Functional expression of zeaxanthin glucosyltransferase from Erwinia herbicola and a proposed uridine diphosphate binding site.
Erwinia herbicola, a nonphotosynthetic bacterium, is yellow colored due to the accumulation of unusually polar carotenoids, primarily mono- and diglucosides of zeaxanthin. We have cloned and expressed the gene for the enzyme that catalyzes the glucosylation of zeaxanthin. The enzyme has an apparent molecular mass of 45 kDa on an SDS/polyacrylamide gel, which is consistent with its calculated molecular mass. In vitro enzymatic activity was demonstrated using UDP-[14C]glucose and zeaxanthin as substrates. The product zeaxanthin diglucoside and its intermediate monoglucoside were identified by thin layer chromatography. The optimum pH and temperature ranges of the enzyme are 7.0-7.5 and 32-37 degrees C, respectively. A hydropathy plot indicates no apparent membrane-spanning regions, and biochemical experiments suggest that the enzyme is weakly membrane-associated. The amino acid sequence derived from the zeaxanthin glucosyltransferase gene shows a small region of high similarity with other glucuronosyl- and glucosyltransferases that use either UDP-activated glucuronic acid or a sugar as one of their substrates. Based on these similarities, we propose that this conserved sequence is part of the UDP binding site.
Hundle
B S
BS
Department of Chemistry, University of California, Berkeley.
O'Brien
D A
DA
Alberti
M
M
Beyer
P
P
Hearst
J E
JE
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D004885:Erwinia / Q000201:enzymology* / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D005798:Genes, Bacterial; D005964:Glucosyltransferases / Q000235:genetics* / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D010957:Plasmids; D016133:Polymerase Chain Reaction / Q000379:methods; D017386:Sequence Homology, Amino Acid; D013728:Terminator Regions, Genetic; D014532:Uridine Diphosphate Glucose / Q000378:metabolism*
D009838:Oligodeoxyribonucleotides; D005964:Glucosyltransferases; C077175:zeaxanthin glucosyltransferase; D014532:Uridine Diphosphate Glucose