Unusual substrate specificity in GH family 12: structure–function analysis of glucanases Bgh12A and Xgh12B from Aspergillus cervinus, and Egh12 from Thielavia terrestris

In this study, we compared the properties and structures of three fungal GH12 enzymes: the strict endoglucanase Bgh12A and the xyloglucanase Xgh12B from Aspergillus cervinus, and the endoglucanase Egh12 from Thielavia terrestris combining activity on linear β-glucan and branched xyloglucan. Egh12 fr...

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Veröffentlicht in:Applied microbiology and biotechnology 2022-02, Vol.106 (4), p.1493-1509
Hauptverfasser: Rykov, Sergey V., Selimzyanova, Alina I., Nikolaeva, Alena Y., Lazarenko, Vladimir A., Tsurin, Nikita V., Akentyev, Philipp I., Zverlov, Vladimir V., Liebl, Wolfgang, Schwarz, Wolfgang H., Berezina, Oksana V.
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container_end_page 1509
container_issue 4
container_start_page 1493
container_title Applied microbiology and biotechnology
container_volume 106
creator Rykov, Sergey V.
Selimzyanova, Alina I.
Nikolaeva, Alena Y.
Lazarenko, Vladimir A.
Tsurin, Nikita V.
Akentyev, Philipp I.
Zverlov, Vladimir V.
Liebl, Wolfgang
Schwarz, Wolfgang H.
Berezina, Oksana V.
description In this study, we compared the properties and structures of three fungal GH12 enzymes: the strict endoglucanase Bgh12A and the xyloglucanase Xgh12B from Aspergillus cervinus, and the endoglucanase Egh12 from Thielavia terrestris combining activity on linear β-glucan and branched xyloglucan. Egh12 from T. terrestris was produced in Pichia pastoris , purified, and characterized as a thermostable enzyme with maximal activity at 70 ºC and a half-life time of 138 min at 65 °C. We for the first time demonstrated that the GH12 endoglucanases Egh12 and Bgh12A, but not the strict xyloglucanase Xgh12B, hydrolyzed (1,3)-β-linkages in (1,3;1,4)-β-D-glucooligosaccharides and had transglycosylase activity on (1,3)-β-D-glucooligosaccharides. Phylogenetic analysis indicated that Egh12 from T. terrestris and Bgh12A from A. cervinus are more related than Bgh12A and Xgh12B isolated from one strain. The X-ray structure of Bgh12A was determined with 2.17 Å resolution and compared with 3D-homology models of Egh12 and Xgh12B. The enzymes have a β-jelly roll structure with a catalytic cleft running across the protein. Comparative analysis and a docking study demonstrated the importance of endoglucanase-specific loop 1 partly covering the catalytic cleft for correct placement of the linear substrates. Variability in substrate specificity between the GH12 endoglucanases is determined by non-conservative residues in structural loops framing the catalytic cleft. A residue responsible for the thermostability of Egh12 was predicted. The key structural elements and residues described in this study may serve as potential targets for modification aimed at the improvement of enzymatic properties. Key points • Thermostable endoglucanase Egh12 from T. terrestris was produced in P. pastoris, purified, and characterized • The X-ray structure of GH12 endoglucanase Bgh12A from A. cervinus was resolved • GH12 endoglucanases, but not GH12 xyloglucanases, hydrolyze (1,3)-β-linkages in (1,3;1,4)-β-D-glucooligosaccharides
doi_str_mv 10.1007/s00253-022-11811-7
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Egh12 from T. terrestris was produced in Pichia pastoris , purified, and characterized as a thermostable enzyme with maximal activity at 70 ºC and a half-life time of 138 min at 65 °C. We for the first time demonstrated that the GH12 endoglucanases Egh12 and Bgh12A, but not the strict xyloglucanase Xgh12B, hydrolyzed (1,3)-β-linkages in (1,3;1,4)-β-D-glucooligosaccharides and had transglycosylase activity on (1,3)-β-D-glucooligosaccharides. Phylogenetic analysis indicated that Egh12 from T. terrestris and Bgh12A from A. cervinus are more related than Bgh12A and Xgh12B isolated from one strain. The X-ray structure of Bgh12A was determined with 2.17 Å resolution and compared with 3D-homology models of Egh12 and Xgh12B. The enzymes have a β-jelly roll structure with a catalytic cleft running across the protein. Comparative analysis and a docking study demonstrated the importance of endoglucanase-specific loop 1 partly covering the catalytic cleft for correct placement of the linear substrates. Variability in substrate specificity between the GH12 endoglucanases is determined by non-conservative residues in structural loops framing the catalytic cleft. A residue responsible for the thermostability of Egh12 was predicted. The key structural elements and residues described in this study may serve as potential targets for modification aimed at the improvement of enzymatic properties. Key points • Thermostable endoglucanase Egh12 from T. terrestris was produced in P. pastoris, purified, and characterized • The X-ray structure of GH12 endoglucanase Bgh12A from A. cervinus was resolved • GH12 endoglucanases, but not GH12 xyloglucanases, hydrolyze (1,3)-β-linkages in (1,3;1,4)-β-D-glucooligosaccharides</description><identifier>ISSN: 0175-7598</identifier><identifier>EISSN: 1432-0614</identifier><identifier>DOI: 10.1007/s00253-022-11811-7</identifier><identifier>PMID: 35129654</identifier><language>eng</language><publisher>Berlin/Heidelberg: Springer Berlin Heidelberg</publisher><subject>Ascomycota ; Aspergillus ; Aspergillus cervinus ; Biomedical and Life Sciences ; Biotechnologically Relevant Enzymes and Proteins ; Biotechnology ; Cellulase - metabolism ; Comparative analysis ; Endoglucanase ; Enzymes ; Function analysis ; Fungi ; Glucan ; Homology ; Life Sciences ; Linkages ; Microbial enzymes ; Microbial Genetics and Genomics ; Microbiology ; Phylogeny ; Physiological aspects ; Pichia pastoris ; Residues ; Sordariales - metabolism ; Structural members ; Structure ; Structure-function relationships ; Substrate Specificity ; Substrates ; Substrates (Biochemistry) ; Thermal stability ; Thielavia terrestri ; Three dimensional models ; Xyloglucan ; Xyloglucanase ; β-Glucan</subject><ispartof>Applied microbiology and biotechnology, 2022-02, Vol.106 (4), p.1493-1509</ispartof><rights>The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature 2022</rights><rights>2022. 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Egh12 from T. terrestris was produced in Pichia pastoris , purified, and characterized as a thermostable enzyme with maximal activity at 70 ºC and a half-life time of 138 min at 65 °C. We for the first time demonstrated that the GH12 endoglucanases Egh12 and Bgh12A, but not the strict xyloglucanase Xgh12B, hydrolyzed (1,3)-β-linkages in (1,3;1,4)-β-D-glucooligosaccharides and had transglycosylase activity on (1,3)-β-D-glucooligosaccharides. Phylogenetic analysis indicated that Egh12 from T. terrestris and Bgh12A from A. cervinus are more related than Bgh12A and Xgh12B isolated from one strain. The X-ray structure of Bgh12A was determined with 2.17 Å resolution and compared with 3D-homology models of Egh12 and Xgh12B. The enzymes have a β-jelly roll structure with a catalytic cleft running across the protein. Comparative analysis and a docking study demonstrated the importance of endoglucanase-specific loop 1 partly covering the catalytic cleft for correct placement of the linear substrates. Variability in substrate specificity between the GH12 endoglucanases is determined by non-conservative residues in structural loops framing the catalytic cleft. A residue responsible for the thermostability of Egh12 was predicted. The key structural elements and residues described in this study may serve as potential targets for modification aimed at the improvement of enzymatic properties. Key points • Thermostable endoglucanase Egh12 from T. terrestris was produced in P. pastoris, purified, and characterized • The X-ray structure of GH12 endoglucanase Bgh12A from A. cervinus was resolved • GH12 endoglucanases, but not GH12 xyloglucanases, hydrolyze (1,3)-β-linkages in (1,3;1,4)-β-D-glucooligosaccharides</description><subject>Ascomycota</subject><subject>Aspergillus</subject><subject>Aspergillus cervinus</subject><subject>Biomedical and Life Sciences</subject><subject>Biotechnologically Relevant Enzymes and Proteins</subject><subject>Biotechnology</subject><subject>Cellulase - metabolism</subject><subject>Comparative analysis</subject><subject>Endoglucanase</subject><subject>Enzymes</subject><subject>Function analysis</subject><subject>Fungi</subject><subject>Glucan</subject><subject>Homology</subject><subject>Life Sciences</subject><subject>Linkages</subject><subject>Microbial enzymes</subject><subject>Microbial Genetics and Genomics</subject><subject>Microbiology</subject><subject>Phylogeny</subject><subject>Physiological aspects</subject><subject>Pichia pastoris</subject><subject>Residues</subject><subject>Sordariales - metabolism</subject><subject>Structural members</subject><subject>Structure</subject><subject>Structure-function relationships</subject><subject>Substrate Specificity</subject><subject>Substrates</subject><subject>Substrates (Biochemistry)</subject><subject>Thermal stability</subject><subject>Thielavia terrestri</subject><subject>Three dimensional models</subject><subject>Xyloglucan</subject><subject>Xyloglucanase</subject><subject>β-Glucan</subject><issn>0175-7598</issn><issn>1432-0614</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2022</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><sourceid>ABUWG</sourceid><sourceid>AFKRA</sourceid><sourceid>AZQEC</sourceid><sourceid>BENPR</sourceid><sourceid>CCPQU</sourceid><sourceid>DWQXO</sourceid><sourceid>GNUQQ</sourceid><recordid>eNp9kk1u1TAQxyMEoo_CBVggS2xYkOKxEydh91qVFqkSm1ZiZzmOnbpykocdV3o77sAxuBUnYdIUKhDCs_DXbz48_mfZS6BHQGn1LlLKSp5TxnKAGiCvHmUbKDjLqYDicbahUJV5VTb1QfYsxhtKgdVCPM0OeAmsEWWxyb5fjSkm5UlMbZyDmg2JO6OdddrNe-JGcnZOrBqc3xNg7wkySc8pmB9fv9k06tlNI1Gj8vvoIpks6X3SuI8mkuP-GtgWbzvyeVkeExumgWwxQeid9ykSbcKtwwre3lGnC7VCl9fOeHXrFJlNCAbTuvg8e2KVj-bF_XyYXX04vTw5zy8-nX082V7kuqT1nCtooKoEAJQWWsV4oepCtG1VdK2uOsZ43TBmgRsQJdNVqevCCG3rojAN0IIfZm_WuLswfUmYWw4uauO9Gs2UomQCjTWcA6Kv_0JvphSwHQvFcTT4Mw9Ur7yRbrQTdlovQeVWNPgXgAUhdfQPCq0zg9PTaKzD8z8c2OqgwxRjMFbughtU2EugclGIXBUiUSHyTiGyQqdX9xWndjDdb5dfkkCAr0DEq7E34eFJ_wn7E8LSxbQ</recordid><startdate>20220201</startdate><enddate>20220201</enddate><creator>Rykov, Sergey V.</creator><creator>Selimzyanova, Alina I.</creator><creator>Nikolaeva, Alena Y.</creator><creator>Lazarenko, Vladimir A.</creator><creator>Tsurin, Nikita V.</creator><creator>Akentyev, Philipp I.</creator><creator>Zverlov, Vladimir V.</creator><creator>Liebl, Wolfgang</creator><creator>Schwarz, Wolfgang H.</creator><creator>Berezina, Oksana V.</creator><general>Springer Berlin Heidelberg</general><general>Springer</general><general>Springer Nature B.V</general><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>3V.</scope><scope>7QL</scope><scope>7T7</scope><scope>7WY</scope><scope>7WZ</scope><scope>7X7</scope><scope>7XB</scope><scope>87Z</scope><scope>88A</scope><scope>88E</scope><scope>88I</scope><scope>8AO</scope><scope>8FD</scope><scope>8FE</scope><scope>8FH</scope><scope>8FI</scope><scope>8FJ</scope><scope>8FK</scope><scope>8FL</scope><scope>ABUWG</scope><scope>AEUYN</scope><scope>AFKRA</scope><scope>AZQEC</scope><scope>BBNVY</scope><scope>BENPR</scope><scope>BEZIV</scope><scope>BHPHI</scope><scope>C1K</scope><scope>CCPQU</scope><scope>DWQXO</scope><scope>FR3</scope><scope>FRNLG</scope><scope>FYUFA</scope><scope>F~G</scope><scope>GHDGH</scope><scope>GNUQQ</scope><scope>HCIFZ</scope><scope>K60</scope><scope>K6~</scope><scope>K9.</scope><scope>L.-</scope><scope>LK8</scope><scope>M0C</scope><scope>M0S</scope><scope>M1P</scope><scope>M2P</scope><scope>M7N</scope><scope>M7P</scope><scope>P64</scope><scope>PQBIZ</scope><scope>PQBZA</scope><scope>PQEST</scope><scope>PQQKQ</scope><scope>PQUKI</scope><scope>Q9U</scope><scope>7X8</scope><orcidid>https://orcid.org/0000-0003-4868-0589</orcidid></search><sort><creationdate>20220201</creationdate><title>Unusual substrate specificity in GH family 12: structure–function analysis of glucanases Bgh12A and Xgh12B from Aspergillus cervinus, and Egh12 from Thielavia terrestris</title><author>Rykov, Sergey V. ; 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Egh12 from T. terrestris was produced in Pichia pastoris , purified, and characterized as a thermostable enzyme with maximal activity at 70 ºC and a half-life time of 138 min at 65 °C. We for the first time demonstrated that the GH12 endoglucanases Egh12 and Bgh12A, but not the strict xyloglucanase Xgh12B, hydrolyzed (1,3)-β-linkages in (1,3;1,4)-β-D-glucooligosaccharides and had transglycosylase activity on (1,3)-β-D-glucooligosaccharides. Phylogenetic analysis indicated that Egh12 from T. terrestris and Bgh12A from A. cervinus are more related than Bgh12A and Xgh12B isolated from one strain. The X-ray structure of Bgh12A was determined with 2.17 Å resolution and compared with 3D-homology models of Egh12 and Xgh12B. The enzymes have a β-jelly roll structure with a catalytic cleft running across the protein. Comparative analysis and a docking study demonstrated the importance of endoglucanase-specific loop 1 partly covering the catalytic cleft for correct placement of the linear substrates. Variability in substrate specificity between the GH12 endoglucanases is determined by non-conservative residues in structural loops framing the catalytic cleft. A residue responsible for the thermostability of Egh12 was predicted. The key structural elements and residues described in this study may serve as potential targets for modification aimed at the improvement of enzymatic properties. Key points • Thermostable endoglucanase Egh12 from T. terrestris was produced in P. pastoris, purified, and characterized • The X-ray structure of GH12 endoglucanase Bgh12A from A. cervinus was resolved • GH12 endoglucanases, but not GH12 xyloglucanases, hydrolyze (1,3)-β-linkages in (1,3;1,4)-β-D-glucooligosaccharides</abstract><cop>Berlin/Heidelberg</cop><pub>Springer Berlin Heidelberg</pub><pmid>35129654</pmid><doi>10.1007/s00253-022-11811-7</doi><tpages>17</tpages><orcidid>https://orcid.org/0000-0003-4868-0589</orcidid></addata></record>
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subjects Ascomycota
Aspergillus
Aspergillus cervinus
Biomedical and Life Sciences
Biotechnologically Relevant Enzymes and Proteins
Biotechnology
Cellulase - metabolism
Comparative analysis
Endoglucanase
Enzymes
Function analysis
Fungi
Glucan
Homology
Life Sciences
Linkages
Microbial enzymes
Microbial Genetics and Genomics
Microbiology
Phylogeny
Physiological aspects
Pichia pastoris
Residues
Sordariales - metabolism
Structural members
Structure
Structure-function relationships
Substrate Specificity
Substrates
Substrates (Biochemistry)
Thermal stability
Thielavia terrestri
Three dimensional models
Xyloglucan
Xyloglucanase
β-Glucan
title Unusual substrate specificity in GH family 12: structure–function analysis of glucanases Bgh12A and Xgh12B from Aspergillus cervinus, and Egh12 from Thielavia terrestris
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