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J. Biol. Chem., Vol. 282, Issue 22, 16288-15294, June 1, 2007

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Crystal Structures and Site-directed Mutagenesis of a Mycothiol-dependent Enzyme Reveal a Novel Folding and Molecular Basis for Mycothiol-mediated Maleylpyruvate Isomerization^*

Rui Wang¹, Ya-Jie Yin^¶1, Feng Wang, Mei Li, Jie Feng^¶, Hong-Mei Zhang, Ji-Ping Zhang, Shuang-Jiang Liu^¶2, and Wen-Rui Chang³

From the National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, the ^¶State Key Laboratory of Microbial Resource, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100080, and the Graduate University of Chinese Academy of Sciences, Beijing 100049, China

Mycothiol (MSH) is the major low molecular mass thiols in many Gram-positive bacteria such as Mycobacterium tuberculosis and Corynebacterium glutamicum. The physiological roles of MSH are believed to be equivalent to those of GSH in Gram-negative bacteria, but current knowledge of MSH is limited to detoxification of alkalating chemicals and protection from host cell defense/killing systems. Recently, an MSH-dependent maleylpyruvate isomerase (MDMPI) was discovered from C. glutamicum, and this isomerase represents one example of many putative MSH-dependent enzymes that take MSH as cofactor. In this report, fourteen mutants of MDMPI were generated. The wild type and mutant (H52A) MDMPIs were crystallized and their structures were solved at 1.75 and 2.05Å resolution, respectively. The crystal structures reveal that this enzyme contains a divalent metal-binding domain and a C-terminal domain possessing a novel folding pattern ( fold). The divalent metal-binding site is composed of residues His⁵², Glu¹⁴⁴, and His¹⁴⁸ and is located at the bottom of a surface pocket. Combining the structural and site-directed mutagenesis studies, it is proposed that this surface pocket including the metal ion and MSH moiety formed the putative catalytic center.

Received for publication, November 6, 2006 , and in revised form, March 30, 2007.

^* This work was supported by National Natural Sciences Foundation of China and Knowledge Innovation Program of the Chinese Academy of Sciences. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Tables S1 and S2.

The atomic coordinates and structure factors (code 2NSF and 2NSG) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

¹ These two authors contributed equally to this work. R. W. contributed to the crystallization and structure determination and analysis, and Y.-J. Y. contributed to the mutagenesis and enzymatic activity determination.

² To whom correspondence may be addressed: Institute of Microbiology, Chinese Academy of Sciences, ZhongGuanCun, Haidian, Beijing 100080, China. Tel.: 86-10-62527118; Fax: 86-10-62652317; E-mail: liusj{at}sun.im.ac.cn. ³ To whom correspondence may be addressed: National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, 15 Datun Rd., Chaoyang District, Beijing 100101, China. Tel.: 86-10-64888512; Fax: 86-10-64889867; E-mail: wrchang{at}sun5.ibp.ac.cn.

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