Identity of cofactor bound to mycothiol conjugate amidase (Mca) influenced by expression and purification conditions

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• 作者：Evren Kocabas ; Hualan Liu ; Marcy Hernick
• 关键词：Mycothiol conjugate amidase ; Metalloamidase ; Zinc ; Iron ; MshB ; Mycothiol
• 刊名：Biometals
• 出版年：2015
• 出版时间：August 2015
• 年：2015
• 卷：28
• 期：4
• 页码：755-763
• 全文大小：866 KB
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• 作者单位：Evren Kocabas (1)
  Hualan Liu (1)
  Marcy Hernick (1) (2)
  
  1. Department of Biochemistry, Virginia Tech, Blacksburg, VA, 24061, USA
  2. Department of Pharmaceutical Sciences, Appalachian College of Pharmacy, Oakwood, VA, 24631, USA
  
• 刊物类别：Biomedical and Life Sciences
• 刊物主题：Life Sciences
  Biochemistry
  Physical Chemistry
  
• 出版者：Springer Netherlands
• ISSN：1572-8773

文摘

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 \(K_{\text{D}}^{{{\text{Zn}}\left( {\text{II}} \right)}}\) values (~10-0?pM). Furthermore, results from pull-down experiments using Halo-Mca indicate that Mca purifies with (stoichiometric) Fe2+ when purified under anaerobic conditions, and Zn2+ when purified under aerobic conditions. Consequently, Mca is likely a Fe2+-dependent enzyme under physiological conditions; with Zn2+-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 Fe2+ form of the enzyme.