title: Molecular characterization of a new N-acetylneuraminate synthase (NeuB1) from Idiomarina loihiensis.
abstract: N-Acetylneuraminate lyase synthase (NeuB; E.C. 2.5.1.56) is a key enzyme in pathogenic microorganisms for producing N-acetylneuraminic acid through the irreversible condensation of N-acetylmannosamine (ManNAc) and phosphoenolpyruvate (PEP). However, nothing is known about this enzyme in non-pathogenic bacteria. This paper describes, for the first time, one of the two putative N-acetylneuraminate synthases from the halophilic non-pathogenic gamma-proteobacterium Idiomarina loihiensis NeuB1 (IlNeuB1). The obtained 95-kDa dimeric enzyme showed maximal activity at pH 7.0 and 40°C and was more stable at pH 8.0 (8 h half-life) than the previously described NeuB. Its catalytic efficiency towards ManNAc and PEP was 10- and 40-fold higher, respectively, than that determined for Campylobacter jejuni NeuB, but only half that found for Neisseria meningitidis NeuB towards PEP. The phylogenetic and structural analyses of NeuB enzymes revealed the new domain architecture 4 has no cystathionine-β-synthase domain (cystathionine-β-synthetase domain), unlike domain architecture 3. In addition, 10 conserved blocks (I-X) were found, and surprisingly, this study showed that the arginine essential for catalysis that is present in antifreeze-like domain (block X) was not fully conserved in NeuB, but is replaced by a serine in a long sequence (>700 residues) NeuB, such as that existing in domain architectures 3 and 4.
authors.affiliation: Department of Biochemistry and Molecular Biology A, Faculty of Biology, Regional Campus of International Excellence "Campus Mare Nostrum", University of Murcia, Campus Espinardo, Murcia E-30100, Spain Murcia Biomedical Research Institute (IMIB-Arrixaca), Murcia 30120, Spain.
authors.forename: María Inmaculada
authors.initials: MI
authors.lastname: García García
authors.affiliation: Institute for Glycomics, Griffith University, Gold Coast Campus, Southport, QLD 4222, Australia.
authors.forename: Kam
authors.initials: K
authors.lastname: Lau
authors.affiliation: Institute for Glycomics, Griffith University, Gold Coast Campus, Southport, QLD 4222, Australia.
authors.forename: Mark
authors.initials: M
authors.lastname: von Itzstein
authors.affiliation: Department of Biochemistry and Molecular Biology A, Faculty of Biology, Regional Campus of International Excellence "Campus Mare Nostrum", University of Murcia, Campus Espinardo, Murcia E-30100, Spain Murcia Biomedical Research Institute (IMIB-Arrixaca), Murcia 30120, Spain.
authors.forename: Francisco
authors.initials: F
authors.lastname: García Carmona
authors.affiliation: Department of Biochemistry and Molecular Biology A, Faculty of Biology, Regional Campus of International Excellence "Campus Mare Nostrum", University of Murcia, Campus Espinardo, Murcia E-30100, Spain Murcia Biomedical Research Institute (IMIB-Arrixaca), Murcia 30120, Spain alvaro@um.es.
authors.forename: Álvaro
authors.initials: Á
authors.lastname: Sánchez Ferrer
mesh_terms: D034241:Alteromonadaceae / Q000737:chemistry* / Q000145:classification / Q000201:enzymology; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D055162:Biocatalysis; D016123:Campylobacter jejuni / Q000737:chemistry / Q000201:enzymology; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D006207:Half-Life; D006595:Hexosamines / Q000737:chemistry* / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D009345:Neisseria meningitidis / Q000737:chemistry / Q000201:enzymology; D007652:Oxo-Acid-Lyases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D010728:Phosphoenolpyruvate / Q000737:chemistry* / Q000378:metabolism; D010802:Phylogeny; D055503:Protein Multimerization; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D013045:Species Specificity; D013379:Substrate Specificity
chemical_list: D001426:Bacterial Proteins; D006595:Hexosamines; D011994:Recombinant Proteins; D010728:Phosphoenolpyruvate; C018730:N-acetylneuraminate synthase; D007652:Oxo-Acid-Lyases; C002022:N-acetylmannosamine
keywords: N-acetylneuraminate synthase; kinetic parameters; phylogenetic analysis; sialic acid