Regulation of pyruvate kinase A activity by HPr in response to glucose in Vibrio vulnificus

Abstract

The bacterial phosphoenolpyruvate:sugar phosphotransferase system (PTS) consists of two general proteins (enzyme I and histidine phosphocarrier protein) and several sugar-specific enzyme IIs (EIIs). In the case of the glucose PTS, EII consists of the cytoplasmic enzyme IIAGlc (EIIAGlc) and the membrane associated enzyme IICBGlc (EIICBGlc). During the uptake of glucose, the phosphoryl group is transferred from phosphoenolpyruvate (PEP) to glucose in order of PEP → enzyme I (EI) → histidine phosphocarrier protein (HPr) → EIIAGlc → EIICBGlc → glucose. In addition to the phosphorylation-coupled transport of sugars, PTS proteins participate in various physiological processes through protein-protein interaction depending on their phosphorylation state. Vibrio vulnificus is an opportunistic human pathogen that causes severe and often fatal infections in susceptible individuals. Although regulatory roles of the PTS have been extensively studied in Escherichia coli, much less is known about the Vibrio vulnificus PTS. To elucidate regulatory roles of the V. vulnificus PTS, ligand fishing was performed using HPr as bait. A HPr-binding protein in V. vulnificus was revealed as an ortholog of E. coli pyruvate kinase A (ePykA), and it was named a vPykA standing for Vibrio pyruvate kinase A. Pyruvate kinase (PK) catalyzes the final step of glycolysis, which is the transfer of a phosphoryl group from PEP to ADP, generating the products ATP and pyruvate for anaerobic and aerobic metabolism. The interaction between HPr and vPykA was strictly dependent on the presence of inorganic phosphate, and only dephosphorylated HPr interacted with vPykA. Experiments involving domain swapping between the PykAs of V. vulnificus and E. coli revealed the requirement for the C-terminal domain of vPykA for a specific interaction with V. vulnificus HPr. Because the binding site for the allosteric effector is located at the C-terminal domain of PKs and the C-terminal domain determines the specificity of the HPr-vPykA interaction, the effect of HPr as an allosteric regulator of vPykA was assessed by a lactate dehydrogenase (LDH)-coupled enzyme assay. Only dephosphorylated HPr decreased the Km of vPykA for PEP by approximately four-fold without affecting Vmax. To elucidate physiological roles of vPykA, the vPykA-deficient mutant (pykA mutant) was constructed. the pykA mutant cells entered the viable but nonculturable (VBNC) state much faster than wild-type cells when 5 x 106cells of each of the two strains were incubated in Luria-Bertani medium containing 2.5% NaCl (LBS) at 4°C. Several studies have provided evidence for the involvement of reactive oxygen species (ROS) in the VBNC state of V. vulnificus by showing that a significant portion of the VBNC population of V. vulnificus can be resuscitated if H2O2 scavenger (catalase or pyruvate) is present in the culture medium. Interestingly, a pykA mutant was more susceptible to H2O2 than wild-type V. vulnificus, and this sensitivity was completely rescued by the addition of pyruvate to the culture medium. Here, it is shown that V. vulnificus dephospho-HPr increases the affinity of vPykA for PEP to confer resistance to H2O2 stress in the presence of a PTS sugar, such as glucose.