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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

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

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

 
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.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

 
Mycothiol, also known as MSH⁴ 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 enzymes that are helpful for the development of new drugs against diseases caused by Mycobacterium spp.

To elucidate the catalytic mechanism of MDMPI and to provide clues to find more MSH-dependent enzymes, we have determined the crystal structure of MDMPI from C. glutamicum at a resolution of 1.75Å. The structure reveals that the MDMPI contains two domains: A metal binding domain binds a divalent metal cofactor (Zn²⁺) and an extended domain contains totally new folding. The site-directed mutagenesis reveals 9 of the 14 conserved amino acid residues are essential for activity. One mutant (H52A) was crystallized. By combination of structural and biochemical results, a putative catalytic center is proposed. So far as we know this is the first structure of an MSH-dependent enzyme, as well as an enzyme involved in gentisate/3-hydroxybenzoate assimilation that is widely existed in bacteria and participate in microbial geocycling of elements of aromatic compounds in nature.

View larger version (24K): [in this window] [in a new window]   FIGURE 1. Isomerization of maleylpyruvate into fumarylpyruvate catalyzed by mycothiol-dependent maleylpyruvate isomerase. Intermediates (i) and (ii) have not been experimentally confirmed.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

Expression and Purification of MDMPI and Its Mutants—Various derivatives of plasmid pET28a (supplemental Table S1) that harbor MDMPI or its mutants were constructed by methods described previously (10). Recombinant proteins were expressed in E. coli BL21 (DE3) cells by inducing with 0.5 mM isopropyl-1-thio--D-galactopyranoside when the culture reached an A₆₀₀ of 0.6–0.8. The culture was harvested after growing for additional 8 h at 25 °C. Protein was purified using a nickel affinity column and a Superdex 75 gel-filtration column (GE Healthcare). The prepared protein sample was checked for purity by SDS-PAGE and by gel-filtration chromatography, which showed only one protein of 28 kDa corresponding to the molecular mass of MDMPI and its mutants. After changing buffer to 10 mM Tris (pH 8.0) by repeatedly concentration and dilution using Amicon Ultra-4 concentrators (molecular weight cutoff 5000), the purified protein was finally concentrated to 17 mg/ml and stored at –80 °C.

Crystallization and Data Collection—The hanging-drop vapor-diffusion method was used for crystallization. The drop was formed by mixing 1 µl of protein solution (17 mg/ml) with an equal volume of reservoir solution containing 1.2 M (NH₄)₂SO₄, 12% glycerol, and 0.1 M Tris (pH 8.5). A 1.75 Å resolution native data set was collected utilizing Rigaku RAXIS IV image plate detector at Institute of Biophysics (Chinese Academy of Sciences). A bromide derivative was prepared by soaking the crystal in cryoprotecting solution containing 1.2 M (NH₄)₂SO₄, 25% glycerol, 1 M NaBr, and 0.1 M Tris (pH 8.5) for 10 min. Single-wavelength anomalous dispersion data were collected at the Beijing Synchrotron Radiation Facility at the peak wavelength of bromine (0.920 Å) with 1° oscillation and an exposure of 15 s per image. A total of 600° of data were collected to a resolution of 1.95 Å with an average of 20-fold redundancy.

The crystallization of mutant H52A was performed similarly to that of native MDMPI. The drops were composed of 1 µl of protein solution (15 mg/ml) and 1 µl of reservoir solution containing 1.4 M (NH₄)₂SO₄, 11% glycerol, and 0.1 M Tris (pH 8.5). A 2.05 Å resolution data set for this mutant was collected at the Institute of Biophysics (Chinese Academy of Sciences).

All the diffraction data (Table 1) were integrated using DENZO and scaled using SCALEPACK (11).

The structure of mutant H52A was solved by molecular replacement with MolRep (17) using the MDMPI wild type structure as a search model. After several cycles of refinement and model rebuilding, a final model with an R-factor of 0.20 and an R_free-factor of 0.22 was obtained.

A structure appraisal of the final model of MDMPI and H52A using PROCHECK (18) indicated that the main-chain torsion angles for both molecules corresponded well with allowed values (96.6% in the most favored regions of the Ramachandran plot of MDMPI and 96.2% in the most favored regions of H52A). A summary of the refinement statistics is given in Table 1.

X-ray Fluorescence Scan Analysis—To determine what kind of metal ion binds to the protein, fluorescence scan analysis of the same crystal used in data collection was performed at beamline, Photon Factory (Tsukuba, Japan). The wavelength ranges of the experiments were selected near the K-edge of Zn²⁺, Ni²⁺,Cu²⁺,Mn²⁺,Fe³⁺, and Ca²⁺, respectively.

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). Primers used for generation of these mutants are provided in supplemental Table S2. Screening for mutants was conducted through enzymatic activity assays, and subsequently the mutations were confirmed by DNA sequencing. MDMPI activity was determined according to Feng et al. (9).

Protein Data Bank Accession Numbers—The coordinates and structure factors for MDMPI wild type and mutant H52A have been deposited in the RCSB Protein Data Bank under the accession numbers of 2NSF and 2NSG, respectively.

	RESULTS AND DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

 
Overall Structure of the MDMPI—The MDMPI of C. glutamicum was purified from recombinant E. coli and its crystal structure was resolved at 1.75 Å. The MDMPI is a monomer and belongs to / folding type (Figs. 2B and Fig. 3). Two domains were clearly observed: An N-terminal domain largely composed of -helices and a C-terminal domain characterized by shorter stretches of -helices and -sheets. The core of the N-terminal domain consists of five long (20 residues) -helices (1, 2, 3, 4, and 5). Also included in the core domain is a metal ion (Fig. 3). The C-terminal domain shows a novel fold with the pattern of fold, which contains three long loops, three -helices (7, 8, and 9), and three -strands (3, 4, and 5). It is noteworthy that 42% of the amino acid residues in the C-terminal domain are located at the three long loops, with the longest loop containing 20 residues (residues 221–241) and are not part of the core C-terminal domain structure (Fig. 3).

The C-terminal Domain Represents a Novel Folding Pattern and Is Necessary for Enzymatic Activity—The folding topology of the C-terminal domain of MDMPI is novel. Structural similarity searches of MDMPI C-terminal domain utilizing the DALI server (20) showed few matches with significant Z score and small root mean square deviation values. The best match was a putative sterol carrier protein (PDB code 1WFR) with Z value of 3.0. The 67 residues of this protein share 15% sequence identity with the C-terminal domain of MDMPI and can be superimposed to this domain with a root mean square deviation of 3.3 Å.

The C-terminal domain is necessary for the enzyme activity and structural stability. When the C-terminal domain was deleted, the truncated MDMPI did not show any enzyme activity (Table 2) and existed as inclusion bodies in E. coli cells (data not shown). Furthermore, site-directed mutagenesis revealed that a conserved Arg²²² residue at the C-terminal domain was necessary for MDMPI activity. When this Arg²²² residue was mutated into Ala²²² residue, the resulting mutant R222A did not show any enzymatic activity. Structural analysis indicated that the side chain of Arg²²² (NH1) at the C-terminal domain interacted with Asp¹⁵¹ (OD2) of the N-terminal domain through a salt bridge (Fig. 4). These two amino acid residues (Arg²²² and Asp¹⁵¹) are highly conserved among the protein family (Fig. 2A). Replacement of either of them with Ala residue resulted in complete loss of enzyme activity. Thus, it is proposed that the interaction between these two residues is vital to enzyme activity and structure stability.

View larger version (39K): [in this window] [in a new window]   FIGURE 2. Alignment of MDMPI from C. glutamicum and its homologous proteins (A) and topology of MDMPI (B). Conserved amino acid residues are highlighted by red, including the metal-binding residues (His52, Glu144, and His148) and residues for structure stability (Gly43, Asn151, Pro162, Gly210, Arg222, Gly221, Gly232, Pro234, and Pro238). Amino acid residues that were mutated for generation of mutant MDMPIs in this study are indicated by asterisk. The sequence alignment was carried out with the EBI server ClustalW (23) and represented by ESPript (24). The abbreviations are as follows: Cg, C. glutamicum; Ce, Corynebacterium efficiens; WA46, Streptomyces sp. strain WA46; Nf, Nocardia farcinica; Sc, S. coelicolor; Sa, S. avermitilis.

View larger version (36K): [in this window] [in a new window]   FIGURE 3. Stereo view of the overall structure of MDMPI. The overall structure of MDMPI consists of two domains that are connected by two loops and one -helix (6). In the N-terminal domain (residues 1–152), the five long -helices (1 to 5), which form the core part, are shown in purple and pink, and the metal ion is shown in green, short -helices ( + 1, + 2, and + 3) are shown in yellow, and -sheets (1 and 2) are shown in blue. The C-terminal domain (residues 163–241) includes helices 7 and 9, a three-strand mixed -sheet (3, 4, and 5), which is connected by a shorter helix 8. The C-terminal domain consisted of two subdomains. The -helices subdomain includes two -helices (7 and 9), and the other subdomain includes three -sheets (3, 4, and 5) and 8. These two subdomains are connected by three loops. A long loop (residues 221–241) is located at the terminal of the C-terminal domain. The image was created by PyMol.

The contribution of the three conserved residues, namely His⁵², Glu¹⁴⁴, and His¹⁴⁸, to the binding of metal atom was examined by individual mutation of each amino acid residue. Enzyme activity assays indicated that all mutants (H52A, E144A, and H148A) were inactive. Moreover, the crystal structure of mutant H52A at resolution of 2.05Å revealed that there was no metal ion bound to the protein (although nickel-affinity chromatography was used in the purification process). The metal atom position at the native MDMPI molecule was occupied by one glycerol moiety (Fig. 5B). Compared with the native MDMPI, the mutant MDMPI (H52A) has the following structural changes: 1) the main chain of the loop (amino acid residues 36–45) moved toward the inner; 2) the side chain of Trp⁴⁴ at the loop is much closer to the former metal binding site; 3) the side chain of Glu¹⁴⁴ had double conformations; and 4) conformational change took place at the side chain of Arg⁸² (Fig. 6). Probably it is the absence of the metal ion that resulted in the significant conformation change around the proposed metal binding region.

View larger version (46K): [in this window] [in a new window]   FIGURE 4. The side chain of Arg222 (NH1) at the C-terminal domain interacts with Asp151 (OD2) of the N-terminal domain through a salt bridge. The N-terminal domain is shown in yellow, C-terminal domain in cyan, and interdomain coil in gray. Asp151 and Arg222 are represented in stick model (carbon atoms are colored the same as the domain they locate at) and the metal ion by a sphere (gray). Salt bridge is shown by dashed line. The figure was created by PyMol.

View larger version (43K): [in this window] [in a new window]   FIGURE 5. Metal binding site of MDMPI. A, stereo view of the metal binding site of wild MDMPI. Amino acid residues, glycerol, and water are shown in ball-and-stick model, and the metal atom is shown as a Corey-Pauling-Koltun model (gray). The metal binding site of MDMPI is comprised of His52 (NE2), Glu144 (OE1), His148 (NE2), two water molecules, and one glycerol. The His52 is located at the 2 helix, and the Glu144 and His148 both are located at 5 helix. This figure was created by Molscript (25) and Raster3D (26). B, the superim-position of the metal binding regions of the native (carbon atoms and the metal atom in cyan) and mutant (carbon atoms in yellow) MDMPI molecules. The position of metal atom at native MDMPI was replaced by one glycerol moiety in mutant H52A. The image was created by PyMol.

View larger version (46K): [in this window] [in a new window]   FIGURE 6. The stereo view of the conformation differences in the putative active pocket between the wild type and the mutant (H52A) MDMPI. Mutant H52A is shown in purple and native MDMPI in green. Conformation differences were found at the side chains of Arg82 and Trp44. In H52A the metal ion was missing and a glycerol occupied the position of the metal ion, and the location of SO2–4 also changed. Furthermore, there is no obvious conformation changes observed, when compared with that of the wild type. The image was created by PyMol.

View larger version (71K): [in this window] [in a new window]   FIGURE 7. The stereo view of the structural elements involved in the putative active pocket. The conserved metal binding residues (His52, Glu144, and His148) and other conserved residues (Asn56, Tyr76, Ser78, Arg82, Arg141, and Trp44) form the putative active pocket. Residues are shown in yellow (stick), the metal ion (Zn2+) is shown in cyan (sphere), and the SO2–4 ion and glycerol in purple (stick). The image was created by PyMol.

Although the catalytic mechanism of MDMPI is still not clear, this and previous studies have revealed several key molecular elements that are necessary for catalysis, including the metal ion (Zn²⁺) and its surrounding amino acid residues (His⁵², Glu¹⁴⁴, and His¹⁴⁸), and the –SH group of the MSH molecule (9). Here, a preliminary and hypothetical process is proposed for description of the MDMPI-catalyzed isomerization (Fig. 1): isomerization of maleylpyruvate is initiated by the attack of –SH of the mycothiol on the -carbon of the maleylpyruvate molecule, which is catalyzed by the MDMPI (most probably by the Zn²⁺ ion on the enzyme). This attack results in the formation of MSH-maleylpyruvate conjugate (Fig. 1, step I). Upon conjugation, the resulting single bond would be freely to rotate (Fig. 1, step II). Finally, the MSH moiety is released and the product (fumarylpyruvate) is generated (Fig. 1, step III). Although this proposed mechanism is similar to that proposed for the GSH-dependent maleylacetoacetate isomerase from humans (22), some sharply different properties between MDMPI and GSH-dependent maleylacetoacetate were observed. The MDMPI is a metalloprotein but the GSH-dependent maleylacetoacetate isomerase is not; the latter belongs to a group of enzymes named zeta class glutathione transferase that catalyze reductive dehalogenation of -hologenated acids (22), but this property was not observed for the MDMPI from C. glutamicum. Currently, works on characterization of more MDMPIs from different bacteria and on details of the catalytic mechanism of MDMPI are in progress.

	FOOTNOTES

 
^* 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.

⁴ The abbreviations used are: MSH, mycothiol; MDMPI, MSH-dependent maleylpyruvate isomerase.

	ACKNOWLEDGMENTS

 
We are grateful to Rui-Fang Wang for protein purification, Yi Han at the Institute of Biophysics (Chinese Academy of Sciences), Yu-Hui Dong and Peng Liu at the Institute of High Energy Physics (Chinese Academy of Sciences) for data collection, and N. Sakabe and A. Iida of the Photon Factory (Tsukuba, Japan) for x-ray fluorescence scanning.

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TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

 

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