An increase in apparent affinity for sucrose of mung bean sucrose synthase is caused by in vitro phosphorylation or directed mutagenesis of Ser11.
A mutational analysis of mung bean (Vigna radiata Wilczek) sucrose synthase was performed by site-directed mutagenesis of the recombinant protein expressed in Escherichia coli, in which two different acidic amino acid residues (Asp or Glu) were introduced at Ser11 (S11D, S11E). Only the wild-type enzyme (Ser11) was phosphorylated in vitro by a Ca(2+)-dependent protein kinase from soybean root nodules, suggesting that this is the specific target residue in mung bean sucrose synthase. The apparent affinity for sucrose was increased in this phosphorylated enzyme and also in the S11D and S11E mutant enzymes, although the affinities for UDP-glucose and fructose were similar in the wild-type, phosphorylated wild-type, and mutant enzymes. These results suggest that a monoanionic (1-) side chain at position 11 mimics the Ser11-P2- residue to bind and cleave sucrose for the synthesis of UDP-glucose. Since the S11E mutant enzyme showed the lowest K(m) (sucrose) and the highest catalytic efficiency of the recombinant proteins, the enzymic properties of this S11E mutant were further characterized. The results showed that replacement of Ser11 with Glu11 modestly protected the sucrose synthesis activity against phenolic glycosides and altered the enzyme nucleotide specificity. We postulate that the introduction of negative charge at Ser11 is possibly involved in the enzymatic perturbation of sucrose synthase.
Nakai
T
T
Wood Research Institute, Kyoto University, Japan.
Konishi
T
T
Zhang
X Q
XQ
Chollet
R
R
Tonouchi
N
N
Tsuchida
T
T
Yoshinaga
F
F
Mori
H
H
Sakai
F
F
Hayashi
T
T
D000595:Amino Acid Sequence; D001224:Aspartic Acid / Q000235:genetics; D007887:Fabaceae / Q000201:enzymology*; D005964:Glucosyltransferases / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D010946:Plants, Medicinal; D011499:Protein Processing, Post-Translational; D012694:Serine / Q000235:genetics; D013379:Substrate Specificity; D013395:Sucrose / Q000378:metabolism*
D001224:Aspartic Acid; D012694:Serine; D013395:Sucrose; D005964:Glucosyltransferases; C022699:sucrose synthase