Sharma Sarita, Bhakuni Vinod
Division of Molecular and Structural Biology, Central Drug Research Institute, Lucknow 226001, India.
Protein Expr Purif. 2007 Sep;55(1):189-97. doi: 10.1016/j.pep.2007.04.017. Epub 2007 Apr 29.
Serine hydroxymethyltransferase (SHMT) plays a key role in cell physiology as it participates in the different interconversion pathway of folate coenzymes, provides almost exclusively folate one carbon fragments for the biosynthesis of a variety of end products. For the first time, Mycobacterium leprae glyA gene, encodes the enzyme serine hydroxymethyltransferase, has been cloned in Escherichia coli, over-expressed and purified the protein product (mlSHMT) for folding and stability studies under various denaturating conditions. The recombinant mlSHMT exists as homo-dimer of molecular mass about 90 kDa under physiological conditions . The studies on catalytic properties of mlSHMT show that the enzyme catalyzes the H(4)-folate dependent retro-aldol cleavage of L-serine, however, D-alanine dependent transaminase activity was absent in the enzyme. Further analysis of the enzyme kinetics for hydroxymethyltransferase reaction for mlSHMT demonstrates a comparable K(m) value for L-serine to SHMTs from other sources but significantly lower catalytic efficiency (k(cat)/K(m)). The mlSHMT is resistant to alkaline denaturation and exist as apo-dimer up to pH 10.5. Urea and guanidinium chloride induces dissociation of mlSHMT dimer into monomer at low denaturant concentrations, and leads to loss of enzymatic activity.
丝氨酸羟甲基转移酶（SHMT）在细胞生理学中起着关键作用，因为它参与叶酸辅酶的不同相互转化途径，几乎只为多种终产物的生物合成提供叶酸一碳片段。首次将编码丝氨酸羟甲基转移酶的麻风分枝杆菌glyA基因克隆到大肠杆菌中，进行过量表达并纯化蛋白质产物（mlSHMT），以研究其在各种变性条件下的折叠和稳定性。重组mlSHMT在生理条件下以分子量约90 kDa的同型二聚体形式存在。对mlSHMT催化特性的研究表明，该酶催化L-丝氨酸的H(4)-叶酸依赖性逆羟醛裂解反应，然而，该酶不存在D-丙氨酸依赖性转氨酶活性。对mlSHMT羟甲基转移酶反应的酶动力学进一步分析表明，其对L-丝氨酸的K(m)值与其他来源的SHMT相当，但催化效率（k(cat)/K(m)）显著较低。mlSHMT对碱性变性具有抗性，在pH值高达10.5时以脱辅基二聚体形式存在。尿素和氯化胍在低变性剂浓度下会诱导mlSHMT二聚体解离为单体，并导致酶活性丧失。
