Active Site Architecture and Reaction Mechanism Determination of Cold Adapted β-d-galactosidase from Arthrobacter sp. 32cB

ArthβDG is a dimeric, cold-adapted β-d-galactosidase that exhibits high hydrolytic and transglycosylation activity. A series of crystal structures of its wild form, as well as its Arth&...

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Main Authors:	Maria Rutkiewicz, Anna Bujacz, Marta Wanarska, Anna Wierzbicka-Wos, Hubert Cieslinski
Format:	Article
Language:	English
Published:	MDPI AG 2019-09-01
Series:	International Journal of Molecular Sciences
Subjects:	galactosidase hydrolysis reaction mechanism complex structures cold-adapted GH2
Online Access:	https://www.mdpi.com/1422-0067/20/17/4301

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spelling	doaj-8b1379d5a8dc441eb0b4d25e42a15a072020-11-25T01:11:35ZengMDPI AGInternational Journal of Molecular Sciences1422-00672019-09-012017430110.3390/ijms20174301ijms20174301Active Site Architecture and Reaction Mechanism Determination of Cold Adapted β-<span style="font-variant: small-caps">d</span>-galactosidase from <i>Arthrobacter</i> sp. 32cBMaria Rutkiewicz0Anna Bujacz1Marta Wanarska2Anna Wierzbicka-Wos3Hubert Cieslinski4Institute of Technical Biochemistry, Faculty of Biotechnology and Food Sciences, Lodz University of Technology, Stefanowskiego 4/10, 90-924 Lodz, PolandInstitute of Technical Biochemistry, Faculty of Biotechnology and Food Sciences, Lodz University of Technology, Stefanowskiego 4/10, 90-924 Lodz, PolandDepartment of Molecular Biotechnology and Microbiology, Faculty of Chemistry, Gdansk University of Technology, Narutowicza 11/12, 80-233 Gdansk, PolandDepartment of Microbiology, Faculty of Biology, University of Szczecin, Felczaka 3c, 71-412 Szczecin, PolandDepartment of Molecular Biotechnology and Microbiology, Faculty of Chemistry, Gdansk University of Technology, Narutowicza 11/12, 80-233 Gdansk, Poland<i>Arth</i>βDG is a dimeric, cold-adapted β-<span style="font-variant: small-caps;">d</span>-galactosidase that exhibits high hydrolytic and transglycosylation activity. A series of crystal structures of its wild form, as well as its <i>Arth</i>βDG_E441Q mutein complexes with ligands were obtained in order to describe the mode of its action. The <i>Arth</i>βDG_E441Q mutein is an inactive form of the enzyme designed to enable observation of enzyme interaction with its substrate. The resulting three-dimensional structures of complexes: <i>Arth</i>βDG_E441Q/LACs and <i>Arth</i>βDG/IPTG (ligand bound in shallow mode) and structures of complexes <i>Arth</i>βDG_E441Q/LACd, <i>Arth</i>βDG/ONPG (ligands bound in deep mode), and galactose <i>Arth</i>βDG/GAL and their analysis enabled structural characterization of the hydrolysis reaction mechanism. Furthermore, comparative analysis with mesophilic analogs revealed the most striking differences in catalysis mechanisms. The key role in substrate transfer from shallow to deep binding mode involves rotation of the F581 side chain. It is worth noting that the 10-aa loop restricting access to the active site in mesophilic GH2 βDGs, in <i>Arth</i>βDG is moved outward. This facilitates access of substrate to active site. Such a permanent exposure of the entrance to the active site may be a key factor for improved turnover rate of the cold adapted enzyme and thus a structural feature related to its cold adaptation.https://www.mdpi.com/1422-0067/20/17/4301galactosidasehydrolysisreaction mechanismcomplex structurescold-adaptedGH2
collection	DOAJ
language	English
format	Article
sources	DOAJ
author	Maria Rutkiewicz Anna Bujacz Marta Wanarska Anna Wierzbicka-Wos Hubert Cieslinski
spellingShingle	Maria Rutkiewicz Anna Bujacz Marta Wanarska Anna Wierzbicka-Wos Hubert Cieslinski Active Site Architecture and Reaction Mechanism Determination of Cold Adapted β-<span style="font-variant: small-caps">d</span>-galactosidase from <i>Arthrobacter</i> sp. 32cB International Journal of Molecular Sciences galactosidase hydrolysis reaction mechanism complex structures cold-adapted GH2
author_facet	Maria Rutkiewicz Anna Bujacz Marta Wanarska Anna Wierzbicka-Wos Hubert Cieslinski
author_sort	Maria Rutkiewicz
title	Active Site Architecture and Reaction Mechanism Determination of Cold Adapted β-<span style="font-variant: small-caps">d</span>-galactosidase from <i>Arthrobacter</i> sp. 32cB
title_short	Active Site Architecture and Reaction Mechanism Determination of Cold Adapted β-<span style="font-variant: small-caps">d</span>-galactosidase from <i>Arthrobacter</i> sp. 32cB
title_full	Active Site Architecture and Reaction Mechanism Determination of Cold Adapted β-<span style="font-variant: small-caps">d</span>-galactosidase from <i>Arthrobacter</i> sp. 32cB
title_fullStr	Active Site Architecture and Reaction Mechanism Determination of Cold Adapted β-<span style="font-variant: small-caps">d</span>-galactosidase from <i>Arthrobacter</i> sp. 32cB
title_full_unstemmed	Active Site Architecture and Reaction Mechanism Determination of Cold Adapted β-<span style="font-variant: small-caps">d</span>-galactosidase from <i>Arthrobacter</i> sp. 32cB
title_sort	active site architecture and reaction mechanism determination of cold adapted β-<span style="font-variant: small-caps">d</span>-galactosidase from <i>arthrobacter</i> sp. 32cb
publisher	MDPI AG
series	International Journal of Molecular Sciences
issn	1422-0067
publishDate	2019-09-01
description	<i>Arth</i>βDG is a dimeric, cold-adapted β-<span style="font-variant: small-caps;">d</span>-galactosidase that exhibits high hydrolytic and transglycosylation activity. A series of crystal structures of its wild form, as well as its <i>Arth</i>βDG_E441Q mutein complexes with ligands were obtained in order to describe the mode of its action. The <i>Arth</i>βDG_E441Q mutein is an inactive form of the enzyme designed to enable observation of enzyme interaction with its substrate. The resulting three-dimensional structures of complexes: <i>Arth</i>βDG_E441Q/LACs and <i>Arth</i>βDG/IPTG (ligand bound in shallow mode) and structures of complexes <i>Arth</i>βDG_E441Q/LACd, <i>Arth</i>βDG/ONPG (ligands bound in deep mode), and galactose <i>Arth</i>βDG/GAL and their analysis enabled structural characterization of the hydrolysis reaction mechanism. Furthermore, comparative analysis with mesophilic analogs revealed the most striking differences in catalysis mechanisms. The key role in substrate transfer from shallow to deep binding mode involves rotation of the F581 side chain. It is worth noting that the 10-aa loop restricting access to the active site in mesophilic GH2 βDGs, in <i>Arth</i>βDG is moved outward. This facilitates access of substrate to active site. Such a permanent exposure of the entrance to the active site may be a key factor for improved turnover rate of the cold adapted enzyme and thus a structural feature related to its cold adaptation.
topic	galactosidase hydrolysis reaction mechanism complex structures cold-adapted GH2
url	https://www.mdpi.com/1422-0067/20/17/4301
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Active Site Architecture and Reaction Mechanism Determination of Cold Adapted β-<span style="font-variant: small-caps">d</span>-galactosidase from <i>Arthrobacter</i> sp. 32cB

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