Anaerobic glycerol-3-phosphate dehydrogenase complex from hyperthermophilic archaeon Thermococcus kodakarensis KOD1

Koga, Yuichi; Konishi, Kanako; Kobayashi, Atsushi; Kanaya, Shigenori; Takano, Kazufumi

doi:10.1016/j.jbiosc.2018.11.012

Cited by 11 publications

(14 citation statements)

References 24 publications

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“…There have been a range of structural analyses of glycerol-3-phosphate dehydrogenases from different organisms that provide important information into the overall structure of the enzyme ,, (including characterizing the flexible loop 292-LNGQKL-297), as well as biochemical and kinetic studies of the catalytic activity of different GPDHs, ,,, and the activation of GPDH by phosphite dianions. , In addition, the examination of primary deuterium kinetic isotope effects (1° DKIEs) of the hydride transfer catalyzed by wild-type and substituted GPDHs show that these values fall within a very narrow range (2.4–3.1) for reactions spanning a 9.1 kcal·mol –1 change in the reaction driving force (ΔΔ G ⧧ ). , This strongly suggests that the rate acceleration for the GPDH-catalyzed reaction is because of the stabilization of the classical TS for hydride transfer, rather than having a large contribution from quantum mechanical tunneling, allowing us to model these hydride transfer reactions using classical EVB simulations.…”

Section: Resultsmentioning

confidence: 99%

Modeling the Role of a Flexible Loop and Active Site Side Chains in Hydride Transfer Catalyzed by Glycerol-3-phosphate Dehydrogenase

et al. 2020

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Glycerol-3-phosphate dehydrogenase is a biomedically important enzyme that plays a crucial role in lipid biosynthesis. It is activated by a ligand-gated conformational change that is necessary for the enzyme to reach a catalytically competent conformation capable of efficient transition-state stabilization. While the human form ( hl GPDH) has been the subject of extensive structural and biochemical studies, corresponding computational studies to support and extend experimental observations have been lacking. We perform here detailed empirical valence bond and Hamiltonian replica exchange molecular dynamics simulations of wild-type hl GPDH and its variants, as well as providing a crystal structure of the binary hl GPDH·NAD R269A variant where the enzyme is present in the open conformation. We estimated the activation free energies for the hydride transfer reaction in wild-type and substituted hl GPDH and investigated the effect of mutations on catalysis from a detailed structural study. In particular, the K120A and R269A variants increase both the volume and solvent exposure of the active site, with concomitant loss of catalytic activity. In addition, the R269 side chain interacts with both the Q295 side chain on the catalytic loop, and the substrate phosphodianion. Our structural data and simulations illustrate the critical role of this side chain in facilitating the closure of hl GPDH into a catalytically competent conformation, through modulating the flexibility of a key catalytic loop (292-LNGQKL-297). This, in turn, rationalizes a tremendous 41,000 fold decrease experimentally in the turnover number, k cat , upon truncating this residue, as loop closure is essential for both correct positioning of key catalytic residues in the active site, as well as sequestering the active site from the solvent. Taken together, our data highlight the importance of this ligand-gated conformational change in catalysis, a feature that can be exploited both for protein engineering and for the design of allosteric inhibitors targeting this biomedically important enzyme.

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Section: Resultsmentioning

confidence: 99%

Modeling the Role of a Flexible Loop and Active Site Side Chains in Hydride Transfer Catalyzed by Glycerol-3-phosphate Dehydrogenase

et al. 2020

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“…The most reliable gene annotations are the ones based on similarity to already characterised gene products, however, not all biochemically characterised proteins are recorded in protein databases and can be used for annotation transfer. In our study we characterised four proteins annotated to EC 1.1.3.15 with alternative activities, and in all cases after a literature search we found articles describing homologous proteins with the same activities (Knorr et al 2018;Koga et al 2019;Weghoff, Bertsch, and Müller 2015;Guo et al 2018) . Only one article postulated for annotation transfer (Knorr et al 2018) which resulted in a recent re-annotation of the protein in UniProt, whereas the remaining proteins are still not recorded in protein databases as being experimentally tested.…”

Section: Discussionmentioning

confidence: 99%

Experimental investigation of enzyme functional annotations reveals extensive annotation error

Rembeza

Engqvist

2020

Preprint

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Only a small fraction of genes deposited to databases has been experimentally characterised. The majority of proteins have their function assigned automatically, which can result in erroneous annotations. The reliability of current annotations in public databases is largely unknown; experimental attempts to validate the accuracy of existing annotations are lacking. In this study we performed an overview of functional annotations to the BRENDA enzyme database. We first applied a high-throughput experimental platform to verify functional annotations to an enzyme class of S-2-hydroxyacid oxidases (EC 1.1.3.15). We chose 122 representative sequences of the class and screened them for their predicted function. Based on the experimental results, predicted domain architecture and similarity to previously characterised S-2-hydroxyacid oxidases, we inferred that at least 78% of sequences in the enzyme class are misannotated. We experimentally confirmed four alternative activities among the misannotated sequences and showed that misannotation in the enzyme class increased over time. Finally, we performed a computational analysis of annotations to all enzyme classes in BRENDA database, and showed that nearly 18% of all sequences are annotated to an enzyme class while sharing no similarity to experimentally characterised representatives. We showed that even well-studied enzyme classes of industrial relevance are affected by the problem of functional misannotation.

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“…It is therefore vital that a complete coverage of functional data is available for automated annotation [52]. In our study we characterised four proteins annotated to EC 1.1.3.15 with alternative activities, and in all cases after a literature search we found articles describing homologous proteins with the same activities [39,41,53,54]. Only one article proposed an annotation transfer [39] which resulted in a recent re-annotation of the protein in UniProt (P37339 protein from E. coli, L-2-hydroxyglutarate dehydrogenase, EC 1.1.5.13).…”

Section: Plos Computational Biologymentioning

confidence: 99%

“…Incorrect gene annotations that accumulate over time might have serious consequences for exploration of novelty and understanding fundamentals of biological functions [23]. As shown by us, a number of enzymes with important biological functions were misannotated to the EC 1.1.3.15, including ones taking part in amino acid [39,41], glycerol [53], or lactate [54] metabolism. Even more proteins with functions yet to be discovered might be hidden among the PLOS COMPUTATIONAL BIOLOGY misannotated sequences.…”

Section: Plos Computational Biologymentioning

confidence: 99%

Experimental and computational investigation of enzyme functional annotations uncovers misannotation in the EC 1.1.3.15 enzyme class

Rembeza

Engqvist

2021

PLoS Comput Biol

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Only a small fraction of genes deposited to databases have been experimentally characterised. The majority of proteins have their function assigned automatically, which can result in erroneous annotations. The reliability of current annotations in public databases is largely unknown; experimental attempts to validate the accuracy within individual enzyme classes are lacking. In this study we performed an overview of functional annotations to the BRENDA enzyme database. We first applied a high-throughput experimental platform to verify functional annotations to an enzyme class of S-2-hydroxyacid oxidases (EC 1.1.3.15). We chose 122 representative sequences of the class and screened them for their predicted function. Based on the experimental results, predicted domain architecture and similarity to previously characterised S-2-hydroxyacid oxidases, we inferred that at least 78% of sequences in the enzyme class are misannotated. We experimentally confirmed four alternative activities among the misannotated sequences and showed that misannotation in the enzyme class increased over time. Finally, we performed a computational analysis of annotations to all enzyme classes in the BRENDA database, and showed that nearly 18% of all sequences are annotated to an enzyme class while sharing no similarity or domain architecture to experimentally characterised representatives. We showed that even well-studied enzyme classes of industrial relevance are affected by the problem of functional misannotation.

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Anaerobic glycerol-3-phosphate dehydrogenase complex from hyperthermophilic archaeon Thermococcus kodakarensis KOD1

Cited by 11 publications

References 24 publications

Modeling the Role of a Flexible Loop and Active Site Side Chains in Hydride Transfer Catalyzed by Glycerol-3-phosphate Dehydrogenase

Modeling the Role of a Flexible Loop and Active Site Side Chains in Hydride Transfer Catalyzed by Glycerol-3-phosphate Dehydrogenase

Experimental investigation of enzyme functional annotations reveals extensive annotation error

Experimental and computational investigation of enzyme functional annotations uncovers misannotation in the EC 1.1.3.15 enzyme class

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