Identification of native co-factors of MshB and MCA from Mycobacterium species

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dc.contributor.authorKocabas, Evrenen
dc.contributor.committeechairHernick, Marcyen
dc.contributor.committeememberHelm, Richard F.en
dc.contributor.committeememberKlemba, Michaelen
dc.contributor.departmentBiochemistryen
dc.date.accessioned2014-03-14T21:43:54Zen
dc.date.adate2010-09-21en
dc.date.available2014-03-14T21:43:54Zen
dc.date.issued2010-08-11en
dc.date.rdate2010-09-21en
dc.date.sdate2010-08-26en
dc.description.abstractMycothiol (MSH), a low-molecular- weight thiol, is a primary reducing agent and essential for the survival of mycobacteria. The full pathway of MSH biosynthesis and detoxification includes various promising drug targets. Several metalloenzymes are involved in this pathway, such as a deacetylase (MshB) and mycothiol S-conjugate amidase (MCA). MshB catalyzes the deacetylation of GlcNAc-Ins to form GlcN-Ins and acetate. Mycothiol S-conjugate amidase (MCA) cleaves the amide bond of mycothiol S-conjugates of various drugs and toxins. The identification of the native co-factor is critical for the design of potent and effective inhibitors. Therefore, in this study, we identified the possible native co-factors of MshB and MCA from M. smegmatis and M. tuberculosis. To reach our aim, we used a pull-down method to rapidly purify halo-MshB and halo-MCA under anaerobic conditions. Our data indicates that the metal bound to MshB and MCA anaerobically purified from E. coli grown in minimal medium is mainly Fe(II), while proteins purified under aerobic conditions contain bound Zn (II) and Fe(II) that varies with the metal content of the medium. For a further clarification of the metal ion preferences of MshB and MCA, we determined the MshB and MCA affinity for Zn(II) to be in the picomolar range and Ms MshB affinity for Fe(II) in nanomolar range. These results indicate that MshB and MCA can be found bound with either iron or zinc and this is independent to their affinities for these metal ions.en
dc.description.degreeMaster of Scienceen
dc.identifier.otheretd-08262010-130942en
dc.identifier.sourceurlhttp://scholar.lib.vt.edu/theses/available/etd-08262010-130942/en
dc.identifier.urihttp://hdl.handle.net/10919/44457en
dc.publisherVirginia Techen
dc.relation.haspartKocabas_E_T_2010.pdfen
dc.rightsIn Copyrighten
dc.rights.urihttp://rightsstatements.org/vocab/InC/1.0/en
dc.subjectMycobacterium tuberculosisen
dc.subjectmetal-dependent deacetylaseen
dc.subjectMCAen
dc.subjectMshBen
dc.subjectironen
dc.subjectzincen
dc.subjectMycobacterium smegmatisen
dc.titleIdentification of native co-factors of MshB and MCA from Mycobacterium speciesen
dc.typeThesisen
thesis.degree.disciplineBiochemistryen
thesis.degree.grantorVirginia Polytechnic Institute and State Universityen
thesis.degree.levelmastersen
thesis.degree.nameMaster of Scienceen

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