@article {1597, title = {Crystal structures and kinetics of N-acetylneuraminate lyase from Fusobacterium nucleatum.}, journal = {Acta Crystallogr F Struct Biol Commun}, volume = {74}, year = {2018}, month = {2018 Nov 01}, pages = {725-732}, abstract = {

N-Acetyl-D-neuraminic acid lyase (NanA) catalyzes the breakdown of sialic acid (Neu5Ac) to N-acetyl-D-mannosamine (ManNAc) and pyruvate. NanA plays a key role in Neu5Ac catabolism in many pathogenic and bacterial commensals where sialic acid is available as a carbon and nitrogen source. Several pathogens or commensals decorate their surfaces with sialic acids as a strategy to escape host innate immunity. Catabolism of sialic acid is key to a range of host-pathogen interactions. In this study, atomic resolution structures of NanA from Fusobacterium nucleatum (FnNanA) in ligand-free and ligand-bound forms are reported at 2.32 and 1.76 {\r A} resolution, respectively. F. nucleatum is a Gram-negative pathogen that causes gingival and periodontal diseases in human hosts. Like other bacterial N-acetylneuraminate lyases, FnNanA also shares the triosephosphate isomerase (TIM)-barrel fold. As observed in other homologous enzymes, FnNanA forms a tetramer. In order to characterize the structure-function relationship, the steady-state kinetic parameters of the enzyme are also reported.

}, keywords = {Bacterial Proteins, Crystallography, X-Ray, Fusobacterium nucleatum, Hydrogen Bonding, Models, Molecular, N-Acetylneuraminic Acid, Oxo-Acid-Lyases, Protein Conformation, Protein Folding, Pyruvic Acid, Schiff Bases, Sequence Alignment, Tyrosine}, issn = {2053-230X}, doi = {10.1107/S2053230X18012992}, author = {Kumar, Jay Prakash and Rao, Harshvardhan and Nayak, Vinod and Ramaswamy, S} } @article {1185, title = {Crystal structure of N-acetylmannosamine kinase from Fusobacterium nucleatum.}, journal = {Acta Crystallogr F Struct Biol Commun}, volume = {73}, year = {2017}, month = {2017 Jun 01}, pages = {356-362}, abstract = {

Sialic acids comprise a varied group of nine-carbon amino sugars that are widely distributed among mammals and higher metazoans. Some human commensals and bacterial pathogens can scavenge sialic acids from their environment and degrade them for use as a carbon and nitrogen source. The enzyme N-acetylmannosamine kinase (NanK; EC 2.7.1.60) belongs to the transcriptional repressors, uncharacterized open reading frames and sugar kinases (ROK) superfamily. NanK catalyzes the second step of the sialic acid catabolic pathway, transferring a phosphate group from adenosine 5{\textquoteright}-triphosphate to the C6 position of N-acetylmannosamine to generate N-acetylmannosamine 6-phosphate. The structure of NanK from Fusobacterium nucleatum was determined to 2.23 {\r A} resolution by X-ray crystallography. Unlike other NanK enzymes and ROK family members, F. nucleatum NanK does not have a conserved zinc-binding site. In spite of the absence of the zinc-binding site, all of the major structural features of enzymatic activity are conserved.

}, keywords = {Adenosine Triphosphate, Amino Acid Sequence, Bacterial Proteins, Binding Sites, Cloning, Molecular, Crystallography, X-Ray, Escherichia coli, Fusobacterium nucleatum, Gene Expression, Genetic Vectors, Hexosamines, Models, Molecular, Phosphotransferases (Alcohol Group Acceptor), Protein Binding, Protein Conformation, alpha-Helical, Protein Conformation, beta-Strand, Protein Interaction Domains and Motifs, Protein Multimerization, Recombinant Proteins, Sequence Alignment, Sequence Homology, Amino Acid, Substrate Specificity}, issn = {2053-230X}, doi = {10.1107/S2053230X17007439}, author = {Caing-Carlsson, Rhawnie and Goyal, Parveen and Sharma, Amit and Ghosh, Swagatha and Setty, Thanuja Gangi and North, Rachel A and Friemann, Rosmarie and Ramaswamy, S} } @article {1204, title = {Horse Liver Alcohol Dehydrogenase: Zinc Coordination and Catalysis.}, journal = {Biochemistry}, volume = {56}, year = {2017}, month = {2017 07 18}, pages = {3632-3646}, abstract = {

During catalysis by liver alcohol dehydrogenase (ADH), a water bound to the catalytic zinc is replaced by the oxygen of the substrates. The mechanism might involve a pentacoordinated zinc or a double-displacement reaction with participation by a nearby glutamate residue, as suggested by studies of human ADH3, yeast ADH1, and some other tetrameric ADHs. Zinc coordination and participation of water in the enzyme mechanism were investigated by X-ray crystallography. The apoenzyme and its complex with adenosine 5{\textquoteright}-diphosphoribose have an open protein conformation with the catalytic zinc in one position, tetracoordinated by Cys-46, His-67, Cys-174, and a water molecule. The bidentate chelators 2,2{\textquoteright}-bipyridine and 1,10-phenanthroline displace the water and form a pentacoordinated zinc. The enzyme-NADH complex has a closed conformation similar to that of ternary complexes with coenzyme and substrate analogues; the coordination of the catalytic zinc is similar to that found in the apoenzyme, except that a minor, alternative position for the catalytic zinc is \~{}1.3 {\r A} from the major position and closer to Glu-68, which could form the alternative coordination to the catalytic zinc. Complexes with NADH and N-1-methylhexylformamide or N-benzylformamide (or with NAD and fluoro alcohols) have the classical tetracoordinated zinc, and no water is bound to the zinc or the nicotinamide rings. The major forms of the enzyme in the mechanism have a tetracoordinated zinc, where the carboxylate group of Glu-68 could participate in the exchange of water and substrates on the zinc. Hydride transfer in the Michaelis complexes does not involve a nearby water.

}, keywords = {2,2{\textquoteright}-Dipyridyl, Adenosine Diphosphate Ribose, Alcohol Dehydrogenase, Animals, Catalytic Domain, Crystallography, X-Ray, Formamides, Horses, Kinetics, Liver, Models, Molecular, NAD, Phenanthrolines, Protein Binding, Protein Conformation, Water, Zinc}, issn = {1520-4995}, doi = {10.1021/acs.biochem.7b00446}, author = {Plapp, Bryce V and Savarimuthu, Baskar Raj and Ferraro, Daniel J and Rubach, Jon K and Brown, Eric N and Ramaswamy, S} } @article {1167, title = {Blue protein with red fluorescence.}, journal = {Proc Natl Acad Sci U S A}, volume = {113}, year = {2016}, month = {2016 10 11}, pages = {11513-11518}, abstract = {

The walleye (Sander vitreus) is a golden yellow fish that inhabits the Northern American lakes. The recent sightings of the blue walleye and the correlation of its sighting to possible increased UV radiation have been proposed earlier. The underlying molecular basis of its adaptation to increased UV radiation is the presence of a protein (Sandercyanin)-ligand complex in the mucus of walleyes. Degradation of heme by UV radiation results in the formation of Biliverdin IXα (BLA), the chromophore bound to Sandercyanin. We show that Sandercyanin is a monomeric protein that forms stable homotetramers on addition of BLA to the protein. A structure of the Sandercyanin-BLA complex, purified from the fish mucus, reveals a glycosylated protein with a lipocalin fold. This protein-ligand complex absorbs light in the UV region (λ of 375 nm) and upon excitation at this wavelength emits in the red region (λ of 675 nm). Unlike all other known biliverdin-bound fluorescent proteins, the chromophore is noncovalently bound to the protein. We provide here a molecular rationale for the observed spectral properties of Sandercyanin.

}, keywords = {Biliverdine, Crystallography, X-Ray, Fluorescence, Models, Molecular, Proteins, Recombinant Proteins}, issn = {1091-6490}, doi = {10.1073/pnas.1525622113}, author = {Ghosh, Swagatha and Yu, Chi-Li and Ferraro, Daniel J and Sudha, Sai and Pal, Samir Kumar and Schaefer, Wayne F and Gibson, David T and Ramaswamy, S} } @article {616, title = {Mechanistic implications from structures of yeast alcohol dehydrogenase complexed with coenzyme and an alcohol.}, journal = {Arch Biochem Biophys}, volume = {591}, year = {2016}, month = {2016 Feb 1}, pages = {35-42}, abstract = {

Yeast alcohol dehydrogenase I is a homotetramer of subunits with 347 amino acid residues, catalyzing the oxidation of alcohols using NAD(+) as coenzyme. A new X-ray structure was determined at 3.0 {\r A} where both subunits of an asymmetric dimer bind coenzyme and trifluoroethanol. The tetramer is a pair of back-to-back dimers. Subunit A has a closed conformation and can represent a Michaelis complex with an appropriate geometry for hydride transfer between coenzyme and alcohol, with the oxygen of 2,2,2-trifluoroethanol ligated at 2.1 {\r A} to the catalytic zinc in the classical tetrahedral coordination with Cys-43, Cys-153, and His-66. Subunit B has an open conformation, and the coenzyme interacts with amino acid residues from the coenzyme binding domain, but not with residues from the catalytic domain. Coenzyme appears to bind to and dissociate from the open conformation. The catalytic zinc in subunit B has an alternative, inverted coordination with Cys-43, Cys-153, His-66 and the carboxylate of Glu-67, while the oxygen of trifluoroethanol is 3.5 {\r A} from the zinc. Subunit B may represent an intermediate in the mechanism after coenzyme and alcohol bind and before the conformation changes to the closed form and the alcohol oxygen binds to the zinc and displaces Glu-67.

}, keywords = {Alcohol Dehydrogenase, Binding Sites, Catalysis, Coenzymes, Computer Simulation, Enzyme Activation, Models, Chemical, Models, Molecular, NAD, Protein Binding, Protein Conformation, Saccharomyces cerevisiae Proteins, Substrate Specificity, Trifluoroethanol}, issn = {1096-0384}, doi = {10.1016/j.abb.2015.12.009}, author = {Plapp, Bryce V and Charlier, Henry A and Ramaswamy, S} } @article {399, title = {Mechanistic heterogeneity in contractile properties of α-tropomyosin (TPM1) mutants associated with inherited cardiomyopathies.}, journal = {J Biol Chem}, volume = {290}, year = {2015}, month = {2015 Mar 13}, pages = {7003-15}, abstract = {

The most frequent known causes of primary cardiomyopathies are mutations in the genes encoding sarcomeric proteins. Among those are 30 single-residue mutations in TPM1, the gene encoding α-tropomyosin. We examined seven mutant tropomyosins, E62Q, D84N, I172T, L185R, S215L, D230N, and M281T, that were chosen based on their clinical severity and locations along the molecule. The goal of our study was to determine how the biochemical characteristics of each of these mutant proteins are altered, which in turn could provide a structural rationale for treatment of the cardiomyopathies they produce. Measurements of Ca(2+) sensitivity of human β-cardiac myosin ATPase activity are consistent with the hypothesis that hypertrophic cardiomyopathies are hypersensitive to Ca(2+) activation, and dilated cardiomyopathies are hyposensitive. We also report correlations between ATPase activity at maximum Ca(2+) concentrations and conformational changes in TnC measured using a fluorescent probe, which provide evidence that different substitutions perturb the structure of the regulatory complex in different ways. Moreover, we observed changes in protein stability and protein-protein interactions in these mutants. Our results suggest multiple mechanistic pathways to hypertrophic and dilated cardiomyopathies. Finally, we examined a computationally designed mutant, E181K, that is hypersensitive, confirming predictions derived from in silico structural analysis.

}, keywords = {Actins, Adenosine Triphosphatases, Calcium, Cardiomyopathies, Humans, Models, Molecular, Myosins, Point Mutation, Protein Stability, Tropomyosin}, issn = {1083-351X}, doi = {10.1074/jbc.M114.596676}, author = {Gupte, Tejas M and Haque, Farah and Gangadharan, Binnu and Sunitha, Margaret S and Mukherjee, Souhrid and Anandhan, Swetha and Rani, Deepa Selvi and Mukundan, Namita and Jambekar, Amruta and Thangaraj, Kumarasamy and Sowdhamini, Ramanathan and Sommese, Ruth F and Nag, Suman and Spudich, James A and Mercer, John A} }