Krupenko S A, Vlasov A P, Wagner C
Department of Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston, South Carolina 29425, USA.
J Biol Chem. 2001 Jun 29;276(26):24030-7. doi: 10.1074/jbc.M009257200. Epub 2001 Apr 24.
The enzyme, 10-formyltetrahydrofolate dehydrogenase (FDH), converts 10-formyltetrahydrofolate (10-formyl-THF) to tetrahydrofolate in an NADP(+)-dependent dehydrogenase reaction or an NADP(+)-independent hydrolase reaction. The hydrolase reaction occurs in a 310-amino acid long amino-terminal domain of FDH (N(t)-FDH), whereas the dehydrogenase reaction requires the full-length enzyme. The amino-terminal domain of FDH shares some sequence identity with several other enzymes utilizing 10-formyl-THF as a substrate. These enzymes have two strictly conserved residues, aspartate and histidine, in the putative catalytic center. We have shown recently that the conserved aspartate is involved in FDH catalysis. In the present work we studied the role of the conserved histidine, His(106), in FDH function. Site-directed mutagenesis experiments showed that replacement of the histidine with alanine, asparagine, aspartate, glutamate, glutamine, or arginine in N(t)-FDH resulted in expression of insoluble proteins. Replacement of the histidine with another positively charged residue, lysine, produced a soluble mutant with no hydrolase activity. The insoluble mutants refolded from inclusion bodies adopted a conformation inherent to the wild-type N(t)-FDH, but they did not exhibit any hydrolase activity. Substitution of alanine for three non-conserved histidines located close to the conserved one did not reveal any significant changes in the hydrolase activity of N(t)-FDH. Expressed full-length FDH with the substitution of lysine for the His(106) completely lost both the hydrolase and dehydrogenase activities. Thus, our study showed that His(106), besides being an important structural residue, is also directly involved in both the hydrolase and dehydrogenase mechanisms of FDH. Modeling of the putative hydrolase catalytic center/folate-binding site suggested that the catalytic residues, aspartate and histidine, are unlikely to be adjacent to the catalytic cysteine in the aldehyde dehydrogenase catalytic center. We hypothesize that 10-formyl-THF dehydrogenase reaction is not an independent reaction but is a combination of hydrolase and aldehyde dehydrogenase reactions.
10-甲酰四氢叶酸脱氢酶（FDH）可通过依赖烟酰胺腺嘌呤二核苷酸磷酸（NADP⁺）的脱氢酶反应或不依赖NADP⁺的水解酶反应，将10-甲酰四氢叶酸（10-甲酰-THF）转化为四氢叶酸。水解酶反应发生在FDH的一个由310个氨基酸组成的氨基末端结构域（N(t)-FDH）中，而脱氢酶反应则需要全长酶。FDH的氨基末端结构域与其他几种以10-甲酰-THF为底物的酶具有一定的序列同源性。这些酶在假定的催化中心有两个严格保守的残基，即天冬氨酸和组氨酸。我们最近发现保守的天冬氨酸参与FDH的催化作用。在本研究中，我们研究了保守的组氨酸His(106)在FDH功能中的作用。定点诱变实验表明，在N(t)-FDH中用丙氨酸、天冬酰胺、天冬氨酸、谷氨酸、谷氨酰胺或精氨酸取代组氨酸会导致不溶性蛋白质的表达。用另一个带正电荷的残基赖氨酸取代组氨酸产生了一种没有水解酶活性的可溶性突变体。从不溶性包涵体中复性的突变体采用了野生型N(t)-FDH固有的构象，但它们没有表现出任何水解酶活性。用丙氨酸取代靠近保守组氨酸的三个非保守组氨酸，未发现N(t)-FDH的水解酶活性有任何显著变化。用赖氨酸取代His(106)表达的全长FDH完全丧失了水解酶和脱氢酶活性。因此，我们的研究表明，His(106)除了是一个重要的结构残基外，还直接参与FDH的水解酶和脱氢酶机制。对假定的水解酶催化中心/叶酸结合位点的建模表明，催化残基天冬氨酸和组氨酸不太可能与醛脱氢酶催化中心的催化半胱氨酸相邻。我们推测10-甲酰-THF脱氢酶反应不是一个独立的反应，而是水解酶和醛脱氢酶反应的组合。
