title: Functional characterization of Corynebacterium glutamicum mycothiol S-conjugate amidase.
abstract: The present study focuses on the genetic and biochemical characterization of mycothiol S-conjugate amidase (Mca) of Corynebacterium glutamicum. Recombinant C. glutamicum Mca was heterologously expressed in Escherichia coli and purified to apparent homogeneity. The molecular weight of native Mca protein determined by gel filtration chromatography was 35 kDa, indicating that Mca exists as monomers in the purification condition. Mca showed amidase activity with mycothiol S-conjugate of monobromobimane (MSmB) in vivo while mca mutant lost the ability to cleave MSmB. In addition, Mca showed limited deacetylase activity with N-acetyl-D-glucosamine (GlcNAc) as substrate. Optimum pH for amidase activity was between 7.5 and 8.5, while the highest activity in the presence of Zn2+ confirmed Mca as a zinc metalloprotein. Amino acid residues conserved among Mca family members were located in C. glutamicum Mca and site-directed mutagenesis of these residues indicated that Asp14, Tyr137, His139 and Asp141 were important for activity. The mca deletion mutant showed decreased resistance to antibiotics, alkylating agents, oxidants and heavy metals, and these sensitive phenotypes were recovered in the complementary strain to a great extent. The physiological roles of Mca in resistance to various toxins were further supported by the induced expression of Mca in C. glutamicum under various stress conditions, directly under the control of the stress-responsive extracytoplasmic function-sigma (ECF-σ) factor SigH.
authors.affiliation: State Key Laboratory of Crop Stress Biology for Arid Areas and College of Life Sciences, Northwest A&F University, Yangling, Shaanxi, China.
authors.forename: Meiru
authors.initials: M
authors.lastname: Si
authors.affiliation: College of Animal Science and Technology, Northwest A&F University, Yangling, Shaanxi, China.
authors.forename: Mingxiu
authors.initials: M
authors.lastname: Long
authors.affiliation: Environmental Analytical Laboratory, National Physical & Standards Laboratory, PCSIR, Islamabad, Pakistan.
authors.forename: Muhammad Tausif
authors.initials: MT
authors.lastname: Chaudhry
authors.affiliation: State Key Laboratory of Crop Stress Biology for Arid Areas and College of Life Sciences, Northwest A&F University, Yangling, Shaanxi, China.
authors.forename: Yixiang
authors.initials: Y
authors.lastname: Xu
authors.affiliation: State Key Laboratory of Crop Stress Biology for Arid Areas and College of Life Sciences, Northwest A&F University, Yangling, Shaanxi, China.
authors.forename: Pan
authors.initials: P
authors.lastname: Zhang
authors.affiliation: State Key Laboratory of Crop Stress Biology for Arid Areas and College of Life Sciences, Northwest A&F University, Yangling, Shaanxi, China.
authors.forename: Lei
authors.initials: L
authors.lastname: Zhang
authors.affiliation: State Key Laboratory of Crop Stress Biology for Arid Areas and College of Life Sciences, Northwest A&F University, Yangling, Shaanxi, China.
authors.forename: Xihui
authors.initials: X
authors.lastname: Shen
mesh_terms: D000477:Alkylating Agents / Q000378:metabolism; D000581:Amidohydrolases / Q000235:genetics* / Q000378:metabolism*; D000900:Anti-Bacterial Agents / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D001643:Bridged Bicyclo Compounds / Q000378:metabolism; D048230:Corynebacterium glutamicum / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D024881:Drug Resistance, Bacterial / Q000235:genetics; D017353:Gene Deletion; D019216:Metals, Heavy / Q000378:metabolism; D008826:Microbial Sensitivity Tests; D016297:Mutagenesis, Site-Directed; D016877:Oxidants / Q000378:metabolism; D012808:Sigma Factor / Q000378:metabolism
chemical_list: D000477:Alkylating Agents; D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D001643:Bridged Bicyclo Compounds; D019216:Metals, Heavy; D016877:Oxidants; D012808:Sigma Factor; D000581:Amidohydrolases; C413523:mycothiol S-conjugate amidase; C028381:monobromobimane