identified at an explosive rate. ► Understanding of PKS chemistry has improved, but its implications remain untested. ► DNA assembly of hybrid PKSs is easier than before, but heterologous expression and product analysis remain challenging. ► A CASP-like competition could propel combinatorial biosynthesis into mainstream engineering.
Co-reporter:Satoshi Yuzawa, Shiven Kapur, David E. Cane, and Chaitan Khosla
Biochemistry 2012 Volume 51(Issue 18) pp:3708-3710
Publication Date(Web):April 17, 2012
DOI:10.1021/bi300399u
The role of interdomain linkers in modular polyketide synthases is poorly understood. Analysis of the 6-deoxyerythronolide B synthase (DEBS) has yielded a model in which chain elongation is governed by interactions between the acyl carrier protein domain and the ketosynthase domain plus an adjacent linker. Alanine scanning mutagenesis of the conserved residues of this linker in DEBS module 3 led to the identification of the R513A mutant with a markedly reduced rate of chain elongation. Limited proteolysis supported a structural role for this Arg. Our findings highlight the importance of domain–linker interactions in assembly line polyketide biosynthesis.
Co-reporter:Louise K. Charkoudian, Bailey P. Farrell and Chaitan Khosla
MedChemComm 2012 vol. 3(Issue 8) pp:926-931
Publication Date(Web):23 Apr 2012
DOI:10.1039/C2MD20008B
Inhibitors of glucose-6-phosphate translocase (G6P T1) control aberrant glucose levels and show promise as anticancer and anti-diabetic agents. This mini-review provides a concise overview of natural product and natural product analogs that inhibit G6P T1. The discovery and development of these inhibitors, as well as their efficacy in cell-based assays and in vivo, are discussed. Finally, we acknowledge the need for improved G6P T1 inhibitors and the potential to harness the programmable chemistry of microorganisms to meet this need.
Co-reporter:Dr. Jianhao Wang;Xi Jin;Jiahui Liu; Chaitan Khosla; Jiang Xia
Chemistry – An Asian Journal 2012 Volume 7( Issue 5) pp:992-999
Publication Date(Web):
DOI:10.1002/asia.201101041
Abstract
Techniques that can effectively separate protein–peptide complexes from free peptides have shown great value in major histocompatibility complex (MHC)–peptide binding studies. However, most of the available techniques are limited to measuring the binding of a single peptide to an MHC molecule. As antigen presentation in vivo involves both endogenous ligands and exogenous antigens, the deconvolution of multiple binding events necessitates the implementation of a more powerful technique. Here we show that capillary electrophoresis coupled to fluorescence detection (CE–FL) can resolve multiple MHC–peptide binding events owing to its superior resolution and the ability to simultaneously monitor multiple emission channels. We utilized CE–FL to investigate competition and displacement of endogenous peptides by an immunogenic gluten peptide for binding to HLA-DQ2. Remarkably, this immunogenic peptide could displace CLIP peptides from the DQ2 binding site at neutral but not acidic pH. This unusual ability of the gluten peptide supports a direct loading mechanism of antigen presentation in extracellular environment, a property that could explain the antigenicity of dietary gluten in celiac disease.
Co-reporter:Shiven Kapur;Brian Lowry;Satoshi Yuzawa;Sanketha Kenthirapalan;Alice Y. Chen;David E. Cane
PNAS 2012 109 (11 ) pp:
Publication Date(Web):2012-03-13
DOI:10.1073/pnas.1118734109
Multimodular polyketide synthases (PKSs) have an assembly line architecture in which a set of protein domains, known as a
module, participates in one round of polyketide chain elongation and associated chemical modifications, after which the growing
chain is translocated to the next PKS module. The ability to rationally reprogram these assembly lines to enable efficient
synthesis of new polyketide antibiotics has been a long-standing goal in natural products biosynthesis. We have identified
a ratchet mechanism that can explain the observed unidirectional translocation of the growing polyketide chain along the 6-deoxyerythronolide
B synthase. As a test of this model, module 3 of the 6-deoxyerythronolide B synthase has been reengineered to catalyze two
successive rounds of chain elongation. Our results suggest that high selectivity has been evolutionarily programmed at three
types of protein–protein interfaces that are present repetitively along naturally occurring PKS assembly lines.
Co-reporter:Cornelius Klöck;Thomas R. DiRaimondo
Seminars in Immunopathology 2012 Volume 34( Issue 4) pp:513-522
Publication Date(Web):2012 July
DOI:10.1007/s00281-012-0305-0
A number of lines of evidence suggest that transglutaminase 2 (TG2) may be one of the earliest disease-relevant proteins to encounter immunotoxic gluten in the celiac gut. These and other investigations also suggest that the reaction catalyzed by TG2 on dietary gluten peptides is essential for the pathogenesis of celiac disease. If so, several questions are of critical significance. How is TG2 activated in the celiac gut? What are the disease-specific and general consequences of activating TG2? Can local inhibition of TG2 in the celiac intestine suppress gluten induced pathogenesis in a dose-responsive manner? And what are the long-term consequences of suppressing TG2 activity in the small intestinal mucosa? Answers to these questions will depend upon the development of judicious models and chemical tools. They also have the potential of yielding powerful next-generation drug candidates for treating this widespread but overlooked chronic disease.
Co-reporter:Ping-Hui Szu, Sridhar Govindarajan, Michael J. Meehan, Abhirup Das, Don D. Nguyen, Pieter C. Dorrestein, Jeremy Minshull, Chaitan Khosla
Chemistry & Biology 2011 Volume 18(Issue 8) pp:1021-1031
Publication Date(Web):26 August 2011
DOI:10.1016/j.chembiol.2011.07.015
The pentadecaketide fredericamycin has the longest carbon chain backbone among polycyclic aromatic polyketide antibiotics whose biosynthetic genes have been sequenced. This backbone is synthesized by the bimodular fdm polyketide synthase (PKS). Here, we demonstrate that the bimodular fdm PKS as well as its elongation module alone synthesize undecaketides and dodecaketides. Thus, unlike other homologs, the fdm ketosynthase-chain length factor (KS-CLF) heterodimer does not exclusively control the backbone length of its natural product. Using sequence- and structure-based approaches, 48 CLF multiple mutants were engineered and analyzed. Unexpectedly, the I134F mutant was unable to turn over but could initiate and partially elongate the polyketide chain. This unprecedented mutant suggests that the KS-CLF heterodimer harbors an as yet uncharacterized chain termination mechanism. Together, our findings reveal fundamental mechanistic differences between the fdm PKS and its well-studied homologs.Graphical AbstractFigure optionsDownload full-size imageDownload high-quality image (177 K)Download as PowerPoint slideHighlights► The fdm PKS synthesizes dodecaketides, shorter than the natural product backbone ► Therefore, its chain length specificity must be modulated by an unknown factor ► We have identified a PKS mutant that can initiate and extend, but not terminate ► This suggests that the KS-CLF has an uncharacterized chain termination mechanism
Co-reporter:Laila Dafik, Chaitan Khosla
Chemistry & Biology 2011 Volume 18(Issue 1) pp:58-66
Publication Date(Web):28 January 2011
DOI:10.1016/j.chembiol.2010.11.004
We report the synthesis and preliminary characterization of “clickable” inhibitors of human transglutaminase 2 (TG2). These inhibitors possess the 3-halo-4,5-dihydroisoxazole warhead along with bioorthogonal groups such as azide or alkyne moieties that enable subsequent covalent modification with fluorophores. Their mechanism for inhibition of TG2 is based on halide displacement, resulting in the formation of a stable imino thioether. Inhibition assays against recombinant human TG2 revealed that some of the clickable inhibitors prepared in this study have comparable specificity as benchmark dihydroisoxazole inhibitors reported earlier. At low micromolar concentrations they completely inhibited transiently activated TG2 in a WI-38 fibroblast scratch assay and could subsequently be used to visualize the active enzyme in situ. The potential use of these inhibitors to probe the role of TG2 in celiac sprue as well as other diseases is discussed.Graphical AbstractFigure optionsDownload full-size imageDownload high-quality image (145 K)Download as PowerPoint slideHighlights► Nonradioactive small molecule probes for imaging catalytically active transglutaminase 2 (TG2) ► Electrophilic dihydroisoxazole compounds with alkyne and azide groups further modifiable by “click chemistry” ► In cell culture, these compounds completely inhibited transiently activated TG2
Co-reporter:Younjoo Lee ; Jun Yong Choi ; Hong Fu ; Colin Harvey ; Sandeep Ravindran ; William R. Roush ; John C. Boothroyd
Journal of Medicinal Chemistry 2011 Volume 54(Issue 8) pp:2792-2804
Publication Date(Web):March 23, 2011
DOI:10.1021/jm101593u
Macrolide antibacterial agents inhibit parasite proliferation by targeting the apicoplast ribosome. Motivated by the long-term goal of identifying antiparasitic macrolides that lack antibacterial activity, we have systematically analyzed the structure−activity relationships among erythromycin analogues and have also investigated the mechanism of action of selected compounds. Two lead compounds, N-benzylazithromycin (11) and N-phenylpropylazithromycin (30), were identified with significantly higher antiparasitic activity and lower antibacterial activity than erythromycin or azithromycin. Molecular modeling based on the cocrystal structure of azithromycin bound to the bacterial ribosome suggested that a substituent at the N-9 position of desmethylazithromycin could improve selectivity because of species-specific interactions with the ribosomal L22 protein. Like other macrolides, these lead compounds display a strong “delayed death phenotype”; however, their early effects on T. gondii replication are more pronounced.
Co-reporter:Fong T. Wong, Xi Jin, Irimpan I. Mathews, David E. Cane, and Chaitan Khosla
Biochemistry 2011 Volume 50(Issue 30) pp:
Publication Date(Web):June 27, 2011
DOI:10.1021/bi200632j
The 1.51 Å resolution X-ray crystal structure of the trans-acyltransferase (AT) from the “AT-less” disorazole synthase (DSZS) and that of its acetate complex at 1.35 Å resolution are reported. Separately, comprehensive alanine-scanning mutagenesis of one of its acyl carrier protein substrates (ACP1 from DSZS) led to the identification of a conserved Asp45 residue on the ACP, which contributes to the substrate specificity of this unusual enzyme. Together, these experimental findings were used to derive a model for the selective association of the DSZS AT and its ACP substrate. With a goal of structurally characterizing the AT–ACP interface, a strategy was developed for covalently cross-linking the active site Ser → Cys mutant of the DSZS AT to its ACP substrate and for purifying the resulting AT–ACP complex to homogeneity. The S86C DSZS AT mutant was found to be functional, albeit with a transacylation efficiency 200-fold lower than that of its wild-type counterpart. Our findings provide new insights as well as new opportunities for high-resolution analysis of an important protein–protein interface in polyketide synthases.
Co-reporter:Cornelius Klöck, Xi Jin, Kihang Choi, Chaitan Khosla, Peter B. Madrid, Andrew Spencer, Brian C. Raimundo, Paul Boardman, Guido Lanza, John H. Griffin
Bioorganic & Medicinal Chemistry Letters 2011 Volume 21(Issue 9) pp:2692-2696
Publication Date(Web):1 May 2011
DOI:10.1016/j.bmcl.2010.12.037
Inhibitors of human transglutaminase 2 (TG2) are anticipated to be useful in the therapy of a variety of diseases including celiac sprue as well as certain CNS disorders and cancers. A class of 3-acylidene-2-oxoindoles was identified as potent reversible inhibitors of human TG2. Structure–activity relationship analysis of a lead compound led to the generation of several potent, competitive inhibitors. Analogs with significant non-competitive character were also identified, suggesting that the compounds bind at one or more allosteric regulatory sites on this multidomain enzyme. The most active compounds had Ki values below 1.0 μM in two different kinetic assays for human TG2, and may therefore be suitable for investigations into the role of TG2 in physiology and disease in animals.
Co-reporter:Xingye Yu;Tiangang Liu;Fayin Zhu
PNAS 2011 Volume 108 (Issue 46 ) pp:
Publication Date(Web):2011-11-15
DOI:10.1073/pnas.1110852108
Microbial fatty acid derivatives are emerging as promising alternatives to fossil fuel derived transportation fuels. Among
bacterial fatty acid synthases (FAS), the Escherichia coli FAS is perhaps the most well studied, but little is known about its steady-state kinetic behavior. Here we describe the reconstitution
of E. coli FAS using purified protein components and report detailed kinetic analysis of this reconstituted system. When all ketosynthases
are present at 1 μM, the maximum rate of free fatty acid synthesis of the FAS exceeded 100 μM/ min. The steady-state turnover
frequency was not significantly inhibited at high concentrations of any substrate or cofactor. FAS activity was saturated
with respect to most individual protein components when their concentrations exceeded 1 μM. The exceptions were FabI and FabZ,
which increased FAS activity up to concentrations of 10 μM; FabH and FabF, which decreased FAS activity at concentrations
higher than 1 μM; and holo-ACP and TesA, which gave maximum FAS activity at 30 μM concentrations. Analysis of the S36T mutant of the ACP revealed that
the unusual dependence of FAS activity on holo-ACP concentration was due, at least in part, to the acyl-phosphopantetheine moiety. MALDI-TOF mass spectrometry analysis
of the reaction mixture further revealed medium and long chain fatty acyl-ACP intermediates as predominant ACP species. We
speculate that one or more of such intermediates are key allosteric regulators of FAS turnover. Our findings provide a new
basis for assessing the scope and limitations of using E. coli as a biocatalyst for the production of diesel-like fuels.
Co-reporter:Jay T Fitzgerald, Christian P Ridley and Chaitan Khosla
The Journal of Antibiotics 2011 64(12) pp:759-762
Publication Date(Web):September 21, 2011
DOI:10.1038/ja.2011.86
The polyketide antibiotic frenolicin B harbors a biosynthetically intriguing benzoisochromanequinone core, and has been shown to exhibit promising antiparasitic activity against Eimeria tenella. To facilitate further exploration of its chemistry and biology, we constructed a biosynthetic route to frenolicin B in the heterologous host Streptomyces coelicolor CH999, despite the absence of key enzymes in the identified frenolicin gene cluster. Together with our understanding of the underlying polyketide biosynthetic pathway, this heterologous production system was exploited to produce analogs modified at the C15 position. Both the natural product and these analogs inhibited the growth of Toxoplasma gondii in a manner that reveals sensitivity to the length of the C15 substituent. The ability to construct a functional biosynthetic pathway, despite a lack of genetic information, illustrates the feasibility of a modular approach to engineering medicinally relevant polyketide products.
Co-reporter:Ho Young Lee, Colin J B Harvey, David E Cane and Chaitan Khosla
The Journal of Antibiotics 2011 64(1) pp:59-64
Publication Date(Web):November 17, 2010
DOI:10.1038/ja.2010.129
Erythromycin and related macrolide antibiotics are widely used polyketide natural products. We have evolved an engineered biosynthetic pathway in Escherichia coli that yields erythromycin analogs from simple synthetic precursors. Multiple rounds of mutagenesis and screening led to the identification of new mutant strains with improved efficiency for precursor-directed biosynthesis. Genetic and biochemical analysis suggested that the phenotypically relevant alterations in these mutant strains were localized exclusively to the host-vector system, and not to the polyketide synthase. We also demonstrate the utility of this improved system through engineered biosynthesis of a novel alkynyl erythromycin derivative with comparable antibacterial activity to its natural counterpart. In addition to reinforcing the power of directed evolution for engineering macrolide biosynthesis, our studies have identified a new lead substance for investigating structure–function relationships in the bacterial ribosome.
Co-reporter:Abhirup Das ; Ping-Hui Szu ; Jay T. Fitzgerald
Journal of the American Chemical Society 2010 Volume 132(Issue 26) pp:8831-8833
Publication Date(Web):June 11, 2010
DOI:10.1021/ja102517q
The ability to incorporate atypical primer units through the use of dedicated initiation polyketide synthase (PKS) modules offers opportunities to expand the molecular diversity of polyketide natural products. Here we identify the initiation PKS module responsible for hexadienyl priming of the antibiotic fredericamycin and investigate its biochemical properties. We also exploit this PKS module for the design and in vivo biosynthesis of unusually primed analogues of a representative polyketide product, thereby emphasizing its utility to the metabolic engineer.
Co-reporter:Kathia Zaleta-Rivera ; Louise K. Charkoudian ; Christian P. Ridley
Journal of the American Chemical Society 2010 Volume 132(Issue 26) pp:9122-9128
Publication Date(Web):June 15, 2010
DOI:10.1021/ja102519v
A-74528 is a recently discovered natural product of Streptomyces sp. SANK 61196 that inhibits 2′,5′-oligoadenylate phosphodiesterase (2′-PDE), a key regulatory enzyme of the interferon pathway. Inhibition of 2′-PDE by A-74528 reduces viral replication, and therefore shows promise as a new type of antiviral drug. The complete A-74528 gene cluster, comprising 29 open reading frames, was cloned and sequenced, and shown to possess a type II polyketide synthase (PKS) at its core. Its identity was confirmed by analysis of a mutant generated by targeted disruption of a PKS gene, and by functional expression in a heterologous Streptomyces host. Remarkably, it showed exceptional end-to-end sequence identity to the gene cluster responsible for biosynthesis of fredericamycin A, a structurally unrelated antitumor antibiotic with a distinct mode of action. Whereas the fredericamycin producing strain, Streptomyces griseus, produced undetectable quantities of A-74528, the A-74528 gene cluster was capable of producing both antibiotics. The biosynthetic roles of three genes, including one that represents the only qualitative difference between the two gene clusters, were investigated by targeted gene disruption. The implications for the evolution of antibiotics with different biological activities from the same gene cluster are discussed.
Co-reporter:Fong T. Wong, Alice Y. Chen, David E. Cane and Chaitan Khosla
Biochemistry 2010 Volume 49(Issue 1) pp:
Publication Date(Web):November 18, 2009
DOI:10.1021/bi901826g
Acyltransferase (AT) domains of multimodular polyketide synthases are the primary gatekeepers for stepwise incorporation of building blocks into a growing polyketide chain. Each AT domain has two substrates, an α-carboxylated CoA thioester (e.g., malonyl-CoA or methylmalonyl-CoA) and an acyl carrier protein (ACP). Whereas the acyl-CoA specificity of AT domains has been extensively investigated, little is known about their ACP specificity. Guided by recent high-resolution structural insights, we have systematically probed the protein−protein interactions between AT domains, ACP domains, and the linkers that flank AT domains. Representative AT domains of the 6-deoxyerythronolide B synthase (DEBS) have greater than 10-fold specificity for their cognate ACP substrates as compared to other ACP domains from the same synthase. Both of the flanking (N- and C-terminal) linkers of an AT domain contributed to the efficiency and specificity of transacylation. As a frame of reference, the activity and specificity of a stand-alone AT domain from the “AT-less” disorazole synthase (DSZS) were also quantified. The activity (kcat/KM) of this AT was >250-fold higher than the corresponding values for DEBS AT domains. Although the AT from DSZS discriminated modestly against ACP domains from DEBS, it exhibited >40-fold higher activity in trans in the presence of these heterologous substrates than their natural AT domains. Our results highlight the opportunity for regioselective modification of a polyketide backbone by in trans complementation of inactivated AT domains. They also reinforce the need for more careful consideration of protein−protein interactions in the engineering of these assembly line enzymes.
Co-reporter:Shiven Kapur;Alice Y. Chen;David E. Cane
PNAS 2010 Volume 107 (Issue 51 ) pp:22066-22071
Publication Date(Web):2010-12-21
DOI:10.1073/pnas.1014081107
Every polyketide synthase module has an acyl carrier protein (ACP) and a ketosynthase (KS) domain that collaborate to catalyze
chain elongation. The same ACP then engages the KS domain of the next module to facilitate chain transfer. Understanding the
mechanism for this orderly progress of the growing polyketide chain represents a fundamental challenge in assembly line enzymology.
Using both experimental and computational approaches, the molecular basis for KS–ACP interactions in the 6-deoxyerythronolide
B synthase has been decoded. Surprisingly, KS–ACP recognition is controlled at different interfaces during chain elongation
versus chain transfer. In fact, chain elongation is controlled at a docking site remote from the catalytic center. Not only
do our findings reveal a new principle in the modular control of polyketide antibiotic biosynthesis, they also provide a rationale
for the mandatory homodimeric structure of polyketide synthases, in contrast to the monomeric nonribosomal peptide synthetases.
Co-reporter:Abhirup Das and Chaitan Khosla
Accounts of Chemical Research 2009 Volume 42(Issue 5) pp:631
Publication Date(Web):March 17, 2009
DOI:10.1021/ar8002249
Natural products, produced chiefly by microorganisms and plants, can be large and structurally complex molecules. These molecules are manufactured by cellular assembly lines, in which enzymes construct the molecules in a stepwise fashion. The means by which enzymes interact and work together in a modular fashion to create diverse structural features has been an active area of research; the work has provided insight into the fine details of biosynthesis. A number of polycyclic aromatic natural products—including several noteworthy anticancer, antibacterial, antifungal, antiviral, antiparasitic, and other medicinally significant substances—are synthesized by polyketide synthases (PKSs) in soil-borne bacteria called actinomycetes. Concerted biosynthetic, enzymological, and structural biological investigations into these modular enzyme systems have yielded interesting mechanistic insights. A core module called the minimal PKS is responsible for synthesizing a highly reactive, protein-bound poly-β-ketothioester chain. In the absence of other enzymes, the minimal PKS also catalyzes chain initiation and release, yielding an assortment of polycyclic aromatic compounds. In the presence of an initiation PKS module, polyketide backbones bearing additional alkyl, alkenyl, or aryl primer units are synthesized, whereas a range of auxiliary PKS enzymes and tailoring enzymes convert the product of the minimal PKS into the final natural product. In this Account, we summarize the knowledge that has been gained regarding this family of PKSs through recent investigations into the biosynthetic pathways of two natural products, actinorhodin and R1128 (A-D). We also discuss the practical relevance of these fundamental insights for the engineered biosynthesis of new polycyclic aromatic compounds. With a deeper understanding of the biosynthetic process in hand, we can assert control at various stages of molecular construction and thus introduce unnatural functional groups in the process. The metabolic engineer affords a number of new avenues for creating novel molecular structures that will likely have properties akin to their fully natural cousins.
Co-reporter:Chaitan Khosla, Shiven Kapur, David E Cane
Current Opinion in Chemical Biology 2009 Volume 13(Issue 2) pp:135-143
Publication Date(Web):April 2009
DOI:10.1016/j.cbpa.2008.12.018
Modularity is a highly sought after feature in engineering design. A modular catalyst is a multi-component system whose parts can be predictably interchanged for functional flexibility and variety. Nearly two decades after the discovery of the first modular polyketide synthase (PKS), we critically assess PKS modularity in the face of a growing body of atomic structural and in vitro biochemical investigations. Both the architectural modularity and the functional modularity of this family of enzymatic assembly lines are reviewed, and the fundamental challenges that lie ahead for the rational exploitation of their full biosynthetic potential are discussed.
Co-reporter:Michael T. Bethune, Mónica Crespo-Bosque, Elin Bergseng, Kaushiki Mazumdar, Lara Doyle, Karol Sestak, Ludvig M. Sollid, Chaitan Khosla
Chemistry & Biology 2009 Volume 16(Issue 8) pp:868-881
Publication Date(Web):28 August 2009
DOI:10.1016/j.chembiol.2009.07.009
New tools are needed for managing celiac sprue, a lifelong immune disease of the small intestine. Ongoing drug trials are also prompting a search for noninvasive biomarkers of gluten-induced intestinal change. We have synthesized and characterized noninflammatory gluten peptide analogs in which key Gln residues are replaced by Asn or His. Like their proinflammatory counterparts, these biomarkers are resistant to gastrointestinal proteases, susceptible to glutenases, and permeable across enterocyte barriers. Unlike gluten peptides, however, they are not appreciably recognized by transglutaminase, HLA-DQ2, or disease-specific T cells. In vitro and animal studies show that the biomarkers can detect intestinal permeability changes as well as glutenase-catalyzed gastric detoxification of gluten. Accordingly, controlled clinical studies are warranted to evaluate the use of these peptides as probes for abnormal intestinal permeability in celiac patients and for glutenase efficacy in clinical trials and practice.
Co-reporter:Abhirup Das, Chaitan Khosla
Chemistry & Biology 2009 Volume 16(Issue 11) pp:1197-1207
Publication Date(Web):25 November 2009
DOI:10.1016/j.chembiol.2009.11.005
Hedamycin is an antitumor polyketide antibiotic with unusual biosynthetic features. Earlier sequence analysis of the hedamycin biosynthetic gene cluster implied a role for type I and type II polyketide synthases (PKSs). We demonstrate that the hedamycin minimal PKS can synthesize a dodecaketide backbone. The ketosynthase (KS) subunit of this PKS has specificity for both type I and type II acyl carrier proteins (ACPs) with which it collaborates during chain initiation and chain elongation, respectively. The KS receives a C6 primer unit from the terminal ACP domain of HedU (a type I PKS protein) directly and subsequently interacts with the ACP domain of HedE (a type II PKS protein) during the process of chain elongation. HedE is a bifunctional protein with both ACP and aromatase activity. Its aromatase domain can modulate the chain length specificity of the minimal PKS. Chain length can also be influenced by HedA, the C-9 ketoreductase. While co-expression of the hedamycin minimal PKS and a chain-initiation module from the R1128 PKS yields an isobutyryl-primed decaketide, the orthologous PKS subunits from the hedamycin gene cluster itself are unable to prime the minimal PKS with a nonacetyl starter unit. Our findings provide new insights into the mechanism of chain initiation and elongation by type II PKSs.
Co-reporter:Shiven Kapur, Andrew Worthington, Yinyan Tang, David E. Cane, Michael D. Burkart, Chaitan Khosla
Bioorganic & Medicinal Chemistry Letters 2008 Volume 18(Issue 10) pp:3034-3038
Publication Date(Web):15 May 2008
DOI:10.1016/j.bmcl.2008.01.073
The critical role of protein–protein interactions in the chemistry of polyketide synthases is well established. However, the transient and weak nature of these interactions, in particular those involving the acyl carrier protein (ACP), has hindered efforts to structurally characterize these interactions. We describe a chemo-enzymatic approach that crosslinks the active sites of ACP and their cognate ketosynthase (KS) domains, resulting in the formation of a stable covalent adduct. This process is driven by specific protein–protein interactions between KS and ACP domains. Suitable manipulation of the reaction conditions enabled complete crosslinking of a representative KS and ACP, allowing isolation of a stable, conformationally constrained adduct suitable for high-resolution structural analysis.
Co-reporter:Christian P. Ridley;Ho Young Lee
PNAS 2008 Volume 105 (Issue 12 ) pp:4595-4600
Publication Date(Web):2008-03-25
DOI:10.1073/pnas.0710107105
The emergence of resistant strains of human pathogens to current antibiotics, along with the demonstrated ability of polyketides
as antimicrobial agents, provides strong motivation for understanding how polyketide antibiotics have evolved and diversified
in nature. Insights into how bacterial polyketide synthases (PKSs) acquire new metabolic capabilities can guide future laboratory
efforts in generating the next generation of polyketide antibiotics. Here, we examine phylogenetic and structural evidence
to glean answers to two general questions regarding PKS evolution. How did the exceptionally diverse chemistry of present-day
PKSs evolve? And what are the take-home messages for the biosynthetic engineer?
Co-reporter:Yinyan Tang, Alice Y. Chen, Chu-Young Kim, David E. Cane, Chaitan Khosla
Chemistry & Biology 2007 Volume 14(Issue 8) pp:931-943
Publication Date(Web):24 August 2007
DOI:10.1016/j.chembiol.2007.07.012
We report the 2.6 Å X-ray crystal structure of a 190 kDa homodimeric fragment from module 3 of the 6-deoxyerthronolide B synthase covalently bound to the inhibitor cerulenin. The structure shows two well-organized interdomain linker regions in addition to the full-length ketosynthase (KS) and acyltransferase (AT) domains. Analysis of the substrate-binding site of the KS domain suggests that a loop region at the homodimer interface influences KS substrate specificity. We also describe a model for the interaction of the catalytic domains with the acyl carrier protein (ACP) domain. The ACP is proposed to dock within a deep cleft between the KS and AT domains, with interactions that span both the KS homodimer and AT domain. In conjunction with other recent data, our results provide atomic resolution pictures of several catalytically relevant protein interactions in this remarkable family of modular megasynthases.
Co-reporter:Alice Y. Chen, David E. Cane, Chaitan Khosla
Chemistry & Biology 2007 Volume 14(Issue 7) pp:784-792
Publication Date(Web):30 July 2007
DOI:10.1016/j.chembiol.2007.05.015
Individual modules of modular polyketide synthases (PKSs) such as 6-deoxyerythronolide B synthase (DEBS) consist of conserved, covalently linked domains separated by unconserved intervening linker sequences. To better understand the protein-protein and enzyme-substrate interactions in modular catalysis, we have exploited recent structural insights to prepare stand-alone domains of selected DEBS modules. When combined in vitro, ketosynthase (KS), acyl transferase (AT), and acyl carrier protein (ACP) domains of DEBS module 3 catalyzed methylmalonyl transfer and diketide substrate elongation. When added to a minimal PKS, ketoreductase domains from DEBS modules 1, 2, and 6 showed specificity for the β-ketoacylthioester substrate, but not for either the ACP domain carrying the polyketide substrate or the KS domain that synthesized the substrate. With insights into catalytic efficiency and specificity of PKS modules, our results provide guidelines for constructing optimal hybrid PKS systems.
Co-reporter:Christian P. Ridley and Chaitan Khosla
ACS Chemical Biology 2007 Volume 2(Issue 2) pp:104
Publication Date(Web):January 26, 2007
DOI:10.1021/cb600382j
The 4-hydroxy-2-pyrone moiety is commonly observed in polyketides generated via biosynthetic engineering of type II polyketide synthases. To explore the synthetic utility of these 2-pyrones, four engineered polyketides (mutactin, SEK4, SEK15, and SEK15b) were isolated from appropriate derivatives of Streptomyces coelicolor CH999. As a test case, we prepared nine novel pyranopyrones through condensation reactions with either citral, 1-cyclohexene-carboxaldehyde, or S-perillaldehyde. Synthetic tricyclic pyranopyrones with simple aromatic substituents are known to possess anticancer properties. We therefore investigated whether pyranopyrone derivatives of aromatic polyketides exhibited bioactivity in a panel of three cancer cell lines. Pyranopyrones generated from SEK4 had activity comparable to that of H10, a pyranopyrone with a 3-pyridyl substituent, whereas other analogues were less active. These results suggest that the diverse library of carbo- and heterocycles available through the genetic engineering of type II polyketide synthases can serve as useful building blocks to generate unique bioactive molecules.
Co-reporter:Jiang Xia, Elin Bergseng, Burkhard Fleckenstein, Matthew Siegel, Chu-Young Kim, Chaitan Khosla, Ludvig M. Sollid
Bioorganic & Medicinal Chemistry 2007 Volume 15(Issue 20) pp:6565-6573
Publication Date(Web):15 October 2007
DOI:10.1016/j.bmc.2007.07.001
Celiac disease is an immune mediated enteropathy elicited by gluten ingestion. The disorder has a strong association with HLA-DQ2. This HLA molecule is involved in the disease pathogenesis by presenting gluten peptides to T cells. Blocking the peptide-binding site of DQ2 may be a way to treat celiac disease. In this study, two types of peptide analogues, modeled after natural gluten antigens, were studied as DQ2 blockers. (a) Cyclic peptides. Cyclic peptides containing the DQ2-αI gliadin epitope LQPFPQPELPY were synthesized with flanking cysteine residues introduced and subsequently crosslinked via a disulfide bond. Alternatively, cyclic peptides were prepared with stable polyethylene glycol bridges across internal lysine residues of modified antigenic peptides such as KQPFPEKELPY and LQLQPFPQPEKPYPQPEKPY. The effect of cyclization as well as the length of the spacer in the cyclic peptides on DQ2 binding and T cell recognition was analyzed. Inhibition of peptide-DQ2 recognition by the T cell receptor was observed in T cell proliferation assays. (b) Dimeric peptides. Previously we developed a new type of peptide blocker with much enhanced affinity for DQ2 by dimerizing LQLQPFPQPEKPYPQPELPY through the lysine side chains. Herein, the effect of linker length on both DQ2 binding and T cell inhibition was investigated. One dimeric peptide analogue with an intermediate linker length was found to be especially effective at inhibiting DQ2 mediated antigen presentation. The implications of these findings for the treatment of celiac disease are discussed.Peptide cyclization and dimerization turned a potent gluten antigen into effective blockers of HLA-DQ2, an attractive target for celiac disease therapy.
Co-reporter:Matthew Siegel, Jiang Xia, Chaitan Khosla
Bioorganic & Medicinal Chemistry 2007 Volume 15(Issue 18) pp:6253-6261
Publication Date(Web):15 September 2007
DOI:10.1016/j.bmc.2007.06.020
Complete, life-long exclusion of gluten containing foods from the diet is the only available treatment for celiac sprue, a widespread immune disease of the small intestine. Investigations into the molecular pathogenesis of celiac sprue have identified the major histocompatibility complex protein HLA-DQ2 and the multi-functional enzyme transglutaminase 2 as potential pharmacological targets. Based upon the crystal structure of HLA-DQ2, we rationally designed an aldehyde-functionalized, gluten peptide analogue as a tight-binding HLA-DQ2 ligand. Aldehyde-bearing gluten peptide analogues were also designed as high-affinity, reversible inhibitors of transglutaminase 2. By varying the side-chain length of the aldehyde-functionalized amino acid, we found that the optimal transglutaminase 2 inhibitor was 5 methylene units in length, 2 carbon atoms longer than its natural glutamine substrate.A series of aldehyde-functionalized gluten peptides were synthesized as inhibitors of HLA-DQ2 and transglutaminase 2, two human proteins that play a critical role in gluten induced pathogenesis of celiac sprue.
Co-reporter:Taek Soon Lee, Abhirup Das, Chaitan Khosla
Bioorganic & Medicinal Chemistry 2007 Volume 15(Issue 15) pp:5207-5218
Publication Date(Web):1 August 2007
DOI:10.1016/j.bmc.2007.05.019
Mumbaistatin (1), a new anthraquinone natural product, is one of the most potent known inhibitors of hepatic glucose-6-phosphate translocase, an important target for the treatment of type II diabetes. Its availability, however, has been limited due to its extremely low yield from the natural source. Starting from DMAC (5, 3,8-dihydroxyanthraquinone-2-carboxylic acid), a structurally related polyketide product of engineered biosynthesis, we developed a facile semisynthetic method that afforded a variety of mumbaistatin analogs within five steps. This work was facilitated by the initial development of a DMAC overproduction system. In addition to reinforcing the biological significance of the anthraquinone moiety of mumbaistatin, several semisynthetic analogs were found to have low micromolar potency against the translocase in vitro. Two of them were also active in glucose release assays from primary hepatocytes. The synergistic combination of biosynthesis and synthesis is a promising avenue for the discovery of new bioactive substances.
Co-reporter:J. Gass;C. Khosla
Cellular and Molecular Life Sciences 2007 Volume 64( Issue 3) pp:345-355
Publication Date(Web):2007 February
DOI:10.1007/s00018-006-6317-y
This review describes the structure and function of prolyl endopeptidase (PEP) enzymes and how they are being evaluated as drug targets and therapeutic agents. The most well studied PEP family has a two-domain structure whose unique seven-blade β-propeller domain works with the catalytic domain to hydrolyze the peptide bond on the carboxyl side of internal proline residues of an oligopeptide substrate. Structural and functional studies on this protease family have elucidated the mechanism for peptide entry between the two domains. Other structurally unrelated PEPs have been identified, but have not been studied in detail. Human PEP has been evaluated as a pharmacological target for neurological diseases due to its high brain concentration and ability to cleave neuropeptides in vitro. Recently, microbial PEPs have been studied as potential therapeutics for celiac sprue, an inflammatory disease of the small intestine triggered by proline-rich gluten.
Co-reporter:Michael T. Bethune, Pavel Strop, Yinyan Tang, Ludvig M. Sollid, Chaitan Khosla
Chemistry & Biology 2006 Volume 13(Issue 6) pp:637-647
Publication Date(Web):June 2006
DOI:10.1016/j.chembiol.2006.04.008
We describe the heterologous expression in Escherichia coli of the proenzyme precursor to EP-B2, a cysteine endoprotease from germinating barley seeds. High yields (50 mg/l) of recombinant proEP-B2 were obtained from E. coli inclusion bodies in shake flask cultures following purification and refolding. The zymogen was rapidly autoactivated to its mature form under acidic conditions at a rate independent of proEP-B2 concentration, suggesting a cis mechanism of autoactivation. Mature EP-B2 was stable and active over a wide pH range and efficiently hydrolyzed a recombinant wheat gluten protein, α2-gliadin, at sequences with known immunotoxicity in celiac sprue patients. The X-ray crystal structure of mature EP-B2 bound to leupeptin was solved to 2.2 Å resolution and provided atomic insights into the observed subsite specificity of the endoprotease. Our findings suggest that orally administered proEP-B2 may be especially well suited for treatment of celiac sprue.
Co-reporter:Matthew Siegel, Michael T. Bethune, Jonathan Gass, Jennifer Ehren, Jiang Xia, Alexandre Johannsen, Tor B. Stuge, Gary M. Gray, Peter P. Lee, Chaitan Khosla
Chemistry & Biology 2006 Volume 13(Issue 6) pp:649-658
Publication Date(Web):June 2006
DOI:10.1016/j.chembiol.2006.04.009
Celiac sprue (also known as celiac disease) is an inheritable, gluten-induced enteropathy of the upper small intestine with an estimated prevalence of 0.5%–1% in most parts of the world. The ubiquitous nature of food gluten, coupled with inadequate labeling regulations in most countries, constantly poses a threat of disease exacerbation and relapse for patients. Here, we demonstrate that a two-enzyme cocktail comprised of a glutamine-specific cysteine protease (EP-B2) that functions under gastric conditions and a PEP, which acts in concert with pancreatic proteases under duodenal conditions, is a particularly potent candidate for celiac sprue therapy. At a gluten:EP-B2:PEP weight ratio of 75:3:1, grocery store gluten is fully detoxified within 10 min of simulated duodenal conditions, as judged by chromatographic analysis, biopsy-derived T cell proliferation assays, and a commercial antigluten antibody test.
Co-reporter:M A Rude and C Khosla
The Journal of Antibiotics 2006 59(8) pp:464-470
Publication Date(Web):
DOI:10.1038/ja.2006.65
For the heterologous production of ansamycin polyketides such as rifamycin and geldanamycin in Escherichia coli, a number of unusual but important tools must be engineered into the bacterium. Here we demonstrate efficient production of the starter unit 3-amino-5-hydroxybenzoic acid (AHBA) and the methoxymalonyl extender unit in E. coli. Previous work has demonstrated the production of the ansamycin starter unit AHBA in E. coli in low yield. It was shown that the low yield is primarily due to acetylation of AHBA into N-acetyl-AHBA. Three methods for minimizing this side reaction were evaluated. First, a putative N-arylamine-acetyltransferase (NAT) was deleted from the E. coli chromosome, although this did not alter N-acetyl-AHBA production. Next, E. coli grown in media devoid of glucose yielded a nearly equal mixture of AHBA and N-acetyl-AHBA. Lastly, the NAT inhibitor glycyrrhizic acid was shown to further inhibit the acetylation reaction. The entire set of genes for synthesizing the methoxymalonyl extender unit was transferred from the geldanamycin producer Streptomyces hygroscopicus into E. coli. The pathway specific ACP isolated from the resulting recombinant strain was found to predominantly occur as methyoxymalonyl-ACP. Together, these findings set the stage for engineered biosynthesis of ansamycin polyketides in E. coli.
Co-reporter:Taek Soon Lee, Chaitan Khosla and Yi Tang
The Journal of Antibiotics 2005 58(10) pp:663-666
Publication Date(Web):
DOI:10.1038/ja.2005.91
The actinomycetes produce antibiotics as well as spore pigments during their life cycle by using Type II polyketide synthases (PKSs). Each PKS minimally consists of a ketosynthase heterodimer and an acyl carrier protein. The acyl carrier protein has been shown to be interchangeable among different antibiotic producing Type II PKSs. Surprisingly, we have discovered a fundamental incompatibility between the ketosynthases and acyl carrier proteins from antibiotic producing pathways and those from spore pigment pathways. Although antibiotic PKSs can interact with acyl carrier proteins from spore pigment pathways, spore pigment PKSs are unable to recognize acyl carrier proteins from polyketide antibiotic pathways. This observation provides an insight into a critical mechanism by which natural product biosynthetic specificity is exercised by members of this bacterial family.
Co-reporter:Lu Shan;Irimpan I. Mathews
PNAS 2005 Volume 102 (Issue 10 ) pp:3599-3604
Publication Date(Web):2005-03-08
DOI:10.1073/pnas.0408286102
Prolyl endopeptidases (PEPs) are a unique class of serine proteases with considerable therapeutic potential for the treatment
of celiac sprue. The crystal structures of two didomain PEPs have been solved in alternative configurations, thereby providing
insights into the mode of action of these enzymes. The structure of the Sphingomonas capsulata PEP, solved and refined to 1.8-Å resolution, revealed an open configuration of the active site. In contrast, the inhibitor-bound
PEP from Myxococcus xanthus was crystallized (1.5-Å resolution) in a closed form. Comparative analysis of the two structures highlights a critical role
for the domain interface in regulating interdomain dynamics and substrate specificity. Structure-based mutagenesis of the
M. xanthus PEP confirms an important role for several interfacial residues. A salt bridge between Arg-572 and Asp-196/Glu-197 appears
to act as a latch for opening or closing the didomain enzyme, and Arg-572 and Ile-575 may also help secure the incoming peptide
substrate to the open form of the enzyme. Arg-618 and Asp-145 are responsible for anchoring the invariant proline residue
in the active site of this postproline-cleaving enzyme. A model is proposed for the docking of a representative substrate
PQPQLPYPQPQLP in the active site, where the N-terminal substrate residues interact extensively with the catalytic domain,
and the C-terminal residues stretch into the propeller domain. Given the promise of the M. xanthus PEP as an oral therapeutic enzyme for treating celiac sprue, our results provide a strong foundation for further optimization
of the PEP's clinically useful features.
Co-reporter:Xuefeng Lu, Harmit Vora, Chaitan Khosla
Metabolic Engineering (November 2008) Volume 10(Issue 6) pp:333-339
Publication Date(Web):1 November 2008
DOI:10.1016/j.ymben.2008.08.006
Whereas microbial fermentation processes for producing ethanol and related alcohol biofuels are well established, biodiesel (methyl esters of fatty acids) is exclusively derived from plant oils. Slow cycle times for engineering oilseed metabolism and the excessive accumulation of glycerol as a byproduct are two major drawbacks of deriving biodiesel from plants. Although most bacteria produce fatty acids as cell envelope precursors, the biosynthesis of fatty acids is tightly regulated at multiple levels. By introducing four distinct genetic changes into the E. coli genome, we have engineered an efficient producer of fatty acids. Under fed-batch, defined media fermentation conditions, 2.5 g/L fatty acids were produced by this metabolically engineered E. coli strain, with a specific productivity of 0.024 g/h/g dry cell mass and a peak conversion efficiency of 4.8% of the carbon source into fatty acid products. At least 50% of the fatty acids produced were present in the free acid form.
Co-reporter:Matthew Siegel, Chaitan Khosla
Pharmacology & Therapeutics (August 2007) Volume 115(Issue 2) pp:232-245
Publication Date(Web):1 August 2007
DOI:10.1016/j.pharmthera.2007.05.003
Transglutaminase 2 (TG2) is a multi-domain, multi-functional enzyme that post-translationally modifies proteins by catalyzing the formation of intermolecular isopeptide bonds between glutamine and lysine side-chains. It plays a role in diverse biological functions, including extracellular matrix formation, integrin-mediated signaling, and signal transduction involving 7-transmembrane receptors. While some of the roles of TG2 under normal physiological conditions remain obscure, the protein is believed to participate in the pathogenesis of several unrelated diseases, including celiac sprue, neurodegenerative diseases, and certain types of cancer. A variety of small molecule and peptidomimetic inhibitors of the TG2 active site have been identified. Here, we summarize the biochemistry, biology, pharmacology and medicinal chemistry of human TG2.
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