Flexible glycine rich motif of Escherichia coli deoxyuridine triphosphate nucleotidohydrolase is important for functional but not for structural integrity of the enzyme

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Abstract

Deoxyuridine triphosphate nucleotidohydrolase (dUTPase), a ubiquitous enzyme of DNA metabolism, has been implicated as a novel target of anticancer and antiviral drug design. This task is most efficiently accomplished by X- ray crystallography of the relevant protein-inhibitor complexes. However, the topic of the present investigation, a glycine-rich strictly conserved structural motif of dUTPases, could not be located in the crystal structure of the Escherichia coli enzyme, probably due to its increased flexibility. The present work shows that removal of a C-terminal 11-residue fragment, including this motif, by limited trypsinolysis strongly impairs catalytic activity. Kinetic analysis of the intact and digested variants showed that κ(cat) decreases 40-fold, while K(M) increases less than twofold upon digestion. The tryptic site was identified by mass spectrometry, amino acid analysis and N-terminal sequencing. The shortened enzyme variant retains the secondary, tertiary, and quaternary (trimeric) structure of the intact species as suggested by UV absorption, fluorescence and circular dichroism spectroscopy, and analytical gel filtration. Moreover, binding affinity of the shortened variant toward the substrate analogue MgdUDP is identical to the one displayed by the intact enzyme. I conclude that the glycine-rich motif is functionally relevant for E. coli dUTPase. It may play a role in enzymatic catalysis by contributing to the formation of the catalytically potent enzyme-substrate complex.

Original languageEnglish
Pages (from-to)568-579
Number of pages12
JournalProteins: Structure, Function and Genetics
Volume28
Issue number4
DOIs
Publication statusPublished - Aug 19 1997

Keywords

  • Circular dichroism spectroscopy
  • Gly-rich motif
  • Limited trypsinolysis
  • MgdUDP binding
  • Motif 5 of dUTPases
  • Phosphate binding P-loop
  • Secondary structure determination

ASJC Scopus subject areas

  • Structural Biology
  • Biochemistry
  • Molecular Biology

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