Schauer Stefan, Chaturvedi Shalini, Randau Lennart, Moser Jürgen, Kitabatake Makoto, Lorenz Stefan, Verkamp Elizabeth, Schubert Wolf-Dieter, Nakayashiki Toru, Murai Masatoshi, Wall Kristen, Thomann Hans-Ulrich, Heinz Dirk W, Inokuchi Hachiro, Söll Dieter, Jahn Dieter
Institute of Microbiology, Technical University Braunschweig, Spielmannstrasse 7, Germany.
J Biol Chem. 2002 Dec 13;277(50):48657-63. doi: 10.1074/jbc.M206924200. Epub 2002 Oct 4.
In the first step of tetrapyrrole biosynthesis in Escherichia coli, glutamyl-tRNA reductase (GluTR, encoded by hemA) catalyzes the NADPH-dependent reduction of glutamyl-tRNA to glutamate-1-semialdehyde. Soluble homodimeric E. coli GluTR was made by co-expressing the hemA gene and the chaperone genes dnaJK and grpE. During Mg(2+)-stimulated catalysis, the reactive sulfhydryl group of Cys-50 in the E. coli enzyme attacks the alpha-carbonyl group of the tRNA-bound glutamate. The resulting thioester intermediate was trapped and detected by autoradiography. In the presence of NADPH, the end product, glutamate-1-semialdehyde, is formed. In the absence of NADPH, E. coli GluTR exhibited substrate esterase activity. The in vitro synthesized unmodified glutamyl-tRNA was an acceptable substrate for E. coli GluTR. Eight 5-aminolevulinic acid auxotrophic E. coli hemA mutants were genetically selected, and the corresponding mutations were determined. Most of the recombinant purified mutant GluTR enzymes lacked detectable activity. Based on the Methanopyrus kandleri GluTR structure, the positions of the amino acid exchanges are close to the catalytic domain (G7D, E114K, R314C, S22L/S164F, G44C/S105N/A326T, G106N, S145F). Only GluTR G191D (affected in NADPH binding) revealed esterase but no reductase activity.
在大肠杆菌四吡咯生物合成的第一步中，谷氨酰 - tRNA还原酶（GluTR，由hemA编码）催化依赖NADPH的谷氨酰 - tRNA还原为谷氨酸 - 1 - 半醛。通过共表达hemA基因以及伴侣基因dnaJK和grpE制备了可溶性同型二聚体大肠杆菌GluTR。在Mg(2 +)刺激的催化过程中，大肠杆菌酶中Cys - 50的活性巯基攻击与tRNA结合的谷氨酸的α - 羰基。通过放射自显影捕获并检测所得硫酯中间体。在NADPH存在下，形成终产物谷氨酸 - 1 - 半醛。在没有NADPH的情况下，大肠杆菌GluTR表现出底物酯酶活性。体外合成的未修饰谷氨酰 - tRNA是大肠杆菌GluTR可接受的底物。通过基因筛选得到了8个5 - 氨基乙酰丙酸营养缺陷型大肠杆菌hemA突变体，并确定了相应的突变。大多数重组纯化的突变体GluTR酶缺乏可检测的活性。基于嗜甲烷嗜热栖热菌GluTR的结构，氨基酸交换的位置靠近催化结构域（G7D、E114K、R314C、S22L/S164F、G44C/S105N/A326T、G106N、S145F）。只有GluTR G191D（在NADPH结合方面受到影响）显示出酯酶活性但没有还原酶活性。
