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A Flavin-dependent Monooxygenase from Mycobacterium tuberculosis Involved in Cholesterol Catabolism

Dresen, Carola and Lin, Leo Y.-C. and D'Angelo, Igor and Tocheva, Elitza I. and Strynadka, Natalie and Eltis, Lindsay D. (2010) A Flavin-dependent Monooxygenase from Mycobacterium tuberculosis Involved in Cholesterol Catabolism. Journal of Biological Chemistry, 285 (29). 22264-22275 . ISSN 0021-9258. PMCID PMC2903365. http://resolver.caltech.edu/CaltechAUTHORS:20100812-092558448

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Abstract

Mycobacterium tuberculosis (Mtb) and Rhodococcus jostii RHA1 have similar cholesterol catabolic pathways. This pathway contributes to the pathogenicity of Mtb. The hsaAB cholesterol catabolic genes have been predicted to encode the oxygenase and reductase, respectively, of a flavin-dependent mono-oxygenase that hydroxylates 3-hydroxy-9,10-seconandrost-1,3,5(10)-triene-9,17-dione (3-HSA) to a catechol. An hsaA deletion mutant of RHA1 did not grow on cholesterol but transformed the latter to 3-HSA and related metabolites in which each of the two keto groups was reduced: 3,9-dihydroxy-9,10-seconandrost-1,3,5(10)-triene-17-one (3,9-DHSA) and 3,17-dihydroxy-9,10-seconandrost-1,3,5(10)-triene-9-one (3,17-DHSA). Purified 3-hydroxy-9,10-seconandrost-1,3,5(10)-triene-9,17-dione 4-hydroxylase (HsaAB) from Mtb had higher specificity for 3-HSA than for 3,17-DHSA (apparent k_(cat)/K_m = 1000 ± 100 M^(−1) s^(−1) versus 700 ± 100 M^(−1) s^(−1)). However, 3,9-DHSA was a poorer substrate than 3-hydroxybiphenyl (apparent k_(cat)/K_m = 80 ± 40 M^(−1) s^(−1)). In the presence of 3-HSA the K_(mapp) for O_2 was 100 ± 10 μM. The crystal structure of HsaA to 2.5-Å resolution revealed that the enzyme has the same fold, flavin-binding site, and catalytic residues as p-hydroxyphenyl acetate hydroxylase. However, HsaA has a much larger phenol-binding site, consistent with the enzyme's substrate specificity. In addition, a second crystal form of HsaA revealed that a C-terminal flap (Val^(367)–Val^(394)) could adopt two conformations differing by a rigid body rotation of 25° around Arg^(366). This rotation appears to gate the likely flavin entrance to the active site. In docking studies with 3-HSA and flavin, the closed conformation provided a rationale for the enzyme's substrate specificity. Overall, the structural and functional data establish the physiological role of HsaAB and provide a basis to further investigate an important class of monooxygenases as well as the bacterial catabolism of steroids.


Item Type:Article
Related URLs:
URLURL TypeDescription
http://dx.doi.org/10.1074/jbc.M109.099028 DOIArticle
http://www.jbc.org/content/285/29/22264PublisherArticle
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2903365/PubMed CentralArticle
ORCID:
AuthorORCID
Tocheva, Elitza I.0000-0002-4869-8319
Additional Information:© 2010 American Society for Biochemistry and Molecular Biology. Received December 24, 2009. Revision received April 6, 2010. The atomic coordinates and structure factors (codes 3AFE and 3AFF) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/). This work was supported by grants from the Canadian Institute for Health Research (to L. D. E. and N. S., respectively) and the Michael Smith Foundation for Health Research (MSFHR) Infrastructure (to N. S.) and Emerging Team programs. We thank the Canadian Light Source (Saskatoon, Saskatchewan, Canada) for access to beamline CMCF1 for X-ray synchrotron data collection. Jie Liu, Mark Okon, Gord Stewart, and Christine Florizone provided skilled technical assistance. Dr. Victor Snieckus and Katherine Yam provided 2,3-dihydroxybiphenyl and HsaC, respectively.
Funders:
Funding AgencyGrant Number
Canadian Institutes of Health Research (CIHR)UNSPECIFIED
Michael Smith Foundation for Health ResearchUNSPECIFIED
Deutsche Forschungsgemeinschaft (DFG)UNSPECIFIED
PubMed Central ID:PMC2903365
Record Number:CaltechAUTHORS:20100812-092558448
Persistent URL:http://resolver.caltech.edu/CaltechAUTHORS:20100812-092558448
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:19408
Collection:CaltechAUTHORS
Deposited By: Tony Diaz
Deposited On:13 Aug 2010 16:56
Last Modified:27 Sep 2017 17:33

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