Cholesterol Ester Oxidation by Mycobacterial Cytochrome P450

Daniel J Frank,Yarrow Madrona,Paul R Ortiz De Montellano

doi:10.1074/jbc.m114.602771

Daniel J Frank, Yarrow Madrona + Show 1 more

Open Access

https://doi.org/10.1074/jbc.m114.602771

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Abstract

Mycobacteria share a common cholesterol degradation pathway initiated by oxidation of the alkyl side chain by enzymes of cytochrome P450 (CYP) families 125 and 142. Structural and sequence comparisons of the two enzyme families revealed two insertions into the N-terminal region of the CYP125 family (residues 58-67 and 100-109 in the CYP125A1 sequence) that could potentially sterically block the oxidation of the longer cholesterol ester molecules. Catalytic assays revealed that only CYP142 enzymes are able to oxidize cholesteryl propionate, and although CYP125 enzymes could oxidize cholesteryl sulfate, they were much less efficient at doing so than the CYP142 enzymes. The crystal structure of CYP142A2 in complex with cholesteryl sulfate revealed a substrate tightly fit into a smaller active site than was previously observed for the complex of CYP125A1 with 4-cholesten-3-one. We propose that the larger CYP125 active site allows for multiple binding modes of cholesteryl sulfate, the majority of which trigger the P450 catalytic cycle, but in an uncoupled mode rather than one that oxidizes the sterol. In contrast, the more unhindered and compact CYP142 structure enables enzymes of this family to readily oxidize cholesteryl esters, thus providing an additional source of carbon for mycobacterial growth.

Highlights

Mycobacterial cytochrome P450 families 125 and 142 initiate metabolism of host cholesterol
A fit of the spectral titrations to a single site binding model revealed that M. tuberculosis CYP142A1 bound both substrates more tightly, with KS ϭ 17.6 Ϯ 3.6 versus 32.8 Ϯ 6.6 ␮M for cholesteryl sulfate and KS ϭ 0.16 Ϯ 0.09 versus 1.5 Ϯ 0.1 ␮M for cholesteryl propionate for CYP142A1 and CYP142A2, respectively, whereas M. smegmatis CYP142A2 produced a larger change in the amplitude of the spectral response (Fig. 1, A and B)
Acid by CYP125A1 and CYP142A1, which in turn, can be catabolized via the ␤-oxidation pathway [7,8,9]. This pathway is conserved in the soil dwelling relative M. smegmatis, whose genome encodes CYP125A3 and CYP142A2, enzymes that share ϳ70% sequence identity with their M. tuberculosis paralogs [10]

Summary

Background

Mycobacterial cytochrome P450 families 125 and 142 initiate metabolism of host cholesterol. This cholesterol degradation pathway is conserved in the soil-dwelling relative of M. tuberculosis, Mycobacterium smegmatis, in which CYP125A3 and CYP142A2 serve as paralogous enzymes [10] Both sets of paralogs share ϳ70% sequence identity, similar kinetic activities toward 4-cholesten-3-one, and overall structural similarity. Structural and sequence comparisons revealed two ϳ10 residue insertions in the CYP125 sequences, which are not present in the CYP142 sequences, (residues 58 – 67 and 100 –109 in the CYP125A1 sequence; Table 1) that form a cap over the active site in the CYP125 enzymes We hypothesized that this structural difference might create a steric barrier that prevents CYP125 from catabolizing the larger cholesterol ester molecules, compounds that have been shown to accumulate in M. tuberculosis infected macrophages [15, 16].

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION