title: Structural insights into the mechanism defining substrate affinity in Arabidopsis thaliana dUTPase: the role of tryptophan 93 in ligand orientation.
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.
authors.affiliation: School of Biological Sciences, University of Nebraska-Lincoln, Lincoln, NE, USA. ninoguchi@huskers.unl.edu.
authors.forename: Noriko
authors.initials: N
authors.lastname: Inoguchi
authors.affiliation: Department of Chemistry, University of Nebraska-Lincoln, Lincoln, NE, USA. kittichai@gmail.com.
authors.forename: Kittichai
authors.initials: K
authors.lastname: Chaiseeda
authors.affiliation: Department of Chemistry, University of Nebraska-Lincoln, Lincoln, NE, USA. myamanishi3@me.com.
authors.forename: Mamoru
authors.initials: M
authors.lastname: Yamanishi
authors.affiliation: School of Mechanical Engineering, Sungkyunkwan University, 300 Cheoncheon, Suwon, South Korea. mkkim@me.skku.ac.kr.
authors.forename: Moon Ki
authors.initials: MK
authors.lastname: Kim
authors.affiliation: Department of Mechanical and Industrial Engineering, University of Massachusetts, Amherst, MA, USA. jang.yunho@gmail.com.|Virology, Surveillance and Diagnostic Branch, Influenza Division, Centers for Disease Control and Prevention, Atlanta, GA, USA. jang.yunho@gmail.com.
authors.forename: Yunho
authors.initials: Y
authors.lastname: Jang
authors.affiliation: School of Biological Sciences, University of Nebraska-Lincoln, Lincoln, NE, USA. mamtajgd@gmail.com.
authors.forename: Mamta
authors.initials: M
authors.lastname: Bajaj
authors.affiliation: School of Biological Sciences, University of Nebraska-Lincoln, Lincoln, NE, USA. cchia1@unl.edu.
authors.forename: Catherine P
authors.initials: CP
authors.lastname: Chia
authors.affiliation: Department of Biochemistry, University of Nebraska-Lincoln, Lincoln, NE, USA. dbecker3@unl.edu.
authors.forename: Donald F
authors.initials: DF
authors.lastname: Becker
authors.affiliation: School of Biological Sciences, University of Nebraska-Lincoln, Lincoln, NE, USA. hmoriyama2@unl.edu.
authors.forename: Hideaki
authors.initials: H
authors.lastname: Moriyama
mesh_terms: D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000201:enzymology / Q000235:genetics; D029681:Arabidopsis Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites / Q000235:genetics; D001667:Binding, Competitive; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D057927:Hydrophobic and Hydrophilic Interactions; D008024:Ligands; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011485:Protein Binding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011755:Pyrophosphatases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D014364:Tryptophan / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D029681:Arabidopsis Proteins; D008024:Ligands; D014364:Tryptophan; D011755:Pyrophosphatases; C020020:dUTP pyrophosphatase