Helmstaedt Kerstin, Heinrich Gabriele, Merkl Rainer, Braus Gerhard H
Abteilung Molekulare Mikrobiologie und Genetik, Institut für Mikrobiologie und Genetik, Grisebachstrasse 8, 37077 Göttingen, Germany.
Arch Microbiol. 2004 Mar;181(3):195-203. doi: 10.1007/s00203-003-0639-z. Epub 2004 Jan 15.
aroG, encoding the monofunctional chorismate mutase (TtCM) of the thermophilic gram-negative bacterium Thermus thermophilus, was cloned and its gene product characterized. TtCM was purified to homogeneity on an SDS polyacrylamide gel as a His-fusion protein with a deduced molecular mass of 15.8 kDa. The enzyme belongs to the rare group of AroH-type chorismate mutases which are mainly found in gram-positive bacteria of the Bacillus/ Clostridia group and have recently also been described for gram-negative organisms. The native molecular mass is consistent with a pseudo-alpha/beta barrel enzyme that is organized as a trimer. Comparison of the enzyme's structure with that of its mesophilic counterpart from Bacillus revealed an increase in hydrophilicity on the protein's surface, greater hydrophobicity in cavities within the protein, and greater restriction of conformational freedom, features that contribute to the thermal stability of this chorismate mutase. The kinetic data show Michaelis-Menten substrate saturation with a Km of 290 microM, and a kcat/ Km value of 180 s(-1) mM(-1). TtCM was inhibited by tyrosine with a Ki =34 microM, possibly in a competitive manner.
编码嗜热革兰氏阴性菌嗜热栖热菌单功能分支酸变位酶（TtCM）的aroG基因被克隆，并对其基因产物进行了表征。TtCM作为一种His融合蛋白在SDS聚丙烯酰胺凝胶上纯化至同质，推导分子量为15.8 kDa。该酶属于罕见的AroH型分支酸变位酶组，主要存在于芽孢杆菌/梭菌属革兰氏阳性菌中，最近也在革兰氏阴性菌中有所描述。天然分子量与组织为三聚体的假α/β桶状酶一致。将该酶的结构与其来自芽孢杆菌的嗜温对应物的结构进行比较，发现蛋白质表面亲水性增加，蛋白质内部腔中疏水性更强，构象自由度限制更大，这些特征有助于该分支酸变位酶的热稳定性。动力学数据显示米氏底物饱和，Km为290 microM，kcat/Km值为180 s(-1) mM(-1)。TtCM受到酪氨酸抑制，Ki = 34 microM，可能是以竞争性方式。
