Sucrose metabolism in cyanobacteria: sucrose synthase from Anabaena sp. strain PCC 7119 is remarkably different from the plant enzymes with respect to substrate affinity and amino-terminal sequence

Abstract

The pathway of sucrose metabolism in cyanobacteria is just starting to be elucidated. The present study describes the first isolation and biochemical characterization of a prokaryotic sucrose synthase (SS, EC 2.4.1.13). Two SS forms (SS-I and SS-II) were detected in Anabaena sp. strain PCC 7119. The isoform SS-II was purified 457-fold and its amino-terminal portion sequenced. Substrate specificity, kinetic constants, native protein and subunit molecular masses, and the effect of different ions and metabolites were studied for SS-II. Anabaena SS was shown to be a tetramer with a 92-kDa polypeptide that was recognized by maize SS polyclonal antibodies. Some striking differences from plant enzymes were demonstrated with respect to substrate affinities, regulation by metal ions and ATP, and the amino-acid sequence of the N-terminal region.

Sucrose synthase activity in the sus1/sus2/sus3/sus4 Arabidopsis mutant is sufficient to support normal cellulose and starch production

“…Fourth, we measured SUS activity under optimum pH conditions for the enzyme (Fig. 2), whereas Bieniawska et al (42) and Barratt et al (43) measured SUS activity at pH 9.4, which is far too basic with respect to previously reported SUS activity optimum pH values of 7.0-8.2 (10,(45)(46)(47)(48).…”

“…Furthermore, the SUS reaction assay mixture used in refs. 42 and 43 contained 6 mM fructose, a concentration that is comparable or even lower than the reported K m values for fructose in SUS from many species (10,11,44). Therefore, Bieniawska et al (42) and Barratt et al (43) measured SUS activity under conditions of substrate (fructose and UDPG) deficiency and/or instability.…”

“…Although UDP is generally considered to be the preferred nucleoside diphosphate for SUS, numerous studies have shown that ADP serves as an effective acceptor molecule to produce ADP-glucose (ADPG) (5)(6)(7)(8)(9)(10)(11)(12)(13)(14). SUS is highly regulated both at transcriptional and posttranslational levels (15)(16)(17)(18)(19)(20)(21), and plays a predominant role in the entry of carbon into metabolism in nonphotosynthetic cells, and in determining both sink strength and phloem loading (22)(23)(24)(25).…”

“…Several assay conditions that departed from optimal were: First, the SUS reaction assay mixture used by the authors contained 6 mM fructose, a concentration comparable to or lower than the reported K m values for fructose in SUS from many species (10,11,44). Second, the pH of the SUS reaction assay mixture used by Bieniawska et al (42) and Barratt et al (43) was 9.4, which is far too basic with respect to both cytosolic pH and to previously reported optimum pH values for SUS activity in the synthetic direction (10,(45)(46)(47)(48). Third, SUS assay reactions were stopped after 20 min of incubation, a condition that does not allow the attainment of reliable data under SUS initial velocity conditions.…”

Most of ADP·glucose linked to starch biosynthesis occurs outside the chloroplast in source leaves

“…In this respect, and based on the capacity of cytosolic sucrose synthase (SuSy) (IUBMB accession no. EC 2.4.1.13) to produce ADPG from sucrose and ADP (21)(22)(23)(24)(25), an ''alternative model'' of starch biosynthesis was proposed in which SuSy catalyzes the de novo production of ADPG, which is subsequently imported into the stromal phase of the chloroplast (Fig. 1B) (18,19).…”