title: First functional and mutational analysis of group 3 N-acetylneuraminate lyases from Lactobacillus antri and Lactobacillus sakei 23K.
abstract: N-acetyl neuraminate lyases (NALs) catalyze the reversible aldol cleavage of N-acetyl neuraminic acid (Neu5Ac) to pyruvate and N-acetyl-D-mannosamine (ManNAc). Previous phylogenetic studies divided NALs into four different groups. Groups 1 and 2 have been well characterized at both kinetic and molecular levels, but no NAL from group 3 has been studied to date. In this work, a functional characterization of two group 3 members was performed using the recombinant NALs from Lactobacillus antri and Lactobacillus sakei 23K, revealing an optimal pH of between 6.0 and 7.0, low stability at basic pHs (>8.0), low optimal temperatures and, especially, low catalytic efficiency compared with their counterparts in group 1 and 2. The mutational analysis carried out showed that a plausible molecular reason for the low activity shown by Lactobacillus antri and Lactobacillus sakei 23k NALs compared with group 1 and 2 NALs could be the relatively small sugar-binding pocket they contain. A functional divergence analysis concluding that group 3 is more closely related to group 2 than to group 1.
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, Espinardo, Murcia, Spain; Murcia Biomedical Research Institute (IMIB), El Palmar, Murcia, Spain.
authors.forename: María Inmaculada
authors.initials: MI
authors.lastname: García-García
authors.affiliation: CELLS-ALBA Synchrotron Light Source, Cerdanyola del Vallès, Barcelona, Spain.
authors.forename: Fernando
authors.initials: F
authors.lastname: Gil-Ortiz
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, Espinardo, Murcia, Spain; Murcia Biomedical Research Institute (IMIB), El Palmar, Murcia, 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, Espinardo, Murcia, Spain; Murcia Biomedical Research Institute (IMIB), El Palmar, Murcia, Spain.
authors.forename: Alvaro
authors.initials: A
authors.lastname: Sánchez-Ferrer
mesh_terms: D000595:Amino Acid Sequence; D000890:Anti-Infective Agents / Q000378:metabolism; D055162:Biocatalysis; D003001:Cloning, Molecular; D004252:DNA Mutational Analysis; D004795:Enzyme Stability; D006595:Hexosamines / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007778:Lactobacillus / Q000201:enzymology* / Q000235:genetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016296:Mutagenesis; D007652:Oxo-Acid-Lyases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D055503:Protein Multimerization; D020836:Protein Structure, Quaternary; D019289:Pyruvic Acid / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013696:Temperature
chemical_list: D000890:Anti-Infective Agents; D006595:Hexosamines; D019289:Pyruvic Acid; D007652:Oxo-Acid-Lyases; C018728:N-acetylneuraminate lyase; C002022:N-acetylmannosamine