Abstract
SENSITIVE TO FREEZING 2 (SFR2) is classified as a family I glycosyl hydrolase but has recently been shown to have galactosyltransferase activity in Arabidopsis thaliana. Natural occurrences of apparent glycosyl hydrolases acting as transferases are interesting from a biocatalysis standpoint, and knowledge about the interconversion can assist in engineering SFR2 in crop plants to resist freezing. To understand how SFR2 evolved into a transferase, the relationship between its structure and function are investigated by activity assay, molecular modeling, and site-directed mutagenesis. SFR2 has no detectable hydrolase activity, although its catalytic site is highly conserved with that of family 1 glycosyl hydrolases. Three regions disparate from glycosyl hydrolases are identified as required for transferase activity as follows: a loop insertion, the C-terminal peptide, and a hydrophobic patch adjacent to the catalytic site. Rationales for the effects of these regions on the SFR2 mechanism are discussed.
Highlights
SENSITIVE TO FREEZING 2 (SFR2) is classified as a glycosyl hydrolase, and by using glycosyltransferase activity, it modifies membrane lipids to promote freeze tolerance
The temperature that resulted in the highest activity was ϳ24 °C, consistent with the role of SFR2 during freezing, activity was detectable at 0 °C (Fig. 1C)
We have demonstrated that SFR2 is a GH1 member that performs little or no hydrolase activity, instead acting as a glycosyltransferase
Summary
SENSITIVE TO FREEZING 2 (SFR2) is classified as a glycosyl hydrolase, and by using glycosyltransferase activity, it modifies membrane lipids to promote freeze tolerance. GH1s are a structurally related group of presumed functionally similar enzymes that catalyze removal of a sugar group while retaining the anomeric configuration of the sugar at carbon 1 (C1) (EC 3.2.1) [5] They adopt a (/␣) or “␣/” barrel protein fold, known as a triose-phosphate isomerase barrel, with loop regions conferring substrate specificity and modulating activity [6]. The SFR2 model surprisingly yields a catalytic site identical in sequence and similar in architecture to that of GH1s with hydrolase but no transferase activity. Three regions of SFR2 dissimilar from other GH1s are shown to be necessary for transferase activity on its native galactosyldiacylglycerol substrates Their relationship to galactolipid transferase activity and processivity is analyzed by modeling the substrate-enzyme complex
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