Mapping Functionally Important Residues in the Na+/Dicarboxylate Cotransporter, NaDC1

August 10th, 2017 by Claire Colas, Avner Schlessinger and Ana M. Pajor

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Biochemistry
DOI: 10.1021/acs.biochem.7b00503

Efficient reduction of CO2 by the molybdenum-containing formate dehydrogenase from Cupriavidus necator (Ralstonia eutropha). [Molecular Biophysics]

August 7th, 2017 by Xuejun Yu, Dimitri Niks, Ashok Mulchandani, Russ Hille

The ability of the FdsABG formate dehydrogenase from Cupriavidus necator (formerly known as Ralstonia eutropha) to catalyze the reverse of the physiological reaction, the reduction of CO2 to formate utilizing NADH as electron donor, has been investigated. Contrary to previous studies of this enzyme, we demonstrate that it is in fact effective in catalyzing the reverse reaction, with a kcat of 11 ± 0.4 s-1. We also quantify the stoichiometric accumulation of formic acid as the product of the reaction and demonstrate that the observed kinetic parameters for catalysis in the forward and reverse reaction are thermodynamically consistent, complying with the expected Haldane relationships. Finally, we demonstrate the reaction conditions necessary for gauging the ability of a given formate dehydrogenase or other CO2-utilizing enzyme to catalyze the reverse direction so as to avoid false negative results. In conjunction with our earlier studies on the reaction mechanism of this enzyme (Niks et al. (2016) J. Biol. Chem. 291, 1162- 1174), and on the basis of the present work we conclude that all molybdenum- and tungsten-containing formate dehydrogenases and related enzymes likely operate via a simple hydride transfer mechanism and are effective in catalysing the reversible interconversion of CO2 and formate under the appropriate experimental conditions.
  • Posted in Journal of Biological Chemistry, Publications
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Colibactin assembly line enzymes use S-adenosylmethionine to build a cyclopropane ring

August 7th, 2017 by Li Zha

Nature Chemical Biology 13, 1063 (2017). doi:10.1038/nchembio.2448

Authors: Li Zha, Yindi Jiang, Matthew T Henke, Matthew R Wilson, Jennifer X Wang, Neil L Kelleher & Emily P Balskus

Despite containing an α-amino acid, the versatile cofactor S-adenosylmethionine (SAM) is not a known building block for nonribosomal peptide synthetase (NRPS) assembly lines. Here we report an unusual NRPS module from colibactin biosynthesis that uses SAM for amide bond formation and subsequent cyclopropanation. Our findings showcase a new use for SAM and reveal a novel biosynthetic route to a functional group that likely mediates colibactin's genotoxicity.

  • Posted in Nat Chem Biol, Publications
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Bisphosphoglycerate mutase controls serine pathway flux via 3-phosphoglycerate

August 7th, 2017 by Rob C Oslund

Nature Chemical Biology 13, 1081 (2017). doi:10.1038/nchembio.2453

Authors: Rob C Oslund, Xiaoyang Su, Michael Haugbro, Jung-Min Kee, Mark Esposito, Yael David, Boyuan Wang, Eva Ge, David H Perlman, Yibin Kang, Tom W Muir & Joshua D Rabinowitz

  • Posted in Nat Chem Biol, Publications
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A genetically encoded tool for manipulation of NADP+/NADPH in living cells

August 7th, 2017 by Valentin Cracan

Nature Chemical Biology 13, 1088 (2017). doi:10.1038/nchembio.2454

Authors: Valentin Cracan, Denis V Titov, Hongying Shen, Zenon Grabarek & Vamsi K Mootha

  • Posted in Nat Chem Biol, Publications
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Molecular basis for the unusual ring reconstruction in fungal meroterpenoid biogenesis

July 31st, 2017 by Takahiro Mori

Nature Chemical Biology 13, 1066 (2017). doi:10.1038/nchembio.2443

Authors: Takahiro Mori, Taiki Iwabuchi, Shotaro Hoshino, Hang Wang, Yudai Matsuda & Ikuro Abe

  • Posted in Nat Chem Biol, Publications
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Modulating the DNA polymerase β reaction equilibrium to dissect the reverse reaction

July 31st, 2017 by David D Shock

Nature Chemical Biology 13, 1074 (2017). doi:10.1038/nchembio.2450

Authors: David D Shock, Bret D Freudenthal, William A Beard & Samuel H Wilson

  • Posted in Nat Chem Biol, Publications
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Enabling Graded and Large-Scale Multiplex of Desired Genes Using a Dual-Mode dCas9 Activator in Saccharomyces cerevisiae

July 27th, 2017 by Matthew Deaner, Julio Mejia and Hal S. Alper

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ACS Synthetic Biology
DOI: 10.1021/acssynbio.7b00163
  • Posted in ACS Synthetic Biology, Publications
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Palmitoylation is a prerequisite for dimerization-dependent raftophilicity of rhodopsin [Membrane Biology]

July 26th, 2017 by Keiji Seno, Fumio Hayashi

The visual photopigment rhodopsin (Rh) is a prototypical G protein-coupled receptor (GPCR) responsible for initiation of the phototransduction cascade in rod photoreceptors. Similar to other GPCRs, Rh can form dimers or even higher oligomers, and tends to have a supramolecular organization that is likely important in the dim light response. Rh also exhibits high affinity for lipid rafts (raftophilicity) upon light-dependent binding with cognate G protein transducin (Gt), suggesting the presence of lipid raft-like domains in the retinal disk membrane and their importance in phototransduction. However, the relationship between Rh oligomerization and lipid rafts in the disk membrane remains to be explored. Given previous findings that Gt binds to dimeric Rh and Rh is post-translationally modified with two highly raftophilic palmitoyl moieties, we hypothesized that Rh becomes raftophilic upon dimerization. Here, we tested this hypothesis biochemically. First, we found that Rh*-Gt complexes in the detergent-resistant membrane (DRM) are partially resistant to cholesterol depletion by methyl-β-cyclodextrin (MCD), and the stoichiometry of Rh to Gt in this MCD-resistant complex is 2:1. We then found that IgG-crosslinking renders Rh highly raftophilic, supporting the premise that Rh becomes raftophilic upon dimerization. Depalmitoylation of Rh with the reduction of thioester linkages by dithiothreitol blocked the translocation of IgG-crosslinked Rh to the DRM, highlighting the importance of the two palmitoyl moieties in the dimerization-dependent raftophilicity of Rh. These results indicate that palmitoylated GPCRs, such as Rh, may acquire raftophilicity upon G protein-stabilized dimerization and thereby organize receptor-cluster rafts by recruiting raftophilic lipids.

Unique Thermal Stability of Unnatural Hydrophobic Ds Bases in Double-Stranded DNAs

July 26th, 2017 by Michiko Kimoto and Ichiro Hirao

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ACS Synthetic Biology
DOI: 10.1021/acssynbio.7b00165