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Substrate recognition by the multifunctional cytochrome P450 MycG in mycinamicin hydroxylation and epoxidation reactions.

ABSTRACT: The majority of characterized cytochrome P450 enzymes in actinomycete secondary metabolic pathways are strictly substrate-, regio-, and stereo-specific. Examples of multifunctional biosynthetic cytochromes P450 with broader substrate and regio-specificity are growing in number and are of particular interest for biosynthetic and chemoenzymatic applications. MycG is among the first P450 monooxygenases characterized that catalyzes both hydroxylation and epoxidation reactions in the final biosynthetic steps, leading to oxidative tailoring of the 16-membered ring macrolide antibiotic mycinamicin II in the actinomycete Micromonospora griseorubida. The ordering of steps to complete the biosynthetic process involves a complex substrate recognition pattern by the enzyme and interplay between three tailoring modifications as follows: glycosylation, methylation, and oxidation. To understand the catalytic properties of MycG, we structurally characterized the ligand-free enzyme and its complexes with three native metabolites. These include substrates mycinamicin IV and V and their biosynthetic precursor mycinamicin III, which carries the monomethoxy sugar javose instead of the dimethoxylated sugar mycinose. The two methoxy groups of mycinose serve as sensors that mediate initial recognition to discriminate between closely related substrates in the post-polyketide oxidative tailoring of mycinamicin metabolites. Because x-ray structures alone did not explain the mechanisms of macrolide hydroxylation and epoxidation, paramagnetic NMR relaxation measurements were conducted. Molecular modeling based on these data indicates that in solution substrate may penetrate the active site sufficiently to place the abstracted hydrogen atom of mycinamicin IV within 6 Å of the heme iron and ~4 Å of the oxygen of iron-ligated water.

SUBMITTER: Li S

PROVIDER: S-EPMC3488060 | biostudies-literature | 2012 Nov

REPOSITORIES: biostudies-literature

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Substrate recognition by the multifunctional cytochrome P450 MycG in mycinamicin hydroxylation and epoxidation reactions.

Li Shengying S Tietz Drew R DR Rutaganira Florentine U FU Kells Petrea M PM Anzai Yojiro Y Kato Fumio F Pochapsky Thomas C TC Sherman David H DH Podust Larissa M LM

The Journal of biological chemistry 20120905 45

The majority of characterized cytochrome P450 enzymes in actinomycete secondary metabolic pathways are strictly substrate-, regio-, and stereo-specific. Examples of multifunctional biosynthetic cytochromes P450 with broader substrate and regio-specificity are growing in number and are of particular interest for biosynthetic and chemoenzymatic applications. MycG is among the first P450 monooxygenases characterized that catalyzes both hydroxylation and epoxidation reactions in the final biosynthet ...[more]

PMID: 22952225

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Project description:Our previous study showed that CYP105D7, a substrate-promiscuous P450, catalyzes the hydroxylation of 1-deoxypentalenic acid, diclofenac, naringenin, and compactin. In this study, 14 steroid compounds were screened using recombinant Escherichia coli cells harboring genes encoding CYP105D7 and redox partners (Pdx/Pdr, RhFRED, and FdxH/FprD), and the screening identified steroid A-ring 2?- and D-ring 16?-hydroxylation activity. Wild-type CYP105D7 was able to catalyze the hydroxylation of five steroids (testosterone, progesterone, 4-androstene-3,17-dione, adrenosterone, and cortisone) with low (<10%) conversion rates. Structure-guided site-directed mutagenesis of arginine residues around the substrate entrance and active site showed that the R70A and R190A single mutants and an R70A/R190A double mutant exhibited greatly enhanced conversion rates for steroid hydroxylation. For the conversion of testosterone in particular, the R70A/R190A mutant's k cat/Km values increased 1.35-fold and the in vivo conversion rates increased significantly by almost 9-fold with high regio- and stereoselectivity. Molecular docking analysis revealed that when Arg70 and Arg190 were replaced with alanine, the volume of the substrate access and binding pocket increased 1.08-fold, which might facilitate improvement of the hydroxylation efficiency of steroids.IMPORTANCE Cytochrome P450 monooxygenases (P450s) are able to introduce oxygen atoms into nonreactive hydrocarbon compounds under mild conditions, thereby offering significant advantages compared to chemical catalysts. Promiscuous P450s with broad substrate specificity and reaction diversity have significant potential for applications in various fields, including synthetic biology. The study of the function, molecular mechanisms, and rational engineering of substrate-promiscuous P450s from microbial sources is important to fulfill this potential. Here, we present a microbial substrate-promiscuous P450, CYP105D7, which can catalyze hydroxylation of steroids. The loss of the bulky side chains of Arg70 and Arg190 in the active site and substrate entrance resulted in an up to 9-fold increase in the substrate conversion rate. These findings will support future rational and semirational engineering of P450s for applications as biocatalysts.

Dataset Information

Substrate recognition by the multifunctional cytochrome P450 MycG in mycinamicin hydroxylation and epoxidation reactions.

Publications

Substrate recognition by the multifunctional cytochrome P450 MycG in mycinamicin hydroxylation and epoxidation reactions.

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