Dataset Information

Heterologous expression, purification and biochemical characterization of a glutamate racemase (MurI) from Streptococcus mutans UA159.

ABSTRACT:

Background

Glutamate racemase (MurI) is a cofactor-independent enzyme that is essential to the bacterial peptidoglycan biosynthesis pathway and has therefore been considered an attractive target for the development of antimicrobial drugs. While in our previous study the essentiality of the murI gene was shown in Streptococcus mutans, the primary aetiologic agent of human dental caries, studies on S. mutans MurI have not yet provided definitive results. This study aimed to produce and characterize the biochemical properties of the MurI from the S. mutans UA159 genome.

Methods

Structure characterization prediction and multiple sequence alignment were performed by bioinformatic analysis. Recombinant His₆-tagged S. mutans MurI was overexpressed in the expression vector pColdII and further purified using a Ni²⁺ affinity chromatography method. Protein solubility, purity and aggregation state were analyzed by SDS-PAGE, Western blotting, native PAGE and SEC-HPLC. Kinetic parameters were assessed by a circular dichroism (CD) assay. Kinetic constants were calculated based on the curve fit for the Michaelis-Menten equation. The effects of temperature and pH on enzymatic activity were determined by a series of coupled enzyme reaction mixtures.

Results

The glutamate racemase gene from S. mutans UA159 was amplified by PCR, cloned and expressed in Escherichia coli BL21 (DE3). The 264-amino-acid protein, as a mixture of dimeric and monomeric enzymes, was purified to electrophoretic homogeneity. In the CD assay, S. mutans MurI displayed unique kinetic parameters (K _{m, d-Glu?l-Glu} = 0.3631 ± 0.3205 mM, V _{max, d-Glu?l-Glu} = 0.1963 ± 0.0361 mM min^-1, k _{cat, d-Glu?l-Glu} = 0.0306 ± 0.0065 s^-1, k _cat/K _m, _d-Glu?l-Glu = 0.0844 ± 0.0128 s^-1 mM^-1, with d-glutamate as substrate; K _{m, l-Glu?d-Glu} = 0.8077 ± 0.5081 mM, V _{max, l-Glu?d-Glu} = 0.2421 ± 0.0418 mM min^-1, k _cat _, _l _- _Glu?d-Glu = 0.0378 ± 0.0056 s^-1, k _cat/K _m, _l-Glu?d-Glu = 0.0468 ± 0.0176 s^-1 mM^-1, with l-glutamate as substrate). S. mutans MurI possessed an assay temperature optimum of 37.5 °C and its optimum pH was 8.0.

Conclusion

The findings of this study provide insight into the structure and biochemical traits of the glutamate racemase in S. mutans and supply a conceivable guideline for employing glutamate racemase in anti-caries drug design.

SUBMITTER: Wang X

PROVIDER: S-EPMC6927343 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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Publications

Heterologous expression, purification and biochemical characterization of a glutamate racemase (MurI) from <i>Streptococcus mutans</i> UA159.

Wang Xiangzhu X Chen Chanchan C Shen Ting T Zhang Jiangying J

PeerJ 20191220

<h4>Background</h4>Glutamate racemase (MurI) is a cofactor-independent enzyme that is essential to the bacterial peptidoglycan biosynthesis pathway and has therefore been considered an attractive target for the development of antimicrobial drugs. While in our previous study the essentiality of the <i>murI</i> gene was shown in <i>Streptococcus mutans</i>, the primary aetiologic agent of human dental caries, studies on <i>S. mutans</i> MurI have not yet provided definitive results. This study aim ...[more]

PMID: 31875162

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Project description:In Streptococcus mutans, an oral colonizer associated with dental caries, development of competence for natural genetic transformation is triggered by either of two types of peptide pheromones, competence-stimulating peptides (CSPs) (18 amino acids [aa]) or SigX-inducing peptides (XIPs) (7 aa). Competence induced by CSP is a late response to the pheromone that requires the response regulator ComE and the XIP-encoding gene comS. XIP binds to ComR to allow expression of the alternative sigma factor SigX and the effector genes it controls. While these regulatory links are established, the precise set of effectors controlled by each regulator is poorly defined. To improve the definition of all three regulons, we used a high-resolution tiling array to map global changes in gene expression in the early and late phases of the CSP response. The early phase of the CSP response was limited to increased gene expression at four loci associated with bacteriocin production and immunity. In the late phase, upregulated regions expanded to a total of 29 loci, including comS and genes required for DNA uptake and recombination. The results indicate that the entire late response to CSP depends on the expression of comS and that the immediate transcriptional response to CSP, mediated by ComE, is restricted to just four bacteriocin-related loci. Comparison of the new data with published transcriptome data permitted the identification of all of the operons in each regulon: 4 for ComE, 2 for ComR, and 21 for SigX. Finally, a core set of 27 panstreptococcal competence genes was identified within the SigX regulon by comparison of transcriptome data from diverse streptococcal species. IMPORTANCES. mutans has the hard surfaces of the oral cavity as its natural habitat, where it depends on its ability to form biofilms in order to survive. The comprehensive identification of S. mutans regulons activated in response to peptide pheromones provides an important basis for understanding how S. mutans can transition from individual to social behavior. Our study placed 27 of the 29 transcripts activated during competence within three major regulons and revealed a core set of 27 panstreptococcal competence-activated genes within the SigX regulon.