The contribution of two isozymes to the pyruvate kinase activity of Vibrio cholerae: One K+-dependent constitutively active and another K+-independent with essential allosteric activation

Abstract

In a previous phylogenetic study of the family of pyruvate kinase EC (2.7.1.40), a cluster with Glu117 and another with Lys117 were found (numbered according to the rabbit muscle enzyme). The sequences with Glu117 have been found to be K+-dependent, whereas those with Lys117 were K+-independent. Interestingly, only γ-proteobacteria exhibit sequences in both branches of the tree. In this context, it was explored whether these phylogenetically distinct pyruvate kinases were both expressed and contribute to the pyruvate kinase activity in Vibrio cholerae. The main findings of this work showed that the isozyme with Glu117 is an active K+-dependent enzyme. At the same substrate concentration, its Vmax in the absence of fructose 1,6 bisphosphate was 80% of that with its effector. This result is in accordance with the non-essential activation described by allosteric ligands for most pyruvate kinases. In contrast, the pyruvate kinase with Lys117 was a K+-independent enzyme displaying an allosteric activation by ribose 5-phosphate. At the same substrate concentration, its activity without the effector was 0.5% of the one obtained in the presence of ribose 5-phosphate, indicating that this sugar monophosphate is a strong activator of this enzyme. This absolute allosteric dependence is a novel feature of pyruvate kinase activity. Interestingly, in the K+-independent enzyme, Mn2+ may “mimic” the allosteric effect of Rib 5-P. Despite their different allosteric behavior, both isozymes display a rapid equilibrium random order kinetic mechanism. The intracellular concentrations of fructose 1,6-bisphosphate and ribose 5-phosphate in Vibrio cholerae have been experimentally verified to be sufficient to induce maximal activation of both enzymes. In addition, Western blot analysis indicated that both enzymes were co-expressed. Therefore, it is concluded that VcIPK and VcIIPK contribute to the activity of pyruvate kinase in this γ-proteobacterium.

New views on an old enzyme: allosteric regulation and evolution of archaeal pyruvate kinases

“…Domain‐specific classification of organisms is indicated by different colors, green for eukarya, blue for bacteria, and red for archaea. UniProt entries and references of PK s from eukarya and bacteria: Saccharomyces cerevisiae , P00549 ; Homo sapiens M2, P14618 ; Trypanosoma brucei , P30615 ; Leishmania mexicana , Q27686 ; Vibrio cholerae I and II , Q9KUN0 and Q9KQJ0 ; Yersinia enterocolitica I and II , A1JPB0 and A1JRG5 ; Salmonella typhimurium I and II , P77983 and Q8ZNW0 ; Escherichia coli I and II , P0AD61 and P21599 ; Lactococcus lactis , Q07637 ; Streptococcus sanguinis , F2CE05 ; Streptococcus mutans , Q8DTX7 ; Lactobacillus bulgaricus , P34038 ; Staphylococcus aureus , Q6GG09 ; Geobacillus stearothermophilus , Q02499 ; Bacillus licheniformis , P51181 ; Mycobacterium tuberculosis , P9WKE5 ; Corynebacterium glutamicum , Q46078 ; Synechococcus PCC6301, A0A0H3K2W0 ; Paracoccus denitrificans , A1B4C4 ; Thermotoga maritima , Q9WY51 . Gene identifiers of archaeal PK s are given in Table .…”

An overview of structure, function, and regulation of pyruvate kinases

“…Cluster I PyKs are regulated by FBP or FDP while AMP or sugar monophosphates modulate the enzymes in cluster II . PyKs belonging to both clusters are expressed in gamma‐proteobacteria, and in apicomplexan parasites in which cluster I and cluster II PyKs are found in cytosolic and apicoplast compartments, respectively . Organisms carrying both families of PyKs include a number of clinically significant pathogens such as Escherichia coli , Vibrio cholera , Salmonella typhimurium , Toxoplasma gondii , and Plasmodium falciparum …”

“…40,41 PyKs belonging to both clusters are expressed in gamma-proteobacteria, 42 and in apicomplexan parasites in which cluster I and cluster II PyKs are found in cytosolic and apicoplast compartments, respectively. 43,44 Organisms carrying both families of PyKs include a number of clinically significant pathogens such as Escherichia coli, 32,40 Vibrio cholera, 42 Salmonella typhimurium, 45 Toxoplasma gondii, 43,46 and Plasmodium falciparum. 44 PyKs of hyperthermophilic archaea Thermoproteales (but not thermophilic bacteria) are activated by 3-phosphoglycerate (3PG) and not by FBP, FDP, or AMP.…”

Exploring the differences between the three pyruvate kinase isozymes from Vibrio cholerae in a heterologous expression system

“…The vcIIIpk gene (1461 bp) was cloned into plasmid pET3a-HisTEVP. vcIpk and vcIIpk genes [ 5 ] were transformed into several BL21(DE3) strains to compare against the transformation of vcIIIpk . DE3 strains have a chromosomal copy of the phage T7 RNA polymerase gene that is compatible with the pET vectors, for which the genes are cloned downstream of the strong T7 promoter being used for recombinant protein expression [ 6 , 8 – 10 ].…”

“…It was also identified by Western blot analyses that both enzymes are present in cell extracts of CVD103 V. cholerae strain. Since they co-express, and their catalytic requirements are present in the bacterium, it was concluded that both enzymes contribute to the activity of pyruvate kinase in V. cholerae [ 5 ]. However, vcIIIpk gene failed to transform in BL21-CodonPlus(DE3)-RIL.…”