Selectivity of fungal sesquiterpene synthases: role of the active site's H-1 alpha loop in catalysis

Appl Environ Microbiol. 2010 Dec;76(23):7723-33. doi: 10.1128/AEM.01811-10. Epub 2010 Oct 1.

Abstract

Sesquiterpene synthases are responsible for the cyclization of farnesyl pyrophosphate into a myriad of structurally diverse compounds with various biological activities. We examine here the role of the conserved active site H-α1 loop in catalysis in three previously characterized fungal sesquiterpene synthases. The H-α1 loops of Cop3, Cop4, and Cop6 from Coprinus cinereus were altered by site-directed mutagenesis and the resultant product profiles were analyzed by gas chromatography-mass spectrometry and compared to the wild-type enzymes. In addition, we examine the effect of swapping the H-α1 loop from the promiscuous enzyme Cop4 with the more selective Cop6 and the effect of acidic or basic conditions on loop mutations in Cop4. Directed mutations of the H-α1 loop had a marked effect on the product profile of Cop3 and Cop4, while little to no change was shown in Cop6. Swapping of the Cop4 and Cop6 loops with one another was again shown to influence the product profile of Cop4, while the product profile of Cop6 remained identical to the wild-type enzyme. The loop mutations in Cop4 also implicate specific residues responsible for the pH sensitivity of the enzyme. These results affirm the role of the H-α1 loop in catalysis and provide a potential target to increase the product diversity of terpene synthases.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Alkyl and Aryl Transferases / chemistry
  • Alkyl and Aryl Transferases / genetics
  • Alkyl and Aryl Transferases / metabolism*
  • Amino Acid Substitution
  • Catalytic Domain
  • Coprinus / enzymology*
  • Fungal Proteins / chemistry
  • Fungal Proteins / genetics
  • Fungal Proteins / metabolism*
  • Gas Chromatography-Mass Spectrometry
  • Models, Molecular
  • Mutagenesis, Site-Directed
  • Mutant Proteins / chemistry
  • Mutant Proteins / genetics
  • Mutant Proteins / metabolism
  • Protein Structure, Tertiary
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / genetics
  • Recombinant Proteins / metabolism
  • Recombination, Genetic
  • Sesquiterpenes / metabolism*
  • Substrate Specificity

Substances

  • Fungal Proteins
  • Mutant Proteins
  • Recombinant Proteins
  • Sesquiterpenes
  • Alkyl and Aryl Transferases
  • terpene synthase