Increasing loop flexibility affords low-temperature adaptation of a moderate thermophilic malate dehydrogenase from Geobacillus stearothermophilus
Abstract Malate dehydrogenase (MDH) catalyzes the reversible reduction of nicotinamide adenine dinucleotide from oxaloacetate to L-malate. MDH from moderate thermophilic Geobacillus stearothermophilus (gs-MDH) has high thermal stability and substrate specificity and is used as a diagnostic reagent....
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Veröffentlicht in: | Protein engineering, design and selection design and selection, 2021-02, Vol.34 |
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creator | Shimozawa, Yuya Himiyama, Tomoki Nakamura, Tsutomu Nishiya, Yoshiaki |
description | Abstract
Malate dehydrogenase (MDH) catalyzes the reversible reduction of nicotinamide adenine dinucleotide from oxaloacetate to L-malate. MDH from moderate thermophilic Geobacillus stearothermophilus (gs-MDH) has high thermal stability and substrate specificity and is used as a diagnostic reagent. In this study, gs-MDH was engineered to increase its catalytic activity at low temperatures. Based on sequential and structural comparison with lactate dehydrogenase from G. stearothermophilus, we selected G218 as a mutation site to increase the loop flexibility pivotal for MDH catalysis. The G218 mutants showed significantly higher specific activities than the wild type at low temperatures and maintained thermal stability. The crystal structure of the G218Y mutant, which had the highest catalytic efficiency among all the G218 mutants, suggested that the flexibility of the mobile loop was successfully increased by the bulky side chain. Therefore, this study demonstrated the low-temperature adaptation of MDH by facilitating conformational changes during catalysis. |
doi_str_mv | 10.1093/protein/gzab026 |
format | Article |
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Malate dehydrogenase (MDH) catalyzes the reversible reduction of nicotinamide adenine dinucleotide from oxaloacetate to L-malate. MDH from moderate thermophilic Geobacillus stearothermophilus (gs-MDH) has high thermal stability and substrate specificity and is used as a diagnostic reagent. In this study, gs-MDH was engineered to increase its catalytic activity at low temperatures. Based on sequential and structural comparison with lactate dehydrogenase from G. stearothermophilus, we selected G218 as a mutation site to increase the loop flexibility pivotal for MDH catalysis. The G218 mutants showed significantly higher specific activities than the wild type at low temperatures and maintained thermal stability. The crystal structure of the G218Y mutant, which had the highest catalytic efficiency among all the G218 mutants, suggested that the flexibility of the mobile loop was successfully increased by the bulky side chain. Therefore, this study demonstrated the low-temperature adaptation of MDH by facilitating conformational changes during catalysis.</description><identifier>ISSN: 1741-0126</identifier><identifier>EISSN: 1741-0134</identifier><identifier>DOI: 10.1093/protein/gzab026</identifier><identifier>PMID: 34850194</identifier><language>eng</language><publisher>England: Oxford University Press</publisher><subject>Geobacillus stearothermophilus - genetics ; Geobacillus stearothermophilus - metabolism ; Kinetics ; L-Lactate Dehydrogenase - metabolism ; Malate Dehydrogenase - genetics ; Malate Dehydrogenase - metabolism ; Temperature</subject><ispartof>Protein engineering, design and selection, 2021-02, Vol.34</ispartof><rights>The Author(s) 2021. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com 2021</rights><rights>The Author(s) 2021. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.</rights><lds50>peer_reviewed</lds50><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c398t-b92e43cb1ae4a27aea2905ff63665e7fd8bb576e40f8329a2e59c81ef8464dd63</citedby><cites>FETCH-LOGICAL-c398t-b92e43cb1ae4a27aea2905ff63665e7fd8bb576e40f8329a2e59c81ef8464dd63</cites><orcidid>0000-0001-5712-0669 ; 0000-0001-5252-1834</orcidid></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><link.rule.ids>314,776,780,1578,27901,27902</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/34850194$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Shimozawa, Yuya</creatorcontrib><creatorcontrib>Himiyama, Tomoki</creatorcontrib><creatorcontrib>Nakamura, Tsutomu</creatorcontrib><creatorcontrib>Nishiya, Yoshiaki</creatorcontrib><title>Increasing loop flexibility affords low-temperature adaptation of a moderate thermophilic malate dehydrogenase from Geobacillus stearothermophilus</title><title>Protein engineering, design and selection</title><addtitle>Protein Eng Des Sel</addtitle><description>Abstract
Malate dehydrogenase (MDH) catalyzes the reversible reduction of nicotinamide adenine dinucleotide from oxaloacetate to L-malate. MDH from moderate thermophilic Geobacillus stearothermophilus (gs-MDH) has high thermal stability and substrate specificity and is used as a diagnostic reagent. In this study, gs-MDH was engineered to increase its catalytic activity at low temperatures. Based on sequential and structural comparison with lactate dehydrogenase from G. stearothermophilus, we selected G218 as a mutation site to increase the loop flexibility pivotal for MDH catalysis. The G218 mutants showed significantly higher specific activities than the wild type at low temperatures and maintained thermal stability. The crystal structure of the G218Y mutant, which had the highest catalytic efficiency among all the G218 mutants, suggested that the flexibility of the mobile loop was successfully increased by the bulky side chain. Therefore, this study demonstrated the low-temperature adaptation of MDH by facilitating conformational changes during catalysis.</description><subject>Geobacillus stearothermophilus - genetics</subject><subject>Geobacillus stearothermophilus - metabolism</subject><subject>Kinetics</subject><subject>L-Lactate Dehydrogenase - metabolism</subject><subject>Malate Dehydrogenase - genetics</subject><subject>Malate Dehydrogenase - metabolism</subject><subject>Temperature</subject><issn>1741-0126</issn><issn>1741-0134</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2021</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNqFkU1r3DAQhkVIaT7ac29FxxBwVl-W7WMJbRII5JKezdga7SrIlivJtJufkV9cL7vZa04zzDzvCzMvId84u-GskasphoxuXK1foWNCn5BzXileMC7V6bEX-oxcpPTCFqLi_DM5k6ouGW_UOXl7GPuIkNy4pj6EiVqP_1znvMtbCtaGaNKy-FtkHCaMkOeIFAxMGbILIw2WAh2C2a2Q5g3GIUybRd7TAfxuZnCzNTGscYSE1MYw0DsMHfTO-znRlBGWI47COX0hnyz4hF8P9ZL8_vXz-fa-eHy6e7j98Vj0sqlz0TUClew7DqhAVIAgGlZaq6XWJVbW1F1XVhoVs7UUDQgsm77maGullTFaXpKrve_yxD8zptwOLvXoPYwY5tQKzUohGa_5gq72aB9DShFtO0U3QNy2nLW7INpDEO0hiEXx_WA-dwOaI__--QW43gNhnj50-w8kvptK</recordid><startdate>20210215</startdate><enddate>20210215</enddate><creator>Shimozawa, Yuya</creator><creator>Himiyama, Tomoki</creator><creator>Nakamura, Tsutomu</creator><creator>Nishiya, Yoshiaki</creator><general>Oxford University Press</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>7X8</scope><orcidid>https://orcid.org/0000-0001-5712-0669</orcidid><orcidid>https://orcid.org/0000-0001-5252-1834</orcidid></search><sort><creationdate>20210215</creationdate><title>Increasing loop flexibility affords low-temperature adaptation of a moderate thermophilic malate dehydrogenase from Geobacillus stearothermophilus</title><author>Shimozawa, Yuya ; Himiyama, Tomoki ; Nakamura, Tsutomu ; Nishiya, Yoshiaki</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c398t-b92e43cb1ae4a27aea2905ff63665e7fd8bb576e40f8329a2e59c81ef8464dd63</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2021</creationdate><topic>Geobacillus stearothermophilus - genetics</topic><topic>Geobacillus stearothermophilus - metabolism</topic><topic>Kinetics</topic><topic>L-Lactate Dehydrogenase - metabolism</topic><topic>Malate Dehydrogenase - genetics</topic><topic>Malate Dehydrogenase - metabolism</topic><topic>Temperature</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Shimozawa, Yuya</creatorcontrib><creatorcontrib>Himiyama, Tomoki</creatorcontrib><creatorcontrib>Nakamura, Tsutomu</creatorcontrib><creatorcontrib>Nishiya, Yoshiaki</creatorcontrib><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>MEDLINE - Academic</collection><jtitle>Protein engineering, design and selection</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Shimozawa, Yuya</au><au>Himiyama, Tomoki</au><au>Nakamura, Tsutomu</au><au>Nishiya, Yoshiaki</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Increasing loop flexibility affords low-temperature adaptation of a moderate thermophilic malate dehydrogenase from Geobacillus stearothermophilus</atitle><jtitle>Protein engineering, design and selection</jtitle><addtitle>Protein Eng Des Sel</addtitle><date>2021-02-15</date><risdate>2021</risdate><volume>34</volume><issn>1741-0126</issn><eissn>1741-0134</eissn><abstract>Abstract
Malate dehydrogenase (MDH) catalyzes the reversible reduction of nicotinamide adenine dinucleotide from oxaloacetate to L-malate. MDH from moderate thermophilic Geobacillus stearothermophilus (gs-MDH) has high thermal stability and substrate specificity and is used as a diagnostic reagent. In this study, gs-MDH was engineered to increase its catalytic activity at low temperatures. Based on sequential and structural comparison with lactate dehydrogenase from G. stearothermophilus, we selected G218 as a mutation site to increase the loop flexibility pivotal for MDH catalysis. The G218 mutants showed significantly higher specific activities than the wild type at low temperatures and maintained thermal stability. The crystal structure of the G218Y mutant, which had the highest catalytic efficiency among all the G218 mutants, suggested that the flexibility of the mobile loop was successfully increased by the bulky side chain. Therefore, this study demonstrated the low-temperature adaptation of MDH by facilitating conformational changes during catalysis.</abstract><cop>England</cop><pub>Oxford University Press</pub><pmid>34850194</pmid><doi>10.1093/protein/gzab026</doi><orcidid>https://orcid.org/0000-0001-5712-0669</orcidid><orcidid>https://orcid.org/0000-0001-5252-1834</orcidid></addata></record> |
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source | Oxford University Press Journals All Titles (1996-Current); MEDLINE; Elektronische Zeitschriftenbibliothek - Frei zugängliche E-Journals; Alma/SFX Local Collection |
subjects | Geobacillus stearothermophilus - genetics Geobacillus stearothermophilus - metabolism Kinetics L-Lactate Dehydrogenase - metabolism Malate Dehydrogenase - genetics Malate Dehydrogenase - metabolism Temperature |
title | Increasing loop flexibility affords low-temperature adaptation of a moderate thermophilic malate dehydrogenase from Geobacillus stearothermophilus |
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