The Crystal Structure of the Hydrolase Domain of 10-Formyltetrahydrofolate Dehydrogenase: MECHANISM OF HYDROLYSIS AND ITS INTERPLAY WITH THE DEHYDROGENASE DOMAIN

Alexander A Chumanevich,Sergey A Krupenko,Christopher Davies

doi:10.1074/jbc.m313934200

Alexander A Chumanevich, Sergey A Krupenko + Show 1 more

Open Access

https://doi.org/10.1074/jbc.m313934200

Copy DOI

Abstract

10-Formyltetrahydrofolate dehydrogenase (FDH) converts 10-formyltetrahydrofolate, a precursor for nucleotide biosynthesis, to tetrahydrofolate. The protein comprises two functional domains: a hydrolase domain that removes a formyl group from 10-formyltetrahydrofolate and a NADP(+)-dependent dehydrogenase domain that reduces the formyl to carbon dioxide. As a first step toward deciphering the catalytic mechanism of the enzyme, we have determined the crystal structure of the hydrolase domain of FDH from rat, solved to 2.3-A resolution. The structure comprises two domains. As expected, domain 1 shares the same Rossmann fold as the related enzymes, methionyl-tRNA-formyltransferase and glycinamide ribonucleotide formyltransferase, but, unexpectedly, the structural similarity between the amino-terminal domain of 10-formyltetrahydrofolate dehydrogenase and methionyl-tRNA-formyltransferase extends to the C terminus of both proteins. The active site contains a molecule of beta-mercaptoethanol that is positioned between His-106 and Asp-142 and that appears to mimic the formate product. We propose a catalytic mechanism for the hydrolase reaction in which Asp-142 polarizes the catalytic water molecule and His-106 orients the carbonyl group of formyl. The structure also provides clues as to how, in the native enzyme, the hydrolase domain transfers its product to the dehydrogenase domain.

Highlights

10-Formyltetrahydrofolate dehydrogenase (FDH) converts 10-formyltetrahydrofolate, a precursor for nucleotide biosynthesis, to tetrahydrofolate
The protein comprises two functional domains: a hydrolase domain that removes a formyl group from 10-formyltetrahydrofolate and a NADP؉-dependent dehydrogenase domain that reduces the formyl to carbon dioxide
Domain 1 shares the same Rossmann fold as the related enzymes, methionyl-tRNA-formyltransferase and glycinamide ribonucleotide formyltransferase, but, unexpectedly, the structural similarity between the amino-terminal domain of 10-formyltetrahydrofolate dehydrogenase and methionyl-tRNA-formyltransferase extends to the C terminus of both proteins

Summary

Introduction

10-Formyltetrahydrofolate dehydrogenase (FDH) converts 10-formyltetrahydrofolate, a precursor for nucleotide biosynthesis, to tetrahydrofolate. The binding site for folate is located in the N-terminal region of the enzyme (termed Nt-FDH, residues 1–310) [11] This domain shows sequence similarity with folate-binding domains in other proteins, including methionyl-tRNA- formyltransferase (FMT) and glycinamide ribonucleotide formyltransferase (GART) [9, 11]. When the N-terminal domain is expressed separately, it functions as a 10-formyl-THF hydrolase, resulting in the production of formate [11], whereas the dehydrogenase reaction is catalyzed only by the full-length FDH [11, 12]. This suggests that the hydrolase activity of the N-terminal domain contributes to the overall dehydrogenase reaction and, some functional communication between the two domains must exist. FDH is an interesting example of a natural fusion of two unrelated proteins that creates a new enzymatic activity

Methods

Results

Conclusion