Sucrose biosynthesis in a prokaryotic organism: presence of two sucrose-phosphate synthases in Anabaena with remarkable differences compared with the plant … (original) (raw)
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Proceedings of the National Academy of Sciences, 1996
Biosynthesis of sucrose-6-P catalyzed by sucrose-phosphate synthase (SPS), and the presence of sucrosephosphate phosphatase (SPP) leading to the formation of sucrose, have both been ascertained in a prokaryotic organism: Anabaena 7119, a filamentous heterocystic cyanobacterium. Two SPS activities (SPS-I and SPS-II) were isolated by ion-exchange chromatography and partially purified. Four remarkable differences between SPSs from Anabaena and those from higher plants were shown: substrate specificity, effect of divalent cations, native molecular mass, and oligomeric composition. Both SPS-I and SPS-II accept Fru-6-P (K m for SPS-I ؍ 0.8 ؎ 0.1 mM; K m for SPS-II ؍ 0.7 ؎ 0.1 mM) and UDP-Glc as substrates (K m for SPS-I ؍ 1.3 ؎ 0.4 mM; K m for SPS-II ؍ 4.6 ؎ 0.4 mM), but unlike higher plant enzymes, they are not specific for UDP-Glc. GDP-Glc and TDP-Glc are also SPS-I substrates (K m for GDP-Glc ؍ 1.2 ؎ 0.2 mM and K m for TDP-Glc ؍ 4.0 ؎ 0.4 mM), and ADP-Glc is used by SPS-II (K m for ADP-Glc ؍ 5.7 ؎ 0.7 mM). SPS-I has an absolute dependence toward divalent metal ions (Mg 2؉ or Mn 2؉) for catalytic activity, not found in plants. A strikingly smaller native molecular mass (between 45 and 47 kDa) was determined by gel filtration for both SPSs, which, when submitted to SDS͞PAGE, showed a monomeric composition. Cyanobacteria are, as far as the authors know, the most primitive organisms that are able to biosynthesize sucrose as higher plants do.
First evidence of sucrose biosynthesis by single cyanobacterial bimodular proteins
FEBS Letters, 2013
The net synthesis of sucrose (Suc) is catalysed by the sequential action of Suc-phosphate synthase (SPS) and Suc-phosphate phosphatase (SPP). SPS and SPP from Anabaena sp. PCC 7120 (7120-SPS and 7120-SPP) define minimal catalytic units. Bidomainal SPSs, where both units are fused, occur in plants and cyanobacteria, but they display only SPS activity. Using recombinant proteins that have fused 7120-SPS and 7120-SPP, we demonstrated that they are bifunctional chimeras and that the arrangement 7120-SPS/SPP is the most efficient to catalyse the sequential reactions to yield Suc. Moreover, we present the first evidence of a bidomainal SPS present in the cyanobacterium Synechococcus elongatus PCC 7942 with both, SPS and SPP activity.
Planta, 1999
The pathway of sucrose metabolism in cyanobacteria is just starting to be elucidated. The present study describes the ®rst 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 puri®ed 457-fold and its amino-terminal portion sequenced. Substrate speci®city, kinetic constants, native protein and subunit molecular masses, and the eect of dierent 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 dierences from plant enzymes were demonstrated with respect to substrate anities, regulation by metal ions and ATP, and the amino-acid sequence of the N-terminal region.
Plant molecular biology, 1999
Sucrose is one of several low-molecular-weight compounds that cyanobacteria accumulate in response to osmotic stress and which are believed to act as osmoprotectants. The genome of the cyanobacterium Synechocystis sp. PCC 6803 contains a 2163 bp open reading frame (ORF) that shows similarity to genes from higher plants encoding sucrose-phosphate synthase (SPS), the enzyme responsible for sucrose synthesis. The deduced amino acid sequence shows 35-39% identity with known higher-plant SPS sequences. The putative Synechocystis sps gene was cloned from genomic DNA by PCR amplification and expressed as a His6-tagged amino-terminal fusion protein in Escherichia coli. The expressed protein was purified and shown to be a functional SPS enzyme, confirming the identity of the ORF, which is the first sps gene to be cloned from a prokaryotic organism. The Synechocystis SPS has a molecular mass of 81.5 kDa, which is smaller than the typical higher-plant SPS subunit (117-119 kDa), and lacks the p...
FEBS Letters, 2002
Based on the functional characterization of sucrose biosynthesis related proteins [SBP: sucrose-phosphate synthase (SPS), sucrose-phosphate phosphatase (SPP), and sucrose synthase (SuS)] in Anabaena sp. PCC7120 and sequence analysis, we have shown that SBP are restricted to cyanobacterium species and plants, and that they are multidomain proteins with modular architecture. Anabaena SPS, a minimal catalytic SPS unit, defines a glucosyltransferase domain present in all SPSs and SuSs. Similarly, Anabaena SPP defines a phosphohydrolase domain characteristic of all SPPs and some SPSs. Phylogenetic analysis points towards the evolution of modern cyanobacterial and plant SBP from a bidomainal common ancestral SPS-like gene.
Planta, 2001
The present study describes the ®rst isolation and characterization of a prokaryotic protein and gene for sucrose-phosphate phosphatase (SPP), the enzyme that catalyzes the terminal step in sucrose synthesis. For gene isolation, a 2,015-bp DNA fragment containing an open reading frame with about 31% amino acid identity to Synechocystis SPS was ampli®ed from Anabaena sp. PCC 7120 DNA. Surprisingly, expression of the putative gene in Escherichia coli demonstrated that it encoded an SPP protein. The expressed protein crossreacted with antibodies against the native form of Anabaena SPP and its biochemical properties were identical to those of the enzyme puri®ed from the cyanobacterial cells. Comparisons of the Anabaena SPP with the higher-plant enzyme revealed important dierences in the C-terminal region, molecular mass, subunit composition and immunoreactivity. Nevertheless, two conserved motifs, including four invariant aspartate residues similar to those found in members of the phosphohydrolase superfamily, were identi®ed in the Anabaena SPP deduced amino acid sequence.