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

Susan Andersen, Birte Svensson, Marie Sofie Møller*

*Corresponding author for this work

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Abstract

Barley 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 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 Phe620Ala/Asp621Ala retained only 7% activity on pullulan albeit showed 25% activity towards BPNPG3G3. This outcome supports that the outer substrate binding area harbour preference determinants for the branched substrates amylopectin and β-limit dextrin.
Original languageEnglish
Article number140294
JournalB B A - Proteins and Proteomics
Volume1868
Issue number1
Number of pages24
ISSN1570-9639
DOIs
Publication statusPublished - 2020

Keywords

  • Glycoside hydrolase family 13 subfamily 13
  • Carbohydrate binding module 21-like
  • Pullulanase
  • Amylopectin
  • β-Limit dextrin
  • α-Limit dextrin

Cite this

@article{17611b9d46b44242a81ced2a6c59bc0d,
title = "Roles of the N-terminal domain and remote substrate binding subsites in activity of the debranching barley limit dextrinase",
abstract = "Barley 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 {\AA} 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 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 Phe620Ala/Asp621Ala retained only 7{\%} activity on pullulan albeit showed 25{\%} activity towards BPNPG3G3. This outcome supports that the outer substrate binding area harbour preference determinants for the branched substrates amylopectin and β-limit dextrin.",
keywords = "Glycoside hydrolase family 13 subfamily 13, Carbohydrate binding module 21-like, Pullulanase, Amylopectin, β-Limit dextrin, α-Limit dextrin",
author = "Susan Andersen and Birte Svensson and M{\o}ller, {Marie Sofie}",
year = "2020",
doi = "10.1016/j.bbapap.2019.140294",
language = "English",
volume = "1868",
journal = "B B A - Proteins and Proteomics",
issn = "1570-9639",
publisher = "Elsevier",
number = "1",

}

Roles of the N-terminal domain and remote substrate binding subsites in activity of the debranching barley limit dextrinase. / Andersen, Susan; Svensson, Birte; Møller, Marie Sofie.

In: B B A - Proteins and Proteomics, Vol. 1868, No. 1, 140294, 2020.

Research output: Contribution to journalJournal articleResearchpeer-review

TY - JOUR

T1 - Roles of the N-terminal domain and remote substrate binding subsites in activity of the debranching barley limit dextrinase

AU - Andersen, Susan

AU - Svensson, Birte

AU - Møller, Marie Sofie

PY - 2020

Y1 - 2020

N2 - Barley 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 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 Phe620Ala/Asp621Ala retained only 7% activity on pullulan albeit showed 25% activity towards BPNPG3G3. This outcome supports that the outer substrate binding area harbour preference determinants for the branched substrates amylopectin and β-limit dextrin.

AB - Barley 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 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 Phe620Ala/Asp621Ala retained only 7% activity on pullulan albeit showed 25% activity towards BPNPG3G3. This outcome supports that the outer substrate binding area harbour preference determinants for the branched substrates amylopectin and β-limit dextrin.

KW - Glycoside hydrolase family 13 subfamily 13

KW - Carbohydrate binding module 21-like

KW - Pullulanase

KW - Amylopectin

KW - β-Limit dextrin

KW - α-Limit dextrin

U2 - 10.1016/j.bbapap.2019.140294

DO - 10.1016/j.bbapap.2019.140294

M3 - Journal article

VL - 1868

JO - B B A - Proteins and Proteomics

JF - B B A - Proteins and Proteomics

SN - 1570-9639

IS - 1

M1 - 140294

ER -