Details

Autor(en) / Beteiligte

• Bose, Sucharita
• Purkait, Debayan
• Joseph, Deepthi
• Nayak, Vinod
• Subramanian, Ramaswamy

Titel

Structural and functional characterization of CMP‐N‐acetylneuraminate synthetase from Vibrio cholerae

Ist Teil von

• Acta crystallographica. Section D, Biological crystallography., 2019-06, Vol.75 (6), p.564-577

Ort / Verlag

5 Abbey Square, Chester, Cheshire CH1 2HU, England: International Union of Crystallography

Erscheinungsjahr

2019

Link zum Volltext

Quelle

Alma/SFX Local Collection

Beschreibungen/Notizen

• 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 Mg2+ 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. CMP‐N‐acetylneuraminate synthetase (CMAS) is a key enzyme in the sialic acid incorporation pathway and plays a crucial role in the virulence and survival of several pathogenic bacteria. Here, the structural and functional properties of CMAS from the pathogenic bacterium Vibrio cholerae are reported. Upon CDP binding, a partial domain closure is observed that was previously unreported in homologous structures. Kinetic studies reveal that the enzyme shows substrate promiscuity and can activate both Neu5Ac and Neu5Gc.