Plant glycerol-3-phosphate-1-acyltransferase (GPAT): structure selectivity studies.
Squash glycerol-3-phosphate-1-acyltransferase has been crystallized and the structure of the enzyme determined, at 1.9-A resolution, using multiple isomorphous replacement of the wild type and a series of individual cysteine mutants. Competitive in vitro substrate selectivity assays have been established that differentiate between selective and non-selective forms of the enzyme. Particular care was taken to use near-physiological concentrations of both substrates. Clear substrate selectivity can be demonstrated with the natural substrate acyl-acyl carrier protein but not with the substrate analogue acyl-CoA. The use of site-directed mutagenesis, coupled to three-dimensional structural determinations, should provide a rational basis for elucidating structural components important in determining the substrate selectivity of this enzyme.
Slabas
A R
AR
Department of Biological Sciences, University of Durham, Science Laboratories, South Road, Durham DH1 3LE, UK. A.R.Slabas@Durham.ac.uk
Simon
W R
WR
Schierer
T
T
Kroon
J
J
Fawcett
T
T
Hayman
M
M
Gilroy
J
J
Nishida
I
I
Murata
N
N
Rafferty
J
J
Turnbull
A
A
Rice
D
D
D017360:Arabidopsis / Q000201:enzymology; D001937:Brassica / Q000201:enzymology; D003001:Cloning, Molecular; D018360:Crystallography, X-Ray; D019662:Cucurbitaceae / Q000201:enzymology; D004926:Escherichia coli; D005992:Glycerol-3-Phosphate O-Acyltransferase / Q000737:chemistry* / Q000378:metabolism*; D007700:Kinetics; D008958:Models, Molecular; D018532:Pisum sativum / Q000201:enzymology; D010944:Plants / Q000201:enzymology*; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D018724:Spinacia oleracea / Q000201:enzymology; D013379:Substrate Specificity
D011994:Recombinant Proteins; D005992:Glycerol-3-Phosphate O-Acyltransferase