Sucrose-phosphate synthase from Synechocystis sp. strain PCC 6803: identification of the spsA gene and characterization of the enzyme expressed in Escherichia … (original) (raw)
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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...
Journal of Experimental Botany, 2003
Sucrose-phosphate synthase (SPS) from the cyanobacterium Synechocystis sp. PCC 6803 lacks all of the Ser residues known to be involved in the regulation of higher plant SPS by protein phosphorylation. The Synechocystis SPS is also not allosterically regulated by glucose 6-phosphate or orthophosphate. To investigate the effects of expressing a potentially unregulated SPS in plants, the Synechocystis sps gene was introduced into tobacco, rice and tomato under the control of constitutive promoters. The Synechocystis SPS protein was expressed at a high level in the plants, which should have been suf®cient to increase overall SPS activity 2±8-fold in the leaves. However, SPS activities and carbon partitioning in leaves from transgenic and wild-type plants were not signi®cantly different. The maximal light-saturated rates of photosynthesis in leaves from tomato plants expressing the Synechocystis SPS were the same as those from wild-type plants. Tomato plants expressing the maize SPS showed 2±3-fold increases in SPS activity, increased partitioning of photoassimilate to sucrose and up to 58% higher maximal rates of photosynthesis. To investigate the apparent inactivity of the Synechocystis SPS the enzyme was puri®ed from transgenic tobacco and rice plants. Surprisingly, the puri®ed enzyme was found to have full catalytic activity. It is proposed that some other protein in plant cells binds to the Synechocystis SPS resulting in inhibition of the enzyme.
Identification of the spsA Gene and Characterization of the Enzyme Expressed in Escherichia coli
1998
The first identification and characterization of a prokaryotic gene (spsA) encoding sucrose-phosphate synthase (SPS) is reported for Synechocystis sp. strain PCC 6803, a unicellular non-nitrogen-fixing cyanobacterium. Comparisons of the deduced amino acid sequence and some relevant biochemical properties of the enzyme with those of plant SPSs revealed important differences in the N-terminal and UDP-glucose binding site regions, substrate specificities, molecular masses, subunit compositions, and regulatory properties.
FEBS Letters, 2010
It has been reported that higher plants and cyanobacteria synthesize sucrose (Suc) by a similar sequential action of sucrose-phosphate synthase (SPS) and sucrose-phosphate phosphatase (SPP). In the genome of the marine unicellular cyanobacterium Synechococcus sp. PCC 7002 there is a sequence that was not annotated as a putative SPP encoding gene (sppA), although the sequence was available. In this study, we functionally characterize the sppA gene of that strain and demonstrate that it is cotranscribed with spsA, the SPS encoding gene. This is the first report on the coordination of Suc synthesis gene expression in an oxygenic-photosynthetic organism.
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.
Proceedings of the National …, 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.
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.