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

Barley limit dextrinase (HvLD) of glycoside hydrolase family 13 is the sole enzyme hydrolysing alpha-1,6-glucosidic linkages from starch in the germinating seed. Surprisingly, HvLD shows 150- and 7-fold higher activity towards pullulan and beta-limit dextrin, respectively, than amylopectin. This is investigated by mutational analysis of residues in the N-terminal CBM-21-like domain (Serl4Arg, His108Arg, Serl4Arg/His108Arg) and at the outer subsites +2 (Phe553Gly) and +3 (Phe620Ala, Asp621Ala, Phe620A1a/Asp621A1a) 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 angstrom 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 13-limit dextrin and 64% for pullulan and amylopectin. These three mutants hydrolysed 4,6-0-benzylidene-4-nitropheny1-63-a-omaltotriosyl-maltotriose (BPNPG3G3) with 51-109% of wild-type activity. The results highlight that the Nterminal 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 subsite + 3 are less prominent. Phe553Gly showed 47% and 25% activity on pullulan and BPNPG3G3, respectively having a main role at subsite + 2. By contrast at subsite + 3, Asp621Ala increased activity on pullulan by 2.4 -fold, while Phe620A1a/Asp621Ala retained only 7% activity on pullulan albeit showed 25% activity towards BPNPG3G3. This outcome supports that the outer substrate binding area harbours preference determinants for the branched substrates amylopectin and beta-limit dextrin.