Research Article: Structural and functional characterization of CMP-N-acetylneuraminate synthetase from Vibrio cholerae

Date Published: June 01, 2019

Publisher: International Union of Crystallography

Author(s): Sucharita Bose, Debayan Purkait, Deepthi Joseph, Vinod Nayak, Ramaswamy Subramanian.


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.

Partial Text

Sialic acids are a family of nine-carbon α-keto sugar acids that are present at the terminal positions of various glycoconjugates on the eukaryotic cell surface and are functionally significant in several physiological and pathological processes (Varki, 2008 ▸). N-Acetylneuraminic acid (Neu5Ac) is the most abundant form of sialic acid. The bacterial pathogens that colonize the heavily sialylated niche of the mammalian gut and respiratory tract have co-evolved to scavenge sialic acid from the eukaryotic host and use it as a carbon and nitrogen source (Severi et al., 2007 ▸). Several opportunistic bacteria including Neisseria meningitidis, Escherichia coli K1, Haemophilus influenzae, Haemophilus ducreyi and Pasteurella multocida ‘sugar-coat’ themselves with host-derived Neu5Ac on various glycoconjugates on their surface to evade the host innate immune system (McDonald et al., 2016 ▸). Sialylated glycoconjugates protect Neisseria gonorrhoae from phagocytosis (Rest & Frangipane, 1992 ▸). A study comparing the virulence of Campylobacter jejuni showed that the sialylated form (as compared with the nonsialylated form) of the organism is capable of invading human epithelial cell lines more effectively (Louwen et al., 2008 ▸). These studies suggested that the enzymes of the Neu5Ac incorporation pathway play a critical role in bacterial virulence, pathogenesis and colonization, and hold promise as potential drug targets. The work reported here is part of our ongoing investigations into the structure–activity relationship of the enzymes involved in the uptake and incorporation of sialic acid into lipooligosaccharides (LOS) and lipopolysaccharides (LPS) from several pathogenic bacteria including H. influenzae, P. multocida, Fusobacterium nucleatum and Vibrio cholerae.

In this study, we have characterized the structural and functional properties of the CMAS enzyme (WP_000064388.1) from V. cholerae. Both the apo and nucleotide-bound structures possess all five of the structural motifs responsible for ligand binding in CMAS enzymes. In this work, we have identified the Mg2+ ion that interacts with the α-phosphate of CDP and the invariant Asp215 in VcCMAS. Upon CDP binding, the VcCMAS structure shows conformational changes in the dimerization domain, which lead to partial closure of the active site and place the catalytic Arg169 near the Neu5Ac binding pocket. We hypothesize that the partially closed CDP–VcCMAS structure represents an intermediate form, fully recognizing that this conformation could also have resulted owing to crystal packing. However, this intermediate conformation of the enzyme (Supplementary Fig. 3) does support a structural basis for the sequential order of binding of CTP prior to sialic acid in CMAS enzymes.

Danio rerio CMAS, NP_001035342.2; Homo sapiens CMAS, NP_061156.1; Mus musculus CMAS, NP_034038.2; Streptococcus agalactiae CMAS, AAD53077.1; Escherichia coli CMAS, WP_001259305.1; Pasteurella multocida CMAS, WP_005723432.1; Vibrio cholerae CMAS, WP_000064388.1; Campylobacter jejuni CMAS, WP_002894653.1; Clostridium thermocellum CMAS, WP_003512903.1; Neisseria meningitidis CMAS, WP_061726245.1; Haemophilus ducreyi CMAS, SEW08179.1; H. influenzae CMAS, WP_080316015.1; Pasteurella haemolytica CMAS, WP_006251815.1.

The following references are cited in the supporting information for this article: Bravo et al. (2001 ▸), Mizanur & Pohl (2007 ▸) and Vann et al. (1987 ▸).




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