title: Structural determinants allowing transferase activity in SENSITIVE TO FREEZING 2, classified as a family I glycosyl hydrolase.
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.
authors.affiliation: Departments of Biochemistry and Molecular Biology and Michigan State University, East Lansing, Michigan 48824. Electronic address: rroston@unl.edu.
authors.forename: Rebecca L
authors.initials: RL
authors.lastname: Roston
authors.affiliation: Departments of Biochemistry and Molecular Biology and Michigan State University, East Lansing, Michigan 48824.
authors.forename: Kun
authors.initials: K
authors.lastname: Wang
authors.affiliation: Departments of Biochemistry and Molecular Biology and Michigan State University, East Lansing, Michigan 48824; Departments of Computer Science and Engineering, Michigan State University, East Lansing, Michigan 48824.
authors.forename: Leslie A
authors.initials: LA
authors.lastname: Kuhn
authors.affiliation: Departments of Biochemistry and Molecular Biology and Michigan State University, East Lansing, Michigan 48824.
authors.forename: Christoph
authors.initials: C
authors.lastname: Benning
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000201:enzymology*; D029681:Arabidopsis Proteins / Q000737:chemistry* / Q000145:classification; D020134:Catalytic Domain; D017124:Conserved Sequence; D006863:Hydrogen-Ion Concentration; D057927:Hydrophobic and Hydrophilic Interactions; D064267:Intrinsically Disordered Proteins; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011485:Protein Binding; D017386:Sequence Homology, Amino Acid; D040681:Structural Homology, Protein; D001617:beta-Glucosidase / Q000737:chemistry* / Q000145:classification
chemical_list: D029681:Arabidopsis Proteins; D064267:Intrinsically Disordered Proteins; C492903:SFR2 protein, Arabidopsis; D001617:beta-Glucosidase
keywords: Chloroplast; Freezing; Glycosidase; Glycosyl Hydrolase; Glycosyltransferase; Hydrolase; Membrane Turnover; Oligogalactolipid; Stress Response; Transferase
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