Journal Article

An Increase in Apparent Affinity for Sucrose of Mung Bean Sucrose Synthase Is Caused by In Vitro Phosphorylation or Directed Mutagenesis of Ser<sup>11</sup>

Tomonori Nakai, Teruko Konishi, Xiu-Qing Zhang, Raymond Chollet, Naoto Tonouchi, Takayasu Tsuchida, Fumihiro Yoshinaga, Hitoshi Mori, Fukumi Sakai and Takahisa Hayashi

in Plant and Cell Physiology

Published on behalf of Japanese Society of Plant Physiologists

Volume 39, issue 12, pages 1337-1341
Published in print December 1998 | ISSN: 0032-0781
Published online December 1998 | e-ISSN: 1471-9053 | DOI: http://dx.doi.org/10.1093/oxfordjournals.pcp.a029339
An Increase in Apparent Affinity for Sucrose of Mung Bean Sucrose Synthase Is Caused by In Vitro Phosphorylation or Directed Mutagenesis of Ser11

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  • Molecular and Cell Biology
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A mutational analysis of mung bean (Vigna radiata Wilczek) sucrose synthase was performed by site-directed mutagenesis of the recombinant protein expressed in Esche-richia 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 Ca2+-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 Km (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.

Keywords: Directed mutagenesis; Protein phosphorylation; Sucrose synthase; Vigna radiata Wilczek

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Subjects: Biochemistry ; Molecular and Cell Biology ; Plant Sciences and Forestry

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