Roles of the N-terminal domain and remote substrate binding subsites in activity of the debranching barley limit dextrinase

Publication date: Available online 30 October 2019Source: Biochimica et Biophysica Acta (BBA) - Proteins and ProteomicsAuthor(s): Susan Andersen, Birte Svensson, Marie Sofie MøllerAbstractBarley limit dextrinase (HvLD) of glycoside hydrolase family 13 is the sole enzyme hydrolysing α-1,6-glucosidic linkages from starch in the germinating seed. Surprisingly, HvLD shows 150- and 7-fold higher activity towards pullulan and β-limit dextrin, respectively, than amylopectin. This is investigated by mutational analysis of residues in the N-terminal CBM-21-like domain (Ser14Arg, His108Arg, Ser14Arg/His108Arg) and at the outer subsites +2 (Phe553Gly) and +3 (Phe620Ala, Asp621Ala, Phe620Ala/Asp621Ala) of the active site. The Ser14 and His108 mutants mimic natural LD variants from sorghum and rice with elevated enzymatic activity. Although situated about 40 Å from the active site, the single mutants had 15–40% catalytic efficiency compared to wild type for the three polysaccharides and the double mutant retained 27% activity for β-limit dextrin and 64% for pullulan and amylopectin. These three mutants hydrolysed 4,6-O-benzylidene-4-nitrophenyl-63-α-d-maltotriosyl-maltotriose (BPNPG3G3) with 51–109% of wild-type activity. The results highlight that the N-terminal CBM21-like domain plays a role in activity. Phe553 and the highly conserved Trp512 sandwich a substrate main chain glucosyl residue at subsite +2 of the active site, while substrate contacts of Phe620 and Asp621 at su...

Source: Biochimica et Biophysica Acta (BBA) Proteins and Proteomics - October 31, 2019 Category: Biochemistry Source Type: research