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

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 Gramnegative 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 Å 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 His 52 , Glu 144 , and His 148 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.Mycothiol, also known as MSH 4 and chemically 1D-myo-inosityl-2-(N-acetyl-L-cysteinyl)-amido-2-deoxy-␣-D-glucopyranoiside (1-4), is the major low molecule mass thiol in many groups of Gram-positive bacteria such as coryneform bacteria, mycobacteria, and streptomycetes (5, 6). These bacteria synthesize MSH but lack GSH molecule that plays important roles in many physiological processes. It is believed that MSH functions similarly to GSH in many microbial activities (6). However, 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). Very recently, a novel physiological role of MSH in assimilation of aromatic compounds was described and an MSH-dependent maleylpyruvate isomerase (MDMPI) was identified in Corynebacterium glutamicum (9). This MDMPI catalyzes the conversion of maleylpyruvate (substrate) to fumarylpyruvate (product) (Fig. 1).BLAST searches with MDMPI sequence against GenBank TM 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). Earlier research on mycothiol biochemistry and biosynthesis has been conducted with mycobacteria such as M. tuberculosis, with the ultimate aim of defining new targets against tuberculosis, the resurgence of which has been a growing health concern in both developed and developing nations. The understanding of MSH-dependent enzyme structure is the key to the characterization of MSH-dependent enzyme...

Arsenate Reductase, Mycothiol, and Mycoredoxin Concert Thiol/Disulfide Exchange

“…Since its discovery in 1993 (6,48), MSH has been shown to be involved in many processes. Next to its role as a storage form of cysteine in Mycobacterium smegmatis (49) and its detoxification role for alkylating agents (50), there are only two enzymes described that depend on MSH for functioning: formaldehyde dehydrogenase MscR (51), later identified as nitrosomycothiol reductase, with a role in the protection against oxidative stress (52), and maleylpyruvate isomerase (22,53). We present the first enzymes, arsenate reductase 1 and 2, from C. glutamicum, which depend for their function on mycothiol as well as on mycoredoxin with a clear link to the mycothione reductase pathway.…”

Biosynthesis and Functions of Mycothiol, the Unique Protective Thiol of Actinobacteria

“…It appears that the dominant activity is that of an MSH-dependent maleylpyruvate isomerase but that a lower-level, thiol-independent activity may also be present. The MSH-dependent maleylpyruvate isomerase from C. glutamicum has been crystallized and, unlike the human GSH-dependent enzyme, was found to be a zinc metalloprotein with a novel fold (153). The MSH-dependent maleylpyruvate isomerase also lacked the dehalogenation activity found for the human enzyme.…”

Degradation and assimilation of aromatic compounds by Corynebacterium glutamicum: another potential for applications for this bacterium?

“…The C-terminal domain is necessary for the enzyme activity and structural stability. Furthermore, site-directed mutagenesis revealed that the Arg 222 residue at the C-terminal domain was necessary for MDMPI activity (Wang et al 2007). The discovery of a mycothiol-dependent maleylpyruvate isomerase (MDMPI) in C. glutamicum introduced a new category of maleylpyruvate isomerase, and constitutes significant progress on the way to understanding both the gentisate pathway of aromatic assimilation and the MSH physiology (Rawat and Av-Gay 2007).…”