title: Identity of cofactor bound to mycothiol conjugate amidase (Mca) influenced by expression and purification conditions.
abstract: Mycothiol serves as the primary reducing agent in Mycobacterium species, and is also a cofactor for the detoxification of xenobiotics. Mycothiol conjugate amidase (Mca) is a metalloamidase that catalyzes the cleavage of MS-conjugates to form a mercapturic acid, which is excreted from the mycobacterium, and 1-D-myo-inosityl-2-amino-2-deoxy-α-D-glucopyranoside. Herein we report on the metal cofactor preferences of Mca from Mycobacterium smegmatis and Mycobacterium tuberculosis. Importantly, results from homology models of Mca from M. smegmatis and M. tuberculosis suggest that the metal binding site of Mca is identical to that of the closely related protein N-acetyl-1-D-myo-inosityl-2-amino-2-deoxy-α-D-glucopyranoside deacetylase (MshB). This finding is supported by results from zinc ion affinity measurements that indicate Mca and MshB have comparable K(D)(ZnII) values (~10-20 pM). Furthermore, results from pull-down experiments using Halo-Mca indicate that Mca purifies with (stoichiometric) Fe(2+) when purified under anaerobic conditions, and Zn(2+) when purified under aerobic conditions. Consequently, Mca is likely a Fe(2+)-dependent enzyme under physiological conditions; with Zn(2+)-Mca an experimental artifact that could become biologically relevant under oxidatively stressed conditions. Importantly, these findings suggest that efforts towards the design of Mca inhibitors should include targeting the Fe(2+) form of the enzyme.
authors.affiliation: Department of Biochemistry, Virginia Tech, Blacksburg, VA, 24061, USA.
authors.forename: Evren
authors.initials: E
authors.lastname: Kocabas
authors.forename: Hualan
authors.initials: H
authors.lastname: Liu
authors.forename: Marcy
authors.initials: M
authors.lastname: Hernick
mesh_terms: D000581:Amidohydrolases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D003067:Coenzymes / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D005296:Ferrous Compounds / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D020102:Mycobacterium smegmatis / Q000201:enzymology; D009169:Mycobacterium tuberculosis / Q000201:enzymology; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D015032:Zinc / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism
chemical_list: D003067:Coenzymes; D005296:Ferrous Compounds; D011994:Recombinant Proteins; D000581:Amidohydrolases; C413523:mycothiol S-conjugate amidase; D015032:Zinc