Cholesterol metabolism in Mycobacterium smegmatis

Degradation of the cholesterol side-chain in Mycobacterium tuberculosis is initiated by two cytochromes P450, CYP125A1 and CYP142A1, that sequentially oxidize C26 to the alcohol, aldehyde and acid metabolites. Here we report characterization of the homologous enzymes CYP125A3 and CYP142A2 from Mycobacterium smegmatis mc(2) 155. Heterologously expressed, purified CYP125A3 and CYP142A2 bound cholesterol, 4-cholesten-3-one, and antifungal azole drugs. CYP125A3 or CYP142A2 reconstituted with spinach ferredoxin and ferredoxin reductase efficiently hydroxylated 4-cholesten-3-one to the C-26 alcohol and subsequently to the acid. The X-ray structures of both substrate-free CYP125A3 and CYP142A2 and of cholest-4-en-3-one-bound CYP142A2 reveal significant differences in the substrate binding sites compared with the homologous M. tuberculosis proteins. Deletion only of cyp125A3 causes a reduction of both the alcohol and acid metabolites and a strong induction of cyp142 at the mRNA and protein levels, indicating that CYP142A2 serves as a functionally redundant back up enzyme for CYP125A3. In contrast to M. tuberculosis, the M. smegmatis Δcyp125Δcyp142 double mutant retains its ability to grow on cholesterol albeit with a diminished capacity, indicating an additional level of redundancy within its genome

A highly conserved mycobacterial cholesterol catabolic pathway

18 p.- 9 fig.- 3 tab.Degradation of the cholesterol side-chain in M. tuberculosis is initiated by two cytochromes P450, CYP125A1 and CYP142A1, that sequentially oxidize C26 to the alcohol, aldehyde and acid metabolites. Here we report characterization of the homologous enzymes CYP125A3 and CYP142A2 from M. smegmatis mc2 155. Heterologously expressed, purified CYP125A3 and CYP142A2 bound cholesterol, 4-cholesten-3-one, and antifungal azole drugs. CYP125A3 or CYP142A2 reconstituted with spinach ferredoxin and ferredoxin reductase efficiently hydroxylated 4-cholesten-3-one to the C-26 alcohol and subsequently to the acid. The X-ray structures of both substrate-free CYP125A3 and CYP142A2 and of cholest-4-en-3-one-bound CYP142A2 reveal significant differences in the substrate binding sites compared with the homologous M. tuberculosis proteins. Deletion of cyp125A3 or cyp142A2 does not impair growth of M. smegmatis mc2 155 on cholesterol. However, deletion only of cyp125A3 causes a reduction of both the alcohol and acid metabolites and a strong induction of cyp142 at the mRNA and protein levels, indicating that CYP142A2 serves as a functionally redundant back up enzyme for CYP125A3. In contrast to M. tuberculosis, the M. smegmatis ∆cyp125∆cyp142 double mutant retains its ability to grow on cholesterol albeit with a diminished capacity, indicating an additional level of redundancy within its genome.Spanish Ministry of Science and Innovation BFU2006-15214-C03-01 and BFU2009-11545-C03-03 (to J. L. G.), an FPU predoctoral fellowship from the Spanish Ministry of Education and Science (to E. G. F.), NIH Grants AI074824 (to P. O. M.), and AI095437 and GM078553 (to L. M. P.). The Advanced Light Source is supported by the Director, Office of Science, Office of Basic Energy Sciences, of the U.S. Department of Energy under Contract No. DE-AC02-05CH11231.Peer reviewe

Directing Group-Controlled Regioselectivity in an Enzymatic C–H Bond Oxygenation

Highly regioselective remote hydroxylation of a natural product scaffold is demonstrated by exploiting the anchoring mechanism of the biosynthetic P450 monooxygenase PikC_D50N-RhFRED. Previous studies have revealed structural and biochemical evidence for the role of a salt bridge between the desosamine <i>N,N</i>-dimethylamino functionality of the natural substrate YC-17 and carboxylate residues within the active site of the enzyme, and selectivity in subsequent C–H bond functionalization. In the present study, a substrate-engineering approach was conducted that involves replacing desosamine with varied synthetic <i>N,N</i>-dimethyl­amino anchoring groups. We then determined their ability to mediate enzymatic total turnover numbers approaching or exceeding that of the natural sugar, while enabling ready introduction and removal of these amino anchoring groups from the substrate. The data establish that the size, stereochemistry, and rigidity of the anchoring group influence the regioselectivity of enzymatic hydroxylation. The natural anchoring group desosamine affords a 1:1 mixture of regioisomers, while synthetic anchors shift YC-17 analogue C-10/C-12 hydroxylation from 20:1 to 1:4. The work demonstrates the utility of substrate engineering as an orthogonal approach to protein engineering for modulation of regioselective C–H functionalization in biocatalysis