title: Mutagenesis of squash (Cucurbita moschata) glycerol-3-phosphate acyltransferase (GPAT) to produce an enzyme with altered substrate selectivity.
abstract: In an attempt to rationalize the relationship between structure and substrate selectivity of glycerol-3-phosphate acyltransferase (GPAT, 1AT, EC 2.3.1.15) we have cloned a number of cDNAs into the pET overexpression system using a PCR-based approach. Following assay of the recombinant enzyme we noted that the substrate selectivity of the squash (Cucurbita moschata) enzyme had altered dramatically. This form of GPAT has now been crystallized and its full three-dimensional structure elucidated. Since we now have two forms of the enzyme that display different substrate selectivities this should provide a powerful tool to determine the basis of the selectivity changes. Kinetic and structural analyses are currently being performed to rationalize the changes which have taken place.
authors.affiliation: Department of Biological Sciences, University of Durham, South Road, Durham DH1 3LE, UK.
authors.forename: M W
authors.initials: MW
authors.lastname: Hayman
authors.forename: T
authors.initials: T
authors.lastname: Fawcett
authors.forename: T F
authors.initials: TF
authors.lastname: Schierer
authors.forename: J W
authors.initials: JW
authors.lastname: Simon
authors.forename: J T
authors.initials: JT
authors.lastname: Kroon
authors.forename: J S
authors.initials: JS
authors.lastname: Gilroy
authors.forename: D W
authors.initials: DW
authors.lastname: Rice
authors.forename: J
authors.initials: J
authors.lastname: Rafferty
authors.forename: A P
authors.initials: AP
authors.lastname: Turnbull
authors.forename: S E
authors.initials: SE
authors.lastname: Sedelnikova
authors.forename: A R
authors.initials: AR
authors.lastname: Slabas
mesh_terms: D017360:Arabidopsis / Q000201:enzymology; D003001:Cloning, Molecular; D018360:Crystallography, X-Ray; D019662:Cucurbitaceae / Q000201:enzymology*; D004926:Escherichia coli; D005992:Glycerol-3-Phosphate O-Acyltransferase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: D011994:Recombinant Proteins; D005992:Glycerol-3-Phosphate O-Acyltransferase