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

Rui Wang,Ya-Jie Yin,Feng Wang,Mei Li,Jie Feng,Hong-Mei Zhang,Ji-Ping Zhang,Shuang-Jiang Liu,Wen-Rui Chang

doi:10.1074/jbc.m610347200

Abstract

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 2.05 A 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 (alphabetaalphabetabetaalpha 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.

Highlights

Mycothiol, known as MSH4 and chemically 1D-myo-inosityl-2-(N-acetyl-L-cysteinyl)-amido-2-deoxy-␣-D-glucopyranoiside [1,2,3,4], is the major low molecule mass thiol in many groups of Gram-positive bacteria such as coryneform bacteria, mycobacteria, and streptomycetes [5, 6]
The understanding of MSH physiological function was limited to detoxification of reactive oxygen/alkalating species and to protection of pathogens such as Mycobacterium tuberculosis from host cell defense systems [1, 7, 8]
BLAST searches with MSH-dependent maleylpyruvate isomerase (MDMPI) sequence against GenBankTM and other protein data bases revealed that MDMPI is not homologous to any functionally identified proteins but showed significant identities (27–36%) to a range of conserved hypothetical proteins from the genomes of Streptomyces coelicolor, Streptomyces avermitilis, Propionibacterium acnes, and Nocardia farcinica [10] (Fig. 2A)

Summary

Introduction

Known as MSH4 and chemically 1D-myo-inosityl-2-(N-acetyl-L-cysteinyl)-amido-2-deoxy-␣-D-glucopyranoiside [1,2,3,4], is the major low molecule mass thiol in many groups of Gram-positive bacteria such as coryneform bacteria, mycobacteria, and streptomycetes [5, 6]. Site-directed Mutagenesis of MDMPI and Enzyme Activity Assay—Fourteen mutants (supplemental Table S1) of MDMPI (W44A, H48A, H52A, N56A, C61A, Y76A, S78A, R82A, E85A, R141A, E144A, H148A, D151A, and R222A) were constructed according to the method described by Sambrook et al [19].

Results

Conclusion