Structural insights into the mechanism defining substrate affinity in Arabidopsis thaliana dUTPase: the role of tryptophan 93 in ligand orientation

Authors

Noriko Inoguchi, University of Nebraska-LincolnFollow
Kittichai Chaiseeda, University of Nebraska-Lincoln
Mamoru Yamanishi, University of Nebraska-Lincoln
Moon Ki Kim, Sungkyunkwan University
Yunho Jang, University of Massachusetts
Mamta Bajaj, University of Nebraska-Lincoln
Catherine P. Chia, University of Nebraska - LincolnFollow
Donald F. Becker, University of Nebraska-LincolnFollow
Hideaki Moriyama, University of Nebraska-LincolnFollow

Document Type

Article

Date of this Version

2015

Citation

Inoguchi et al. BMC Res Notes (2015) 8:784 DOI 10.1186/s13104-015-1760-1

Comments

© 2015 Inoguchi et al. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License

Abstract

Background: Deoxyuridine triphosphate nucleotidohydrolase (dUTPase) hydrolyzes dUTP to dUMP and pyrophosphate to maintain the cellular thymine-uracil ratio. dUTPase is also a target for cancer chemotherapy. However, the mechanism defining its substrate affinity remains unclear. Sequence comparisons of various dUTPases revealed that Arabidopsis thaliana dUTPase has a unique tryptophan at position 93, which potentially contributes to its degree of substrate affinity. To better understand the roles of tryptophan 93, A. thaliana dUTPase was studied.

Results: Enzyme assays showed that A. thaliana dUTPase belongs to a high-affinity group of isozymes, which also includes the enzymes from Escherichia coli and Mycobacterium tuberculosis. Enzymes from Homo sapiens and Saccharomyces cerevisiae are grouped as low-affinity dUTPases. The structure of the homo-trimeric A. thaliana dUTPase showed three active sites, each with a different set of ligand interactions between the amino acids and water molecules. On an α-helix, tryptophan 93 appears to keep serine 89 in place via a water molecule and to specifically direct the ligand. Upon being oriented in the active site, the C-terminal residues close the active site to promote the reaction.

Conclusions: In the high-affinity group, the prefixed direction of the serine residues was oriented by a positively charged residue located four amino acids away, while low-affinity enzymes possess small hydrophobic residues at the corresponding sites.