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Characterization of a C-C hydrolase from Mycobacterium tuberuclosis involved in cholesterol metabolism

DOI: 10.1074/jbc.M109.058081 DOI Help
PMID: 19875455 PMID Help

Authors: Nathan Lack (University of Oxford) , Katherine Yam (University of British Columbia) , Ed Lowe (University of Oxford) , Geoff Horsman (University of British Columbia) , Robin Owen (Diamond Light Source) , Edith Sim (University of Oxford) , Lindsay Eltis (University of British Columbia)
Co-authored by industrial partner: No

Type: Journal Paper
Journal: Journal Of Biological Chemistry

State: Published (Approved)
Published: October 2009

Abstract: In the recently identified cholesterol catabolic pathway of Mycobacterium tuberculosis, 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoate hydrolase (HsaD) is proposed to catalyze the hydrolysis of a carbon-carbon bond in 4,5–9,10-diseco-3-hydroxy-5,9,17-tri-oxoandrosta-1(10),2-diene-4-oic acid (DSHA), the cholesterol meta-cleavage product (MCP) and has been implicated in the intracellular survival of the pathogen. Herein, purified HsaD demonstrated 4–33 times higher specificity for DSHA (kcat/Km = 3.3 ± 0.3 × 104 m?1 s?1) than for the biphenyl MCP 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid (HOPDA) and the synthetic analogue 8-(2-chlorophenyl)-2-hydroxy-5-methyl-6-oxoocta-2,4-dienoic acid (HOPODA), respectively. The S114A variant of HsaD, in which the active site serine was substituted with alanine, was catalytically impaired and bound DSHA with a Kd of 51 ± 2 ?m. The S114A·DSHA species absorbed maximally at 456 nm, 60 nm red-shifted versus the DSHA enolate. Crystal structures of the variant in complex with HOPDA, HOPODA, or DSHA to 1.8–1.9 Åindicate that this shift is due to the enzyme-induced strain of the enolate. These data indicate that the catalytic serine catalyzes tautomerization. A second role for this residue is suggested by a solvent molecule whose position in all structures is consistent with its activation by the serine for the nucleophilic attack of the substrate. Finally, the ?-helical lid covering the active site displayed a ligand-dependent conformational change involving differences in side chain carbon positions of up to 6.7 Å, supporting a two-conformation enzymatic mechanism. Overall, these results provide novel insights into the determinants of specificity in a mycobacterial cholesterol-degrading enzyme as well as into the mechanism of MCP hydrolases.

Journal Keywords: Biocatalysis; Cholesterol; Crystallography; X-Ray; Fatty; Unsaturated; Hydrolases; Kinetics; Models; Biological; Mutant; Mycobacterium; Protein; Secondary; Solutions; Spectrophotometry; Ultraviolet; Static; Substrate; Torsion; Mechanical

Subject Areas: Biology and Bio-materials

Instruments: I03-Macromolecular Crystallography