title: Cloning, expression and characterization of histidine-tagged biotin synthase of Mycobacterium tuberculosis.
abstract: The emergence of Mycobacterium tuberculosis strains that are resistant to the current anti-tuberculosis (TB) drugs necessitates a need to develop a new class of drugs whose targets are different from the current ones. M. tuberculosis biotin synthase (MtbBS) is one such target that is essential for the survival of the bacteria. In this study, MtbBS was cloned, overexpressed and purified to homogeneity for biochemical characterization. It is likely to be a dimer in its native form. Its pH and temperature optima are 8.0 and 37 °C, respectively. Km for DTB and SAM was 2.81 ± 0.35 and 9.95 ± 0.98 μM, respectively. The enzyme had a maximum velocity of 0.575 ± 0.015 μM min(-1), and a turn-over of 0.0935 min(-1). 5'-deoxyadenosine (dAH), S-(5'-Adenosyl)-l-cysteine (AdoCy) and S-(5'-Adenosyl)-l-homocysteine (AdoHcy) were competitive inhibitors of MtbBS with the following inactivation parameters: Ki = 24.2 μM, IC50 = 267.4 μM; Ki = 0.84 μM, IC50 = 9.28 μM; and Ki = 0.592 μM, IC50 = 6.54 μM for dAH, AdoCy and AdoHcy respectively. dAH could inhibit the growth of M. tuberculosis H37Ra with an MIC of 392.6 μg/ml. This information should be useful for the discovery of inhibitors of MtbBS.
authors.affiliation: Department of Microbiology, Faculty of Science, Mahidol University, Rama 6 Road, Bangkok 10400, Thailand; Ministry of Health, Private Bag 0038, Gaborone, Botswana. Electronic address: cmagwamba@yahoo.com.
authors.forename: Clement Chedza
authors.initials: CC
authors.lastname: Magwamba
authors.affiliation: Mahidol University, Amnatcharoen Campus, Muang, Amnatcharoen 37000, Thailand. Electronic address: rukseree@gmail.com.
authors.forename: Kamolchanok
authors.initials: K
authors.lastname: Rukseree
authors.affiliation: Department of Microbiology, Faculty of Science, Mahidol University, Rama 6 Road, Bangkok 10400, Thailand; National Center for Genetic Engineering and Biotechnology, National Science and Technology Development Agency, Pathumthani 12120, Thailand. Electronic address: prasit.pal@mahidol.ac.th.
authors.forename: Prasit
authors.initials: P
authors.lastname: Palittapongarnpim
mesh_terms: D000995:Antitubercular Agents / Q000494:pharmacology; D001426:Bacterial Proteins / Q000037:antagonists & inhibitors / Q000096:biosynthesis* / Q000235:genetics; D001710:Biotin / Q000031:analogs & derivatives / Q000378:metabolism; D003001:Cloning, Molecular; D003545:Cysteine / Q000031:analogs & derivatives / Q000494:pharmacology; D003839:Deoxyadenosines / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004795:Enzyme Stability; D006639:Histidine / Q000096:biosynthesis* / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008826:Microbial Sensitivity Tests; D009169:Mycobacterium tuberculosis / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics; D015202:Protein Engineering; D055503:Protein Multimerization; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D012435:S-Adenosylhomocysteine / Q000494:pharmacology; D012436:S-Adenosylmethionine / Q000378:metabolism; D013379:Substrate Specificity; D013466:Sulfurtransferases / Q000037:antagonists & inhibitors / Q000096:biosynthesis* / Q000235:genetics; D013696:Temperature
chemical_list: D000995:Antitubercular Agents; D001426:Bacterial Proteins; D003839:Deoxyadenosines; D004791:Enzyme Inhibitors; D011993:Recombinant Fusion Proteins; C033223:polyhistidine; D006639:Histidine; D001710:Biotin; C004749:desthiobiotin; D012436:S-Adenosylmethionine; D012435:S-Adenosylhomocysteine; D013466:Sulfurtransferases; C057985:biotin synthetase; D003545:Cysteine; C058118:2'-deoxyadenosine
keywords: Biotin synthase; Drug target; Mycobacterium tuberculosis