Cargando…

Characterization of an Unknown Region Linked to the Glycoside Hydrolase Family 17 β-1,3-Glucanase of Vibrio vulnificus Reveals a Novel Glucan-Binding Domain

The glycoside hydrolase family 17 β-1,3-glucanase of Vibrio vulnificus (VvGH17) has two unknown regions in the N- and C-termini. Here, we characterized these domains by preparing mutant enzymes. VvGH17 demonstrated hydrolytic activity of β-(1→3)-glucan, mainly producing laminaribiose, but not of β-(...

Descripción completa

Detalles Bibliográficos
Autores principales: Kumagai, Yuya, Kishimura, Hideki, Lang, Weeranuch, Tagami, Takayoshi, Okuyama, Masayuki, Kimura, Atsuo
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9026390/
https://www.ncbi.nlm.nih.gov/pubmed/35447923
http://dx.doi.org/10.3390/md20040250
_version_ 1784691109751947264
author Kumagai, Yuya
Kishimura, Hideki
Lang, Weeranuch
Tagami, Takayoshi
Okuyama, Masayuki
Kimura, Atsuo
author_facet Kumagai, Yuya
Kishimura, Hideki
Lang, Weeranuch
Tagami, Takayoshi
Okuyama, Masayuki
Kimura, Atsuo
author_sort Kumagai, Yuya
collection PubMed
description The glycoside hydrolase family 17 β-1,3-glucanase of Vibrio vulnificus (VvGH17) has two unknown regions in the N- and C-termini. Here, we characterized these domains by preparing mutant enzymes. VvGH17 demonstrated hydrolytic activity of β-(1→3)-glucan, mainly producing laminaribiose, but not of β-(1→3)/β-(1→4)-glucan. The C-terminal-truncated mutants (ΔC466 and ΔC441) showed decreased activity, approximately one-third of that of the WT, and ΔC415 lost almost all activity. An analysis using affinity gel containing laminarin or barley β-glucan revealed a shift in the mobility of the ΔC466, ΔC441, and ΔC415 mutants compared to the WT. Tryptophan residues showed a strong affinity for carbohydrates. Three of four point-mutations of the tryptophan in the C-terminus (W472A, W499A, and W542A) showed a reduction in binding ability to laminarin and barley β-glucan. The C-terminus was predicted to have a β-sandwich structure, and three tryptophan residues (Trp472, Trp499, and Trp542) constituted a putative substrate-binding cave. Linker and substrate-binding functions were assigned to the C-terminus. The N-terminal-truncated mutants also showed decreased activity. The WT formed a trimer, while the N-terminal truncations formed monomers, indicating that the N-terminus contributed to the multimeric form of VvGH17. The results of this study are useful for understanding the structure and the function of GH17 β-1,3-glucanases.
format Online
Article
Text
id pubmed-9026390
institution National Center for Biotechnology Information
language English
publishDate 2022
publisher MDPI
record_format MEDLINE/PubMed
spelling pubmed-90263902022-04-23 Characterization of an Unknown Region Linked to the Glycoside Hydrolase Family 17 β-1,3-Glucanase of Vibrio vulnificus Reveals a Novel Glucan-Binding Domain Kumagai, Yuya Kishimura, Hideki Lang, Weeranuch Tagami, Takayoshi Okuyama, Masayuki Kimura, Atsuo Mar Drugs Article The glycoside hydrolase family 17 β-1,3-glucanase of Vibrio vulnificus (VvGH17) has two unknown regions in the N- and C-termini. Here, we characterized these domains by preparing mutant enzymes. VvGH17 demonstrated hydrolytic activity of β-(1→3)-glucan, mainly producing laminaribiose, but not of β-(1→3)/β-(1→4)-glucan. The C-terminal-truncated mutants (ΔC466 and ΔC441) showed decreased activity, approximately one-third of that of the WT, and ΔC415 lost almost all activity. An analysis using affinity gel containing laminarin or barley β-glucan revealed a shift in the mobility of the ΔC466, ΔC441, and ΔC415 mutants compared to the WT. Tryptophan residues showed a strong affinity for carbohydrates. Three of four point-mutations of the tryptophan in the C-terminus (W472A, W499A, and W542A) showed a reduction in binding ability to laminarin and barley β-glucan. The C-terminus was predicted to have a β-sandwich structure, and three tryptophan residues (Trp472, Trp499, and Trp542) constituted a putative substrate-binding cave. Linker and substrate-binding functions were assigned to the C-terminus. The N-terminal-truncated mutants also showed decreased activity. The WT formed a trimer, while the N-terminal truncations formed monomers, indicating that the N-terminus contributed to the multimeric form of VvGH17. The results of this study are useful for understanding the structure and the function of GH17 β-1,3-glucanases. MDPI 2022-03-31 /pmc/articles/PMC9026390/ /pubmed/35447923 http://dx.doi.org/10.3390/md20040250 Text en © 2022 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Kumagai, Yuya
Kishimura, Hideki
Lang, Weeranuch
Tagami, Takayoshi
Okuyama, Masayuki
Kimura, Atsuo
Characterization of an Unknown Region Linked to the Glycoside Hydrolase Family 17 β-1,3-Glucanase of Vibrio vulnificus Reveals a Novel Glucan-Binding Domain
title Characterization of an Unknown Region Linked to the Glycoside Hydrolase Family 17 β-1,3-Glucanase of Vibrio vulnificus Reveals a Novel Glucan-Binding Domain
title_full Characterization of an Unknown Region Linked to the Glycoside Hydrolase Family 17 β-1,3-Glucanase of Vibrio vulnificus Reveals a Novel Glucan-Binding Domain
title_fullStr Characterization of an Unknown Region Linked to the Glycoside Hydrolase Family 17 β-1,3-Glucanase of Vibrio vulnificus Reveals a Novel Glucan-Binding Domain
title_full_unstemmed Characterization of an Unknown Region Linked to the Glycoside Hydrolase Family 17 β-1,3-Glucanase of Vibrio vulnificus Reveals a Novel Glucan-Binding Domain
title_short Characterization of an Unknown Region Linked to the Glycoside Hydrolase Family 17 β-1,3-Glucanase of Vibrio vulnificus Reveals a Novel Glucan-Binding Domain
title_sort characterization of an unknown region linked to the glycoside hydrolase family 17 β-1,3-glucanase of vibrio vulnificus reveals a novel glucan-binding domain
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9026390/
https://www.ncbi.nlm.nih.gov/pubmed/35447923
http://dx.doi.org/10.3390/md20040250
work_keys_str_mv AT kumagaiyuya characterizationofanunknownregionlinkedtotheglycosidehydrolasefamily17b13glucanaseofvibriovulnificusrevealsanovelglucanbindingdomain
AT kishimurahideki characterizationofanunknownregionlinkedtotheglycosidehydrolasefamily17b13glucanaseofvibriovulnificusrevealsanovelglucanbindingdomain
AT langweeranuch characterizationofanunknownregionlinkedtotheglycosidehydrolasefamily17b13glucanaseofvibriovulnificusrevealsanovelglucanbindingdomain
AT tagamitakayoshi characterizationofanunknownregionlinkedtotheglycosidehydrolasefamily17b13glucanaseofvibriovulnificusrevealsanovelglucanbindingdomain
AT okuyamamasayuki characterizationofanunknownregionlinkedtotheglycosidehydrolasefamily17b13glucanaseofvibriovulnificusrevealsanovelglucanbindingdomain
AT kimuraatsuo characterizationofanunknownregionlinkedtotheglycosidehydrolasefamily17b13glucanaseofvibriovulnificusrevealsanovelglucanbindingdomain