Identification of a Low Molecular Weight Protein Tyrosine Phosphatase and Its Potential Physiological Substrates in Synechocystis sp. PCC 6803

• Main Author: Mukhopadhyay, Archana
• Other Authors: Biochemistry
• Format: Others
• Published: Virginia Tech 2014
• Subjects: Protein tyrosine phosphorylation; Phycocyanin; Protein tyrosine kinases; Low molecular weight protein tyrosine phosphatases
• Online Access: http://hdl.handle.net/10919/26447; http://scholar.lib.vt.edu/theses/available/etd-03172006-054826/

The predicted protein product of open reading frame slr0328 from Synechocystis sp. PCC 6803, SynPTP, possesses significant amino acid sequence similarity with known low molecular weight protein tyrosine phosphatases (PTPs). To determine the gross functional properties of this hypothetical protein, open reading frame slr0328 was cloned, and its predicted protein product was expressed in E. coli. The recombinant protein, SynPTP, was purified by metal ion column chromatography. The catalytic activity of SynPTP was examined toward several exogenous protein substrates that had been phosphorylated on either tyrosine residues or serine residues. SynPTP exhibited phosphatase activity toward tyrosine phosphorylated protein substrates (Vmax toward phosphotyrosyl 32P-casein was 1.5 nmol/min/mg). However, no phosphatase activity was detected toward serine phosphorylated protein substrates. SynPTP displayed phosphohydrolase activity toward several organophosphoesters including para-nitrophenyl phosphate (p-NPP), beta-napthyl phosphate and phosphotyrosine but not toward alpha-napthyl phosphate, phosphoserine, or phosphothreonine. Kinetic analysis indicated that the Km (0.6 mM) and Vmax (3.2 mmole/min/mg) values for SynPTP toward pNPP are similar to those of other known bacterial low molecular weight PTPs. The protein phosphatase activity of SynPTP was inhibited by sodium orthovanadate, a known inhibitor for tyrosine phosphatases, but not by okadaic acid, an inhibitor for many serine/threonine phosphatases. Mutagenic alteration of the predicted catalytic cysteine, Cys7, to serine abolished enzyme activity. Several phosphotyrosine containing proteins were detected from the whole cell extracts of Synechocystis sp. PCC 6803 through immunoreactions using anti-phosphotyrosine antibody. SynPTP was observed to dephosphorylate three of these proteins in vitro. Two of these proteins were identified by peptide-mass fingerprinting analysis, as PsaD (photosystem I subunit II) and CpcD (phycocyanin rod linker protein). In addition, several phosphotyrosine proteins were detected from the soluble and membrane fractions of Synechocystis sp. PCC 6803 cell extracts by in vitro substrate trapping as potential endogenous substrates of SynPTP. Two of these proteins were identified as the alpha and beta subunits of phycocyanin. We therefore speculate that SynPTP might be involved in the regulation of photosynthesis in Synechocystis sp. PCC 6803. === Ph. D.