ADP-dependent phosphofructokinases from the archaeal order Methanosarcinales display redundant glucokinase activity

Zamora, Ricardo A.; Gonzalez-Ordenes, F.; Castro‐Fernández, Víctor; Guixé, Victoria

doi:10.1016/j.abb.2017.09.008

Cited by 6 publications

(9 citation statements)

References 22 publications

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“…To specifically assess the relationship between the catalytic process and structural flexibility, we focused on three conserved motifs involved in the catalytic process described for the ADPdependent sugar kinase family: 27 the GXGD motif, which contains an aspartic acid residue that acts as the catalytic base; the DXXE motif, involved in coordinating the metal cation related to the regulation of the enzyme activity; and the NXNXD motif, involved in F6P and GLC binding during the formation of the ternary complex.…”

Section: Resultsmentioning

confidence: 99%

“…This enzyme belongs to the ribokinase superfamily and has the same catalytic residues as other thermostable bifunctional homologues of the ADPdependent sugar kinases family. 27,28 Then, we assumed that the catalytic adaptability of this enzyme at low temperatures should be linked to structural features other than the active site residues. Through kinetic, biophysical, phylogenetic, and computational methodologies, we traced the evolutionary trajectory of the structural determinants responsible for the differential structural flexibility of the psychrophilic MbPFK/GK compared with its mesophilic homologue, the ADP-dependent PFK/GK from M. maripaludis (MmPFK/GK).…”

Section: Introductionmentioning

confidence: 99%

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Tuning of Conformational Dynamics Through Evolution-Based Design Modulates the Catalytic Adaptability of an Extremophilic Kinase

et al. 2020

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Conformational flexibility plays a critical role in enzyme function and is a key aspect in transitions from an open to a closed state induced by substrate binding and product release. Psychrophilic enzymes display a high catalytic efficiency at low temperatures through the improved flexibility of some regions involved in the catalytic cycle. This flexibility enables an optimal conformational dynamic for the catalytic process, whose conservation in homologous enzymes that perform the same biological function has been highlighted. In this work, we demonstrated that two homologous enzymes adapted to function in niches with different temperatures exhibited different conformational dynamics. The psychrophilic bifunctional ADP-dependent PFK/GK from Methanococcoides burtonii (MbPFK/GK) shows a domain closing/opening dynamic described as a breathing-type, while its mesophilic homologue from Methanococcus maripaludis (MmPFK/GK) shows a twist-type domain closing/opening dynamic. In the psychrophilic MbPFK/GK, these conformational movements are associated with increased structural flexibility of the active site, reflected in the exponential increase of the K m values with increasing temperature, and a greater H/D exchange of regions flanking the active site. Through sequence alignment between extant and ancestral enzymes, we identified two ion pairs outside the active site that were highly conserved in the mesophilic MmPFK/GK branch of the ADP-dependent sugar kinases family but were absent in the psychrophilic MbPFK/GK branch. Incorporation of these two ionic pairs in the psychrophilic MbPFK/GK modified the conformational dynamics of the domain closing/opening transition, the K m dependence on temperature, and the H/D exchange, making them similar to those of its mesophilic homologue. We propose that conformational dynamics are responsible for the catalytic adaptability of this enzyme at low temperatures.

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Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Tuning of Conformational Dynamics Through Evolution-Based Design Modulates the Catalytic Adaptability of an Extremophilic Kinase

et al. 2020

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“…In order to characterize the distribution of the ADP-PFK/GK enzymes present in the order Methanosarcinales , we retrieved the deposited sequences from the non-redundant database and updated a previous phylogenetic tree reported for the same protein family ( Zamora et al, 2017 ). As shown in Figure 1 , ADP-PFK/GKs are present in all organisms from Methanosarcinales sequenced to date (13 in total), and encompasses species inhabiting environments ranging from 0 to 5 M NaCl (Supplementary Table S1 ).…”

Section: Resultsmentioning

confidence: 99%

“…ADP-dependent phosphofructokinase (PFK) from Methano halobium evestigatum (MevePFK/GK) was purified as described previously ( Zamora et al, 2017 ). PFK from Methanosarcina mazei (MmazPFK/GK) was purified as follows: Escherichia coli C41 cells were transformed with the plasmid pET-15b harboring the de novo and codon-optimized gene for the MmazPFK/GK protein sequence (UNIPROT code: Q8PZL9), including a poly-histidine-tag at the N-terminus and ampicillin resistance.…”

Section: Methodsmentioning

confidence: 99%

“…Some of the superfamily members are substrate specific for glucose or fructose-6P (GK-ADP or PFK-ADP) while others are bifunctional enzymes (PFK/GK-ADP). Recently, it was reported that the enzymes from Methasanosarcinales, which have been annotated to be ADP-specific, are in fact bifunctional and therefore able to catalyze the phosphorylation of both substrates ( Zamora et al, 2017 ). By combined bioinformatics and experimental approaches along with ancestral enzyme reconstruction, we determined the structural properties that determine the halophilic character in these proteins and the evolutionarily trajectory of this trait.…”

Section: Introductionmentioning

confidence: 99%

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ADP-Dependent Kinases From the Archaeal Order Methanosarcinales Adapt to Salt by a Non-canonical Evolutionarily Conserved Strategy

et al. 2018

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Halophilic organisms inhabit hypersaline environments where the extreme ionic conditions and osmotic pressure have driven the evolution of molecular adaptation mechanisms. Understanding such mechanisms is limited by the common difficulties encountered in cultivating such organisms. Within the Euryarchaeota, for example, only the Halobacteria and the order Methanosarcinales include readily cultivable halophilic species. Furthermore, only the former have been extensively studied in terms of their component proteins. Here, in order to redress this imbalance, we investigate the halophilic adaptation of glycolytic enzymes from the ADP-dependent phosphofructokinase/glucokinase family (ADP-PFK/GK) derived from organisms of the order Methanosarcinales. Structural analysis of proteins from non-halophilic and halophilic Methanosarcinales shows an almost identical composition and distribution of amino acids on both the surface and within the core. However, these differ from those observed in Halobacteria or Eukarya. Proteins from Methanosarcinales display a remarkable increase in surface lysine content and have no reduction to the hydrophobic core, contrary to the features ubiquitously observed in Halobacteria and which are thought to be the main features responsible for their halophilic properties. Biochemical characterization of recombinant ADP-PFK/GK from M. evestigatum (halophilic) and M. mazei (non-halophilic) shows the activity of both these extant enzymes to be only moderately inhibited by salt. Nonetheless, its activity over time is notoriously stabilized by salt. Furthermore, glycine betaine has a protective effect against KCl inhibition and enhances the thermal stability of both enzymes. The resurrection of the last common ancestor of ADP-PFK/GK from Methanosarcinales shows that the ancestral enzyme displays an extremely high salt tolerance and thermal stability. Structure determination of the ancestral protein reveals unique traits such as an increase in the Lys and Glu content at the protein surface and yet no reduction to the volume of the hydrophobic core. Our results suggest that the halophilic character is an ancient trait in the evolution of this protein family and that proteins from Methanosarcinales have adapted to highly saline environments by a non-canonical strategy, different from that currently proposed for Halobacteria. These results open up new avenues for the search and development of novel salt tolerant biocatalysts.

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Kinetic characterization and phylogenetic analysis of human ADP-dependent glucokinase reveal new insights into its regulatory properties

Herrera-Morandé¹,

Vallejos-Baccelliere²,

Pablo³

et al. 2023

Archives of Biochemistry and Biophysics

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ADP-dependent phosphofructokinases from the archaeal order Methanosarcinales display redundant glucokinase activity

Cited by 6 publications

References 22 publications

Tuning of Conformational Dynamics Through Evolution-Based Design Modulates the Catalytic Adaptability of an Extremophilic Kinase

Tuning of Conformational Dynamics Through Evolution-Based Design Modulates the Catalytic Adaptability of an Extremophilic Kinase

ADP-Dependent Kinases From the Archaeal Order Methanosarcinales Adapt to Salt by a Non-canonical Evolutionarily Conserved Strategy

Kinetic characterization and phylogenetic analysis of human ADP-dependent glucokinase reveal new insights into its regulatory properties

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