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Characterization of Hsac and Hsad, an Oxygenase and a Hydrolase in the Cholesterol Catabolic Pathway of Mycobacterium Tuberculosis

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Characterization of Hsac and Hsad, an Oxygenase and a Hydrolase in the Cholesterol Catabolic Pathway of Mycobacterium Tuberculosis Characterization of HsaC and HsaD, an oxygenase and a hydrolase in the cholesterol catabolic pathway of Mycobacterium tuberculosis by Katherine Yam A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES (Biochemistry & Molecular Biology) THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver) August 2011 © Katherine Yam, 2011 ABSTRACT 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 4 -1 -1 for DSHA (kcat/Km = 3.3 ± 0.3 x 10 M s ) 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 ii 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. iii PREFACE Parts of this thesis have been published in peer-reviewed journals. The identification of the cholesterol catabolic pathway in Rhodococcus jostii RHA1 and Mycobacterium tuberculosis appeared in the Proceedings of the National Academy of Sciences of USA (Van der Geize, R., Yam, K., Heuser, T., Wilbrink, M. H., Hara, H., Anderton, M. C., Sim, E., Dijkhuizen, L., Davies, J. E., Mohn, W. W., and Eltis, L. D. “A gene cluster encoding cholesterol catabolism in a soil actinomycete provides insight into Mycobacterium tuberculosis survival in macrophages” (2007) Proc Natl Acad Sci U S A 104, 1947-1952). In this study, I was responsible for cloning hsaC and hsaD from M. tuberculosis H37Rv genomic DNA and performing the preliminary activity tests with raw extracts. Dr. Van der Geize wrote the paper. This published work is located in section 2.2.1. Characterization of HsaC appeared in PLoS Pathogens (Yam, K. C.*, D'Angelo, I.*, Kalscheuer, R., Zhu, H., Wang, J. X., Snieckus, V., Ly, L. H., Converse, P. J., Jacobs, W. R., Jr., Strynadka, N., and Eltis, L. D. “Studies of a Ring-Cleaving Dioxygenase Illuminate the Role of Cholesterol Metabolism in the Pathogenesis of Mycobacterium tuberculosis” (2009) PLoS Pathog 5, e1000344 (*shared first authorship)). In this study, I was responsible for the HsaC purification, DHSA purification, and all kinetic aspects of this work. I also contributed to interpreting the structural data. Dr. D’Angelo and I wrote the paper. This published work is located in sections 3.1.1-3 and 3.2. Characterization of HsaD appeared in the Journal of Biological Chemistry (Lack, N. A., Yam, K. C., Lowe, E. D., Horsman, G. P., Owen, R. L., Sim, E., and Eltis, L. D. iv "Characterization of a carbon-carbon hydrolase from Mycobacterium tuberculosis involved in cholesterol metabolism." (2010). J Biol Chem 285(1): 434-443). In this study, I was responsible for HsaD purification, purification of meta-cleavage products, DSHA and HOPODA, determination of their chemical and physical properties, and all kinetic characterization. I also contributed to interpreting the structural data. I proposed a revised reaction mechanism for HsaD. Jie Liu helped construct the S114A variant. Dr. Lack and I wrote the paper. This published work is located in sections 3.1.4 and 3.4. The high-throughput screen to identify inhibitors of HsaC was performed in collaboration with Duncan Browman and Tom Pfeifer (Center for Drug Research and Development). These results will be part of a manuscript to be submitted shortly. I was responsible for the design of the high-throughput screening assay, the screening of the KD2 and DiverSet libraries, the data analysis, and the inhibitor studies of PPB sulfonamide and gedunin. This work can be located in section 3.3. v TABLE OF CONTENTS ABSTRACT.............................................................................................................................. ii PREFACE.................................................................................................................................iv TABLE OF CONTENTS ........................................................................................................vi LIST OF TABLES ....................................................................................................................x LIST OF FIGURES .................................................................................................................xi LIST OF ABBREVIATIONS ...............................................................................................xiv ACKNOWLEDGEMENTS ..................................................................................................xvi CHAPTER 1: INTRODUCTION............................................................................................1 1.1 Steroids..............................................................................................................................1 1.1.1 Cholesterol .................................................................................................................1 1.2 Bacterial catabolism of steroids ........................................................................................2 1.2.1 Cholesterol catabolic pathway ...................................................................................3 1.2.2 Side chain degradation...............................................................................................4 1.2.3 A, B ring degradation.................................................................................................5 1.2.4 C, D ring degradation.................................................................................................6 1.2.5 Distribution of steroid catabolic genes in actinomycetes ..........................................9 1.3 Mycobacterium tuberculosis ...........................................................................................10 1.3.1 Mtb strains and other model organisms ...................................................................10 1.3.2 Infection cycle of Mtb..............................................................................................11 1.3.3 Role of cholesterol catabolism in Mtb virulence .....................................................14 1.4 Enzymes of the meta-cleavage pathway .........................................................................18 1.4.1 Extradiol dioxygenases ............................................................................................18 1.4.2 Meta-cleavage product (MCP) hydrolases...............................................................22 1.5 Aim of this study .............................................................................................................27 CHAPTER 2: MATERIALS AND METHODS ..................................................................29 2.1 Chemicals and reagents...................................................................................................29 2.1.1 Commercially and privately sourced .......................................................................29 2.1.2 Preparation of DHSA...............................................................................................30 2.1.3 Preparation and characterization of HOPODA and DSHA .....................................31 vi 2.1.4 Oligonucleotides and DNA sequencing...................................................................32 2.2 Manipulation of DNA .....................................................................................................33 2.2.1 Cloning of Mtb genes...............................................................................................33
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