Recombinant production of Yersinia enterocolitica pyruvate kinase isoenzymes PykA and PykF

Hofmann, Julia; Heider, Christine; Li, Wei; Krausze, J.; Roessle, Manfred; Wilharm, Gottfried

doi:10.1016/j.pep.2013.01.010

Cited by 8 publications

(5 citation statements)

References 25 publications

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“…Many intracellular bacteria, such as Listeria monocytogenes, Shigella, and Mycobacterium tuberculosis, can adapt to the harsh living environment by regulating their own metabolism. In addition, Brucella strains lacking the pyruvate kinase gene pykF were found to have significantly reduced viability in host macrophages [53][54][55]. Meanwhile, the pykF-centric network diagram in the present study demonstrated that ndk was a manipulation of bacterial virulence and adaptive responses of the pleiotropic effector.…”

Section: Discussionmentioning

confidence: 51%

Mechanisms underlying the virulence regulation of new Vibrio alginolyticus ncRNA Vvrr1 with a comparative proteomic analysis

Zuo

Zhao

et al. 2019

Emerging Microbes & Infections

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The incidence of Vibrio alginolyticus infections has increased in recent years due to the influence of climate change and rising sea temperature. Vibrio virulence regulatory RNA 1 (Vvrr1) is a newly found noncoding RNA (ncRNA) predicted to be closely related to the adhesion ability of V. alginolyticus based on the previous RNA-seq. In this study, the target genes of Vvrr1 were fully screened and verified by constructing Vvrr1-overexpressing strains and using the proteome sequencing technology. Pyruvate kinase I (pykF) gene was predicted to be a chief target gene of Vvrr1 involved in virulence regulation. The adhesion ability, biofilm formation and virulence were significantly reduced in the Vvrr1-overexpressing and the pykFsilenced strain compared with the wild strains. Similar to the overexpression of Vvrr1, the silencing of pykF also reduced the expression level of virulence genes, such as ndk, eno, sdhB, glpF, and cysH. Meanwhile, by constructing the "pykF-GFP" fusion expression plasmid and using the GFP reporter gene analysis in Escherichia coli, the fluorescence intensity of the strain containing Vvrr1 whole ncRNA sequence vector was found to be significantly weakened. These indicated that Vvrr1 participated in the virulence regulation mechanism of V. alginolyticus by interacting with the virulence gene pykF.

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Section: Discussionmentioning

confidence: 51%

Mechanisms underlying the virulence regulation of new Vibrio alginolyticus ncRNA Vvrr1 with a comparative proteomic analysis

Zuo

Zhao

et al. 2019

Emerging Microbes & Infections

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“…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 .…”

Section: Resultsmentioning

confidence: 99%

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

et al. 2019

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Pyruvate kinases (PKs) synthesize ATP as the final step of glycolysis in the three domains of life. PKs from most bacteria and eukarya are allosteric enzymes that are activated by sugar phosphates; for example, the feed‐forward regulator fructose‐1,6‐bisphosphate, or AMP as a sensor of energy charge. Archaea utilize unusual glycolytic pathways, but the allosteric properties of PKs from these species are largely unknown. Here, we present an analysis of 24 PKs from most archaeal clades with respect to allosteric properties, together with phylogenetic analyses constructed using a novel mode of rooting protein trees. We find that PKs from many Thermoproteales, an order of crenarchaeota, are allosterically activated by 3‐phosphoglycerate (3PG). We also identify five conserved amino acids that form the binding pocket for 3PG. 3PG is generated via an irreversible reaction in the modified glycolytic pathway of these archaea and therefore functions as a feed‐forward regulator. We also show that PKs from hyperthermophilic Methanococcales, an order of euryarchaeota, are activated by AMP. Phylogenetic analyses indicate that 3PG‐activated PKs form an evolutionary lineage that is distinct from that of sugar‐phosphate activated PKs, and that sugar phosphate‐activated PKs originated as AMP‐regulated PKs in hyperthermophilic Methanococcales. Since the phospho group of sugar phosphates and 3PG overlap in the allosteric site, our data indicate that the allostery in PKs first started from a progenitor phosphate‐binding site that evolved in two spatially distinct directions: one direction generated the canonical site that responds to sugar phosphates and the other gave rise to the 3PG site present in Thermoproteales. Overall, our data suggest an intimate connection between the allosteric properties and evolution of PKs.

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“…Cluster I is mainly comprised of PKs modulated by bi-phosphorylated sugars such as fructose 1,6 bisphosphate (Fru 1,6-BP) [6, 7] or fructose 2,6 bisphosphate (Fru 2,6-BP) [8,9] whereas cluster II includes enzymes largely regulated either by AMP or monophosphate sugars [10–12]. Members of the γ-proteobacteria group such as Escherichia coli [10], Salmonella typhimurium [13] and Yersinia enterocolitica [14] also have both type I and type II PKs which are located, respectively in Cluster I and Cluster II of the phylogenetic tree. These enterobacteriaceae type I PKs are modulated by Fru 1,6-BP, and the type II are regulated principally by AMP.…”

Section: Introductionmentioning

confidence: 99%

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

Guerrero-Mendiola¹,

García-Trejo²,

Encalada³

et al. 2017

PLoS ONE

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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.

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Recombinant production of Yersinia enterocolitica pyruvate kinase isoenzymes PykA and PykF

Cited by 8 publications

References 25 publications

Mechanisms underlying the virulence regulation of new Vibrio alginolyticus ncRNA Vvrr1 with a comparative proteomic analysis

Mechanisms underlying the virulence regulation of new Vibrio alginolyticus ncRNA Vvrr1 with a comparative proteomic analysis

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

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

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