Disruption of the Proton Relay Network in the Class 2 Dihydroorotate Dehydrogenase from Escherichia coli
Disruption of the Proton Relay Network in the Class 2 Dihydroorotate Dehydrogenase from Escherichia coli
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DOI：
10.1021/bi901024m
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发表时间：
2009-10-20
期刊：
影响因子：
2.9
通讯作者：
Fagan, Rebecca L.
中科院分区：
文献类型：
--
作者：
Kow, Rebecca L.;Whicher, Jonathan R.;Fagan, Rebecca L.
二氢羊角酸脱氢酶（DHODs）是一种含fmn的酶，在嘧啶的从头合成中催化二氢羊角酸（DHO）转化为羊角酸。在反应过程中，一个质子从DHO的C5转移到活性位点碱基上，DHO的C6上的氢作为氢化物转移到黄素的异alloxazine环的N5上。在2类DHODs中，在晶体结构中观察到一个氢键网络，使dhod的C5原子去质子化。活性位点碱基（大肠杆菌酶中的Ser175）氢键与苯基丙氨酸（大肠杆菌酶中的Phe115）上的结晶水分子相连，氢键与苏氨酸（大肠杆菌酶中的Thr178）相连，这些残基在2类酶中是保守的。这些残基在DHO氧化中的重要性用定点诱变进行了研究。将Ser175突变为丙氨酸对黄素的还原速度有严重影响，减缓了3个数量级以上。改变氢键网络Thr178和Phe115残基的大小和/或疏水性，可使黄素还原速度减慢2个数量级，表明活性位点碱基和氢键网络起作用。一起进行DHO的有效去质子化。
Dihydroorotate dehydrogenases (DHODs) are FMN-containing enzymes that catalyze the conversion of dihydroorotate (DHO) to orotate in the de novo synthesis of pyrimidines. During the reaction, a proton is transferred from C5 of DHO to an active site base and the hydrogen at C6 of DHO is transferred to N5 of the isoalloxazine ring of the flavin as a hydride. In class 2 DHODs, a hydrogen bond network observed in crystal structures has been proposed to deprotonate the C5 atom of DHO. The active site base (Ser175 in the Escherichia coli enzyme) hydrogen bonds to a crystallographic water molecule that sits on a phenylalanine (Phe115 in the E. coli enzyme) and hydrogen bonds to a threonine (Thr178 in the E. coli enzyme), residues that are conserved in class 2 enzymes. The importance of these residues in the oxidation of DHO was investigated using site-directed mutagenesis. Mutating Ser175 to alanine had severe effects on the rate of flavin reduction, slowing it by more than 3 orders of magnitude. Changing the size and/or hydrophobicity of the residues of the hydrogen bond network, Thr178 and Phe115, slowed flavin reduction as much as 2 orders of magnitude, indicating that the active site base and the hydrogen bond network work. together for efficient deprotonation of DHO.