While the enzymes responsible for the initial step of glycerolipid synthesis, known as glycerol 3-phosphate acyltransferases, are present in all organisms, a family of GPATs with unusual sn-2 regiospecificity are present only in land plants. These produce precursors of the hydrophobic polyesters cutin and suberin, as sn-2 lysophosphatidic acid or sn-2 monoacylglycerols in cases where their N-terminal phosphatase domain is functional. As this latter lipid is a substrate for mammalian monoacylglycerol acyltransferases/diacylglycerol acyltransferases, our research group has attempted to utilise these sn-2 GPATs to reconstitute a two enzyme metabolic pathway to triacylglycerol biosynthesis. However, sn-2 phosphatase active GPATs, such as A. thaliana GPAT4, reportedly exhibit little activity with fatty acids typically found in triacylglycerols, displaying greater activity with the ω-oxidised fatty acids that comprise the majority of cutin. Our research has investigated the effect of site directed mutagenesis on the activity and substrate preference of AtGPAT4. Consistent with their predicted role in acyl transfer, substitution of conserved residues in the C-terminal domain of GPAT4 prevented formation of glycerolipids in assays of the recombinantly expressed protein in S. cerevisae microsomes. Mutation of phosphatase motif residues in the N-terminal domain reduced monoacylglycerol formation, with concomitant increases in phospholipids indicating preserved acyltransferase activity. Within assays of recombinant GPAT4, the hydrolysis of the nascent MAG was observed to vary with the identity of the fatty acyl moiety. GPAT4 displayed substantial activity with non-preferred substrate oleoyl CoA when expressed in a yeast strain lacking the endogenous monoacylglycerol lipase Yju3p, suggesting that enzymatic monoacylglycerol hydrolysis may have biased previous reports of GPAT substrate preference. This research indicates the sn-2 GPAT AtGPAT4 may have appropriate substrate preference for inclusion in a novel triacylglycerol biosynthetic pathway.