Si Meiru, Long Mingxiu, Chaudhry Muhammad Tausif, Xu Yixiang, Zhang Pan, Zhang Lei, Shen Xihui
State Key Laboratory of Crop Stress Biology for Arid Areas and College of Life Sciences, Northwest A&F University, Yangling, Shaanxi, China.
College of Animal Science and Technology, Northwest A&F University, Yangling, Shaanxi, China.
PLoS One. 2014 Dec 16;9(12):e115075. doi: 10.1371/journal.pone.0115075. eCollection 2014.
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.
本研究聚焦于谷氨酸棒杆菌的麦硫因S-共轭酰胺酶（Mca）的遗传和生化特性。重组谷氨酸棒杆菌Mca在大肠杆菌中异源表达并纯化至表观均一性。通过凝胶过滤色谱法测定的天然Mca蛋白的分子量为35 kDa，表明Mca在纯化条件下以单体形式存在。Mca在体内对单溴双马来酰亚胺的麦硫因S-共轭物（MSmB）表现出酰胺酶活性，而mca突变体失去了切割MSmB的能力。此外，Mca以N-乙酰-D-葡萄糖胺（GlcNAc）为底物时表现出有限的脱乙酰酶活性。酰胺酶活性的最适pH在7.5至8.5之间，而在Zn2+存在下的最高活性证实Mca是一种锌金属蛋白。Mca家族成员中保守的氨基酸残基存在于谷氨酸棒杆菌Mca中，对这些残基进行定点诱变表明，Asp14、Tyr137、His139和Asp141对活性很重要。mca缺失突变体对抗生素、烷基化剂、氧化剂和重金属的抗性降低，而这些敏感表型在互补菌株中在很大程度上得以恢复。在各种应激条件下，谷氨酸棒杆菌中Mca在应激反应胞外功能-σ（ECF-σ）因子SigH的直接控制下诱导表达，进一步支持了Mca在抵抗各种毒素中的生理作用。
