Structural and functional characterization of CMP-N-acetylneuraminate synthetase from Vibrio cholerae.
|Title||Structural and functional characterization of CMP-N-acetylneuraminate synthetase from Vibrio cholerae.|
|Publication Type||Journal Article|
|Year of Publication||2019|
|Authors||Bose S, Purkait D, Joseph D, Nayak V, Subramanian R|
|Journal||Acta Crystallogr D Struct Biol|
|Date Published||2019 Jun 01|
|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|
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.
|Alternate Journal||Acta Crystallogr D Struct Biol|
|PubMed Central ID||PMC6580227|
|Grant List||BT/IN/SWEDEN/06/SR/2017-2018 / / Department of Biotechnology, Ministry of Science and Technology / |
BT/PR5081/INF/156/2012 / / Department of Biotechnology, Ministry of Science and Technology /
BT/PR12422/MED/31/287/214 / / Tata Institute of Fundamental Research /