Conserved Calcium-Binding Residues at the Ca-I Site Involved in Fructooligosaccharide Synthesis by Lactobacillus reuteri 121 Inulosucrase.

Inulosucrase is an enzyme that synthesizes inulin-type ß-2,1-linked fructooligosaccharides (IFOS) from sucrose. Previous studies have shown that calcium is important for the activity and stability of Lactobacillus reuteri 121 inulosucrase (LrInu). Here, mutational analyses of four conserved calcium-binding site I (Ca-I) residues of LrInu, Asp418, Gln449, Asn488, and Asp520 were performed. Alanine substitution for these residues not only reduced the stability and activity of LrInu, but also modulated the pattern of the IFOS produced. Circular dichroism spectroscopy and molecular dynamics simulation indicated that these mutations had limited impact on the overall conformation of the enzyme. One of Ca-I residues most critical for controlling LrInu-mediated polymerization of IFOS, Asp418, was also subjected to mutagenesis, generating D418E, D418H, D418L, D418N, D418S, and D418W. The activity of these mutants demonstrated that the IFOS chain length could be controlled by a single mutation at the Ca-I site.

Molecular dynamics reveals insight into how N226P and H227Y mutations affect maltose binding in the active site of α-glucosidase II from European honeybee, Apis mellifera.

European honeybee, Apis mellifera, produces α-glucosidase (HBGase) that catalyzes the cleavage of an α-glycosidic bond of the non-reducing end of polysaccharides and has potential applications for malt hydrolysis in brewing industry. Characterized by their substrate specificities, HBGases have three isoforms including HBGase II, which prefers maltose to sucrose as a substrate. Previous study found that the catalytic efficiency of maltose hydrolysis of N226P mutant of HBGase II was higher than that of the wild type (WT), and the catalytic efficiency of maltose hydrolysis of WT was higher than those of H227Y and N226P-H227Y mutants. We hypothesized that N226P mutation probably caused maltose to bind with better affinity and position/orientation for hydrolysis than WT, while H227Y and N226P-H227Y mutations caused maltose to bind with worse affinity and position/orientation for hydrolysis than WT. Using this hypothesis, we performed molecular dynamics on the catalytically competent binding conformations of maltose/WT, maltose/N226P, maltose/H227Y, and maltose/N226P-H227Y complexes to elucidate effects of N226P and H227Y mutations on maltose binding in HBGase II active site. Our results reasonably support this hypothesis because the N226P mutant had better binding affinity, higher number of important binding residues, strong and medium hydrogen bonds as well as shorter distance between atoms necessary for hydrolysis than WT, while the H227Y and N226P-H227Y mutants had worse binding affinities, lower number of important binding residues and strong hydrogen bonds as well as longer distances between atoms necessary for hydrolysis than WT. Moreover, results of binding free energy and hydrogen bond interaction of residue 227 support the role of H227 as a maltose preference residue, as proposed by previous studies. Our study provides important and novel insight into how N226P and H227Y mutations affect maltose binding in HBGase II active site. This knowledge could potentially be used to engineer HBGase II to improve its efficiency.