Pentalenic acid is a shunt metabolite in the biosynthesis of the pentalenolactone family of metabolites: hydroxylation of 1-deoxypentalenic acid mediated by CYP105D7 (SAV_7469) of Streptomyces avermitilis

Takamatsu, Satoshi; Xu, Lian‐Hua; Fushinobu, Shinya; Shoun, Hirofumi; Komatsu, Mamoru; Cane, David E.; Ikeda, Haruo

doi:10.1038/ja.2010.135

Cited by 37 publications

(29 citation statements)

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“…The in vivo formation of the epimeric epoxylactone 18 as a shunt metabolite is therefore most likely due to oxidation of the intermediate α-methylenelactone pentalenolactone E ( 15 ) by an oxygenase that is distinct from both PenD or PntD and that is encoded by a gene that is external to either the pen or pnt biosynthetic gene clusters (Scheme 5). In a similar manner, we have recently reported that the hydroxylation of the normal biosynthetic pathway intermediate 1-deoxypentalenic acid ( 5) to the commonly occurring shunt metabolite pentalenic acid ( 20 ) is catalyzed by CYP105D7, a typical heme-dependent monooxygenase that is encoded by the sav7469 gene, located in the 9.05-Mb S. avermitilis linear chromosome more than 5 Mb from the ptl biosynthetic gene cluster (77). …”

Section: Discussionmentioning

confidence: 79%

Genome Mining in Streptomyces. Elucidation of the Role of Baeyer−Villiger Monooxygenases and Non-Heme Iron-Dependent Dehydrogenase/Oxygenases in the Final Steps of the Biosynthesis of Pentalenolactone and Neopentalenolactone

et al. 2011

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The pentalenolactone biosynthetic gene clusters have been cloned and sequenced from two known producers of the sesquiterpenoid antibiotic pentalenolactone, Streptomyces exfoliatus UC5319 and S. 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 S. avermitilis, each catalyze the Fe 2+ -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).Terpenoid compounds are ubiquitous in Nature, being widely distributed in terrestial and marine plants, fungi, liverworts, and, as is becoming increasingly common, many bacteria. Of the tens of thousands of known monoterpenes, sequiterpenes, and diterpenes, the several hundred parent cyclic hydrocarbon and alcohol products are formed from the universal acyclic C 10 , C 15 , and C 20 precursors geranyl, farnesyl, and geranylgeranyl diphosphate. More than a thousand presumptive terpene cyclase genes from plants and microorganisms have been identified, of which about 10% have been experimentally assigned a confirmed † This work was supported by National Institutes of Health Grant GM30301 (D.E.C.) and by a Grant-in-Aid for Scientific Research on Innovative Areas from MEXT Japan, from JSPS 20310122 and from the Institute for Fermentation, Osaka, Japan (H.I.). * Address correspondence to: David E. Cane, Department of Chemistry, Box H, Brown University, Providence, RI 02912-9108, USA; Tel: +1-401-863-3588; David_Cane@brown.edu. Haruo Ikeda, Laboratory of Microbial Engineering, Kitasato Institute for Life Sciences, Kitasato University, 1-15-1 Kitasato, Sagamihara, Minami-ku, Kanagawa 252-0373, Japan; Tel: .jp. ¶ These authors contributed equally to this work. Table of strains and plasmids, GC-MS and LC-MS data, and construction of deletion mutants and complemented bacterial mutants. This material is available free of charge at http://pubs.acs.org. SUPPORTING INFORMATION AVAILABLE NIH Public Access Author ManuscriptBiochemistry. Author manuscript; available in PMC 2012 March 15. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscriptbiochemical function by identification of their native substra...

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Section: Discussionmentioning

confidence: 79%

Genome Mining in Streptomyces. Elucidation of the Role of Baeyer−Villiger Monooxygenases and Non-Heme Iron-Dependent Dehydrogenase/Oxygenases in the Final Steps of the Biosynthesis of Pentalenolactone and Neopentalenolactone

et al. 2011

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“…5 The responsible P450, CYP105D7 (SAV_7469) has been cloned and characterized from S. avermitilis . 27 Although the recently determined structure of CYP105D7 (PDB ID 4UBS) has two molecules of diclofenac, rather than the natural substrate 11 , bound in the active site, 28 alignment with the structure of PntM carrying bound pentalenolactone F ( 2 ) reveals that the binding region for the carboxylate of 11 lies on the wall of the active site cavity of CYP105D7 that is opposite to that established for carboxylate binding by PntM. Binding of 1-deoxypentalenic acid ( 11 ) to CYP105D7 could therefore readily be accommodated by a ca.…”

Section: Resultsmentioning

confidence: 99%

The Cytochrome P450-Catalyzed Oxidative Rearrangement in the Final Step of Pentalenolactone Biosynthesis: Substrate Structure Determines Mechanism

2016

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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 orthologues 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 orthologues 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.

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“…There are only a limited number of reports on k cat /K m values for P450s. Examples are CYP105D7 (0.0006 M Ϫ1 s Ϫ1 ) from Streptomyces avermitilis (38), which is involved in the biosynthesis of pentalenolactone, and CYP107L (2.3 M Ϫ1 s Ϫ1 ) from Streptomyces platensis (39), which hydroxylates terfenadine, a kind of antihistamine agent. Compared to these values, the k cat /K m value (5.6 M Ϫ1 s Ϫ1 ) of the CYP154C3-catalyzed hydroxylation of ⌬ 4 -androstene-3,17-dione seems to be in an appropriate range.…”

Section: Discussionmentioning

confidence: 99%

Regio- and Stereospecific Hydroxylation of Various Steroids at the 16α Position of the D Ring by the Streptomyces griseus Cytochrome P450 CYP154C3

Makino

Katsuyama

Otomatsu³

et al. 2014

Appl Environ Microbiol

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c Cytochrome P450 monooxygenases (P450s), which constitute a superfamily of heme-containing proteins, catalyze the direct oxidation of a variety of compounds in a regio-and stereospecific manner; therefore, they are promising catalysts for use in the oxyfunctionalization of chemicals. In the course of our comprehensive substrate screening for all 27 putative P450s encoded by the Streptomyces griseus genome, we found that Escherichia coli cells producing an S. griseus P450 (CYP154C3), which was fused C terminally with the P450 reductase domain (RED) of a self-sufficient P450 from Rhodococcus sp., could transform various steroids (testosterone, progesterone, ⌬ 4 -androstene-3,17-dione, adrenosterone, 1,4-androstadiene-3,17-dione, dehydroepiandrosterone, 4-pregnane-3,11,20-trione, and deoxycorticosterone) into their 16␣-hydroxy derivatives as determined by nuclear magnetic resonance and high-resolution mass spectrometry analyses. The purified CYP154C3, which was not fused with RED, also catalyzed the regio-and stereospecific hydroxylation of these steroids at the same position with the aid of ferredoxin and ferredoxin reductase from spinach. The apparent equilibrium dissociation constant (K d ) values of the binding between CYP154C3 and these steroids were less than 8 M as determined by the heme spectral change, indicating that CYP154C3 strongly binds to these steroids. Furthermore, kinetic parameters of the CYP154C3-catalyzed hydroxylation of ⌬ 4 -androstene-3,17-dione were determined (K m , 31.9 ؎ 9.1 M; k cat , 181 ؎ 4.5 s ؊1 ). We concluded that CYP154C3 is a steroid D-ring 16␣-specific hydroxylase which has considerable potential for industrial applications. This is the first detailed enzymatic characterization of a P450 enzyme that has a steroid D-ring 16␣-specific hydroxylation activity. C ytochrome P450 monooxygenases (P450s or CYPs) are found in all three domains of life, i.e., archaea, bacteria, and eukarya, and are classified into more than 1,000 families by their amino acid sequence homology (1). They have the ability to hydroxylate inactive carbons of hydrocarbons or aromatic compounds regioand stereospecifically (2, 3). In animals, P450s are mainly involved in xenobiotic metabolism and steroid biosynthesis (4, 5). In plants, many P450s are involved in the biosynthesis of plant hormones and secondary metabolites (6). In contrast, the functions of bacterial P450s are largely unknown, although some bacterial P450s are required for secondary metabolite biosynthesis and assimilation of terpenoids, such as cineol or terpineol (7-10). In spite of their unknown physiological functions, however, bacterial P450s have attracted much attention as a rich resource for new biocatalysts for use in the oxyfunctionalization of chemicals (11, 12). For example, microbial conversion of steroid precursors is widely used for large-scale synthesis of steroid drugs (13-17).CYP154 family members are widely found in actinomycetes, and many putative CYP154 genes have been found in the genomes of Streptomyces species. The structu...

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Pentalenic acid is a shunt metabolite in the biosynthesis of the pentalenolactone family of metabolites: hydroxylation of 1-deoxypentalenic acid mediated by CYP105D7 (SAV_7469) of Streptomyces avermitilis

Cited by 37 publications

References 27 publications

Genome Mining in Streptomyces. Elucidation of the Role of Baeyer−Villiger Monooxygenases and Non-Heme Iron-Dependent Dehydrogenase/Oxygenases in the Final Steps of the Biosynthesis of Pentalenolactone and Neopentalenolactone

Genome Mining in Streptomyces. Elucidation of the Role of Baeyer−Villiger Monooxygenases and Non-Heme Iron-Dependent Dehydrogenase/Oxygenases in the Final Steps of the Biosynthesis of Pentalenolactone and Neopentalenolactone

The Cytochrome P450-Catalyzed Oxidative Rearrangement in the Final Step of Pentalenolactone Biosynthesis: Substrate Structure Determines Mechanism

Regio- and Stereospecific Hydroxylation of Various Steroids at the 16α Position of the D Ring by the Streptomyces griseus Cytochrome P450 CYP154C3

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