Allostery Wiring Map for Kinesin Energy Transduction and its Evolution [Enzymology]

August 8th, 2016 by Richard, J., Kim, E. D., Nguyen, H., Kim, C. D., Kim, S.

How signals between the kinesin active- and cytoskeletal-binding sites are transmitted is an open question and an allosteric question. By extracting correlated evolutionary changes within 700+ sequences, we built a model of residues that are energetically coupled and that define molecular routes for signal transmission. Typically, these coupled residues are located at multiple distal sites and, thus, are predicted to form a complex, nonlinear network that wires together different functional sites in the protein. Of note, our model connected the site for ATP hydrolysis with sites that ultimately utilize its free energy, such as the microtubule-binding site, drug-binding loop-5, and necklinker. To confirm the calculated energetic connectivity between non-adjacent residues, double-mutant cycle analysis was conducted with 22 kinesin mutants. There was a direct correlation between thermodynamic coupling in experiment and evolutionarily-derived energetic coupling. We conclude that energy transduction is coordinated by multiple distal sites in the protein, rather than only being relayed through adjacent residues. Moreover, this allosteric map forecasts how energetic orchestration gives rise to different nanomotor behaviors within the superfamily.

Different Divalent Cations Alter the Kinetics and Fidelity of DNA Polymerases [DNA and Chromosomes]

August 8th, 2016 by Vashishtha, A. K., Wang, J., Konigsberg, W. H.

Divalent metal ions are essential components of DNA polymerases both for catalysis of the nucleotidyl transfer reaction and for base excision. They occupy two sites, A and B for DNA synthesis. Recently, a third metal ion was shown to be essential for phosphoryl transfer reaction. The metal ion in the A site is coordinated by the carboxylate of two highly conserved acidic residues, water molecules, and the 3' hydroxyl group of the primer so that the A metal is in an octahedral complex. Its catalytic function is to lower the pKa of the hydroxyl group making it a highly effective nucleophile that can attack the alpha phosphorous atom of the incoming dNTP. The metal ion in the B site is coordinated by the same two carboxylates that are affixed to the A metal ion as well as the non-bridging oxygen atoms of the incoming dNTP. The carboxyl oxygen of an adjacent peptide bond serves as the sixth ligand that completes the octahedral coordination geometry of the B metal ion. Similarly two metal ions are required for proof-reading, one helps to lower the pKa of the attacking water molecule and the other helps to stabilize the transition state for nucleotide excision. The role of different divalent cations are discussed in relation to these two activities as well as their influence on base selectivity and misincorporation by DNA polymerases. Some, but not all of the effects of these different metal ions can be rationalized based on their intrinsic properties which are tabulated in this review.

Rice endosperm starch phosphorylase (Pho1) assembles with disproportionating enzyme (Dpe1) to form a protein complex that enhances synthesis of malto-oligosaccharides [Metabolism]

August 8th, 2016 by Hwang, S.-K., Koper, K., Satoh, H., Okita, T. W.

Starch synthesis in cereal grain endosperm is dependent on the concerted actions of many enzymes. The starch plastidial phosphorylase (Pho1) plays an important role in the initiation of starch synthesis and in the maturation of starch granule in developing rice seeds. Prior evidence has suggested that the rice enzyme, OsPho1, may have a physical/functional interaction with other starch biosynthetic enzymes. Pull-down experiments showed that OsPho1 as well as OsPho1 devoid of its L80 region, a peptide unique to higher plant phosphorylases, captures disproportionating enzyme (OsDpe1). Interaction of the latter enzyme form with OsDpe1 indicates that the putative regulatory L80 is not responsible for multi-enzyme assembly. This heterotypic enzyme complex, determined at a molar ratio of 1:1, was validated by reciprocal co-immunoprecipitation studies of native seed proteins and by co-elution chromatographic and co-migration electrophoretic patterns of these enzymes in rice seed extracts. The OsPho1-OsDpe1 complex utilized a broader range of substrates for enhanced synthesis of larger malto-oligosaccharides than each individual enzyme and significantly elevated the substrate affinities of OsPho1 at 30oC. Moreover, the assembly with OsDpe1 enables OsPho1 to utilize products of transglycosylation reactions involving G1 and G3, sugars that it cannot catalyze directly.
  • Posted in Journal of Biological Chemistry, Publications
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Functional Annotation of a Presumed Nitronate Monooxygenase Reveals a New Class of NADH:quinone Reductases [Enzymology]

August 8th, 2016 by Ball, J., Salvi, F., Gadda, G.

The protein PA1024 from Pseudomonas aeruginosa PAO1 is currently classified as 2-nitropropane dioxygenase, the previous name for nitronate monooxygenase in the GenBankTM and PDB databases, but the enzyme was not kinetically characterized. In this study, PA1024 was purified to high levels and the enzymatic activity was investigated by spectroscopic and polarographic techniques. Purified PA1024 did not exhibit nitronate monooxygenase activity; however, it displayed NADH:quinone reductase and a small NADH:oxidase activity. The enzyme preferred NADH to NADPH as a reducing substrate. PA1024 could reduce a broad spectrum of quinone substrates via a Ping Pong Bi-Bi steady-state kinetic mechanism, generating the corresponding hydroquinones. The reductive half reaction with NADH showed a kred value of 24 s-1 and an apparent Kd value estimated in the low μM range. The enzyme was not able to reduce the azo dye methyl red, routinely used in the kinetic characterization of azoreductases. Finally, we revisited and modified the existing six conserved motifs of PA1024, which define a new class of NADH:quinone reductases and are present in more than 490 hypothetical proteins in the GenBankTM, the vast majority of which are currently misannotated as nitronate monooxygenase.

FRET binding antenna reports spatiotemporal dynamics of GDI–Cdc42 GTPase interactions

August 8th, 2016 by Louis Hodgson

Nature Chemical Biology 12, 802 (2016). doi:10.1038/nchembio.2145

Authors: Louis Hodgson, Désirée Spiering, Mohsen Sabouri-Ghomi, Onur Dagliyan, Céline DerMardirossian, Gaudenz Danuser & Klaus M Hahn

  • Posted in Nat Chem Biol, Publications
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Inhibiting androgen receptor nuclear entry in castration-resistant prostate cancer

August 8th, 2016 by Julie A Pollock

Nature Chemical Biology 12, 795 (2016). doi:10.1038/nchembio.2131

Authors: Julie A Pollock, Suzanne E Wardell, Alexander A Parent, David B Stagg, Stephanie J Ellison, Holly M Alley, Christina A Chao, Scott A Lawrence, James P Stice, Ivan Spasojevic, Jennifer G Baker, Sung Hoon Kim, Donald P McDonnell, John A Katzenellenbogen & John D Norris

  • Posted in Nat Chem Biol, Publications
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A hybrid polyketide–nonribosomal peptide in nematodes that promotes larval survival

August 8th, 2016 by Qingyao Shou

Nature Chemical Biology 12, 770 (2016). doi:10.1038/nchembio.2144

Authors: Qingyao Shou, Likui Feng, Yaoling Long, Jungsoo Han, Joshawna K Nunnery, David H Powell & Rebecca A Butcher

Polyketides and nonribosomal peptides are two important types of natural products that are produced by many species of bacteria and fungi but are exceedingly rare in metazoans. Here, we elucidate the structure of a hybrid polyketide–nonribosomal peptide from Caenorhabditis elegans that is produced in the canal-associated neurons (CANs) and promotes survival during starvation-induced larval arrest. Our results uncover a novel mechanism by which animals respond to nutrient fluctuations to extend survival.

  • Posted in Nat Chem Biol, Publications
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Enhanced Nitrite Reductase Activity and Its Correlation with Oxygen Affinity in Hemoglobin Bis-Tetramers

August 5th, 2016 by Aizhou Wang and Ronald Kluger

TOC Graphic

Biochemistry
DOI: 10.1021/acs.biochem.6b00542

Fluorescence-Activated Cell Sorting of Human l-asparaginase Mutant Libraries for Detecting Enzyme Variants with Enhanced Activity

August 1st, 2016 by Christos S. Karamitros and Manfred Konrad

TOC Graphic

ACS Chemical Biology
DOI: 10.1021/acschembio.6b00283
  • Posted in ACS Chemical Biology, Publications
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Metabolic plasticity underpins innate and acquired resistance to LDHA inhibition

August 1st, 2016 by Aaron Boudreau

Nature Chemical Biology 12, 779 (2016). doi:10.1038/nchembio.2143

Authors: Aaron Boudreau, Hans E Purkey, Anna Hitz, Kirk Robarge, David Peterson, Sharada Labadie, Mandy Kwong, Rebecca Hong, Min Gao, Christopher Del Nagro, Raju Pusapati, Shuguang Ma, Laurent Salphati, Jodie Pang, Aihe Zhou, Tommy Lai, Yingjie Li, Zhongguo Chen, Binqing Wei, Ivana Yen, Steve Sideris, Mark McCleland, Ron Firestein, Laura Corson, Alex Vanderbilt, Simon Williams, Anneleen Daemen, Marcia Belvin, Charles Eigenbrot, Peter K Jackson, Shiva Malek, Georgia Hatzivassiliou, Deepak Sampath, Marie Evangelista & Thomas O'Brien

  • Posted in Nat Chem Biol, Publications
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