Characterization of HsaC and HsaD, an oxygenase and a hydrolase in the cholesterol catabolic pathway of Mycobacterium tuberculosis
| Autor: | Yam, Katherine |
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| Jazyk: | angličtina |
| Rok vydání: | 2011 |
| Druh dokumentu: | Text |
| Popis: | Mycobacterium tuberculosis (Mtb) is the leading cause of mortality from bacterial infection. A cholesterol degradation pathway identified in Mtb is implicated in the pathogen’s survival in the host. This pathway includes an Fe(II)-containing extradiol dioxygenase, HsaC, and a meta-cleavage product (MCP) hydrolase, HsaD, which are predicted to catalyze the cleavage of DHSA (3,4-dihydroxy-9,10-seconandrost-1,3,5(10)-triene-9,17-dione) and subsequent hydrolysis of DSHA (4,5-9,10-diseco-3-hydroxy-5,9,17-trioxoandrosta-1(10),2-diene-4-oic acid), respectively. HsaC and HsaD were expressed in E. coli, purified, and characterized. Substrates were obtained by biotransformation of cholesterol using a ΔhsaC mutant of Rhodococcus jostii RHA1. From steady-state kinetic studies, purified HsaC efficiently cleaved the proposed steroid metabolite, DHSA (kcat/Km = 15 ± 2 μM-¹s-¹), better than the biphenyl catechol, DHB, or a synthetic analogue, DHDS. Two halogenated substrates, 2’,6’-diCl DHB and 4-Cl DHDS, inactivated HsaC with partition coefficients < 50. Structures of HsaC:DHSA at 2.1 Å revealed predominantly bidentate binding of the catechol to the active site iron, as has been reported in similar enzymes. A high-throughput colorimetric assay was developed to screen for small molecular inhibitors of HsaC. 4-chloro-N-methyl-N-(4-[(4-methylpiperidin-1-yl) carbonyl] phenyl) benzene-1-sulfonamide and gedunin were identified as potent inhibitors of HsaC with Kic values of 450 ± 50 nM and 80 ± 10 nM, respectively. Purified HsaD had higher specificity for DSHA (kcat/Km = 3.3 ± 0.3 x 104 M-¹s-¹) than for the biphenyl metabolite and a synthetic analogue. The catalytically impaired S114A variant of HsaD bound DSHA with a Kd of 51 ± 2 μM. The S114A:DSHA species absorbed maximally at 456 nm, 60 nm red-shifted versus the DSHA enolate. Crystal structures of the S114A:DSHA complex at 1.9 Å identified the trapped intermediate as a 2-oxo, 6-oxido species. These data indicate that the catalytic serine catalyzes enol-to-keto tautomerization as well as C-C bond hydrolysis. While both ΔhsaC and ΔhsaD mutants of M. bovis BCG did not grow on cholesterol, the presence of an additional carbon source restored growth of the ΔhsaD mutant but only partial growth of the ΔhsaC mutant, likely due to toxic oxidized metabolites. Overall, the kinetic and structural characterization of these mycobacterial cholesterol-degrading enzymes provides novel insights into a disease of global importance. Medicine, Faculty of Biochemistry and Molecular Biology, Department of Graduate |
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