A low molecular weight protein tyrosine phosphatase from Synechocystis sp. strain PCC 6803: enzymatic characterization and identification of its potential substrates

Reversible protein phosphorylation is one of the most fundamental regulatory mechanisms in signal transduction and information processing in cells for modulating the functional properties of proteins. This mechanism is evolutionarily conserved in every living cell among all three domains of life. This reversible phosphorylation/dephosphorylation process is catalysed by opposing reactions of protein kinases and protein phosphatases (1, 2). It has been well studied that in eukaryotes protein phosphorylation on tyrosine residues plays a key role in the regulatory mechanism of various cellular processes including growth, differentiation, cell cycle regulation and cytoskeletal function (3, 4), and as well as controls many diseases (5, 6) including infectious diseases (7, 8). In comparison to eukaryotes, the occurrence of protein tyrosine kinases (PTKs)/protein tyrosine phosphatases (PTPs) in bacteria including photosynthetic cyanobacteria, was suggested much later (9–15). The PTPs are characterized by the presence of a common active site signature motif (CX5RS/T), namely phosphate binding loop, and an essential aspartate that serves as a catalytic acid/base (16, 17). The PTPs are classified into three superfamilies: (i) conventional PTPs, where the active site sequence preceding to histidine (HCX5RS/T) is located in the central portion of the catalytic domain and conserved aspartate is present in 25 to 45 residues to the N-terminal side of the catalytic cysteine (18); (ii) low molecular weight (LMW) PTPs, where the active site sequence precedes by either a leucine or valine residue and resides near the extreme N-terminus of the catalytic domain and the conserved aspartate is located 85 to 105 residues to the C-terminal side of catalytic cysteine followed by a proline (19); and (iii) in dual specific phosphatases (DSPs), which can catalyse phosphoesters from both tyrosine and serine/threonine residues, the active site sequence closely resembles to that of conventional PTPs, D-X45-CX5RS/T (16–19). In photosynthetic cyanobacterium Synechocystis sp. strain PCC 6803, genome analysis (20–22) revealed that this organism contains three ORFs, which encode LMW protein tyrosine PTPs (23). One is slr0946, whose predicted protein product was characterized as arsenate reductase (24). The functional properties of the other two ORFs, slr0328 and slr1617, have not been reported yet. In Synechocystis sp. PCC 6803, a number of serine/threonine kinases (25, 26), protein serine/threonine phosphatases (PP2A) (27, 28), and serine/threonine phosphoproteins (29–32) are found. However, no specific PTKs, not any member of conventional or LMW PTPs, or any tyrosine-phosphorylated proteins have been detected yet. In this study, we have characterized one open reading frame, slr0328, which encodes a hypothetical LMW PTP in Synechocystis sp. PCC 6803. We have described here that Slr0328, named as SynPTP functions as a PTP, not as a DSP. To understand the biological function of this phosphatase, we have identified several tyrosyl-phosphorylated proteins, which include PsaD, photosystem I subunit II, and CpcD, phycocyanin rod linker protein from whole cell extracts and phycocyanin alpha and beta subunits from soluble fractions of this organism as potential substrates of SynPTP. Results suggest that this tyrosine phosphatase in Synechocystis sp. may be involved in the regulation of photosynthesis.