Reaction mechanism of Escherichia coli dihydrodipicolinate synthase investigated by X-ray crystallography and NMR spectroscopy
Reaction mechanism of Escherichia coli dihydrodipicolinate synthase investigated by X-ray crystallography and NMR spectroscopy
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DOI：
10.1021/bi962272d
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发表时间：
1997-01-07
期刊：
影响因子：
2.9
通讯作者：
Huber, R
中科院分区：
文献类型：
--
作者：
Blickling, S;Renner, C;Huber, R
二氢二吡啶甲酸合成酶催化丙酮酸与L-天冬氨酸β-半醛的缩合反应。它是赖氨酸生物合成途径中第一个独有的酶。在这里，我们介绍了五种大肠杆菌DHDPS与底物、底物类似物和抑制剂的络合物的晶体结构。这些化合物包括DHDPS与(1)丙酮酸，(2)丙酮酸和L-天冬氨酸-β-半醛类似物琥珀酸β-半醛的络合物，(3)抑制剂α-酮戊二酸，(4)二吡啶甲酸，和(5)天然反馈抑制剂L-赖氨酸。测定了抑制动力学，确定了L与赖氨酸的结合部位。为了阐明DHDPS催化反应产物的性质，进行了核磁共振实验。通过这种方法，(4S)-4-hydroxy-2，3，4，5-tetrahydro(2S)-dipicolinic酸被鉴定为唯一的产物。提出了DHDPS的反应机理，并确定了抑制的重要特征。
Dihydrodipicolinate synthase (DHDPS) catalyzes the condensation of pyruvate with L-aspartate beta-semialdehyde. It is the first enzyme unique to the diaminopimelate pathway of lysine biosynthesis. Here we present the crystal structures of five complexes of Escherichia coli DHDPS with substrates, substrate analogs, and inhibitors. These include the complexes of DHDPS with (1) pyruvate, (2) pyruvate and the L-aspartate beta-semialdehyde analog succinate beta-semialdehyde, (3) the inhibitor alpha-ketopimelic acid, (4) dipicolinic acid, and (5) the natural feedback inhibitor L-lysine. The kinetics of inhibition were determined, and the binding site of the L-lysine was identified. NMR experiments were conducted order to elucidate the nature of the product of the reaction catalyzed by DHDPS. By this method, (4S)-4-hydroxy-2,3,4,5-tetrahydro(2S)-dipicolinic acid is identified as the only product. A reaction mechanism for DHDPS is proposed, and important features for inhibition are identified.