Mycobacterial cytochrome P450 125 (Cyp125) catalyzes the terminal hydroxylation of C27 steroids

Jenna K. Capyk, Rainer Kalscheuer, Gordon R. Stewart, Jie Liu, Hyukin Kwon, Rafael Zhao, Sachi Okamoto, William R. Jacobs, Lindsay D. Eltis, William W. Mohn

Research output: Contribution to journalArticle

94 Citations (Scopus)

Abstract

Cyp125 (Rv3545c), a cytochrome P450, is encoded as part of the cholesterol degradation gene cluster conserved among members of the Mycobacterium tuberculosis complex. This enzyme has been implicated in mycobacterial pathogenesis, and a homologue initiates cholesterol catabolism in the soil actinomycete Rhodococcus jostii RHA1. In Mycobacterium bovis BCG, cyp125 was up-regulated 7.1-fold with growth on cholesterol. A cyp125 deletion mutant of BCG did not grow on cholesterol and accumulated 4-cholesten-3-one when incubated in the presence of cholesterol. Wild-type BCG grew on this metabolite. By contrast, a parallel cyp125 deletion mutation of M. tuberculosis H37Rv did not affect growth on cholesterol. Purified Cyp125 from M. tuberculosis, heterologously produced in R. jostii RHA1, bound cholesterol and 4-cholesten-3-one with apparent dissociation constants of 0.20 ± 0.02 μM and 0.27 ± 0.05 μM, respectively. When reconstituted with KshB, the cognate reductase of the ketosteroid 9α-hydroxylase, Cyp125 catalyzed the hydroxylation of these steroids. MS and NMR analyses revealed that hydroxylation occurred at carbon 26 of the steroid side chain, allowing unambiguous classification of Cyp125 as a steroid C26-hydroxylase. This study establishes the catalytic function of Cyp125 and, in identifying an important difference in the catabolic potential of M. bovis and M. tuberculosis, suggests that Cyp125 may have an additional function in pathogenesis.

Original languageEnglish
Pages (from-to)35534-35542
Number of pages9
JournalJournal of Biological Chemistry
Volume284
Issue number51
DOIs
Publication statusPublished - Dec 18 2009

    Fingerprint

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Capyk, J. K., Kalscheuer, R., Stewart, G. R., Liu, J., Kwon, H., Zhao, R., ... Mohn, W. W. (2009). Mycobacterial cytochrome P450 125 (Cyp125) catalyzes the terminal hydroxylation of C27 steroids. Journal of Biological Chemistry, 284(51), 35534-35542. https://doi.org/10.1074/jbc.M109.072132