Probing the essential catalytic residues and substrate affinity in thermophilic L-arabinose isomerase by homology modeling and site-directed mutagenesis

The L-arabinose isomerase from Lactobacillus fermentum CGMCC2921 (named LFAI) was distinguished from other L-AIs by its outstanding thermostability, and was defined as a potential candidate for industrial D-tagatose production. By means of homologous modeling and structure analysis, some important amino acid residues influencing D-galactose isomerization of LFAI were selected and mutated. The results showed that when residues Q16, M311, K423, and Q438 mutated to alanine, the K _m value of the mutant LFAI decreased. Among them, mutant enzyme M311A retained half of its original K _m value, and the conversion rate for D-galactose raised approximately 20%. Furthermore, by comparing mutants K423R, K423N, K423A, and native LFAI, it was found that the side-chain length of residue K423 may determine the substrate affinity and D-galactose conversion rate of these mutated enzymes. Through computer molecular modeling, it was also found mutation M311A had an enhancement on hydrogen bonding with D-galactose, thus resulting in an enhancement on its substrate affinity and enzyme activity.