TY - JOUR T1 - Structural and functional characterization of CMP-N-acetylneuraminate synthetase from Vibrio cholerae. JF - Acta Crystallogr D Struct Biol Y1 - 2019 A1 - Bose, Sucharita A1 - Purkait, Debayan A1 - Joseph, Deepthi A1 - Nayak, Vinod A1 - Subramanian, Ramaswamy KW - Amino Acid Sequence KW - Bacterial Proteins KW - Binding Sites KW - Catalytic Domain KW - Crystallization KW - Crystallography, X-Ray KW - Cytidine Diphosphate KW - Cytidine Monophosphate N-Acetylneuraminic Acid KW - Cytidine Triphosphate KW - N-Acylneuraminate Cytidylyltransferase KW - Protein Interaction Domains and Motifs KW - Protein Structure, Quaternary KW - Sialic Acids KW - Vibrio cholerae AB -

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.

VL - 75 IS - Pt 6 ER - TY - JOUR T1 - Automation aided optimization of cloning, expression and purification of enzymes of the bacterial sialic acid catabolic and sialylation pathways enzymes for structural studies. JF - Microb Biotechnol Y1 - 2018 A1 - Bairy, Sneha A1 - Gopalan, Lakshmi Narayanan A1 - Setty, Thanuja Gangi A1 - Srinivasachari, Sathya A1 - Manjunath, Lavanyaa A1 - Kumar, Jay Prakash A1 - Guntupalli, Sai R A1 - Bose, Sucharita A1 - Nayak, Vinod A1 - Ghosh, Swagatha A1 - Sathyanarayanan, Nitish A1 - Caing-Carlsson, Rhawnie A1 - Wahlgren, Weixiao Yuan A1 - Friemann, Rosmarie A1 - Ramaswamy, S A1 - Neerathilingam, Muniasamy AB -

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.

VL - 11 IS - 2 ER - TY - JOUR T1 - Crystal structures and kinetics of N-acetylneuraminate lyase from Fusobacterium nucleatum. JF - Acta Crystallogr F Struct Biol Commun Y1 - 2018 A1 - Kumar, Jay Prakash A1 - Rao, Harshvardhan A1 - Nayak, Vinod A1 - Ramaswamy, S KW - Bacterial Proteins KW - Crystallography, X-Ray KW - Fusobacterium nucleatum KW - Hydrogen Bonding KW - Models, Molecular KW - N-Acetylneuraminic Acid KW - Oxo-Acid-Lyases KW - Protein Conformation KW - Protein Folding KW - Pyruvic Acid KW - Schiff Bases KW - Sequence Alignment KW - Tyrosine AB -

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 Å 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.

VL - 74 IS - Pt 11 ER -