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

Abstract

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...

A Peroxygenase from Chaetomium globosum Catalyzes the Selective Oxygenation of Testosterone

“…Its formation was shown to be catalyzed by membrane‐bound cytochrome P450s in human and rat liver microsomes;50 beyond that, the hydroxylation of testosterone at the 16α‐position is an important step in the formation of estriol in late pregnancy 51, 52. Soluble P450s of Gram‐positive bacteria, such as CYP154C3 from Streptomyces griseus , P450 BM3 (CYP102A1, variant M01 A82W S72I) from Bacillus megaterium , and CYP145C5 from Nocardia farcinica , were reported to selectively hydroxylate testosterone and related steroids at the 16α‐position 8, 53, 54. Crude preparations of the latter enzyme were particularly efficient (as an isolated P450) and achieved TTNs of 500 to 2300 for this reaction 54.…”

Substrate Hunting for the Myxobacterial CYP260A1 Revealed New 1α‐Hydroxylated Products from C‐19 Steroids

“…Hydroxylation of testosterone by human CYPsr eviewed by Agematu et al,[43] and Niwa et al,[49] and by the more recently reported human,[50][51][52] and bacterial[44,[53][54][55][56] CYPs is shown. Hydroxylation of testosterone by human CYPsr eviewed by Agematu et al,[43] and Niwa et al,[49] and by the more recently reported human,[50][51][52] and bacterial[44,[53][54][55][56] CYPs is shown.…”

Cytochromes P450 for natural product biosynthesis in Streptomyces: sequence, structure, and function

“…While each P450 behaves differently, streptomycete P450s have been shown to accept redox proteins from other Streptomyces species, 36–38 other bacterial genera including putidaredoxin reductase and putidaredoxin from the P450 cam system in Pseudomonas putida 39,40 and flavodoxin reductase and flavodoxin from Escherichia coli , 41,42 and eukaryotes such as the commercially available spinach ferredoxin reductase and ferredoxin. 43,44 Inspired by the one-component systems, Streptomyces P450s have also been engineered into redox self-sufficient enzymes by the fusion of the P450 protein to the reductase domains of P450 RhF 45–47 or CYP102D1. 48 Finally, some P450s, are able to utilize H 2 O 2 as a surrogate for the oxygens, electrons, and proton needed to directly generate Cpd 0 from the substrate-bound high-spin ferric P450 (i.e., peroxide shunt or peroxygenation; Fig.…”