A novel NAD-binding protein revealed by the crystal structure of 2,3-diketo-L-gulonate reductase (YiaK)
A novel NAD-binding protein revealed by the crystal structure of 2,3-diketo-L-gulonate reductase (YiaK)
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DOI：
10.1074/jbc.m313580200
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发表时间：
2004-03-26
影响因子：
4.8
通讯作者：
Tong, L
中科院分区：
文献类型：
--
作者：
Forouhar, F;Lee, I;Tong, L
大肠杆菌YiaK在NADH存在下催化2，3-二酮基-L-古洛糖酸的还原。它属于氧化还原酶的一个大家族，在古细菌、细菌和真核生物中保守，但与其他蛋白质没有序列同源性。我们在这里报告的晶体结构在高达2.0埃分辨率的YiaK单独和与NAD-酒石酸盐的复合物。YiaK具有新的多肽骨架折叠和识别NAD辅因子的新模式。此外，NAD在酶的二聚体的界面处以不寻常的构象结合。晶体学分析意外地揭示了酒石酸盐在活性位点中的结合。酶动力学研究证实，酒石酸盐和相关的D-苹果酸盐是YiaK的抑制剂。与底物结合产生更封闭构象的大多数其他酶相比，NAD-酒石酸盐与YiaK的结合产生更开放的活性位点。游离酶构象与NAD结合不相容。His（44）可能是酶的催化残基。
Escherichia coli YiaK catalyzes the reduction of 2,3-diketo-L-gulonate in the presence of NADH. It belongs to a large family of oxidoreductases that is conserved in archaea, bacteria, and eukaryotes but shows no sequence homology to other proteins. We report here the crystal structures at up to 2.0-Angstrom resolution of YiaK alone and in complex with NAD-tartrate. YiaK has a new polypeptide backbone fold and a novel mode of recognizing the NAD cofactor. In addition, NAD is bound in an unusual conformation, at the interface of a dimer of the enzyme. The crystallographic analysis unexpectedly revealed the binding of tartrate in the active site. Enzyme kinetics studies confirm that tartrate and the related D-malate are inhibitors of YiaK. In contrast to most other enzymes where substrate binding produces a more closed conformation, the binding of NAD-tartrate to YiaK produces a more open active site. The free enzyme conformation is incompatible with NAD binding. His(44) is likely the catalytic residue of the enzyme.