Analysis of the structure, substrate specificity, and mechanism of squash glycerol-3-phosphate (1)-acyltransferase

Background: Glycerol-3-phosphate (1)-acyltransferase (G3PAT) catalyzes the incorporation of an acyl group from either acyl-acyl carrier proteins (acyl ACPs) or acylCoAs into the sn-1 position of glycerol 3-phosphate to yield 1 -acylglycerol-3-phosphate. G3PATs can either be selective, preferentially u sing the unsaturated fatty acid, oleate (C18:1), as the acyl donor, or nons elective, using either oleate or the saturated fatty acid, palmitate (C16:0 ), at comparable rates. The differential substrate specificity for saturate d versus unsaturated fatty acids seen within this enzyme family has been im plicated in the sensitivity of plants to chilling temperatures. Results: The three-dimensional structure of recombinant G3PAT from squash c hloroplast has been determined to 1.9 Angstrom resolution by X-ray crystall ography using the technique of multiple isomorphous replacement and provide s the first representative structure of an enzyme of this class. Conclusions: The tertiary structure of G3PAT comprises two domains, the lar ger of which, domain II, features an extensive cleft lined by hydrophobic r esidues and contains at one end a cluster of positively charged residues fl anked by a H(X)(4)D motif, which is conserved amongst many glycerolipid acy ltransferases. We predict that these hydrophobic and positively charged res idues represent the binding sites for the fatty acyl substrate and the phos phate moiety of the glycerol 3-phosphate, respectively, and that the H(X)(4 )D motif is a critical component of the enzyme's catalytic machinery.