Correction: New Insights on the Mechanism of the K+-Independent Activity of Crenarchaeota Pyruvate Kinases

Vega-Ruíz, Gustavo De la; Domínguez-Ramírez, Lenin; Riveros‐Rosas, Héctor; Guerrero-Mendiola, Carlos; Torres‐Larios, Alfredo; Hernández-Alcántara, Gloria; García-Trejo, José J.; Ramírez‐Silva, Leticia

doi:10.1371/journal.pone.0129757

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“…Vc IPK exhibited lower activities in the presence of Mn 2+ ( Fig 5A, 5C and 5E ) compared to those in the presence of Mg 2+ ( Fig 4A, 4C and 4E ) either with or without Fru 1,6-BP. This result is in agreement with other PKs [ 15 , 46 , 54 , 55 ]. Although the maximum activity for Vc IPK was 1.4-fold higher with Mg 2+ than with Mn 2+ , the K 0 .…”

Section: Resultssupporting

confidence: 93%

“…In the absence of K + , RMPK changes its kinetic mechanism to ordered rapid equilibrium with PEP being the first substrate [18]. Similar studies have been conducted in the K + -dependent PK of the muscle of the sea mollusk Concholepas concholepas [61], and in the K + -independent PK of the crenarchaeota Thermophilum pendens [15]. It was found that both enzymes follow a rapid equilibrium random order kinetic mechanism.…”

Section: Resultsmentioning

confidence: 88%

“…The genes were amplified by PCR using the following primers for Vc IPK: FW and RV and for Vc IIPK: FW and RV . Each insert was introduced into the pMCSG7 vector as described in [ 15 ]. The plasmids were isolated and sequenced to verify the absence of mutations.…”

Section: Methodsmentioning

confidence: 99%

“…coli BL21-CodonPlus (DE3)-RIL competent cells. Both proteins were then overexpressed in four liters of Luria Bertani (LB) medium then growth, induced, harvested and lysed as described previously [ 15 ]. The suspensions were centrifuged at 13,250 × g , the supernatants loaded on a His Trap FF column (GE Healthcare), and the enzymes eluted with a linear gradient of imidazole (10–500 mM).…”

Section: Methodsmentioning

confidence: 99%

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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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Section: Resultssupporting

confidence: 93%

Section: Resultsmentioning

confidence: 88%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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