Steroid conversion with CYP106A2 – production of pharmaceutically interesting DHEA metabolites

Schmitz, Daniela; Zapp, Josef; Bernhardt, Rita

doi:10.1186/1475-2859-13-81

Cited by 43 publications

(36 citation statements)

References 45 publications

(59 reference statements)

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“…Recently, the genomes of Bacillus megaterium DSM319 and ATCC 13368 were sequenced, allowing exploration of their cytochrome P450 complement. Its analysis has resulted in the identification and characterization of several novel steroid‐converting P450s . Here, we report the functional and structural properties of CYP109E1 from B. megaterium DSM319.…”

Section: Introductionmentioning

confidence: 99%

Structural basis of steroid binding and oxidation by the cytochrome P450 CYP109E1 from Bacillus megaterium

et al. 2016

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Cytochrome P450 monooxygenases (P450s) are attractive enzymes for the pharmaceutical industry, in particular, for applications in steroidal drug synthesis. Here, we report a comprehensive functional and structural characterization of CYP109E1, a novel steroid‐converting cytochrome P450 enzyme identified from the genome of Bacillus megaterium DSM319. In vitro and whole‐cell in vivo turnover experiments, combined with binding assays, revealed that CYP109E1 is able to hydroxylate testosterone at position 16β. Related steroids with bulky substituents at carbon C17, like corticosterone, bind to the enzyme without being converted. High‐resolution X‐ray structures were solved of a steroid‐free form of CYP109E1 and of complexes with testosterone and corticosterone. The structural analysis revealed a highly dynamic active site at the distal side of the heme, which is wide open in the absence of steroids, can bind four ordered corticosterone molecules simultaneously, and undergoes substantial narrowing upon binding of single steroid molecules. In the crystal structures, the single bound steroids adopt unproductive binding modes coordinating the heme‐iron with their C3‐keto oxygen. Molecular dynamics (MD) simulations suggest that the steroids may also bind in ~180° reversed orientations with the C16 carbon and C17‐substituents pointing toward the heme, leading to productive binding of testosterone explaining the observed regio‐ and stereoselectivity. The X‐ray structures and MD simulations further identify several residues with important roles in steroid binding and conversion, which could be confirmed by site‐directed mutagenesis. Taken together, our results provide unique insights into the CYP109E1 activity, substrate specificity, and regio/stereoselectivity.DatabaseThe atomic coordinates and structure factors have been deposited in the Protein Data Bank with accession codes 5L90 (steroid‐free CYP109E1), 5L91 (CYP109E1‐COR4), 5L94 (CYP109E1‐TES), and 5L92 (CYP109E1‐COR).EnzymesCytochrome P450 monooxygenase CYP109E1, EC 1.14.14.1, UniProt ID: D5DKI8, Adrenodoxin reductase EC 1.18.1.6.

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Section: Introductionmentioning

confidence: 99%

Structural basis of steroid binding and oxidation by the cytochrome P450 CYP109E1 from Bacillus megaterium

et al. 2016

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“…In this regard, bacterial P450s could be the best alternatives. However, there are only few bacterial P450s which can convert steroidal molecules including CYP106A1 , CYP106A2 , CYP125 , CYP142 , CYP154C5 , and CYP260 .…”

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Structural characterization of CYP260A1 from Sorangium cellulosum to investigate the 1α‐hydroxylation of a mineralocorticoid

et al. 2016

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In this study, we report the crystal structure of the cytochrome P450 CYP260A1 (PDB 5LIV) from the myxobacterium Sorangium cellulosum So ce56. In addition, we investigated the hydroxylation of 11-deoxycorticosterone by CYP260A1 by reconstituting the enzyme with the surrogate redox partners adrenodoxin and adrenodoxin reductase. The major product of this steroid conversion was identified as 1a-hydroxy-11-deoxycorticosterone, a novel D4 C-21 steroidal derivative. Furthermore, we docked the substrate into the crystal structure and replaced Ser326, the residue responsible for substrate orientation, with asparagine and observed that the mutant S326N displayed higher activity and selectivity for the formation of 1a-hydroxy-11-deoxycorticosterone compared to the wild-type CYP260A1. Thus, our findings highlight the usefulness of the obtained crystal structure of CYP260A1 in identifying biotechnologically more efficient reactions.

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“…It can be expressed with high yields in E. coli and B. megaterium and has already been applied for the preparative hydroxylation of several steroids, including dehydroepiandrosterone, pregnenolone, testosterone, deoxycorticosterone, deoxycortisol, and progesterone [92][93][94]. Recent studies showed that the main hydroxylation position of CYP106A2 is influenced by the character of groups attached to C-3 in the steroid core: 3-oxo-Δ4-steroids are preferentially hydroxylated at the 15β position whereas the 7β-position is favored when converting 3-hydroxy-Δ5-steroids [92]. However, the hydroxylation reaction is not strictly regioselective and the formation of different side products occurs in many CYP106A2-dependent bioconversions as, for instance, in the case of progesterone where 11α-, 6β-, and 9α-hydroxyprogesterone are formed.…”

Section: Steroidsmentioning

confidence: 99%

Terpene Hydroxylation with Microbial Cytochrome P450 Monooxygenases

Janocha

Schmitz

Bernhardt

2015

Biotechnology of Isoprenoids

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Terpenoids comprise a highly diverse group of natural products. In addition to their basic carbon skeleton, they differ from one another in their functional groups. Functional groups attached to the carbon skeleton are the basis of the terpenoids' diverse properties. Further modifications of terpene olefins include the introduction of acyl-, aryl-, or sugar moieties and usually start with oxidations catalyzed by cytochrome P450 monooxygenases (P450s, CYPs). P450s are ubiquitously distributed throughout nature, involved in essential biological pathways such as terpenoid biosynthesis as well as the tailoring of terpenoids and other natural products. Their ability to introduce oxygen into nonactivated C-H bonds is unique and makes P450s very attractive for applications in biotechnology. Especially in the field of terpene oxidation, biotransformation methods emerge as an attractive alternative to classical chemical synthesis. For this reason, microbial P450s depict a highly interesting target for protein engineering approaches in order to increase selectivity and activity, respectively. Microbial P450s have been described to convert industrial and pharmaceutically interesting terpenoids such as ionones, limone, valencene, resin acids, and triterpenes (including steroids) as well as vitamin D3. Highly selective and active mutants have been evolved by applying classical site-directed mutagenesis as well as directed evolution of proteins. As P450s usually depend on electron transfer proteins, mutagenesis has also been applied to improve the interactions between P450s and their respective redox partners. This chapter provides an overview of terpenoid hydroxylation reactions catalyzed by bacterial P450s and highlights the achievements made by protein engineering to establish productive hydroxylation processes.

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Steroid conversion with CYP106A2 – production of pharmaceutically interesting DHEA metabolites

Cited by 43 publications

References 45 publications

Structural basis of steroid binding and oxidation by the cytochrome P450 CYP109E1 from Bacillus megaterium

Structural basis of steroid binding and oxidation by the cytochrome P450 CYP109E1 from Bacillus megaterium

Structural characterization of CYP260A1 from Sorangium cellulosum to investigate the 1α‐hydroxylation of a mineralocorticoid

Terpene Hydroxylation with Microbial Cytochrome P450 Monooxygenases

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