@article {1983, title = {Structural and functional characterization of CMP-N-acetylneuraminate synthetase from Vibrio cholerae.}, journal = {Acta Crystallogr D Struct Biol}, volume = {75}, year = {2019}, month = {2019 Jun 01}, pages = {564-577}, abstract = {

Several pathogenic bacteria utilize sialic acid, including host-derived N-acetylneuraminic acid (Neu5Ac), in at least two ways: they use it as a nutrient source and as a host-evasion strategy by coating themselves with Neu5Ac. Given the significant role of sialic acid in pathogenesis and host-gut colonization by various pathogenic bacteria, including Neisseria meningitidis, Haemophilus influenzae, Pasteurella multocida and Vibrio cholerae, several enzymes of the sialic acid catabolic, biosynthetic and incorporation pathways are considered to be potential drug targets. In this work, findings on the structural and functional characterization of CMP-N-acetylneuraminate synthetase (CMAS), a key enzyme in the incorporation pathway, from Vibrio cholerae are reported. CMAS catalyzes the synthesis of CMP-sialic acid by utilizing CTP and sialic acid. Crystal structures of the apo and the CDP-bound forms of the enzyme were determined, which allowed the identification of the metal cofactor Mg in the active site interacting with CDP and the invariant Asp215 residue. While open and closed structural forms of the enzyme from eukaryotic and other bacterial species have already been characterized, a partially closed structure of V.\ cholerae CMAS (VcCMAS) observed upon CDP binding, representing an intermediate state, is reported here. The kinetic data suggest that VcCMAS is capable of activating the two most common sialic acid derivatives, Neu5Ac and Neu5Gc. Amino-acid sequence and structural comparison of the active site of VcCMAS with those of eukaryotic and other bacterial counterparts reveal a diverse hydrophobic pocket that interacts with the C5 substituents of sialic acid. Analyses of the thermodynamic signatures obtained from the binding of the nucleotide (CTP) and the product (CMP-sialic acid) to VcCMAS provide fundamental information on the energetics of the binding process.

}, keywords = {Amino Acid Sequence, Bacterial Proteins, Binding Sites, Catalytic Domain, Crystallization, Crystallography, X-Ray, Cytidine Diphosphate, Cytidine Monophosphate N-Acetylneuraminic Acid, Cytidine Triphosphate, N-Acylneuraminate Cytidylyltransferase, Protein Interaction Domains and Motifs, Protein Structure, Quaternary, Sialic Acids, Vibrio cholerae}, issn = {2059-7983}, doi = {10.1107/S2059798319006831}, author = {Bose, Sucharita and Purkait, Debayan and Joseph, Deepthi and Nayak, Vinod and Subramanian, Ramaswamy} } @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 {1203, title = {Structural and functional studies of ferredoxin and oxygenase components of 3-nitrotoluene dioxygenase from Diaphorobacter sp. strain DS2.}, journal = {PLoS One}, volume = {12}, year = {2017}, month = {2017}, pages = {e0176398}, abstract = {

3-nitrotoluene dioxygenase (3NTDO) from Diaphorobacter sp. strain DS2 catalyses the conversion of 3-nitrotoluene (3NT) into a mixture of 3- and 4-methylcatechols with release of nitrite. We report here, X-ray crystal structures of oxygenase and ferredoxin components of 3NTDO at 2.9 {\r A} and 2.4 {\r A}, respectively. The residues responsible for nitrite release in 3NTDO were further probed by four single and two double mutations in the catalytic site of α-subunit of the dioxygenase. Modification of Val 350 to Phe, Ile 204 to Ala, and Asn258 to Val by site directed mutagenesis resulted in inactive enzymes revealing the importance of these residues in catalysis. Docking studies of meta nitrotoluene to the active site of 3NTDO suggested possible orientations of binding that favor the formation of 3-methylcatechol (3MC) over 4-methylcatechol energetically. The electron transfer pathway from ferredoxin subunit to the active site of the oxygenase subunit is also proposed.

}, keywords = {Catalytic Domain, Comamonadaceae, Crystallography, X-Ray, Ferredoxins, Molecular Docking Simulation, Mutation, Oxygenases, Substrate Specificity, Toluene}, issn = {1932-6203}, doi = {10.1371/journal.pone.0176398}, author = {Kumari, Archana and Singh, Deepak and Ramaswamy, S and Ramanathan, Gurunath} }