IDENTIFICATION OF THE URIDINE-BINDING DOMAIN OF SUCROSE-PHOSPHATE SYNTHASE - EXPRESSION OF A REGION OF THE PROTEIN THAT PHOTOAFFINITY LABELS WITH 5-AZIDOURIDINE DIPHOSPHATE-GLUCOSE

The uridine diphosphate-glucose (UDP-Glc) binding domain of sucrose-ph osphate synthase (SPS) was identified by overexpressing part of the ge ne from spinach (Spinacia oleracea). Degenerate oligonucleotide primer s corresponding to two tryptic peptides common to both the full-length 120-kD SPS subunit and an 82-kD form that photoaffinity labeled with 5-azidouridine diphosphate-glucose (5-N3UDP-Glc) were used in a polyme rase chain reaction to isolate a partial cDNA clone. Comparison of the deduced amino acid sequence of spinach SPS with the sequences of pota to sucrose synthase showed that the partial cDNA included one region t hat was highly conserved between the proteins. Expression of the parti al cDNA clone of SPS in Escherichia coli produced a 26-kD fusion prote in that photoaffinity labeled with 5-N3UDP-Glc. Photoaffinity labeling of the 26-kD fusion protein was specific, indicating that this portio n of the SPS protein harbors the UDP-Glc-binding domain. Isolation of a modified peptide from the photolabeled protein provided tentative id entification of amino acid residues that make up the uridine-binding d omain of SPS.