Jürgen Rohr

Name: Carmen Méndez

Organization: University of Kentucky

Department: Department of Pharmaceutical Sciences, College of Pharmacy

Title:

Chemicals
References

One-Pot Enzymatic Total Synthesis of Presteffimycinone, an Early Intermediate of the Anthracycline Antibiotic Steffimycin Biosynthesis

Co-reporter:Guojun WangJing Chen, Haining Zhu, Jürgen Rohr

Organic Letters February 3, 2017 Volume 19(Issue 3) pp:

Publication Date(Web):January 19, 2017

DOI:10.1021/acs.orglett.6b03708

Early acting cyclases play critical roles in programming the polyketide biosynthesis toward certain, distinguished scaffolds. Starting from acetyl-CoA and malonyl-CoA, a one-pot enzymatic total synthesis of an anthracyclinone scaffold, presteffimycinone, was achieved by mixing polyketide synthase (PKS) and early post-PKS enzymes from the biosynthetic pathways of three different types of type II-PKS driven anticancer antibiotics, namely, the mithramycin (aureolic acid-type), gilvocarcin (rearranged angucycline-type), and steffimycin (anthracycline) pathways.

Insights into Complex Oxidation during BE-7585A Biosynthesis: Structural Determination and Analysis of the Polyketide Monooxygenase BexE

Co-reporter:David R. Jackson, Xia Yu, Guojung Wang, Avinash B. Patel, Jordi Calveras, Jesus F. Barajas, Eita Sasaki, Mikko Metsä-Ketelä, Hung-wen Liu, Jürgen Rohr, and Shiou-Chuan Tsai

ACS Chemical Biology 2016 Volume 11(Issue 4) pp:1137

Publication Date(Web):January 27, 2016

DOI:10.1021/acschembio.5b00913

Cores of aromatic polyketides are essential for their biological activities. Most type II polyketide synthases (PKSs) biosynthesize these core structures involving the minimal PKS, a PKS-associated ketoreductase (KR) and aromatases/cyclases (ARO/CYCs). Oxygenases (OXYs) are rarely involved. BE-7585A is an anticancer polyketide with an angucyclic core. 13C isotope labeling experiments suggest that its angucyclic core may arise from an oxidative rearrangement of a linear anthracyclinone. Here, we present the crystal structure and functional analysis of BexE, the oxygenase proposed to catalyze this key oxidative rearrangement step that generates the angucyclinone framework. Biochemical assays using various linear anthracyclinone model compounds combined with docking simulations narrowed down the substrate of BexE to be an immediate precursor of aklaviketone, possibly 12-deoxy-aklaviketone. The structural analysis, docking simulations, and biochemical assays provide insights into the role of BexE in BE-7585A biosynthesis and lay the groundwork for engineering such framework-modifying enzymes in type II PKSs.

Synthesis of Psoralidin derivatives and their anticancer activity: first synthesis of Lespeflorin I1

Co-reporter:Pallab Pahari, Ujwal Pratim Saikia, Trinath Prasad Das, Chendil Damodaran, Jürgen Rohr

Tetrahedron 2016 Volume 72(Issue 23) pp:3324-3334

Publication Date(Web):9 June 2016

DOI:10.1016/j.tet.2016.04.066

Synthetic scheme for the preparation of a number of different derivatives of anticancer natural product Psoralidin is described. A convergent synthetic approach is followed using simple starting materials like substituted phenyl acetic esters and benzoic acids. The developed synthetic route leads us to complete the first synthesis of an analogous natural product Lespeflorin I1, a mild melanin synthesis inhibitor. Preliminary bioactivity studies of the synthesized compounds are carried out against two commonly used prostate cancer cell lines. Results show that the bioactivity of the compounds can be manipulated by the simple modification of the functional groups.

Structural Insight into MtmC, a Bifunctional Ketoreductase-Methyltransferase Involved in the Assembly of the Mithramycin Trisaccharide Chain

Co-reporter:Jhong-Min Chen, Caixia Hou, Guojun Wang, Oleg V. Tsodikov, and Jürgen Rohr

Biochemistry 2015 Volume 54(Issue 15) pp:2481-2489

Publication Date(Web):January 14, 2015

DOI:10.1021/bi501462g

More and more post-PKS tailoring enzymes are recognized as being multifunctional and codependent on other tailoring enzymes. One of the recently discovered intriguing examples is MtmC, a bifunctional TDP-4-keto-d-olivose ketoreductase-methyltransferase, which—in codependence with glycosyltransferase MtmGIV—is a key contributor to the biosynthesis of the critical trisaccharide chain of the antitumor antibiotic mithramycin (MTM), produced by Streptomyces argillaceus. We report crystal structures of three binary complexes of MtmC with its methylation cosubstrate SAM, its coproduct SAH, and a nucleotide TDP as well as crystal structures of two ternary complexes, MtmC-SAH-TDP-4-keto-d-olivose and MtmC-SAM-TDP, in the range of 2.2–2.7 Å resolution. The structures reveal general and sugar-specific recognition and catalytic structural features of MtmC. Depending on the catalytic function that is conducted by MtmC, it must bind either NADPH or SAM in the same cofactor binding pocket. A tyrosine residue (Tyr79) appears as a lid covering the sugar moiety of the substrate during the methyl transfer reaction. This residue swings out of the active site by ∼180° in the absence of the substrate. This unique conformational change likely serves to release the methylated product and, possibly, to open the active site for binding the bulkier cosubstrate NADPH prior to the reduction reaction.

Microbispora sp. LGMB259 Endophytic Actinomycete Isolated from Vochysia divergens (Pantanal, Brazil) Producing β-Carbolines and Indoles with Biological Activity

Co-reporter:Daiani C. Savi;Khaled A. Shaaban;Nathalia Vargas

Current Microbiology 2015 Volume 70( Issue 3) pp:345-354

Publication Date(Web):2015 March

DOI:10.1007/s00284-014-0724-3

Endophytic actinomycetes encompass bacterial groups that are well known for the production of a diverse range of secondary metabolites. Vochysia divergens is a medicinal plant, common in the “Pantanal” region (Brazil) and was focus of many investigations, but never regarding its community of endophytic symbionts. During a screening program, an endophytic strain isolated from the V. divergens, was investigated for its potential to show biological activity. The strain was characterized as Microbispora sp. LGMB259 by spore morphology and molecular analyze using nucleotide sequence of the 16S rRNA gene. Strain LGMB259 was cultivated in R5A medium producing metabolites with significant antibacterial activity. The strain produced 4 chemically related β-carbolines, and 3 Indoles. Compound 1-vinyl-β-carboline-3-carboxylic acid displayed potent activity against the Gram-positive bacterial strains Micrococcus luteus NRRL B-2618 and Kocuria rosea B-1106, and was highly active against two human cancer cell lines, namely the prostate cancer cell line PC3 and the non-small-cell lung carcinoma cell line A549, with IC50 values of 9.45 and 24.67 µM, respectively. 1-Vinyl-β-carboline-3-carboxylic acid also showed moderate activity against the yeast Saccharomyces cerevisiae ATCC204508, as well as the phytopathogenic fungi Phyllosticta citricarpa LGMB06 and Colletotrichum gloeosporioides FDC83.

Molecular Insight into Substrate Recognition and Catalysis of Baeyer–Villiger Monooxygenase MtmOIV, the Key Frame-Modifying Enzyme in the Biosynthesis of Anticancer Agent Mithramycin

Co-reporter:Mary A. Bosserman, Theresa Downey, Nicholas Noinaj, Susan K. Buchanan, and Jürgen Rohr

ACS Chemical Biology 2013 Volume 8(Issue 11) pp:2466

Publication Date(Web):August 30, 2013

DOI:10.1021/cb400399b

Baeyer–Villiger monooxygenases (BVMOs) have been shown to play key roles for the biosynthesis of important natural products. MtmOIV, a homodimeric FAD- and NADPH-dependent BVMO, catalyzes the key frame-modifying steps of the mithramycin biosynthetic pathway, including an oxidative C–C bond cleavage, by converting its natural substrate premithramycin B into mithramycin DK, the immediate precursor of mithramycin. The drastically improved protein structure of MtmOIV along with the high-resolution structure of MtmOIV in complex with its natural substrate premithramycin B are reported here, revealing previously undetected key residues that are important for substrate recognition and catalysis. Kinetic analyses of selected mutants allowed us to probe the substrate binding pocket of MtmOIV and also to discover the putative NADPH binding site. This is the first substrate-bound structure of MtmOIV providing new insights into substrate recognition and catalysis, which paves the way for the future design of a tailored enzyme for the chemo-enzymatic preparation of novel mithramycin analogues.

Semi-Synthetic Mithramycin SA Derivatives with Improved AntiCancer Activity

Co-reporter:Daniel Scott;Jhong-Min Chen;Younsoo Bae ;Jürgen Rohr

Chemical Biology & Drug Design 2013 Volume 81( Issue 5) pp:615-624

Publication Date(Web):

DOI:10.1111/cbdd.12107

Mithramycin (MTM) is a potent anti-cancer agent that has recently garnered renewed attention. This manuscript describes the design and development of mithramycin derivatives through a combinational approach of biosynthetic analogue generation followed by synthetic manipulation for further derivatization. Mithramycin SA is a previously discovered analogue produced by the M7W1 mutant strain alongside the improved mithramycin analogues mithramycin SK and mithramycin SDK. Mithramycin SA shows decreased anti-cancer activity compared to mithramycin and has a shorter, two carbon aglycon side chain that is terminated in a carboxylic acid. The aglycon side chain is responsible for an interaction with the DNA-phosphate backbone as mithramycin interacts with its target DNA. It was therefore decided to further functionalize this side chain through reactions with the terminal carboxylic acid in an effort to enhance the interaction with the DNA phosphate backbone and improve the anti-cancer activity. This side chain was modified with a variety of molecules increasing the anti-cancer activity to a comparable level to mithramycin SK. This work shows the ability to transform the previously useless mithramycin SA into a valuable molecule and opens the door to further functionalization and semi-synthetic modification for the development of molecules with increased specificity and/or drug formulation.

Baeyer–Villiger C–C Bond Cleavage Reaction in Gilvocarcin and Jadomycin Biosynthesis

Co-reporter:Nidhi Tibrewal ; Pallab Pahari ; Guojun Wang ; Madan K. Kharel ; Caleb Morris ; Theresa Downey ; Yanpeng Hou ; Tim S. Bugni

Journal of the American Chemical Society 2012 Volume 134(Issue 44) pp:18181-18184

Publication Date(Web):October 27, 2012

DOI:10.1021/ja3081154

GilOII has been unambiguously identified as the key enzyme performing the crucial C–C bond cleavage reaction responsible for the unique rearrangement of a benz[a]anthracene skeleton to the benzo[d]naphthopyranone backbone typical of the gilvocarcin-type natural anticancer antibiotics. Further investigations of this enzyme led to the isolation of a hydroxyoxepinone intermediate, leading to important conclusions regarding the cleavage mechanism.

Roles of the Synergistic Reductive O-Methyltransferase GilM and of O-Methyltransferase GilMT in the Gilvocarcin Biosynthetic Pathway

Co-reporter:Nidhi Tibrewal, Theresa E. Downey, Steven G. Van Lanen, Ehesan Ul Sharif, George A. O’Doherty, and Jürgen Rohr

Journal of the American Chemical Society 2012 Volume 134(Issue 30) pp:12402-12405

Publication Date(Web):July 16, 2012

DOI:10.1021/ja305113d

Two enzymes of the gilvocarcin biosynthetic pathway, GilMT and GilM, with unclear functions were investigated by in vitro studies using purified, recombinant enzymes along with synthetically prepared intermediates. The studies revealed GilMT as a typical S-adenosylmethionine (SAM) dependent O-methyltransferase, but GilM was identified as a pivotal enzyme in the pathway that exhibits dual functionality in that it catalyzes a reduction of a quinone intermediate to a hydroquinone, which goes hand-in-hand with a stabilizing O-methylation and a hemiacetal formation. GilM mediates its reductive catalysis through the aid of GilR that provides FADH2 for the GilM reaction, through which FAD is regenerated for the next catalytic cycle. This unusual synergy eventually completes the biosynthesis of the polyketide-derived defuco-gilvocarcin chromphore.

Delineation of gilvocarcin, jadomycin, and landomycin pathways through combinatorial biosynthetic enzymology

Co-reporter:Madan K Kharel, Jürgen Rohr

Current Opinion in Chemical Biology 2012 Volume 16(1–2) pp:150-161

Publication Date(Web):April 2012

DOI:10.1016/j.cbpa.2012.03.007

The exact sequence of events in biosyntheses of natural products is essential not only to understand and learn from nature's strategies and tricks to assemble complex natural products, but also for yield optimization of desired natural products, and for pathway engineering and muta-synthetic preparation of analogues of bioactive natural products. Biosyntheses of natural products were classically studied applying in vivo experiments, usually by combining incorporation experiments with stable-isotope labeled precursors with cross-feeding experiments of putative intermediates. Later genetic studies were dominant, which consist of gene cluster determination and analysis of gene inactivation experiments. From such studies various biosynthetic pathways were proposed, to a large extent just through in silico analyses of the biosynthetic gene clusters after DNA sequencing. Investigations of the complex biosyntheses of the angucycline group anticancer drugs landomycin, jadomycin and gilvocarcin revealed that in vivo and in silico studies were insufficient to delineate the true biosynthetic sequence of events. Neither was it possible to unambiguously assign enzyme activities, especially where multiple functional enzymes were involved. However, many of the intriguing ambiguities could be solved after in vitro reconstitution of major segments of these pathways, and subsequent systematic variations of the used enzyme mixtures. This method has been recently termed ‘combinatorial biosynthetic enzymology’.Highlights► The exact sequence of biosynthetic events is essential for the preparation of bioactive natural product analogues. ► Biosynthetic investigation of landomycin and gilvocarcin using classical methods led to many ambiguities. ► The combinatorial biosynthetic enzymology approach turned out to be the superior way to delineate the complex post-polyketide tailoring steps of landomycin, gilvocarcin and jadomycin biosyntheses. ► Previously impossible unambiguous assignments could be made for the function of many of the involved enzymes. ► Many of the earlier drawn hypotheses and conclusions were revised.

A Novel Mithramycin Analogue with High Antitumor Activity and Less Toxicity Generated by Combinatorial Biosynthesis

Co-reporter:Luz E. Núñez ; Stephen E. Nybo ; Javier González-Sabín ; María Pérez ; Nuria Menéndez ; Alfredo F. Braña ; Khaled A. Shaaban ; Min He ; Francisco Morís ; José A. Salas ; Jürgen Rohr ;Carmen Méndez

Journal of Medicinal Chemistry 2012 Volume 55(Issue 12) pp:5813-5825

Publication Date(Web):May 14, 2012

DOI:10.1021/jm300234t

Mithramycin is an antitumor compound produced by Streptomyces argillaceus that has been used for the treatment of several types of tumors and hypercalcaemia processes. However, its use in humans has been limited because of its side effects. Using combinatorial biosynthesis approaches, we have generated seven new mithramycin derivatives, which differ from the parental compound in the sugar profile or in both the sugar profile and the 3-side chain. From these studies three novel derivatives were identified, demycarosyl-3D-β-d-digitoxosylmithramycin SK, demycarosylmithramycin SDK, and demycarosyl-3D-β-d-digitoxosylmithramycin SDK, which show high antitumor activity. The first one, which combines two structural features previously found to improve pharmacological behavior, was generated following two different strategies, and it showed less toxicity than mithramycin. Preliminary in vivo evaluation of its antitumor activity through hollow fiber assays, and in subcutaneous colon and melanoma cancers xenografts models, suggests that demycarosyl-3D-β-d-digitoxosylmithramycin SK could be a promising antitumor agent worthy of further investigation.

Saquayamycins G–K, Cytotoxic Angucyclines from Streptomyces sp. Including Two Analogues Bearing the Aminosugar Rednose

Co-reporter:Khaled A. Shaaban, Tamer A. Ahmed, Markos Leggas, and Jürgen Rohr

Journal of Natural Products 2012 Volume 75(Issue 7) pp:1383-1392

Publication Date(Web):July 3, 2012

DOI:10.1021/np300316b

Streptomyces sp. KY40-1, a strain isolated from the Kentucky Appalachian foothills, is the producer of moromycins A (18) and B (19). Further investigations of this strain led to the isolation and structure elucidation of the five new saquayamycins G–K (1–5), along with known compounds. Two of the new compounds bear the unusual aminosugar rednose, which was found here for the first time in angucyclines. The different attachment positions of this aminosugar in these two compounds indicate a high acceptor substrate flexibility of the responsible glycosyl transferase or alternatively the involvement of multiple glycosyl transferases. The cytotoxic activity of the isolated compounds was determined using human prostate cancer (PC-3) and non-small-cell lung cancer (H460) cell lines. Cell viability assays showed that saquayamycins J (4), K (5), A (7), and B (8) were most active in PC3 cells, with saquayamycin B (8) showing the highest activity (GI50 = 0.0075 μM). The aminosugar-containing saquayamycins H (2) and saquayamycin B (8) showed the highest activity against H460 cells, with a GI50 of 3.3 and 3.9 μM, respectively. The results presented here provide more insights into the structure–activity relationship of saquayamycins with respect to the nature, number, and linkage of sugar residues.

Elucidation of post-PKS tailoring steps involved in landomycin biosynthesis

Co-reporter:Madan K. Kharel, Pallab Pahari, Khaled A. Shaaban, Guojun Wang, Caleb Morris and Jürgen Rohr

Organic & Biomolecular Chemistry 2012 vol. 10(Issue 21) pp:4256-4265

Publication Date(Web):05 Mar 2012

DOI:10.1039/C2OB07171A

The functional roles of all proposed enzymes involved in the post-PKS redox reactions of the biosynthesis of various landomycin aglycones were thoroughly studied, both in vivo and in vitro. The results revealed that LanM2 acts as a dehydratase and is responsible for concomitant release of the last PKS-tethered intermediate to yield prejadomycin (10). Prejadomycin (10) was confirmed to be a general pathway intermediate of the biosynthesis. Oxygenase LanE and the reductase LanV are sufficient to convert 10 into 11-deoxylandomycinone (5) in the presence of NADH. LanZ4 is a reductase providing reduced flavin (FMNH) co-factor to the partner enzyme LanZ5, which controls all remaining steps. LanZ5, a bifunctional oxygenase–dehydratase, is a key enzyme directing landomycin biosynthesis. It catalyzes hydroxylation at the 11-position preferentially only after the first glycosylation step, and requires the presence of LanZ4. In the absence of such a glycosylation, LanZ5 catalyzes C5,6-dehydration, leading to the production of anhydrolandomycinone (8) or tetrangulol (9). The overall results provided a revised pathway for the biosynthesis of the four aglycones that are found in various congeners of the landomycin group.

Ketoolivosyl-tetracenomycin C: A new ketosugar bearing tetracenomycin reveals new insight into the substrate flexibility of glycosyltransferase ElmGT

Co-reporter:S. Eric Nybo, Khaled A. Shabaan, Madan K. Kharel, Happy Sutardjo, José A. Salas, Carmen Méndez, Jürgen Rohr

Bioorganic & Medicinal Chemistry Letters 2012 Volume 22(Issue 6) pp:2247-2250

Publication Date(Web):15 March 2012

DOI:10.1016/j.bmcl.2012.01.094

A new tetracenomycin analog, 8-demethyl-8-(4′-keto)-α-l-olivosyl-tetracenomycin C, was generated through combinatorial biosynthesis. Streptomyces lividans TK 24 (cos16F4) was used as a host for expression of a ‘sugar plasmid’ (pKOL) directing the biosynthesis of NDP-4-keto-l-olivose. This strain harbors all of the genes necessary for production of 8-demethyl-tetracenomycin C and the sugar flexible glycosyltransferase ElmGT. To the best of our knowledge, this report represents the first characterization of a tetracenomycin derivative decorated with a ketosugar moiety. Also, as far as we know, 4-keto-l-olivose has only been described as an intermediate of oleandomycin biosynthesis, but has not been described before as an appendage for a polyketide compound. Furthermore, this report gives further insight into the substrate flexibility of ElmGT to include an NDP-ketosugar, which is unusual and is rarely observed among glycosyltransferases from antibiotic biosynthetic pathways.

Enzymatic Total Synthesis of DefucogilvocarcinM and Its Implications for Gilvocarcin Biosynthesis

Co-reporter:Dr. Pallab Pahari;Dr. Madan K. Kharel;Dr. Micah D. Shepherd;Dr. Steven G. vanLanen;Dr. Jürgen Rohr

Angewandte Chemie International Edition 2012 Volume 51( Issue 5) pp:1216-1220

Publication Date(Web):

DOI:10.1002/anie.201105882

Cooperation of Two Bifunctional Enzymes in the Biosynthesis and Attachment of Deoxysugars of the Antitumor Antibiotic Mithramycin

Co-reporter:Dr. Guojun Wang;Dr. Pallab Pahari;Dr. Madan K. Kharel;Dr. Jing Chen;Dr. Haining Zhu;Dr. Steven G. VanLanen;Dr. Jürgen Rohr

Angewandte Chemie International Edition 2012 Volume 51( Issue 42) pp:10638-10642

Publication Date(Web):

DOI:10.1002/anie.201205414

Enzymatic Total Synthesis of DefucogilvocarcinM and Its Implications for Gilvocarcin Biosynthesis

Co-reporter:Dr. Pallab Pahari;Dr. Madan K. Kharel;Dr. Micah D. Shepherd;Dr. Steven G. vanLanen;Dr. Jürgen Rohr

Angewandte Chemie 2012 Volume 124( Issue 5) pp:1242-1246

Publication Date(Web):

DOI:10.1002/ange.201105882

Zusammenwirken zweier difunktionaler Enzyme bei Aufbau und Verknpfung von Desoxyzuckern des Antitumor-Antibiotikums Mithramycin

Co-reporter:Dr. Guojun Wang;Dr. Pallab Pahari;Dr. Madan K. Kharel;Dr. Jing Chen;Dr. Haining Zhu;Dr. Steven G. VanLanen;Dr. Jürgen Rohr

Angewandte Chemie 2012 Volume 124( Issue 42) pp:10790-10794

Publication Date(Web):

DOI:10.1002/ange.201205414

Pyramidamycins A-D and 3-hydroxyquinoline-2-carboxamide; cytotoxic benzamides from Streptomyces sp. DGC1

Co-reporter:Khaled A Shaaban, Micah D Shepherd, Tamer A Ahmed, S Eric Nybo, Markos Leggas and Jürgen Rohr

The Journal of Antibiotics 2012 65(12) pp:615-622

Publication Date(Web):October 10, 2012

DOI:10.1038/ja.2012.81

Four new benzamides, pyramidamycins A-D (2–5) along with the new natural 3-hydroxyquinoline-2-carboxamide (6) were isolated from the crude extract of Streptomyces sp. DGC1. Additionally, five other known compounds, namely 2-aminobenzamide (anthranilamide) (1), 4′,7-dihydroxyisoflavanone (7), 2′-deoxy-thymidine, 2′-deoxy-uridine and adenosine were also isolated and identified. The structures of the new compounds 2–6 were elucidated by 1D and 2D NMR studies along with HR MS analyses. The isolated compounds 1–6 contained the same amide side chain. The isolated compounds 1–7 were biologically evaluated in comparison with landomycin A against a prostate cancer cell line (PC3) and non-small cell lung cancer cell line (H460) for 48 h and against several bacterial strains. Pyramidamycin C (4) was the most active compound against both PC3 and H460 cell lines (GI50=2.473 and 7.339 μM, respectively). Benzamides (1–3) demonstrated inhibitory activity against Kocuria rosea B-1106 (a diameter halo of 13±2 mm for 1; 10±2 mm for 2 and 3). Compound 6 was slightly active against both Escherichia coli DH5α and Micrococcus luteus NRRL B-2618 (diameter halos 8±2 and 9±2 mm, respectively). Taxonomically, the amplified 500-bp 16 S rRNA fragment of the Streptomyces sp. DGC1 had 99% identity (BLAST search) to the 16S rRNA gene of Streptomyces atrovirens strain NRRL B-16357.

Landomycins P−W, Cytotoxic Angucyclines from Streptomyces cyanogenus S-136

Co-reporter:Khaled A. Shaaban, Sowmyalakshmi Srinivasan, Raj Kumar, Chendil Damodaran, and Jürgen Rohr

Journal of Natural Products 2011 Volume 74(Issue 1) pp:2-11

Publication Date(Web):December 28, 2010

DOI:10.1021/np100469y

Streptomyces cyanogenus S-136 is the producer of previously reported landomycins A−D. An analysis of minor products of the strain led to isolation and structure elucidation of eight new congeners, named landomycins P−W (5, 6, 3, 17, 9, 10, 15, 7), along with 10 other known angucyclin(on)es. The structures of the new compounds were established from their NMR and mass spectrometry data. The activity of these angucyclin(on)es was determined using MCF-7 (estrogen responsive) and MDA-231 (estrogen refractory) breast cancer cell lines. Cell viability assays showed that anhydrolandomycinone (2), landomycinone (11), and landomycin A (16) showed the best combined activities in both MCF-7 and MDA-231 assays, with 2 being the most potent in the former and 11 and 16 in the latter. These data reveal that some of the aglycones are equipotent to the principle product 16, which contains the longest saccharide chain. Specifically, anhydrolandomycinone (2) was the most active against MCF-7 cells (IC50 = 1.8 μM). Compounds with shorter saccharidal moieties were less potent against MCF-7. The fact that the most active landomycins have either long penta- or hexasaccharide chains or no sugars at all suggests that the large compounds may act by a different mode of action than their small sugar-free congeners. The results presented here provide more insights into the structure−activity relationship of landomycins.

Characterization of the TDP-D-ravidosamine biosynthetic pathway: one-pot enzymatic synthesis of TDP-D-ravidosamine from thymidine-5-phosphate and glucose-1-phosphate

Co-reporter:Madan K. Kharel, Hui Lian and Jürgen Rohr

Organic & Biomolecular Chemistry 2011 vol. 9(Issue 6) pp:1799-1808

Publication Date(Web):24 Jan 2011

DOI:10.1039/C0OB00854K

Ravidomycin V and related compounds, e.g., FE35A-B, exhibit potent anticancer activities against various cancer cell lines in the presence of visible light. The amino sugar moieties (D-ravidosamine and its analogues, respectively) in these molecules contribute to the higher potencies of ravidomycin and analogues when compared to closely related compounds with neutral or branched sugars. Within the ravidomycin V biosynthetic gene cluster, five putative genes encoding NDP-D-ravidosamine biosynthetic enzymes were identified. Through the activities of the isolated enzymes in vitro, it is demonstrated that ravD, ravE, ravIM, ravAMT and ravNMT encode TDP-D-glucose synthase, TDP-4-keto-6-deoxy-D-glucose-4,6-dehydratase, TDP-4-keto-6-deoxy-D-glucose-3,4-ketoisomerase, TDP-3-keto-6-deoxy-D-galactose-3-aminotransferase, and TDP-3-amino-3,6-dideoxy-D-galactose-N,N-dimethyl-transferase, respectively. A protocol for a one-pot enzymatic synthesis of TDP-D-ravidosamine has been developed. The results presented here now set the stage to produce TDP-D-ravidosamine routinely for glycosylation studies.

Characterization of the Terminal Activation Step Catalyzed by Oxygenase CmmOIV of the Chromomycin Biosynthetic Pathway from Streptomyces griseus

Co-reporter:

Biochemistry 2011 Volume 50(Issue 8) pp:1421-1428

Publication Date(Web):January 18, 2011

DOI:10.1021/bi1016205

Inactivation and initial interrogation of key oxygenase CmmOIV of the biosynthetic pathway of chromomycin A3 in Streptomyces griseus ssp. griseus revealed that a completely methylated and acetylated prechromomycin is the preferred substrate of this enzyme. This suggests that the three sugar decoration reactions, two O-acetylations and an O-methylation, which were previously believed to occur as the final steps of chromomycin A3 biosynthesis, indeed take place prior to the CmmOIV reaction. Upon inactivation of CmmOIV, four new compounds accumulated; the fully decorated prechromomycin and its incompletely acetylated precursor along with a diketoprechromomycin-type compound were fully characterized and assayed with CmmOIV.

Investigating Mithramycin Deoxysugar Biosynthesis: Enzymatic Total Synthesis of TDP-D-Olivose

Co-reporter:Dr. Guojun Wang; Dr. Madan K. Kharel;Dr. Pallab Pahari; Dr. Jürgen Rohr

ChemBioChem 2011 Volume 12( Issue 17) pp:

Publication Date(Web):

DOI:10.1002/cbic.201100540

11-Deoxylandomycinone and landomycins X-Z, new cytotoxic angucyclin(on)es from a Streptomyces cyanogenus K62 mutant strain

Co-reporter:Khaled A Shaaban, Chris Stamatkin, Chendil Damodaran and Jürgen Rohr

The Journal of Antibiotics 2011 64(1) pp:141-150

Publication Date(Web):October 27, 2010

DOI:10.1038/ja.2010.121

Four new angucyclin(on)es, 11-deoxylandomycinone (1) and landomycins X–Z (2–4) were isolated from the crude extract of Streptomyces cyanogenus K62 mutant strain, along with the recently reported landomycins S, T and V (5–7) and five other known compounds. The structures of the new compounds 1–4 were elucidated by 1D and 2D NMR studies along with HR-MS analyses. Unique about the structures is that the fourth sugar moiety (sugar D) in landomycins X–Z (2–4) was β-D-amicetose instead of β-D-olivose, usually found in this position. The new angucyclin(on)es were biologically evaluated in comparison with previously known congeners against a small panel of MCF-7 (estrogen responsive) and MDA 231 (estrogen refractory) breast cancer cell lines. 11-deoxylandomycinone (IC50 2.1±0.3 and 1.2±0.4 μM) and landomycin Y (IC50 1.0±0.1 and 2.0±0.1 μM) showed the highest cytotoxic potencies against both the cell lines.

Enzymatic Total Synthesis of Rabelomycin, an Angucycline Group Antibiotic

Co-reporter:Madan Kumar Kharel, Pallab Pahari, Hui Lian and Jürgen Rohr

Organic Letters 2010 Volume 12(Issue 12) pp:2814-2817

Publication Date(Web):May 20, 2010

DOI:10.1021/ol1009009

A one-pot enzymatic total synthesis of angucycline antibiotic rabelomycin was accomplished, starting from acetyl-CoA and malonyl-CoA, using a mixture of polyketide synthase (PKS) enzymes of the gilvocarcin, ravidomycin, and jadomycin biosynthetic pathways. The in vitro results were compared to in vivo catalysis using analogous sets of enzymes.

Delineating the earliest steps of gilvocarcin biosynthesis: role of GilP and GilQ in starter unit specificity

Co-reporter:Micah D. Shepherd, Madan K. Kharel, Lili L. Zhu, Steven G. van Lanen and Jürgen Rohr

Organic & Biomolecular Chemistry 2010 vol. 8(Issue 17) pp:3851-3856

Publication Date(Web):08 Jul 2010

DOI:10.1039/C0OB00036A

In vivo and in vitro investigations of GilP and GilQ, two acyltransferases encoded by the gilvocarcin gene cluster, show that GilQ confers unique starter unit specificity when catalyzing an early as well as rate limiting step of gilvocarcin biosynthesis.

Cloning and Characterization of the Ravidomycin and Chrysomycin Biosynthetic Gene Clusters

Co-reporter:Madan K. Kharel Dr.;S. Eric Nybo ;Micah D. Shepherd ;Jürgen Rohr Dr.

ChemBioChem 2010 Volume 11( Issue 4) pp:523-532

Publication Date(Web):

DOI:10.1002/cbic.200900673

Abstract

The gene clusters responsible for the biosynthesis of two antitumor antibiotics, ravidomycin and chrysomycin, have been cloned from Streptomyces ravidus and Streptomyces albaduncus, respectively. Sequencing of the 33.28 kb DNA region of the cosmid cosRav32 and the 34.65 kb DNA region of cosChry1-1 and cosChryF2 revealed 36 and 35 open reading frames (ORFs), respectively, harboring tandem sets of type II polyketide synthase (PKS) genes, D-ravidosamine and D-virenose biosynthetic genes, post-PKS tailoring genes, regulatory genes, and genes of unknown function. The isolated ravidomycin gene cluster was confirmed to be involved in ravidomycin biosynthesis through the production of a new analogue of ravidomycin along with anticipated pathway intermediates and biosynthetic shunt products upon heterologous expression of the cosmid, cosRav32, in Streptomyces lividans TK24. The identity of the cluster was further verified through cross complementation of gilvocarcin V (GV) mutants. Similarly, the chrysomycin gene cluster was demonstrated to be indirectly involved in chrysomycin biosynthesis through cross-complementation of gilvocarcin mutants deficient in the oxygenases GilOII, GilOIII, and GilOIV with the respective chrysomycin monooxygenase homologues. The ravidomycin glycosyltransferase (RavGT) appears to be able to transfer both amino- and neutral sugars, exemplified through the structurally distinct 6-membered D-ravidosamine and 5-membered D-fucofuranose, to the coumarin-based polyketide derived backbone. These results expand the library of biosynthetic genes involved in the biosyntheses of gilvocarcin class compounds that can be used to generate novel analogues through combinatorial biosynthesis.

Crystal Structure of Baeyer−Villiger Monooxygenase MtmOIV, the Key Enzyme of the Mithramycin Biosynthetic Pathway,

Co-reporter:Miranda P. Beam, Mary A. Bosserman, Nicholas Noinaj, Marie Wehenkel and Jürgen Rohr

Biochemistry 2009 Volume 48(Issue 21) pp:

Publication Date(Web):April 13, 2009

DOI:10.1021/bi8023509

Baeyer−Villiger monooxygenases (BVMOs), mostly flavoproteins, were shown to be powerful biocatalysts for synthetic organic chemistry applications and were also suggested to play key roles for the biosyntheses of various natural products. Here we present the three-dimensional structure of MtmOIV, a 56 kDa homodimeric FAD- and NADPH-dependent monooxygenase, which catalyzes the key frame-modifying step of the mithramycin biosynthetic pathway and currently the only BVMO proven to react with its natural substrate via a Baeyer−Villiger reaction. MtmOIV’s structure was determined by X-ray crystallography using molecular replacement to a resolution of 2.9 Å. MtmOIV cleaves a C−C bond, essential for the conversion of the biologically inactive precursor, premithramycin B, into the active drug mithramycin. The MtmOIV structure combined with substrate docking calculations and site-directed mutagenesis experiments identifies several residues that participate in cofactor and substrate binding. Future experimentation aimed at broadening the substrate specificity of the enzyme could facilitate the generation of chemically diverse mithramycin analogues through combinatorial biosynthesis.

GilR, an Unusual Lactone-Forming Enzyme Involved in Gilvocarcin Biosynthesis

Co-reporter:Madan Kumar Kharel Dr.;Pallab Pahari Dr.;Hui Lian ;Jürgen Rohr Dr.

ChemBioChem 2009 Volume 10( Issue 8) pp:1305-1308

Publication Date(Web):

DOI:10.1002/cbic.200900130

Inactivation of the Ketoreductase gilU Gene of the Gilvocarcin Biosynthetic Gene Cluster Yields New Analogues with Partly Improved Biological Activity

Co-reporter:Tao Liu Dr.;Madan K. Kharel Dr.;Lili Zhu Dr.;Samuel A. Bright Dr.;Cynthia Mattingly;Val R. Adams Dr.;Jürgen Rohr Dr.

ChemBioChem 2009 Volume 10( Issue 2) pp:278-286

Publication Date(Web):

DOI:10.1002/cbic.200800348

Abstract

Four new analogues of the gilvocarcin-type aryl-C-glycoside antitumor compounds, namely 4′-hydroxy gilvocarcin V (4′-OH-GV), 4′-hydroxy gilvocarcin M, 4′-hydroxy gilvocarcin E and 12-demethyl-defucogilvocarcin V, were produced through inactivation of the gilU gene. The 4′-OH-analogues showed improved activity against lung cancer cell lines as compared to their parent compounds without 4′-OH group (gilvocarcins V and E). The structures of the sugar-containing new mutant products indicate that the enzyme GilU acts as an unusual ketoreductase involved in the biosynthesis of the C-glycosidically linked deoxysugar moiety of the gilvocarcins. The structures of the new gilvocarcins indicate substrate flexibility of the post-polyketide synthase modifying enzymes, particularly the C-glycosyltransferase and the enzyme responsible for the sugar ring contraction. The results also shed light into biosynthetic sequence of events in the late steps of biosynthetic pathway of gilvocarcin V.

Mithramycin Analogues Generated by Combinatorial Biosynthesis Show Improved Bioactivity

Co-reporter:Irfan Baig, María Perez, Alfredo F. Braña, Rohini Gomathinayagam, Chendil Damodaran, Jose A. Salas, Carmen Méndez and Jürgen Rohr

Journal of Natural Products 2008 Volume 71(Issue 2) pp:199-207

Publication Date(Web):January 15, 2008

DOI:10.1021/np0705763

Plasmid pLNBIV was used to overexpress the biosynthetic pathway of nucleoside-diphosphate (NDP)-activated l-digitoxose in the mithramycin producer Streptomyces argillaceus. This led to a “flooding” of the biosynthetic pathway of the antitumor drug mithramycin (MTM) with NDP-activated deoxysugars, which do not normally occur in the pathway, and consequently to the production of the four new mithramycin derivatives 1–4 with altered saccharide patterns. Their structures reflect that NDP sugars produced by pLNBIV, namely, l-digitoxose and its biosynthetic intermediates, influenced the glycosyl transfer to positions B, D, and E, while positions A and C remained unaffected. All four new structures have unique, previously not found sugar decoration patterns, which arise from either overcoming the substrate specificity or inhibition of certain glycosyltransferases (GTs) of the MTM pathway with the foreign NDP sugars expressed by pLNBIV. An apoptosis TUNEL (=terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling) assay revealed that compounds 1 (demycarosyl-3D-β-d-digitoxosyl-MTM) and 3 (deoliosyl-3C-β-d-mycarosyl-MTM) show improved activity (64.8 ± 2% and 50.3 ± 2.5% induction of apoptosis, respectively) against the estrogen receptor (ER)-positive human breast cancer cell line MCF-7 compared with the parent drug MTM (37.8 ± 2.5% induction of apoptosis). In addition, compounds 1 and 4 (3A-deolivosyl-MTM) show significant effects on the ER-negative human breast cancer cell line MDA-231 (63.6 ± 2% and 12.6 ± 2.5% induction of apoptosis, respectively), which is not inhibited by the parent drug MTM itself (2.6 ± 1.5% induction of apoptosis), but for which chemotherapeutic agents are urgently needed.

Moromycins A and B, Isolation and Structure Elucidation of C-Glycosylangucycline-Type Antibiotics from Streptomyces sp. KY002

Co-reporter:Mohamed S. Abdelfattah, Madan Kumar Kharel, John Andrew Hitron, Irfan Baig and Jürgen Rohr

Journal of Natural Products 2008 Volume 71(Issue 9) pp:1569-1573

Publication Date(Web):July 31, 2008

DOI:10.1021/np800281f

Two new anticancer antibiotics of the angucycline class, moromycins A and B (1, 2), along with the known microbial metabolites saquayamycin B (3) and fridamycin D (4) were isolated from the ethyl acetate extract of a culture broth of the terrestrial Streptomyces sp. KY002. The structures consist of a tetrangomycin core and various C- and O-glycosidically linked deoxysugars. The chemical structures of the new secondary metabolites were elucidated by 1D and 2D NMR and by mass spectrometry. Moromycin B (2) showed significant cytotoxicity against H-460 human lung cancer and MCF-7 human breast cancer cells.

On the Acceptor Substrate of C-Glycosyltransferase UrdGT2: Three Prejadomycin C-Glycosides from an Engineered Mutant of Streptomyces globisporus 1912 ΔlndE(urdGT2)

Co-reporter:Irfan Baig Dr.;Madan Kharel Dr.;Anton Kobylyanskyy;Lili Zhu Dr.;Yuriy Rebets Dr.;Bohdan Ostash Dr.;Andriy Luzhetskyy Dr.;Andreas Bechthold Dr.;Victor A. Fedorenko Dr. Dr.

Angewandte Chemie International Edition 2006 Volume 45(Issue 46) pp:

Publication Date(Web):24 OCT 2006

DOI:10.1002/anie.200603176

The transfer of the geneurdGT2, which encodes a C-glycosyltransferase in the urdamycin pathway, into the lnd-minus mutant of the landomycin (lnd) biosynthetic pathway indicates the difference in the first glycosylation step in the urdamycin and landomycin pathways. It also reveals indirectly the acceptor substrate of UrdGT2 (see scheme) and gives evidence of greatly relaxed substrate specificity of all lnd glycosyltransferases except LndGT2.

Premithramycinone G, an Early Shunt Product of the Mithramycin Biosynthetic Pathway Accumulated upon Inactivation of Oxygenase MtmOII

Co-reporter:Mohamed S. Abdelfattah Dr. Dr.

Angewandte Chemie International Edition 2006 Volume 45(Issue 34) pp:

Publication Date(Web):20 JUL 2006

DOI:10.1002/anie.200600511

The unusual dodecaketide premithramycinone G (see formula), which possesses a five-membered ring with two ketide chains in its tetracyclic structure, was accumulated upon inactivation of an early acting oxygenase of the mithramycin biosynthetic pathway. This unexpected structure allows novel hypothetic conclusions to be drawn regarding the early mithramycin pathway and suggestions for the involvement of three previously unassigned oxygenases.

Über das Acceptorsubstrat der C-Glycosyltransferase UrdGT2: drei Prejadomycin-C-glycoside aus einer konstruierten Mutante von Streptomyces globisporus 1912 ΔlndE(urdGT2)

Co-reporter:Irfan Baig Dr.;Madan Kharel Dr.;Anton Kobylyanskyy;Lili Zhu Dr.;Yuriy Rebets Dr.;Bohdan Ostash Dr.;Andriy Luzhetskyy;Andreas Bechthold Dr.;Victor A. Fedorenko Dr. Dr.

Angewandte Chemie 2006 Volume 118(Issue 46) pp:

Publication Date(Web):24 OCT 2006

DOI:10.1002/ange.200603176

Die Transformation des GensurdGT2, das für eine C-Glycosyltransferase des Urdamycin-Weges codiert, in die lnd-minus-Mutante des Landomycin(lnd)-Weges ergab Hinweise auf die unterschiedliche Lage des ersten Glycosylierungsschrittes in diesen Biosynthesewegen, identifizierte indirekt das Acceptorsubstrat von UrdGT2 (siehe Schema) und bestätigte eine stark aufgeweitete Substratspezifität für alle lnd-Glycosyltransferasen außer LndGT2.

Inactivation of gilGT, Encoding a C-Glycosyltransferase, and gilOIII, Encoding a P450 Enzyme, Allows the Details of the Late Biosynthetic Pathway to Gilvocarcin V to be Delineated

Co-reporter:Tao Liu;Madan Kumar Kharel Dr.;Carsten Fischer Dr.;Andrew McCormick Dr.

ChemBioChem 2006 Volume 7(Issue 7) pp:

Publication Date(Web):23 JUN 2006

DOI:10.1002/cbic.200600031

Resequencing of the gilGT gene, which encodes a putative glycosyltransferase (GT) that is 495 amino acids (aa) long, from the Streptomyces griseoflavus Gö3592 gilvocarcin V (GV) gene cluster, revealed that the previously reported gilGT indeed contains two genes. These are the larger gilGT, which encodes the C-glycosyltransferase GilGT (379 aa), and the smaller gilV gene, which encodes an enzyme of unknown function (116 aa). The gene gilV is located immediately upstream of gilGT in the GV gene cluster. In-frame deletion of gilGT created a mutant that accumulated defucogilvocarcin E (defuco-GE). The result proves the function of GilGT as a C-glycosyltransferase. Deletion of gilOIII, which is located immediately downstream of gilGT, led to a mutant that accumulated gilvocarcin E (GE). This confirms that the corresponding P450 enzyme, GilOIII, is involved in the vinyl-group formation of GV. Cross-feeding experiments in which GE, defuco-GE, and defucogilvocarcin V (defuco-GV) were fed to an early blocked mutant of the GV biosynthetic pathway, showed that neither GE nor any of the defuco- compounds was an intermediate of the pathway.

Premithramycinon G, ein frühes Shuntprodukt des Mithramycin-Biosyntheseweges, akkumuliert nach Inaktivierung der Oxygenase MtmOII

Co-reporter:Mohamed S. Abdelfattah Dr. Dr.

Angewandte Chemie 2006 Volume 118(Issue 34) pp:

Publication Date(Web):20 JUL 2006

DOI:10.1002/ange.200600511

Das ungewöhnliche Dodecaketid Premithramycinon G (siehe Formel), das eine tetracyclische Struktur mit fünfgliedrigem Ring und zwei daran gebundenen Ketidketten aufweist, wurde nach Inaktivierung einer früh agierenden Oxygenase des Mithramycin-Biosyntheseweges akkumuliert. Die Bildung dieser Struktur lässt neue Hypothesen zum frühen Mithramycin-Biosyntheseweg zu, einschließlich der Beteiligung dreier Oxygenasen, deren Funktion bislang nicht zugeordnet werden konnte.

Cytotoxic Activities of New Jadomycin Derivatives

Co-reporter:Jian-Ting Zheng, Uwe Rix, Lixia Zhao, Cynthia Mattingly, Val Adams, Quan Chen, Jürgen Rohr and Ke-Qian Yang

The Journal of Antibiotics 2005 58(6) pp:405-408

Publication Date(Web):

DOI:10.1038/ja.2005.51

Cytotoxic activities of jadomycin B and five new jadomycin derivatives against four cancer cell lines (HepG2, IM-9, IM-9/Bcl-2 and H460) were evaluated. Jadomycin S was most potent against HepG2, IM-9 and IM-9/Bcl-2 while jadomycin F was most potent against H460. Their potencies correlated with the degrees of apoptosis induced. Structure-activity-relationship analyses clearly demonstrate that the side chains of the oxazolone ring derived from the incorporated amino acids make a significant impact on biological activity. Therefore, jadomycin offers an ideal scaffold to manipulate structure and could be exploited to make many novel bioactive compounds with altered activities.

Surprising production of a new urdamycin derivative by S. fradiae ΔurdQ/R

Co-reporter:M. Fedoryshyn, M. Nur-e-Alam, L. Zhu, A. Luzhetskyy, J. Rohr, A. Bechthold

Journal of Biotechnology (31 May 2007) Volume 130(Issue 1) pp:32-38

Publication Date(Web):31 May 2007

DOI:10.1016/j.jbiotec.2007.02.018

A strain (S. fradiae ΔurdQ/R) with mutations in urdQ and urdR encoding a dTDP-hexose-3,4-dehydratase and a dTDP-hexose-4-ketoreductase, respectively, produces a new urdamycin analogue (urdamycin X) with changes in the polyketide structure. The structure of urdamycin X has been elucidated by NMR spectroscopy. Urdamycin X was not detectable, even in small amounts, in either S. fradiae ΔurdQ, in S. fradiae ΔurdR or in S. fradiae A0, a mutant lacking all glycosyltransferase genes. Complementation of S. fradiae ΔurdQ/R restored urdamycin A production indicating that the mutations did not cause any polar effect.

Elucidation of post-PKS tailoring steps involved in landomycin biosynthesis

Co-reporter:Madan K. Kharel, Pallab Pahari, Khaled A. Shaaban, Guojun Wang, Caleb Morris and Jürgen Rohr

Organic & Biomolecular Chemistry 2012 - vol. 10(Issue 21) pp:NaN4265-4265

Publication Date(Web):2012/03/05

DOI:10.1039/C2OB07171A

Characterization of the TDP-D-ravidosamine biosynthetic pathway: one-pot enzymatic synthesis of TDP-D-ravidosamine from thymidine-5-phosphate and glucose-1-phosphate

Co-reporter:Madan K. Kharel, Hui Lian and Jürgen Rohr

Organic & Biomolecular Chemistry 2011 - vol. 9(Issue 6) pp:NaN1808-1808

Publication Date(Web):2011/01/24

DOI:10.1039/C0OB00854K

Delineating the earliest steps of gilvocarcin biosynthesis: role of GilP and GilQ in starter unit specificity

Co-reporter:Micah D. Shepherd, Madan K. Kharel, Lili L. Zhu, Steven G. van Lanen and Jürgen Rohr