First functional and mutational analysis of group 3 N-acetylneuraminate lyases from Lactobacillus antri and Lactobacillus sakei 23K

• Published in: PloS one 2014-05, Vol.9 (5), p.e96976
• Main Authors: García-García, María Inmaculada, Gil-Ortiz, Fernando, García-Carmona, Francisco, Sánchez-Ferrer, Alvaro
• Format: Article
• Language: English
• Subjects: Acids; Aldehydes; Amino Acid Sequence; Anti-Infective Agents - metabolism; Bacteria; Bacteroides; Biocatalysis; Biochemistry; Bioinformatics; Biology and Life Sciences; Biomedical research; Carbon; Catalysis; Cloning; Cloning, Molecular; Clostridium perfringens; Divergence; DNA Mutational Analysis; E coli; Enzyme Stability; Enzymes; Escherichia coli; Genes; Hexosamines - metabolism; Hydrogen-Ion Concentration; Influenza; Laboratories; Lactobacillus; Lactobacillus - enzymology; Lactobacillus - genetics; Lactobacillus sakei; Models, Molecular; Molecular biology; Molecular Sequence Data; Mutagenesis; Oxo-Acid-Lyases - chemistry; Oxo-Acid-Lyases - genetics; Oxo-Acid-Lyases - metabolism; Pasteurella multocida; pH effects; Phylogeny; Physical Sciences; Protein Multimerization; Protein Structure, Quaternary; Proteins; Pyruvic acid; Pyruvic Acid - metabolism; Sequence Homology, Amino Acid; Sugar; Temperature
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0096976

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.