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See detail3‑Phosphoglycerate Transhydrogenation Instead of Dehydrogenation Alleviates the Redox State Dependency of Yeast de Novo L‑Serine Synthesis
Paczia, Nicole UL; Becker-Kettern, Julia UL; Conrotte, Jean-François UL et al

in Biochemistry (2019)

The enzymatic mechanism of 3-phosphoglycerate to 3-phosphohydroxypyruvate oxidation, which forms the first step of the main conserved de novo serine synthesis pathway, has been revisited recently in ... [more ▼]

The enzymatic mechanism of 3-phosphoglycerate to 3-phosphohydroxypyruvate oxidation, which forms the first step of the main conserved de novo serine synthesis pathway, has been revisited recently in certain microorganisms. While this step is classically considered to be catalyzed by an NAD-dependent dehydrogenase (e.g., PHGDH in mammals), evidence has shown that in Pseudomonas, Escherichia coli, and Saccharomyces cerevisiae, the PHGDH homologues act as transhydrogenases. As such, they use α-ketoglutarate, rather than NAD+, as the final electron acceptor, thereby producing D-2-hydroxyglutarate in addition to 3-phosphohydroxypyruvate during 3-phosphoglycerate oxidation. Here, we provide a detailed biochemical and sequence−structure relationship characterization of the yeast PHGDH homologues, encoded by the paralogous SER3 and SER33 genes, in comparison to the human and other PHGDH enzymes. Using in vitro assays with purified recombinant enzymes as well as in vivo growth phenotyping and metabolome analyses of yeast strains engineered to depend on either Ser3, Ser33, or human PHGDH for serine synthesis, we confirmed that both yeast enzymes act as transhydrogenases, while the human enzyme is a dehydrogenase. In addition, we show that the yeast paralogs differ from the human enzyme in their sensitivity to inhibition by serine as well as hydrated NADH derivatives. Importantly, our in vivo data support the idea that a 3PGA transhydrogenase instead of dehydrogenase activity confers a growth advantage under conditions where the NAD+:NADH ratio is low. The results will help to elucidate why different species evolved different reaction mechanisms to carry out a widely conserved metabolic step in central carbon metabolism. [less ▲]

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See detailAlternative binding modes of proline-rich peptides binding to the GYF domain
Gu, Wei UL; Kofler, M.; Antes, I. et al

in Biochemistry (2005), 44(17), 6404-6415

Recognition of proline-rich sequences plays an important role for the assembly of multiprotein complexes during the course of eukaryotic signal transduction and is mediated by a set of protein folds that ... [more ▼]

Recognition of proline-rich sequences plays an important role for the assembly of multiprotein complexes during the course of eukaryotic signal transduction and is mediated by a set of protein folds that share characteristic features. The GYF (glycine-tyrosine-phenylalanine) domain is known as a member of the superfamily of recognition domains for proline-rich sequences. Recent studies on the complexation of the CD2BP2-GYF domain with CD2 peptides showed that the peptide adopts an extended conformation and forms a polyproline type-II helix involving residues Pro4-Pro7 [Freund et al. (2002) EMBO J. 21, 5985-5995]. R/K/GxxPPGxR/K is the key signature for the peptides that bind to the GYF domain [Kofler et at. (2004) J. Biol. Chem. 279, 28292-28297]. In our combined theoretical and experimental study, we show that the peptides adopt a polyproline 11 helical conformation in the unbound form as well as in the complex. From molecular dynamics simulations, we identify a novel binding mode for the G8W mutant and the wild-type peptide (shifted by one proline in register). In contrast, the conformation of the peptide mutant H9M remains close to the experimentally derived wild-type GYF-peptide complex. Possible functional implications of this altered conformation of the bound ligand are discussed in the light of our experimental and theoretical results. [less ▲]

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