title: The activity and cofactor preferences of N-acetyl-1-D-myo-inosityl-2-amino-2-deoxy-alpha-D-glucopyranoside deacetylase (MshB) change depending on environmental conditions.
abstract: Actinomycetes, such as Mycobacterium species, are Gram-positive bacteria that utilize the small molecule mycothiol (MSH) as their primary reducing agent. Consequently, the enzymes involved in MSH biosynthesis are targets for drug development. The metal-dependent enzyme N-acetyl-1-D-myo-inosityl-2-amino-2-deoxy-α-D-glucopyranoside deacetylase (MshB) catalyzes the hydrolysis of N-acetyl-1-D-myo-inosityl-2-amino-2-deoxy-α-D-glucopyranoside to form 1-D-myo-inosityl-2-amino-2-deoxy-α-D-glucopyranoside and acetate, the fourth overall step in MSH biosynthesis. Inhibitors of metalloenzymes typically contain a group that binds to the active site metal ion; thus, a comprehensive understanding of the native cofactor(s) of metalloenzymes is critical for the development of biologically effective inhibitors. Herein, we examined the effect of metal ions on the overall activity of MshB and probed the identity of the native cofactor. We found that the activity of MshB follows the trend Fe(2+) > Co(2+) > Zn(2+) > Mn(2+) and Ni(2+). Additionally, our results show that the identity of the cofactor bound to purified MshB is highly dependent on the purification conditions used (aerobic versus anaerobic), as well as the metal ion content of the medium during protein expression. MshB prefers Fe(2+) under anaerobic conditions regardless of the metal ion content of the medium and switches between Fe(2+) and Zn(2+) under aerobic conditions as the metal content of the medium is altered. These results indicate that the cofactor bound to MshB under biological conditions is dependent on environmental conditions, suggesting that MshB may be a cambialistic metallohydrolase that contains a dynamic cofactor. Consequently, biologically effective inhibitors will likely need to dually target Fe(2+)-MshB and Zn(2+)-MshB.
authors.affiliation: Department of Biochemistry, Virginia Tech, Blacksburg, Virginia 24061, USA.
authors.forename: Xinyi
authors.initials: X
authors.lastname: Huang
authors.forename: Evren
authors.initials: E
authors.lastname: Kocabas
authors.forename: Marcy
authors.initials: M
authors.lastname: Hernick
mesh_terms: D000581:Amidohydrolases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D001426:Bacterial Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D003067:Coenzymes / Q000378:metabolism*; D003545:Cysteine / Q000378:metabolism; D006020:Glycopeptides / Q000378:metabolism; D007294:Inositol / Q000378:metabolism; D019216:Metals, Heavy / Q000378:metabolism*; D020102:Mycobacterium smegmatis / Q000201:enzymology* / Q000254:growth & development; D009169:Mycobacterium tuberculosis / Q000201:enzymology* / Q000254:growth & development
chemical_list: D001426:Bacterial Proteins; D003067:Coenzymes; D006020:Glycopeptides; D019216:Metals, Heavy; C089265:mycothiol; D007294:Inositol; D000581:Amidohydrolases; C417275:N-acetyl-1-D-inosityl-2-amino-2-deoxy-alpha-D-glucopyranoside deacetylase; D003545:Cysteine