Characterization of a carbon-carbon hydrolase from Mycobacterium tuberculosis involved in cholesterol metabolism.
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 (k(cat)/K(m) = 3.3 +/- 0.3 x 10(4) 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 K(d) of 51 +/- 2 mum. 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 Aindicate 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 alpha-helical lid covering the active site displayed a ligand-dependent conformational change involving differences in side chain carbon positions of up to 6.7 A, 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.
Lack
Nathan A
NA
Department of Pharmacology, University of Oxford, Oxford OX1 3QT, United Kingdom.
Yam
Katherine C
KC
Lowe
Edward D
ED
Horsman
Geoff P
GP
Owen
Robin L
RL
Sim
Edith
E
Eltis
Lindsay D
LD
D019943:Amino Acid Substitution / Q000235:genetics; D055162:Biocatalysis; D002784:Cholesterol / Q000737:chemistry / Q000378:metabolism*; D018360:Crystallography, X-Ray; D005231:Fatty Acids, Unsaturated / Q000737:chemistry / Q000378:metabolism; D006867:Hydrolases / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D008954:Models, Biological; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism; D009169:Mycobacterium tuberculosis / Q000201:enzymology*; D017433:Protein Structure, Secondary; D012996:Solutions; D013056:Spectrophotometry, Ultraviolet; D055672:Static Electricity; D013379:Substrate Specificity; D054159:Torsion, Mechanical
D005231:Fatty Acids, Unsaturated; D050505:Mutant Proteins; D012996:Solutions; D002784:Cholesterol; D006867:Hydrolases; C527647:HsaD protein, Mycobacterium tuberculosis