Dataset Information

Oleic acid based experimental evolution of Bacillus megaterium yielding an enhanced P450 BM3 variant.

ABSTRACT:

Background

Unlike most other P450 cytochrome monooxygenases, CYP102A1 from Bacillus megaterium (BM3) is both soluble and fused to its redox partner forming a single polypeptide chain. Like other monooxygenases, it can catalyze the insertion of oxygen unto the carbon-hydrogen bond which can result in a wide variety of commercially relevant products for pharmaceutical and fine chemical industries. However, the instability of the enzyme holds back the implementation of a BM3-based biocatalytic industrial processes due to the important enzyme cost it would prompt.

Results

In this work, we sought to enhance BM3's total specific product output by using experimental evolution, an approach not yet reported to improve this enzyme. By exploiting B. megaterium's own oleic acid metabolism, we pressed the evolution of a new variant of BM3, harbouring 34 new amino acid substitutions. The resulting variant, dubbed DE, increased the conversion of the substrate 10-pNCA to its product p-nitrophenolate 1.23 and 1.76-fold when using respectively NADPH or NADH as a cofactor, compared to wild type BM3.

Conclusions

This new DE variant, showed increased organic cosolvent tolerance, increased product output and increased versatility in the use of either nicotinamide cofactors NADPH and NADH. Experimental evolution can be used to evolve or to create libraries of evolved BM3 variants with increased productivity and cosolvent tolerance. Such libraries could in turn be used in bioinformatics to further evolve BM3 more precisely. The experimental evolution results also supports the hypothesis which surmises that one of the roles of BM3 in Bacillus megaterium is to protect it from exogenous unsaturated fatty acids by breaking them down.

SUBMITTER: Vincent T

PROVIDER: S-EPMC9281120 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description: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.