@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 {1192, title = {Automation aided optimization of cloning, expression and purification of enzymes of the bacterial sialic acid catabolic and sialylation pathways enzymes for structural studies.}, journal = {Microb Biotechnol}, volume = {11}, year = {2018}, month = {2018 Mar}, pages = {420-428}, abstract = {

The process of obtaining a well-expressing, soluble and correctly folded constructs can be made easier and quicker by automating the optimization of cloning, expression and purification. While there are many semiautomated pipelines available for cloning, expression and purification, there is hardly any pipeline that involves complete automation. Here, we achieve complete automation of all the steps involved in cloning and in\ vivo expression screening. This is demonstrated using 18 genes involved in sialic acid catabolism and the surface sialylation pathway. Our main objective was to clone these genes into a His-tagged Gateway vector, followed by their small-scale expression optimization in\ vivo. The constructs that showed best soluble expression were then selected for purification studies and scaled up for crystallization studies. Our technique allowed us to quickly find conditions for producing significant quantities of soluble proteins in Escherichia coli, their large-scale purification and successful crystallization of a number of these proteins. The method can be implemented in other cases where one needs to screen a large number of constructs, clones and expression vectors for successful recombinant production of functional proteins.

}, issn = {1751-7915}, doi = {10.1111/1751-7915.13041}, author = {Bairy, Sneha and Gopalan, Lakshmi Narayanan and Setty, Thanuja Gangi and Srinivasachari, Sathya and Manjunath, Lavanyaa and Kumar, Jay Prakash and Guntupalli, Sai R and Bose, Sucharita and Nayak, Vinod and Ghosh, Swagatha and Sathyanarayanan, Nitish and Caing-Carlsson, Rhawnie and Wahlgren, Weixiao Yuan and Friemann, Rosmarie and Ramaswamy, S and Neerathilingam, Muniasamy} } @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} }