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.

Figure 1. Multiple sequence alignment for LaNAL, LsNAL and related N-acetyl neuraminate lyases.

ESPript outputs were obtained with sequences from SWISSPROT and aligned with CLUSTAL-W. Sequences were grouped according to similarity. Residues strictly conserved across NAL enzymes are highlighted against a red background. The secondary structure corresponding to the modeled LaNAL is shown, springs represent helices and arrows represent β-strands. The residues forming the active site are indicated with small triangles. The sequence motifs previously described (13) are C-B (carboxylate-binding motif), A-C (aldol-cleaveage motif) and S-B (sugar-binding motif). ChNAL, NAL from Clostridium hylemonae; SpNAL, NAL from Streptococcus pneumoniae; SfNAL, NAL from Shigella flexneri; SeNAL from Salmonella enterica.

Figure 2. Effects of pH and temperature on the activity of LaNAL and LsNAL.

A) Optimum pH of LaNAL for Neu5Ac synthesis (▪) and hydrolysis (•). Standard reaction conditions for HPLC at 37°C were used with the following buffers: 20 mM sodium acetate pH 4.5–5, sodium phosphate pH 6–7, Tris-HCl pH 8.5 and glycine pH 9. B) Optimum pH of LsNAL for Neu5Ac synthesis (▪) and hydrolysis (•). Assay conditions were the standard reaction medium for LaNAL. (C and D) Optimum temperature of LaNAL and LsNAL for Neu5Ac synthesis (▪) and hydrolysis (•). The activity was determined by HPLC at different temperatures under the corresponding standard reaction medium.

Figure 3. KDO synthesis from D-arabinose and pyruvate using LaNAL.

Activity was assayed at 37°C with 0.6 M D-arabinose, 1.2 M pyruvate and 1 mg mL⁻¹ LaNAL. D-arabinose (•) and KDO (○).

Figure 4. Functional divergence analysis of NALs.

A) Analysis of group 3 and 1. B) Molecular representation of Cat I and Cat II residues in group 3 and group 1. C) Analysis of group 3 and 2. D) Molecular representation of Cat I and Cat II residues of group 3 and 2. The residues forming the active site are indicated with yellow spheres, those forming the Category I are indicated by red spheres and those forming Category II by blue spheres.