Phylogenetic and evolutive analysis of mdh gene in Fructobacillus and related organisms.

MOHAMED, F.; RAYA R.R.; MOZZI F

Congreso; 1st Latin American Congress of Women in Bioinformatics and Data Science.; 2020

Mannitol is a polyol with several technological applications, and is mainly used in the food industry as a low-calorie sweetener. Mannitol synthesis in heterofermentative lactic acid bacteria is performed by a mannitol-2-dehydrogenase (MDH) enzyme that catalyzes the reduction of fructose to mannitol by using NAD(P)H as cofactor. The MDH enzyme is particularly important in Fructobacillus, a genus with a peculiar fructophilic metabolism. We aimed to study the variability of MDH sequences in nine Fructobacillus strains and related organisms through a phylogenetic and evolutive approach. Initially, the complete DNA sequence of mdh genes from the Fructobacillus strains was obtained by PCR and Sanger sequencing. The translated amino acid sequences showed a high sequence similarity (>88%), especially in the NAD(P)H- and Zn-binding sites. In addition, a BLASTp search against the UniprotKB database was performed by using the MDH sequence of the F. tropaeoli CRL 2034 strain as a query. The sequences of MDH homologous with >50% similarity, besides Fructobacillus previously obtained sequences, were used for further analysis. A sequence similarity network (SSN) showed 298 nodes (sequences) grouped into 8 clusters. Fructobacillus MDHs were located in cluster 4 with phylogenetically related genera (Leuconostoc, Weissella and Convivina) organisms. When MDH sequences of this cluster were used to obtain a Maximum Likelihood-phylogenetic tree, Fructobacillus MDHs were located in a subclade, and species distribution was consistent with the previously known phylogeny among organisms. Finally, the identification of conserved and coevolved residues in MDH was performed using the MISTIC tool. Amino acids involved in structural Zn-binding and two residues involved in cofactor-binding sites were the most conserved. Also, two pairs of residues of the NAD(P)H-binding region were highly coevolved. In conclusion, MDH sequence variation in Fructobacillus is minimal and consistent to phylogeny; moreover, amino acids involved in catalytic functions are conserved in MDH enzymes.