title: Altering product outcome in Abies grandis (-)-limonene synthase and (-)-limonene/(-)-alpha-pinene synthase by domain swapping and directed mutagenesis.
abstract: (-)-(4S)-limonene synthase (LS) and (-)-(4S)-limonene/(-)-(1S, 5S)-alpha-pinene synthase (LPS) from grand fir (Abies grandis) exhibit nearly 91% sequence identity (93% similarity) at the amino acid level, yet produce very different mixtures of monoterpene olefins. To elucidate critical amino acids involved in determining monoterpene product distribution, a combination of domain swapping and reciprocal site-directed mutagenesis was carried out between these two enzymes. Exchange of the predicted helix D through F region in LS gave rise to an LPS-like product outcome, whereas reciprocal substitutions of four amino acids in LPS (two in the predicted helix D and two in the predicted helix F) altered the product distribution to that intermediate between LS and LPS, and resulted in a 5-fold increase in relative velocity. These results, in conjunction with modeling of the two enzymes, suggest that amino acids in the predicted D through F helix regions are critical for product determination.
authors.affiliation: Institute of Biological Chemistry, Washington State University, Pullman, Washington 99164-6340, USA.
authors.forename: Sadanobu
authors.initials: S
authors.lastname: Katoh
authors.forename: David
authors.initials: D
authors.lastname: Hyatt
authors.forename: Rodney
authors.initials: R
authors.lastname: Croteau
mesh_terms: D028202:Abies / Q000201:enzymology*; D000595:Amino Acid Sequence; D019757:Carbon-Oxygen Lyases / Q000235:genetics / Q000378:metabolism*; D019753:Intramolecular Lyases / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D017434:Protein Structure, Tertiary; D016415:Sequence Alignment
chemical_list: D019757:Carbon-Oxygen Lyases; C473195:(-)-alpha-pinene synthase; D019753:Intramolecular Lyases; C040265:pinene cyclase I