Catalysis by Glomerella cingulata Cutinase Requires Conformational Cycling between the Active and Inactive States of Its Catalytic Triad

Mun Peak Nyon, David W. Rice, John M. Berrisford, Andrea M. Hounslow, Arthur J G Moir, Huazhang Huang, Sheila Nathan, Nor Muhammad Mahadi, Farah Diba Abu Bakar, C. Jeremy Craven

Research output: Contribution to journalArticle

17 Citations (Scopus)

Abstract

Cutinase belongs to a group of enzymes that catalyze the hydrolysis of esters and triglycerides. Structural studies on the enzyme from Fusarium solani have revealed the presence of a classic catalytic triad that has been implicated in the enzyme's mechanism. We have solved the crystal structure of Glomerella cingulata cutinase in the absence and in the presence of the inhibitors E600 (diethyl p-nitrophenyl phosphate) and PETFP (3-phenethylthio-1,1,1-trifluoropropan-2-one) to resolutions between 2.6 and 1.9 Å. Analysis of these structures reveals that the catalytic triad (Ser136, Asp191, and His204) adopts an unusual configuration with the putative essential histidine His204 swung out of the active site into a position where it is unable to participate in catalysis, with the imidazole ring 11 Å away from its expected position. Solution-state NMR experiments are consistent with the disrupted configuration of the triad observed crystallographically. H204N, a site-directed mutant, was shown to be catalytically inactive, confirming the importance of this residue in the enzyme mechanism. These findings suggest that, during its catalytic cycle, cutinase undergoes a significant conformational rearrangement converting the loop bearing the histidine from an inactive conformation, in which the histidine of the triad is solvent exposed, to an active conformation, in which the triad assumes a classic configuration.

Original languageEnglish
Pages (from-to)226-235
Number of pages10
JournalJournal of Molecular Biology
Volume385
Issue number1
DOIs
Publication statusPublished - 9 Jan 2009

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Phyllachorales
Catalysis
Histidine
Enzymes
Paraoxon
Fusarium
Catalytic Domain
Esters
Triglycerides
Hydrolysis
cutinase

Keywords

  • catalytic triad
  • conformational change
  • cutinase
  • Glomerella cingulata
  • inhibitor complexes

ASJC Scopus subject areas

  • Molecular Biology

Cite this

Catalysis by Glomerella cingulata Cutinase Requires Conformational Cycling between the Active and Inactive States of Its Catalytic Triad. / Nyon, Mun Peak; Rice, David W.; Berrisford, John M.; Hounslow, Andrea M.; Moir, Arthur J G; Huang, Huazhang; Nathan, Sheila; Mahadi, Nor Muhammad; Abu Bakar, Farah Diba; Craven, C. Jeremy.

In: Journal of Molecular Biology, Vol. 385, No. 1, 09.01.2009, p. 226-235.

Research output: Contribution to journalArticle

Nyon, Mun Peak ; Rice, David W. ; Berrisford, John M. ; Hounslow, Andrea M. ; Moir, Arthur J G ; Huang, Huazhang ; Nathan, Sheila ; Mahadi, Nor Muhammad ; Abu Bakar, Farah Diba ; Craven, C. Jeremy. / Catalysis by Glomerella cingulata Cutinase Requires Conformational Cycling between the Active and Inactive States of Its Catalytic Triad. In: Journal of Molecular Biology. 2009 ; Vol. 385, No. 1. pp. 226-235.
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