The sesquiterpene cyclase epi-isozizaene synthase (EIZS) catalyzes the cyclization of farnesyl diphosphate to form the tricyclic hydrocarbon precursor of the antibiotic albaflavenone. The hydrophobic active site pocket of EIZS serves as a template as it binds and chaperones the flexible substrate and carbocation intermediates through the conformations required for a multistep reaction sequence. We previously demonstrated that the substitution of hydrophobic residues with other hydrophobic residues remolds the template and expands product chemodiversity [Li, R., Chou, W. K. W., Himmelberger, J. A., Litwin, K. M., Harris, G. G., Cane, D. E., and Christianson, D. W. (2014) Biochemistry 53, 1155–1168]. Here, we show that the substitution of hydrophobic residues—specifically, Y69, F95, F96, and W203—with polar side chains also yields functional enzyme catalysts that expand product chemodiversity. Fourteen new EIZS mutants are reported that generate product arrays in which eight new sesquiterpene products have been identified. Of note, some mutants generate acyclic and cyclic hydroxylated products, suggesting that the introduction of polarity in the hydrophobic pocket facilitates the binding of water capable of quenching carbocation intermediates. Furthermore, the substitution of polar residues for F96 yields high-fidelity sesquisabinene synthases. Crystal structures of selected mutants reveal that residues defining the three-dimensional contour of the hydrophobic pocket can be substituted without triggering significant structural changes elsewhere in the active site. Thus, more radical nonpolar–polar amino acid substitutions should be considered when terpenoid cyclase active sites are remolded by mutagenesis with the goal of exploring and expanding product chemodiversity.

The dehydratase domain FosDH1 from module 1 of the fostriecin polyketide synthase (PKS) catalyzed the stereospecific interconversion of (3R)-3-hydroxybutyryl-FosACP1 (5) and (E)-2-butenoyl-FosACP1 (11), as established by a combination of direct LC–MS/MS and chiral GC–MS. FosDH1 did not act on either (3S)-3-hydroxybutyryl-FosACP1 (6) or (Z)-2-butenoyl-FosACP1 (12). FosKR2, the ketoreductase from module 2 of the fostriecin PKS that normally provides the natural substrate for FosDH2, was shown to catalyze the NADPH-dependent stereospecific reduction of 3-ketobutyryl-FosACP2 (23) to (3S)-3-hydroxybutyryl-FosACP2 (8). Consistent with this finding, FosDH2 catalyzed the interconversion of the corresponding triketide substrates (3R,4E)-3-hydroxy-4-hexenoyl-FosACP2 (18) and (2Z,4E)-2,4-hexadienoyl-FosACP2 (21). FosDH2 also catalyzed the stereospecific hydration of (Z)-2-butenoyl-FosACP2 (14) to (3S)-3-hydroxybutyryl-FosACP2 (8). Although incubation of FosDH2 with (3S)-3-hydroxybutyryl-FosACP2 (8) did not result in detectable accumulation of (Z)-2-butenoyl-FosACP2 (14), FosDH2 catalyzed the slow exchange of the 3-hydroxy group of 8 with [18O]-water. FosDH2 unexpectedly could also support the stereospecific interconversion of (3R)-3-hydroxybutyryl-FosACP2 (7) and (E)-2-butenoyl-FosACP2 (13).

S-Adenosyl methionine (SAM)-dependent C-methyltransferases are responsible for the C2-methylation of 3-ketoacyl-acyl carrier protein (ACP) intermediates to give the corresponding 2-methy-3-ketoacyl-ACP products during bacterial polyketide biosynthesis mediated by trans-AT polyketide synthases that lack integrated acyl transferase (AT) domains. A coupled ketoreductase (KR) assay was used to assign the stereochemistry of the C-methyltransferase-catalyzed reaction. Samples of chemoenzymatically generated 3-ketopentanoyl-ACP (9) were incubated with SAM and BonMT2 from module 2 of the bongkrekic acid polyketide synthase. The resulting 2-methyl-3-ketopentanoyl-ACP (10) was incubated separately with five (2R)- or (2S)-methyl specific KR domains. Analysis of the derived 2-methyl-3-hydroxypentanoate methyl esters (8) by chiral GC-MS established that the BonMT2-catalyzed methylation generated exclusively (2R)-2-methyl-3-ketopentanoyl-ACP ((2R)-10). Identical results were also obtained with three additional C-methyltransferases—BaeMT9, DifMT1, and MupMT1—from the bacillaene, difficidin, and mupirocin trans-AT polyketide synthases

The polyketide synthases responsible for the biosynthesis of the polyether antibiotics nanchangmycin (1) and salinomycin (4) harbor a number of redox-inactive ketoreductase (KR0) domains that are implicated in the generation of C2-epimerized (2S)-2-methyl-3-ketoacyl-ACP intermediates. Evidence that the natural substrate for the polyether KR0 domains is, as predicted, a (2R)-2-methyl-3-ketoacyl-ACP intermediate, came from a newly developed coupled ketosynthase (KS)-ketoreductase (KR) assay that established that the decarboxylative condensation of methylmalonyl-CoA with S-propionyl-N-acetylcysteamine catalyzed by the Nan[KS1][AT1] didomain from module 1 of the nanchangmycin synthase generates exclusively the corresponding (2R)-2-methyl-3-ketopentanoyl-ACP (7a) product. In tandem equilibrium isotope exchange experiments, incubation of [2-2H]-(2R,3S)-2-methyl-3-hydroxypentanoyl-ACP (6a) with redox-active, epimerase-inactive EryKR6 from module 6 of the 6-deoxyerythronolide B synthase and catalytic quantities of NADP+ in the presence of redox-inactive, recombinant NanKR10 or NanKR50, from modules 1 and 5 of the nanchangmycin synthase, or recombinant SalKR70 from module 7 of the salinomycin synthase, resulted in first-order, time-dependent washout of deuterium from 6a. Control experiments confirmed that this washout was due to KR0-catalyzed isotope exchange of the reversibly generated, transiently formed oxidation product [2-2H]-(2R)-2-methyl-3-ketopentanoyl-ACP (7a), consistent with the proposed epimerase activity of each of the KR0 domains. Although they belong to the superfamily of short chain dehydrogenase-reductases, the epimerase-active KR0 domains from polyether synthases lack one or both residues of the conserved Tyr-Ser dyad that has previously been implicated in KR-catalyzed epimerizations.

Dehydratase (DH) domains of cryptic function are often found in polyketide synthase (PKS) modules that produce epimerized (2S)-2-methyl-3-ketoacyl-ACP (acyl carrier protein) intermediates. A combination of tandem equilibrium isotope exchange (EIX) and a newly developed Tandem Modules Epimerase assay revealed the intrinsic epimerase activity of NanDH1 and NanDH5, from modules 1 and 5, respectively, of the nanchangmycin (1) PKS as well of NigDH1, from module 1 of the nigericin (3) PKS. Unexpectedly, all three epimerase-active DH domains were also found to possess intrinsic dehydratase activity, whereas the conventional DH domains, EryDH4, from module 4 of the erythromycin synthase, and NanDH2 from module 2 of the nanchangmycin synthase, were shown to have cryptic epimerase activity.

In the biosynthesis of pentalenolactone (1), PenE and PntE, orthologous proteins from Streptomyces exfoliatus and S. arenae, respectively, catalyze the flavin-dependent Baeyer–Villiger oxidation of 1-deoxy-11-oxopentalenic acid (4) to the lactone pentalenolactone D (5), in which the less-substituted methylene carbon has migrated. By contrast, the paralogous PtlE enzyme from S. avermitilis catalyzes the oxidation of 4 to neopentalenolactone D (6), in which the more substituted methane substitution has undergone migration. We report the design and analysis of 13 single and multiple mutants of PntE mutants to identify the key amino acids that contribute to the regiospecificity of these two classes of Baeyer–Villiger monooxygenases. The L185S mutation in PntE reversed the observed regiospecificity of PntE such that all recombinant PntE mutants harboring this L185S mutation acquired the characteristic regiospecificity of PtlE, catalyzing the conversion of 4 to 6 as the major product. The recombinant PntE mutant harboring R484L exhibited reduced regiospecificity, generating a mixture of lactones containing more than 17% of 6. These in vitro results were corroborated by analysis of the complementation of the S. avermitilis ΔptlED double deletion mutant with pntE mutants, such that pntE mutants harboring L185S produced 6 as the major product, whereas complemention of the ΔptlED deletion mutant with pntE mutants carrying the R484L mutation gave 6 as more than 33% of the total lactone product mixture.

The final step in the biosynthesis of the sesquiterpenoid antibiotic pentalenolactone (1) is the highly unusual cytochrome P450-catalyzed, oxidative rearrangement of pentalenolactone F (2), involving the transient generation and rearrangement of a neopentyl cation. In Streptomyces arenae this reaction is catalyzed by CYP161C2 (PntM), with highly conserved orthologs being present in at least 10 other Actinomycetes. Crystal structures of substrate-free PntM, as well as PntM with bound substrate 2, product 1, and substrate analogue 6,7-dihydropentalenolactone F (7) revealed interactions of bound ligand with three residues, F232, M77, and M81 that are unique to PntM and its orthologs and absent from essentially all other P450s. Site-directed mutagenesis, ligand-binding measurements, steady-state kinetics, and reaction product profiles established there is no special stabilization of reactive cationic intermediates by these side chains. Reduced substrate analogue 7 did not undergo either oxidative rearrangement or simple hydroxylation, suggesting that the C1 carbocation is not anchimerically stabilized by the 6,7-double bond of 2. The crystal structures also revealed plausible proton relay networks likely involved in the generation of the key characteristic P450 oxidizing species, Compound I, and in mediating stereospecific deprotonation of H-3re of the substrate. We conclude that the unusual carbocation intermediate results from outer shell electron transfer from the transiently generated C1 radical to the tightly paired heme-•Fe3+–OH radical species. The oxidative electron transfer is kinetically dominant as a result of the unusually strong steric barrier to oxygen rebound to the neopentyl center C-1si, which is flanked on each neighboring carbon by syn-axial substituents.

The role of the conserved active site tyrosine and serine residues in epimerization catalyzed by polyketide synthase ketoreductase (PKS KR) domains has been investigated. Both mutant and wild-type forms of epimerase-active KR domains, including the intrinsically redox-inactive EryKR3° and PicKR3° as well as redox-inactive mutants of EryKR1, were incubated with [2-2H]-(2R,3S)-2-methyl-3-hydroxypentanoyl-SACP ([2-2H]-2) and 0.05 equiv of NADP+ in the presence of the redox-active, epimerase-inactive EryKR6 domain. The residual epimerase activity of each mutant was determined by tandem equilibrium isotope exchange, in which the first-order, time-dependent washout of isotope from 2 was monitored by liquid chromatography–tandem mass spectrometry with quantitation of the deuterium content of the diagnostic pantetheinate ejection fragment (4). Replacement of the active site Tyr or Ser residues, alone or together, significantly reduced the observed epimerase activity of each KR domain with minimal effect on substrate binding. Our results demonstrate that the epimerase and reductase activities of PKS KR domains share a common active site, with both reactions utilizing the same pair of Tyr and Ser residues.

The T296V mutant of amorpha-4,11-diene synthase catalyzes the abortive conversion of the natural substrate (E,E)-farnesyl diphosphate mainly into the acyclic product (E)-β-farnesene (88%) instead of the natural bicyclic sesquiterpene amorphadiene (7%). Incubation of the T296V mutant with (3R,6E)-nerolidyl diphosphate resulted in cyclization to amorphadiene. Analysis of additional mutants of amino acid residue 296 and in vitro assays with the intermediate analogue (2Z,6E)-farnesyl diphosphate as well as (3S,6E)-nerolidyl diphosphate demonstrated that the T296V mutant can no longer catalyze the allylic rearrangement of farnesyl diphosphate to the normal intermediate (3R,6E)-nerolidyl diphosphate, while retaining the ability to cyclize (3R,6E)-nerolidyl diphosphate to amorphadiene. The T296A mutant predominantly retained amorphadiene synthase activity, indicating that neither the hydroxyl nor the methyl group of the Thr296 side chain is required for cyclase activity.

Many modular polyketide synthases harbor one or more redox-inactive domains of unknown function that are highly homologous to ketoreductase (KR) domains. A newly developed tandem equilibrium isotope exchange (EIX) assay has now established that such “KR0” domains catalyze the biosynthetically essential epimerization of transient (2R)-2-methyl-3-ketoacyl-ACP intermediates to the corresponding (2S)-2-methyl-3-ketoacyl-ACP diastereomers. Incubation of [2-2H]-(2R,3S)-2-methyl-3-hydroxypentanoyl-SACP ([2-2H]-3b) with the EryKR30 domain from module 3 of the 6-deoxyerythronolide B synthase, and the redox-active, nonepimerizing EryKR6 domain and NADP+ resulted in time- and cofactor-dependent washout of deuterium from 3b, as a result of EryKR30-catalyzed epimerization of transiently generated [2-2H]-2-methyl-3-ketopentanoyl-ACP (4). Similar results were obtained with redox-inactive PicKR30 from module 3 of the picromycin synthase. Four redox-inactive mutants of epimerase-active EryKR1 were engineered by mutagenesis of the NADPH binding site of this enzyme. Tandem EIX established that these EryKR10 mutants retained the intrinsic epimerase activity of the parent EryKR1 domain. These results establish the intrinsic epimerase activity of redox-inactive KR0 domains, rule out any role for the NADPH cofactor in epimerization, and provide a general experimental basis for decoupling the epimerase and reductase activities of a large class of PKS domains.

The class I terpenoid cyclase epi-isozizaene synthase (EIZS) utilizes the universal achiral isoprenoid substrate, farnesyl diphosphate, to generate epi-isozizaene as the predominant sesquiterpene cyclization product and at least five minor sesquiterpene products, making EIZS an ideal platform for the exploration of fidelity and promiscuity in a terpenoid cyclization reaction. The hydrophobic active site contour of EIZS serves as a template that enforces a single substrate conformation, and chaperones subsequently formed carbocation intermediates through a well-defined mechanistic sequence. Here, we have used the crystal structure of EIZS as a guide to systematically remold the hydrophobic active site contour in a library of 26 site-specific mutants. Remolded cyclization templates reprogram the reaction cascade not only by reproportioning products generated by the wild-type enzyme but also by generating completely new products of diverse structure. Specifically, we have tripled the overall number of characterized products generated by EIZS. Moreover, we have converted EIZS into six different sesquiterpene synthases: F96A EIZS is an (E)-β-farnesene synthase, F96W EIZS is a zizaene synthase, F95H EIZS is a β-curcumene synthase, F95M EIZS is a β-acoradiene synthase, F198L EIZS is a β-cedrene synthase, and F96V EIZS and W203F EIZS are (Z)-γ-bisabolene synthases. Active site aromatic residues appear to be hot spots for reprogramming the cyclization cascade by manipulating the stability and conformation of critical carbocation intermediates. A majority of mutant enzymes exhibit only relatively modest 2–100-fold losses of catalytic activity, suggesting that residues responsible for triggering substrate ionization readily tolerate mutations deeper in the active site cavity.

Ketoreductase (KR) domains from modular polyketide synthases (PKSs) catalyze the reduction of 2-methyl-3-ketoacyl acyl carrier protein (ACP) substrates and in certain cases epimerization of the 2-methyl group as well. The structural and mechanistic basis of epimerization is poorly understood, and only a small number of such KRs been studied. In this work, we studied three recombinant KR domains with putative epimerase activity: NysKR1 from module 1 of the nystatin PKS, whose stereospecificity can be predicted from both the protein sequence and the product structure; RifKR7 from module 7 of the rifamycin PKS, whose stereospecificity cannot be predicted from the protein sequence; and RifKR10 from module 10 of the rifamycin PKS, whose specificity is unclear from both the sequence and the structure. Each KR was individually incubated with NADPH and (2R)- or (2RS)-2-methyl-3-ketopentanoyl-ACP generated enzymatically in situ or via chemoenzymatic synthesis, respectively. Chiral GC–MS analysis revealed that each KR stereospecifically produced the corresponding (2S,3S)-2-methyl-3-hydroxypentanoyl-ACP in which the 2-methyl substituent had undergone KR-catalyzed epimerization. Thus, our results have led to the identification of a prototypical set of KR domains that generate (2S,3S)-2-methyl-3-hydroxyacyl products in the course of polyketide biosynthesis.

Incubation of [2-2H]-(2S,3R)-2-methyl-3-hydroxypentanoyl-SACP ([2-2H]-1a) with the epimerizing ketoreductase domain EryKR1 in the presence of a catalytic amount NADP+ (0.05 equiv) resulted in time- and cofactor-dependent washout of deuterium from 1a, as a result of equilibrium isotope exchange of transiently generated [2-2H]-2-methyl-3-ketopentanoyl-ACP. Incubations of [2-2H]-(2S,3S)-2-methyl-3-hydroxy-pentanoyl-SACP with RifKR7 and with NysKR1 also resulted in time-dependent loss of deuterium. By contrast, incubations of [2-2H]-(2R,3S)-2-methyl-3-hydroxypentanoyl-SACP and [2-2H]-(2R,3R)-2-methyl-3-hydroxypentanoyl-SACP with the non-epimerizing ketoreductase domains EryKR6 and TylKR1, respectively, did not result in any significant washout of deuterium. The isotope exchange assay directly establishes that specific polyketide synthase ketoreductase domains also have an intrinsic epimerase activity, thus enabling mechanistic analysis of a key determinant of polyketide stereocomplexity.

RifDH10, the dehydratase domain from the terminal module of the rifamycin polyketide synthase, catalyzes the stereospecific syn dehydration of the model substrate (2S,3S)-2-methyl-3-hydroxypentanoyl-RifACP10, resulting in the exclusive formation of (E)-2-methyl-2-pentenoyl-RifACP10. RifDH10 does not dehydrate any of the other three diastereomeric, RifACP10-bound, diketide thioester substrates. On the other hand, when EryACP6, from the sixth module of the erythromycin polyketide synthase, is substituted for RifACP10, RifDH10 stereospecifically dehydrates only (2R,3R)-2-methyl-3-hydroxypentanoyl-EryACP6 to give exclusively (E)-2-methyl-2-pentenoyl-EryACP6, with no detectable dehydration of any of the other three diastereomeric, EryACP6-bound, diketides. An identical alteration in substrate diastereospecificity was observed for the corresponding N-acetylcysteamine or pantetheine thioester analogues, regardless of acyl chain length or substitution pattern. Incubation of (2RS)-2-methyl-3-ketopentanoyl-RifACP10 with the didomain reductase-dehydratase RifKR10-RifDH10 yielded (E)-2-methyl-2-pentenoyl-RifACP10, the expected product of syn dehydration of (2S,3S)-2-methyl-3-hydroxypentanoyl-RifACP10, while incubation with the corresponding EryACP6-bound substrate, (2RS)-2-methyl-3-ketopentanoyl-EryACP6, gave only the reduction product (2S,3S)-2-methyl-3-hydroxypentanoyl-EryACP6 with no detectable dehydration. These results establish the intrinsic syn dehydration stereochemistry and substrate diastereoselectivity of RifDH10 and highlight the critical role of the natural RifACP10 domain in chaperoning the proper recognition and processing of the natural ACP-bound undecaketide substrate. The 1.82 Å resolution structure of RifDH10 reveals the atomic-resolution details of the active site and allows modeling of the syn dehydration of the (2S,3S)-2-methyl-3-hydroxyacyl-RifACP10 substrate. These results suggest that generation of the characteristic cis double bond of the rifamycins occurs after formation of the full-length RifACP10-bound acyclic trans-unsaturated undecaketide intermediate, most likely during the subsequent macrolactamization catalyzed by the amide synthase RifF.

The crystal structure of 2-methylisoborneol synthase (MIBS) from Streptomyces coelicolor A3(2) has been determined in its unliganded state and in complex with two Mg2+ ions and 2-fluoroneryl diphosphate at 1.85 and 2.00 Å resolution, respectively. Under normal circumstances, MIBS catalyzes the cyclization of the naturally occurring, noncanonical 11-carbon isoprenoid substrate, 2-methylgeranyl diphosphate, which first undergoes an ionization–isomerization–ionization sequence through the tertiary diphosphate intermediate 2-methyllinalyl diphosphate to enable subsequent cyclization chemistry. MIBS does not exhibit catalytic activity with 2-fluorogeranyl diphosphate, and we recently reported the crystal structure of MIBS complexed with this unreactive substrate analogue [Köksal, M., Chou, W. K. W., Cane, D. E., Christianson, D. W. (2012) Biochemistry51, 3011–3020]. However, cocrystallization of MIBS with the fluorinated analogue of the tertiary allylic diphosphate intermediate, 2-fluorolinalyl diphosphate, reveals unexpected reactivity for the intermediate analogue and yields the crystal structure of the complex with the primary allylic diphosphate, 2-fluoroneryl diphosphate. Comparison with the structure of the unliganded enzyme reveals that the crystalline enzyme active site remains partially open, presumably due to the binding of only two Mg2+ ions. Assays in solution indicate that MIBS catalyzes the generation of (1R)-(+)-camphor from the substrate 2-fluorolinalyl diphosphate, suggesting that both 2-fluorolinalyl diphosphate and 2-methyllinalyl diphosphate follow the identical cyclization mechanism leading to 2-substituted isoborneol products; however, the initially generated 2-fluoroisoborneol cyclization product is unstable and undergoes elimination of hydrogen fluoride to yield (1R)-(+)-camphor.

Tens of thousands of terpenoids are present in both terrestrial and marine plants, as well as fungi. In the last 5–10 years, however, it has become evident that terpenes are also produced by numerous bacteria, especially soil-dwelling Gram-positive organisms such as Streptomyces and other Actinomycetes. Although some microbial terpenes, such as geosmin, the degraded sesquiterpene responsible for the smell of moist soil, the characteristic odor of the earth itself, have been known for over 100 years, few terpenoids have been identified by classical structure- or activity-guided screening of bacterial culture extracts. In fact, the majority of cyclic terpenes from bacterial species have only recently been uncovered by the newly developed techniques of “genome mining”. In this new paradigm for biochemical discovery, bacterial genome sequences are first analyzed with powerful bioinformatic tools, such as the BLASTP program or Profile Hidden Markov models, to screen for and identify conserved protein sequences harboring a characteristic set of universally conserved functional domains typical of all terpene synthases. Of particular importance is the presence of variants of two universally conserved domains, the aspartate-rich DDXX(D/E) motif and the NSE/DTE triad, (N/D)DXX(S/T)XX(K/R)(D/E). Both domains have been implicated in the binding of the essential divalent cation, typically Mg2+, that is required for cyclization of the universal acyclic terpene precursors, such as farnesyl and geranyl diphosphate.The low level of overall sequence similarity among terpene synthases, however, has so far precluded any simple correlation of protein sequence with the structure of the cyclized terpene product. The actual biochemical function of a cryptic bacterial (or indeed any) terpene synthase must therefore be determined by direct experiment. Two common approaches are (i) incubation of the expressed recombinant protein with acyclic allylic diphosphate substrates and identification of the resultant terpene hydrocarbon or alcohol and (ii) in vivo expression in engineered bacterial hosts that can support the production of terpene metabolites. One of the most attractive features of the coordinated application of genome mining and biochemical characterization is that the discovery of natural products is directly coupled to the simultaneous discovery and exploitation of the responsible biosynthetic genes and enzymes.Bacterial genome mining has proved highly rewarding scientifically, already uncovering more than a dozen newly identified cyclic terpenes (many of them unique to bacteria), as well as several novel cyclization mechanisms. Moreover, bioinformatic analysis has identified more than 120 presumptive genes for bacterial terpene synthases that are now ripe for exploration. In this Account, we review a particularly rich vein we have mined in the genomes of two model Actinomycetes, Streptomyces coelicolor and Streptomyces avermitilis, from which the entire set of terpenoid biosynthetic genes and pathways have now been elucidated. In addition, studies of terpenoid biosynthetic gene clusters have revealed a wealth of previously unknown oxidative enzymes, including cytochromes P450, non-heme iron-dependent dioxygenases, and flavin monooxygenases. We have shown that these enzymes catalyze a variety of unusual biochemical reactions, including two-step ketonization of methylene groups, desaturation–epoxidation of secondary methyl groups, and pathway-specific Baeyer–Villiger oxidations of cyclic ketones.

Mechanistic proposals for the carbocation cascade reaction leading to the tricyclic sesquiterpene pentalenene are assessed in light of the results of isotopically sensitive branching experiments with the H309A mutant of pentalenene synthase. These experimental results support a mechanism for pentalenene formation involving a 7-protoilludyl cation whose intermediacy was first predicted using quantum-chemical calculations.

Incubation of recombinant module 2 of the polyether nanchangmycin synthase (NANS), carrying an appended thioesterase domain, with the ACP-bound substrate (2RS)-2-methyl-3-ketobutyryl-NANS_ACP1 (2-ACP1) and methylmalonyl-CoA in the presence of NADPH gave diastereomerically pure (2S,4R)-2,4-dimethyl-5-ketohexanoic acid (4a). These results contrast with the previously reported weak discrimination by NANS module 2+TE between the enantiomers of the corresponding N-acetylcysteamine-conjugated substrate analogue (±)-2-methyl-3-ketobutyryl-SNAC (2-SNAC), which resulted in formation of a 5:3 mixture of 4a and its (2S,4S)-diastereomer 4b. Incubation of NANS module 2+TE with 2-ACP1 in the absence of NADPH gave unreduced 3,5,6-trimethyl-4-hydroxypyrone (3) with a kcat of 4.4 ± 0.9 min–1 and a kcat/Km of 67 min–1 mM–1, corresponding to a ∼2300-fold increase compared to the kcat/Km for the diffusive substrate 2-SNAC. Covalent tethering of the 2-methyl-3-ketobutyryl thioester substrate to the NANS ACP1 domain derived from the natural upstream PKS module of the nanchangmycin synthase significantly enhanced both the stereospecificity and the kinetic efficiency of the sequential polyketide chain translocation and condensation reactions catalyzed by the ketosynthase domain of NANS module 2.

The penM and pntM genes from the pentalenolactone biosynthetic gene clusters of Streptomyces exfoliatus UC5319 and Streptomyces arenae TÜ469 were predicted to encode orthologous cytochrome P450s, CYP161C3 and CYP161C2, responsible for the final step in the biosynthesis of the sesquiterpenoid antibiotic pentalenolactone (1). Synthetic genes optimized for expression in Escherichia coli were used to obtain recombinant PenM and PntM, each carrying an N-terminal His6-tag. Both proteins showed typical reduced-CO UV maxima at 450 nm, and each bound the predicted substrate, pentalenolactone F (4), with KD values of 153 ± 14 and 126 ± 11 μM for PenM and PntM, respectively, as determined by UV shift titrations. PenM and PntM both catalyzed the oxidative rearrangement of 4 to 1 when incubated in the presence of NADPH, spinach ferredoxin, ferredoxin reductase, and O2. The steady-state kinetic parameters were kcat = 10.5 ± 1.7 min−1 and Km = 340 ± 100 μM 4 for PenM and kcat = 8.8 ± 0.9 min−1 and Km = 430 ± 100 μM 4 for PntM. The in vivo function of both gene products was confirmed by the finding that the corresponding deletion mutants S. exfoliatus/ΔpenM ZD22 and S. arenae/ΔpntM ZD23 no longer produced pentalenolactone but accumulated the precursor pentalenolactone F. Complementation of each deletion mutant with either penM or pntM restored production of antibiotic 1. Pentalenolactone was also produced by an engineered strain of Streptomyces avermitilis that had been complemented with pntE, pntD, and either pntM or penM, as well as the S. avermitilis electron-transport genes for ferredoxin and ferrodoxin reductase, fdxD and fprD.

Two presumptive terpene synthases of unknown biochemical function encoded by the sscg_02150 and sscg_03688 genes of Streptomyces clavuligerus ATCC 27074 were individually expressed in Escherichia coli as N-terminal-His6-tag proteins, using codon-optimized synthetic genes. Incubation of recombinant SSCG_02150 with farnesyl diphosphate (1, FPP) gave (-)-δ-cadinene (2) while recombinant SSCG_03688 converted FPP to (+)-T-muurolol (3). Individual incubations of (-)-δ-cadinene synthase with [1,1-2H2]FPP (1a), (1S)-[1-2H]-FPP (1b), and (1R)-[1-2H]-FPP (1c) and NMR analysis of the resulting samples of deuterated (-)-δ-cadinene supported a cyclization mechanism involving the intermediacy of nerolidyl diphosphate (4) leading to a helminthogermacradienyl cation 5. Following a 1,3-hydride shift of the original H-1si of FPP, cyclization and deprotonation will give (-)-δ-cadinene. Similar incubations with recombinant SSCG_03688 supported an analogous mechanism for the formation of (+)-T-muurolol (3), also involving a 1,3-hydride shift of the original H-1si of FPP.Graphical AbstractFigure optionsDownload full-size imageDownload high-quality image (22 K)Download as PowerPoint slideHighlights► Recombinant SSCG_02150 protein cyclizes farnesyl diphosphate to (-)-δ-cadinene ► Recombinant SSCG_02150 protein cyclizes farnesyl diphosphate to (+)-T-muurolol ► Both cyclizations involve a 1,3-hydride shift of H-1si of farnesyl diphosphate ► Terpene synthase function cannot be deduced simply from amino acid sequence

The pentalenolactone biosynthetic gene clusters have been cloned and sequenced from two known producers of the sesquiterpenoid antibiotic pentalenolactone, Streptomyces exfoliatus UC5319 and Streptomyces arenae TÜ469. The recombinant enzymes PenE and PntE, from S. exfoliatus and S. arenae, respectively, catalyze the flavin-dependent Baeyer−Villiger oxidation of 1-deoxy-11-oxopentalenic acid (7) to pentalenolactone D (8). Recombinant PenD, PntD, and PtlD, the latter from Streptomyces avermitilis, each catalyze the Fe2+-α-ketoglutarate-dependent oxidation of pentalenolactone D (8) to pentalenolactone E (15) and pentalenolactone F (16). Incubation of PenD, PntD, or PtlD with the isomeric neopentalenolactone D (9) gave PL308 (12) and a compound tentatively identified as neopentalenolactone E (14). These results are corroborated by analysis of the ΔpenD and ΔpntD mutants of S. exfoliatus and S. arenae, respectively, both of which accumulate pentalenolactone D but are blocked in production of pentalenolactone as well as the precursors pentalenolactones E and F. Finally, complementation of the previously described S. avermitilis ΔptlE ΔptlD deletion mutant with either penE or pntE gave pentalenolactone D (8), while complemention of the ΔptlE ΔptlD double mutant with pntE plus pntD or penE plus pntD gave pentalenolactone F (16).

Recombinant nanchangmycin synthase module 2 (NANS module 2), with the thioesterase domain from the 6-deoxyerythronolide B synthase (DEBS TE) appended to the C-terminus, was cloned and expressed in Escherichia coli. Incubation of NANS module 2+TE with (±)-2-methyl-3-keto-butyryl-N-acetylcysteamine thioester (1), the SNAC analog of the natural ACP-bound substrate, with methylmalonyl-CoA (MM-CoA) in the absence of NADPH gave 3,5,6-trimethyl-4-hydroxypyrone (2), identified by direct comparison with synthetic 2 by radio-TLC-phosphorimaging and LC-ESI(+)-MS-MS. The reaction showed kcat 0.5 ± 0.1 min−1 and Km(1) 19 ± 5 mM at 0.5 mM MM-CoA and kcat(app) 0.26 ± 0.02 min−1 and Km(MM-CoA) 0.11 ± 0.02 mM at 8 mM 1. Incubation in the presence of NADPH generated the fully saturated triketide chain elongation product as a 5:3 mixture of (2S,4R)-2,4-dimethyl-5-ketohexanoic acid (3a) and the diastereomeric (2S,4S)-3b. The structure and stereochemistry of each product was established by comparison with synthetic 3a and 3b by a combination of radio-TLC-phosphorimaging and LC-ESI(−)-MS-MS, as well as chiral capillary GC-MS analysis of the corresponding methyl esters 3a-Me and 3b-Me. The recombinant dehydratase domain from NANS module 2, NANS DH2, was shown to catalyze the formation of an (E)-double bond by syn-dehydration of the ACP-bound substrate anti-(2R,3R,4S,5R)-2,4-dimethyl-3,5-dihydroxyheptanoyl-ACP6 (4), generated in situ by incubation of (2S,3R)-2-methyl-3-hydroxypentanoyl-SNAC (5), methylmalonyl-CoA, and NADPH with the recombinant [KS6][AT6] didomain and ACP6 from DEBS module 6 along with the ketoreductase from the tylactone synthase module 1 (TYLS KR1). These results also indirectly establish the stereochemistry of the reactions catalyzed by the KR and enoylreductase (ER) domains of NANS module 2.

The dehydratase (DH) domain of module 4 of the 6-deoxyerythronolide B synthase (DEBS) has been shown to catalyze an exclusive syn elimination/syn addition of water. Incubation of recombinant DH4 with chemoenzymatically prepared anti-(2R,3R)-2-methyl-3-hydroxypentanoyl-ACP (2a-ACP) gave the dehydration product 3-ACP. Similarly, incubation of DH4 with synthetic 3-ACP resulted in the reverse enzyme-catalyzed hydration reaction, giving an ∼3:1 equilbrium mixture of 2a-ACP and 3-ACP. Incubation of a mixture of propionyl-SNAC (4), methylmalonyl-CoA, and NADPH with the DEBS β-ketoacyl synthase-acyl transferase [KS6][AT6] didomain, DEBS ACP6, and the ketoreductase domain from tylactone synthase module 1 (TYLS KR1) generated in situ anti-2a-ACP that underwent DH4-catalyzed syn dehydration to give 3-ACP. DH4 did not dehydrate syn-(2S,3R)-2b-ACP, syn-(2R,3S)-2c-ACP, or anti-(2S,3S)-2d-ACP generated in situ by DEBS KR1, DEBS KR6, or the rifamycin synthase KR7 (RIFS KR7), respectively. Similarly, incubation of a mixture of (2S,3R)-2-methyl-3-hydroxypentanoyl-N-acetylcysteamine thioester (2b-SNAC), methylmalonyl-CoA, and NADPH with DEBS [KS6][AT6], DEBS ACP6, and TYLS KR1 gave anti-(2R,3R)-6-ACP that underwent syn dehydration catalyzed by DEBS DH4 to give (4R,5R)-(E)-2,4-dimethyl-5-hydroxy-hept-2-enoyl-ACP (7-ACP). The structure and stereochemistry of 7 were established by GC-MS and LC-MS comparison of the derived methyl ester 7-Me to a synthetic sample of 7-Me.

The terpene synthase encoded by the sav76 gene of Streptomyces avermtilis was expressed in Escherichia coli as an N-terminal-His6-tag protein, using a codon-optimized synthetic gene. Incubation of the recombinant protein, SAV_76, with farnesyl diphosphate (1, FPP) in the presence of Mg2+ gave a new sesquiterpene alcohol avermitilol (2), whose structure and stereochemistry were determined by a combination of 1H, 13C, COSY, HMQC, HMBC, and NOESY NMR, along with minor amounts of germacrene A (3), germacrene B (4), and viridiflorol (5). The absolute configuration of 2 was assigned by 1H NMR analysis of the corresponding (R)- and (S)-Mosher esters. The steady state kinetic parameters were kcat 0.040 ± 0.001 s−1 and Km 1.06 ± 0.11 μM. Individual incubations of recombinant avermitilol synthase with [1,1-2H2]FPP (1a), (1S)-[1-2H]-FPP (1b), and (1R)-[1-2H]-FPP (1c) and NMR analysis of the resulting avermitilols supported a cyclization mechanism involving the loss of H-1re to generate the intermediate bicyclogermacrene (7), which then undergoes proton-initiated anti-Markovnikov cyclization and capture of water to generate 2. A copy of the sav76 gene was reintroduced into S. avermitilis SUKA17, a large deletion mutant from which the genes for the major endogenous secondary metabolites had been removed, and expressed under control of the native S. avermitilis promoter rpsJp (sav4925). The resultant transformants generated avermitilol (2) as well as the derived ketone, avermitilone (8), along with small amounts of 3, 4, and 5. The biochemical function of all four terpene synthases found in the S. avermtilis genome have now been determined.

The X-ray crystal structure of recombinant epi-isozizaene synthase (EIZS), a sesquiterpene cyclase from Streptomyces coelicolor A3(2), has been determined at 1.60 Å resolution. Specifically, the structure of wild-type EIZS is that of its closed conformation in complex with three Mg2+ ions, inorganic pyrophosphate (PPi), and the benzyltriethylammonium cation (BTAC). Additionally, the structure of D99N EIZS has been determined in an open, ligand-free conformation at 1.90 Å resolution. Comparison of these two structures provides the first view of conformational changes required for substrate binding and catalysis in a bacterial terpenoid cyclase. Moreover, the binding interactions of BTAC may mimic those of a carbocation intermediate in catalysis. Accordingly, the aromatic rings of F95, F96, and F198 appear to be well-oriented to stabilize carbocation intermediates in the cyclization cascade through cation−π interactions. Mutagenesis of aromatic residues in the enzyme active site results in the production of alternative sesquiterpene product arrays due to altered modes of stabilization of carbocation intermediates as well as altered templates for the cyclization of farnesyl diphosphate. Accordingly, the 1.64 Å resolution crystal structure of F198A EIZS in a complex with three Mg2+ ions, PPi, and BTAC reveals an alternative binding orientation of BTAC; alternative binding orientations of a carbocation intermediate could lead to the formation of alternative products. Finally, the crystal structure of wild-type EIZS in a complex with four Hg2+ ions has been determined at 1.90 Å resolution, showing that metal binding triggers a significant conformational change of helix G to cap the active site.

One of the most striking features of complex polyketides is the presence of numerous methyl- and hydroxyl-bearing stereogenic centers. To investigate the biochemical basis for the control of polyketide stereochemistry and to establish the timing and mechanism of the epimerization at methyl-bearing centers, a series of incubations was carried out using reconstituted components from a variety of modular polyketide synthases. In all cases the stereochemistry of the product was directly correlated with the intrinsic stereospecificity of the ketoreductase domain, independent of the particular chain elongation domains that were used, thereby establishing that methyl group epimerization, when it does occur, takes place after ketosynthase-catalyzed chain elongation. The finding that there were only minor differences in the rates of product formation observed for parallel incubations using an epimerizing ketoreductase domain and the nonepimerizing ketoreductase domain supports the proposal that the epimerization is catalyzed by the ketoreductase domain itself.

Presilphiperfolan-8β-ol synthase, encoded by the BcBOT2 gene from the necrotrophic plant pathogen Botrytis cinerea, catalyzes the multistep cyclization of farnesyl diphosphate (2) to the tricyclic sesquiterpene alcohol presilphiperfolan-8β-ol (3), the preursor of the phytotoxin botrydial, a strain-dependent fungal virulence factor. Incubation of (1R)-[1-2H]farnesyl diphosphate (2b) with recombinant presilphiperfolan-8β-ol synthase gave exclusively (5R)-[5α-2H]-3b, while complementary incubation of (1S)-[1-2H]FPP (2c) gave (5S)-[5β-2H]-3c. These results established that cyclization of farnesyl diphosphate involves displacement of the diphosphate group from C-1 with net inversion of configuration and ruled out the proposed intermediacy of the cisoid conformer of nerolidyl diphosphate (9) in the cyclization. While not a mandatory intermediate, (3R)-nerolidyl diphosphate was shown to act as a substrate surrogate. Cyclization of [13,13,13-2H3] farnesyl diphosphate (2d) gave [14,14,14-2H3]-3d, thereby establishing that electrophilic attack takes place exclusively on the si face of the 12,13-double bond of 2. The combined results provide a detailed picture of the conformation of enzyme-bound farnesyl diphosphate at the active site of presilphiperfolan-8β-ol synthase.

Epi-isozizaene synthase from Streptomyces coelicolor catalyzes the multistep cyclization of farnesyl diphosphate (2, FPP) to the tricyclic sesquiterpene hydrocarbon (+)-epi-isozizaene (3), which is converted in turn to the antibiotic albaflavenone (1) in a two-step, cytochrome P450-catalyzed oxidation. Competitive incubation of deuterated and nondeuterated samples of (3S)-NPP and (3RS)-NPP followed by GC−MS analysis of the degree of deuteration in the resulting labeled epi-isozizaene established that (3R)-NPP is the natural cyclization intermediate. Incubation of (3RS)-(Z)-[1-2H]NPP (4b) with epi-isozizaene synthase gave [11anti-2H]epi-isozizaene (3b), indicating that the SN′ cyclization of 4 involves the predicted anti stereochemistry, consistent with the inference from earlier experiments with chirally deuterated FPP. Incubation of separate samples of [12,12,12-2H3]FPP (2d) and [13,13,13-2H3]FPP (2e) gave epi-isozizaenes 3d and 3e, thereby establishing the stereochemical course of the cyclization of the proposed intermediate acorenyl cation 6, as well as the stereochemistry of the successive 1,2-methyl migration and deprotonation that generate the final product. Further insights into the mechanism and the role of the enzyme came from site-directed mutagenesis of active site residues in two universally conserved Mg2+-binding domains and the identification of six minor sesquiterpene products 9−13 and 15 produced by the wild-type and mutant proteins. The aberrant products are believed to result from derailment and premature quenching of the normal intermediates of the cationic cyclization cascade.

Incubation of 1-deoxy-11-oxopentalenic acid (12) with recombinant PtlE protein from Streptomyces avermitilis in the presence of NADPH and catalytic FAD gave the Baeyer−Villiger oxidation product, the previously unknown compound neopentalenolactone D (13), representing a new branch of the pentalenolactone biosynthetic pathway. The structure and stereochemistry of the derived neopentalenolactone D methyl ester (13-Me) were fully assigned by a combination of GC-MS and NMR analysis and confirmed by X-ray crystallography. Neopentalenolactone D (13) was also isolated from engineered cultures of S. avermitilis from which the ptlD gene within the 13.4-kb (neo)-ptl biosynthetic gene cluster had been deleted. The ΔptlEΔptlD double deletion mutant accumulated 12, the substrate for the ptlE gene product, while the corresponding single ΔptlE mutant produced 12 as well as the related oxidation products 14 and 15. Engineered strains of S. avermitilis, SUKA5 and pKU462::ermRp-ptl cluster, harboring the complete (neo)ptl cluster produced the oxidized lactone 18 and the closely related seco acid hydrolysis products 16 and 17.

The polyketide backbone of the polyether ionophore antibiotic nanchangmycin (1) is assembled by a modular polyketide synthase in Streptomyces nanchangensis NS3226. The ACP-bound polyketide is thought to undergo a cascade of oxidative cyclizations to generate the characteristic polyether. Deletion of the glycosyl transferase gene nanG5 resulted in accumulation of the corresponding nanchangmycin aglycone (6). The discrete thioesterase NanE exhibited a nearly 17-fold preference for hydrolysis of 4, the N-acetylcysteamine (SNAC) thioester of nanchangmycin, over 7, the corresponding SNAC derivative of the aglycone, consistent with NanE-catalyzed hydrolysis of ACP-bound nanchangmycin being the final step in the biosynthetic pathway. Site-directed mutagenesis established that Ser96, His261, and Asp120, the proposed components of the NanE catalytic triad, were all essential for thioesterase activity, while Trp97 was shown to influence the preference for polyether over polyketide substrates.

The fungus Botrytis cinerea is the causal agent of the economically important gray mold disease that affects more than 200 ornamental and agriculturally important plant species. B. cinerea is a necrotrophic plant pathogen that secretes nonspecific phytotoxins, including the sesquiterpene botrydial and the polyketide botcinic acid. The region surrounding the previously characterized BcBOT1 gene has now been identified as the botrydial biosynthetic gene cluster. Five genes including BcBOT1 and BcBOT2 were shown by quantitative reverse transcription-PCR to be co-regulated through the calcineurin signaling pathway. Inactivation of the BcBOT2 gene, encoding a putative sesquiterpene cyclase, abolished botrydial biosynthesis, which could be restored by in trans complementation. Inactivation of BcBOT2 also resulted in overproduction of botcinic acid that was observed to be strain-dependent. Recombinant BcBOT2 protein converted farnesyl diphosphate to the parent sesquiterpene of the botrydial biosynthetic pathway, the tricyclic alcohol presilphiperfolan-8β-ol.

A novel variant of 6-deoxyerythronolide B synthase (DEBS) module 2 was constructed to explore the balance between protein–protein-mediated intermodular channeling and intrinsic substrate specificity within DEBS. This construct, termed (N3)Mod2 + TE, was co-incubated with a complementary, donor form of the same module, (N5)Mod2(C2), as well as with a mutant of (N5)Mod2(C2) with an inactive ketosynthase domain, in order to determine the extent of intermediate channeling versus substrate diffusion into the downstream module.Co-incubation of two engineered variants of 6-deoxyerythronolide B synthase module 2 designed to communicate with one another results in inhibition of catalytic processing of free substrate by the downstream, acceptor module. This inhibition was relieved when a catalytically inactive form of the upstream, donor module was used.

The recombinant thioesterase (TE) domain of the picromycin/methymycin synthase (PICS) catalyzes the macrolactonization of 3, the N-acetylcysteamine thioester of seco-10-deoxymethynolide to generate 10-deoxymethynolide (1) with high efficiency. By contrast, 4, the 7-dihydro derivative of seco-thioester 3, undergoes exclusive hydrolysis by PICS TE to seco-acid 5. The recombinant TE domain of 6-deoxyerythronolide B synthase (DEBS TE) shows the same reaction specificity as PICS TE, but with significantly lower activity.The recombinant thioesterase (TE) domain of the picromycin-methymycin synthase (PICS) catalyzes the macrolactonization of 3, the N-acetylcysteamine thioester of seco-10-deoxymethynolide, to generate 10-deoxymethynolide (1) with high efficiency. The recombinant TE domain of 6-deoxyerythronolide B synthase (DEBS TE) shows the same reaction specificity as PICS TE, but with significantly lower activity.

Geosmin (1) is responsible for the characteristic odor of moist soil. The Gram-positive soil bacterium Streptomyces avermitilis produces geosmin (1) as well as its precursor germacradienol (3). The S. avermitilis gene SAV2163 (geoA) is extremely similar to the S. coelicolor A3(2) SCO6073 gene that encodes a germacradienol/geosmin synthase. S. avermitilis mutants with a deleted geoA were unable to produce either germacradienol (3) or geosmin (1). Biosynthesis of both compounds was restored by introducing an intact geoA gene into the mutants. Incubation of recombinant GeoA, encoded by the SAV2163 gene of S. avermitilis, with farnesyl diphosphate (2) in the presence of Mg2+ gave a mixture of (4S,7R)-germacra-1(10)E,5E-diene-11-ol (3) (66%), (7S)-germacrene D (4) (24%), geosmin (1) (8%), and a hydrocarbon, tentatively assigned the structure of octalin 5 (2%). Incubation of this germacradienol/geosmin synthase with [1,1-2H2]FPP (2a) gave geosmin-d1 (1a), as predicted. When recombinant GeoA from either S. avermitilis or S. coelicolor A3(2) was incubated with nerolidyl diphosphate (8), only the acyclic elimination products -farnesene (10), (Z)--farnesene (11), and (E)--farnesene (12) were formed, thereby ruling out nerolidyl diphosphate as an intermediate in the conversion of farnesyl diphosphate to geosmin, germacradienol, and germacrene D.

Precursor-directed polyketide biosynthesis was demonstrated in the heterologous host Escherichia coli. Several diketide and triketide substrates were fed to a recombinant E. coli strain containing a variant form of deoxyerythronolide B synthase (DEBS) from which the first elongation module was deleted resulting in successful macrolactone formation from the diketide, but not the triketide, substrates.Precursor-directed polyketide biosynthesis was demonstrated in the heterologous host Escherichia coli. Diketide and triketide substrates were fed to a recombinant E. coli strain containing a variant form of deoxyerythronolide B synthase (DEBS) from which the first elongation module was deleted resulting in successful macrolactone formation from the diketide, but not the triketide, substrates.

The eryA gene of the bacterial pathogen Brucella abortus has been functionally expressed in Escherichia coli. The resultant EryA was shown to catalyze the ATP-dependent conversion of erythritol to l-erythritol-4-phosphate (l-E4P). The steady state kinetic parameters of this reaction were determined and the enzyme was used to prepare l-E4P which was shown to be a weak inhibitor of 2-C-methyl-d-erythritol-4-phosphate cytidyltransferase (YgbP).The eryA gene has been functionally expressed in Escherichia coli, and the resultant EryA was shown to catalyze the ATP-dependent conversion of erythritol (1) to l-erythritol-4-phosphate (2,l-E4P).

The PCR has been used to amplify a 2,181-bp ORF from Streptomyces coelicolor A3(2), designated SC9B1.20 (= SCO6073), encoding a protein of 726 amino acids and showing significant sequence similarity at the deduced amino acid level in both the N-terminal and C-terminal halves to the known sesquiterpene synthase pentalenene synthase. The full-length recombinant protein was expressed at high levels in Escherichia coli and shown to catalyze the Mg2+-dependent conversion of farnesyl diphosphate to the sesquiterpene alcohol (4S, 7R)-germacra-1 (10)E, 5E-diene-11-ol. The enzymatic cyclization had a kcat of 6.2 ± 0.5 × 10−3 s−1 and a Km for farnesyl diphosphate of 62 ± 8 nM. Expression of the N-terminal (366 amino acids) domain of the SC9B1.20 protein also gave a fully functional cyclase which converted farnesyl diphosphate to the identical sesquiterpene alcohol with a slightly lower kcat of 3.2 ± 0.4 × 10−3 s−1 and a twofold greater km of 115 ± 14 nM. By contrast, the expressed C-terminal domain of SC9B1.20 had no farnesyl diphosphate cyclase activity. The formation of the germacradienol seems to be the committed step in the formation of geosmin, the characteristic odoriferous constituent of Streptomyces species.

Incubation of chirally deuterated NADPH with 6-deoxyerythronolide B synthase (DEBS) modules 5 and module 6 and analysis of the derived triketide lactones established that the two ketoreductase domains, KR5 and KR6, are both specific for the 4-pro-S hydride of the nicotinamide cofactor.The stereochemistry of the ketoreductase-catalyzed reactions of modules 5 and 6 of the 6-deoxyerythronolide B synthase is reported.

Trichodiene synthase from Fusarium sporotrichioides contains two metal ion-binding motifs required for the cyclization of farnesyl diphosphate: the “aspartate-rich” motif D100DXX(D/E) that coordinates to Mg2+A and Mg2+C, and the “NSE/DTE” motif N225DXXSXXXE that chelates Mg2+B (boldface indicates metal ion ligands). Here, we report steady-state kinetic parameters, product array analyses, and X-ray crystal structures of trichodiene synthase mutants in which the fungal NSE motif is progressively converted into a plant-like DDXXTXXXE motif, resulting in a degradation in both steady-state kinetic parameters and product specificity. Each catalytically active mutant generates a different distribution of sesquiterpene products, and three newly detected sesquiterpenes are identified. In addition, the kinetic and structural properties of the Y295F mutant of trichodiene synthase were found to be similar to those of the wild-type enzyme, thereby ruling out a proposed role for Y295 in catalysis.