A PP2C-type phosphatase dephosphorylates the PII signaling protein in the cyanobacterium Synechocystis PCC 6803

Abstract

The family of the PII signal transduction proteins contains the most highly conserved signaling proteins in nature. The cyanobacterial PII-homologue transmits signals of the cellular nitrogen status and carbon status through phosphorylation of a seryl-residue. To identify the enzyme responsible for dephosphorylation of the phosphorylated PII protein in Synechocystis PCC 6803, prospective phosphatase encoding genes were inactivated by targeted insertion of kanamycin resistance cassettes. Disruption of ORF sll1771 generates a mutant unable to dephosphorylate PII under various experimental conditions. On the basis of conserved signature motifs, the sll1771 product (termed PphA) is a member of the protein phosphatase 2C (PP2C) superfamily, which is characterized by Mg 2؉ ͞Mn 2؉ -dependent catalytic activity. Biochemical analysis of overexpressed and purified PphA confirms its PP2C-type enzymatic properties and demonstrated its reactivity toward the phosphorylated PII protein. Thus, PphA is the first protein phosphatase in Synechocystis PCC 6803 for which the physiological substrate and function is known.

Evolution of the PPM-family protein phosphatases in Streptomyces: duplication of catalytic domain and lateral recruitment of additional sensory domains

“…The functions of the microbial PPMs that have been characterized include regulation of spore formation, stress response, cell density during stationary phase, carbon and nitrogen assimilation, vegetative growth, development of fruiting bodies and cell segregation (Beuf et al, 1994;Duncan et al, 1995;Gaidenko et al, 2002;Irmler & Forchhammer, 2001;Rajagopal et al, 2003;Shi et al, 1999;Treuner-Lange et al, 2001;Yang et al, 1996). It is notable that most bacterial species containing relatively large numbers of PPMencoding genes, such as Anabaena sp.…”

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

“…The cells were lysed by sonic disruption with four bursts of 20 S each at 50 watts of a Heat Systems-Ultrasonics Inc, Model W185 Sonifier Cell Disruptor equipped with a microprobe. The resulting lysate was centrifuged for 20 min at 10,000g and 4 C. The supernatant was filtered through a 0.45 mm filter to remove any particulate materials and applied to a metal ion affinity column for purification of the recombinant protein following established protocols (26). In brief, a 1 ml bed volume of column was prepared using Chelating Sepharose Fast Flow (Pharmacia Biotech, Uppsala, Sweden).…”

“…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 tyrosinephosphorylated proteins have been detected yet.…”

From cyanobacteria to plants: conservation of PII functions during plastid evolution

“…With S. elongatus cell-free extracts, phosphorylation could be achieved in the presence of millimolar concentrations of 2-OG and ATP. The PII kinase is still unknown; however, the phosphatase of PII-P, PphA, could be identified in Synechocystis PCC 6803 (Irmler and Forchhammer 2001) and has since then been thoroughly studied (Ruppert et al 2002;Kloft and Forchhammer 2005;Schlicker et al 2008;Forchhammer 2011, 2012). PphA is a Ser/Thr phosphatase of the PP2C family and the crystal structure of the PphA homologue from the thermophilic cyanobacterium Thermosynechococcus has been solved (Schlicker et al 2008).…”