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
Me. Salvucci et Rr. Klein, 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, Plant physiology, 102(2), 1993, pp. 529-536
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.