From DNA sequence to improved functionality: using protein sequence comparisons to rapidly design a thermostable consensus phytase
Naturally-occurring phytases having the required level of thermostability for application in animal feeding have not been found in nature thus far. We decided to de novo construct consensus phytases using primary protein sequence comparisons. A consensus enzyme based on 13 fungal phytase sequences h...
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| Veröffentlicht in: | Protein engineering 2000-01, Vol.13 (1), p.49-57 |
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| creator | Lehmann, Martin Kostrewa, Dirk Wyss, Markus Brugger, Roland D'Arcy, Allan Pasamontes, Luis van Loon, Adolphus P.G.M. |
| description | Naturally-occurring phytases having the required level of thermostability for application in animal feeding have not been found in nature thus far. We decided to de novo construct consensus phytases using primary protein sequence comparisons. A consensus enzyme based on 13 fungal phytase sequences had normal catalytic properties, but showed an unexpected 15–22°C increase in unfolding temperature compared with each of its parents. As a first step towards understanding the molecular basis of increased heat resistance, the crystal structure of consensus phytase was determined and compared with that of Aspergillus niger phytase. Aspergillus niger phytase unfolds at much lower temperatures. In most cases, consensus residues were indeed expected, based on comparisons of both three-dimensional structures, to contribute more to phytase stabilization than non-consensus amino acids. For some consensus amino acids, predicted by structural comparisons to destabilize the protein, mutational analysis was performed. Interestingly, these consensus residues in fact increased the unfolding temperature of the consensus phytase. In summary, for fungal phytases apparently an unexpected direct link between protein sequence conservation and protein stability exists. |
| doi_str_mv | 10.1093/protein/13.1.49 |
| format | Article |
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We decided to de novo construct consensus phytases using primary protein sequence comparisons. A consensus enzyme based on 13 fungal phytase sequences had normal catalytic properties, but showed an unexpected 15–22°C increase in unfolding temperature compared with each of its parents. As a first step towards understanding the molecular basis of increased heat resistance, the crystal structure of consensus phytase was determined and compared with that of Aspergillus niger phytase. Aspergillus niger phytase unfolds at much lower temperatures. In most cases, consensus residues were indeed expected, based on comparisons of both three-dimensional structures, to contribute more to phytase stabilization than non-consensus amino acids. For some consensus amino acids, predicted by structural comparisons to destabilize the protein, mutational analysis was performed. Interestingly, these consensus residues in fact increased the unfolding temperature of the consensus phytase. In summary, for fungal phytases apparently an unexpected direct link between protein sequence conservation and protein stability exists.</description><identifier>ISSN: 0269-2139</identifier><identifier>ISSN: 1741-0126</identifier><identifier>EISSN: 1460-213X</identifier><identifier>EISSN: 1741-0134</identifier><identifier>DOI: 10.1093/protein/13.1.49</identifier><identifier>PMID: 10679530</identifier><language>eng</language><publisher>England: Oxford University Press</publisher><subject>6-Phytase - chemistry ; 6-Phytase - genetics ; 6-Phytase - metabolism ; Amino Acid Sequence ; animal feed pelleting ; Aspergillus niger ; Aspergillus niger - enzymology ; consensus protein design ; Crystallography, X-Ray ; Enzyme Stability ; Fungal Proteins - chemistry ; Fungal Proteins - genetics ; Fungal Proteins - metabolism ; increased heat stability ; Models, Molecular ; Molecular Sequence Data ; Mutation ; phytase ; phytase family ; Protein Conformation ; Protein Engineering - methods ; Recombinant Proteins - chemistry ; Recombinant Proteins - genetics ; Recombinant Proteins - metabolism ; Sequence Alignment - methods ; Sequence Homology, Amino Acid ; three-dimensional structure</subject><ispartof>Protein engineering, 2000-01, Vol.13 (1), p.49-57</ispartof><rights>Oxford University Press 2000</rights><rights>Copyright Oxford University Press(England) Jan 2000</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c562t-e1ac79d36459c81118c1ca4fe5adbfa755a488fff930a139ad241aa08e94ecb03</citedby><cites>FETCH-LOGICAL-c562t-e1ac79d36459c81118c1ca4fe5adbfa755a488fff930a139ad241aa08e94ecb03</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><link.rule.ids>314,780,784,1584,27924,27925</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/10679530$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Lehmann, Martin</creatorcontrib><creatorcontrib>Kostrewa, Dirk</creatorcontrib><creatorcontrib>Wyss, Markus</creatorcontrib><creatorcontrib>Brugger, Roland</creatorcontrib><creatorcontrib>D'Arcy, Allan</creatorcontrib><creatorcontrib>Pasamontes, Luis</creatorcontrib><creatorcontrib>van Loon, Adolphus P.G.M.</creatorcontrib><title>From DNA sequence to improved functionality: using protein sequence comparisons to rapidly design a thermostable consensus phytase</title><title>Protein engineering</title><addtitle>Protein Eng</addtitle><addtitle>Protein Eng</addtitle><description>Naturally-occurring phytases having the required level of thermostability for application in animal feeding have not been found in nature thus far. We decided to de novo construct consensus phytases using primary protein sequence comparisons. A consensus enzyme based on 13 fungal phytase sequences had normal catalytic properties, but showed an unexpected 15–22°C increase in unfolding temperature compared with each of its parents. As a first step towards understanding the molecular basis of increased heat resistance, the crystal structure of consensus phytase was determined and compared with that of Aspergillus niger phytase. Aspergillus niger phytase unfolds at much lower temperatures. In most cases, consensus residues were indeed expected, based on comparisons of both three-dimensional structures, to contribute more to phytase stabilization than non-consensus amino acids. For some consensus amino acids, predicted by structural comparisons to destabilize the protein, mutational analysis was performed. Interestingly, these consensus residues in fact increased the unfolding temperature of the consensus phytase. In summary, for fungal phytases apparently an unexpected direct link between protein sequence conservation and protein stability exists.</description><subject>6-Phytase - chemistry</subject><subject>6-Phytase - genetics</subject><subject>6-Phytase - metabolism</subject><subject>Amino Acid Sequence</subject><subject>animal feed pelleting</subject><subject>Aspergillus niger</subject><subject>Aspergillus niger - enzymology</subject><subject>consensus protein design</subject><subject>Crystallography, X-Ray</subject><subject>Enzyme Stability</subject><subject>Fungal Proteins - chemistry</subject><subject>Fungal Proteins - genetics</subject><subject>Fungal Proteins - metabolism</subject><subject>increased heat stability</subject><subject>Models, Molecular</subject><subject>Molecular Sequence Data</subject><subject>Mutation</subject><subject>phytase</subject><subject>phytase family</subject><subject>Protein Conformation</subject><subject>Protein Engineering - methods</subject><subject>Recombinant Proteins - chemistry</subject><subject>Recombinant Proteins - genetics</subject><subject>Recombinant Proteins - metabolism</subject><subject>Sequence Alignment - methods</subject><subject>Sequence Homology, Amino Acid</subject><subject>three-dimensional structure</subject><issn>0269-2139</issn><issn>1741-0126</issn><issn>1460-213X</issn><issn>1741-0134</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2000</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNqNkb1v1DAYhy1ERY_CzIYsBgak3L2O7SRmq_rBUZ34ECCdulg-x2ldEjvYDuJW_nIS5QSIpUz28Pye9wuhZwSWBARd9cEnY92K0CVZMvEALQgrIMsJ3T5EC8gLMf3FMXoc4x0AVCDyR-iYQFEKTmGBfl4G3-Hzd6c4mm-Dcdrg5LHtRvF3U-NmcDpZ71Rr0_41HqJ1N_hQ9E9C-65XwUbv4pQOqrd1u8e1ifbGYYXTrQmdj0nt2gl20bg4RNzf7pOK5gk6alQbzdPDe4K-XF58Pltnm_dv3p6dbjLNizxlhihdipoWjAtdEUIqTbRijeGq3jWq5FyxqmqaRlBQ48yqzhlRCiojmNE7oCfo5ewd-x8bj0l2NmrTtsoZP0RZggCeC3YvmEPFWFHxe0FScoCSTaVf_APe-SGMWx1lOWdVDlyM0GqGdPAxBtPIPthOhb0kIKdry8PiJaGSSDYlnh-0w64z9V_8fN4ReDUDfuj_w5bNsI3J_PiNq_BVFiUtuVxvr-Xm00d6vb36INf0FzQxx_g</recordid><startdate>200001</startdate><enddate>200001</enddate><creator>Lehmann, Martin</creator><creator>Kostrewa, Dirk</creator><creator>Wyss, Markus</creator><creator>Brugger, Roland</creator><creator>D'Arcy, Allan</creator><creator>Pasamontes, Luis</creator><creator>van Loon, Adolphus P.G.M.</creator><general>Oxford University Press</general><general>Oxford Publishing Limited (England)</general><scope>BSCLL</scope><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>7QL</scope><scope>7QO</scope><scope>7QP</scope><scope>7TK</scope><scope>7TM</scope><scope>7U9</scope><scope>8FD</scope><scope>C1K</scope><scope>FR3</scope><scope>H94</scope><scope>K9.</scope><scope>M7N</scope><scope>P64</scope><scope>7X8</scope></search><sort><creationdate>200001</creationdate><title>From DNA sequence to improved functionality: using protein sequence comparisons to rapidly design a thermostable consensus phytase</title><author>Lehmann, Martin ; Kostrewa, Dirk ; Wyss, Markus ; Brugger, Roland ; D'Arcy, Allan ; Pasamontes, Luis ; van Loon, Adolphus P.G.M.</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c562t-e1ac79d36459c81118c1ca4fe5adbfa755a488fff930a139ad241aa08e94ecb03</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2000</creationdate><topic>6-Phytase - chemistry</topic><topic>6-Phytase - genetics</topic><topic>6-Phytase - metabolism</topic><topic>Amino Acid Sequence</topic><topic>animal feed pelleting</topic><topic>Aspergillus niger</topic><topic>Aspergillus niger - enzymology</topic><topic>consensus protein design</topic><topic>Crystallography, X-Ray</topic><topic>Enzyme Stability</topic><topic>Fungal Proteins - chemistry</topic><topic>Fungal Proteins - genetics</topic><topic>Fungal Proteins - metabolism</topic><topic>increased heat stability</topic><topic>Models, Molecular</topic><topic>Molecular Sequence Data</topic><topic>Mutation</topic><topic>phytase</topic><topic>phytase family</topic><topic>Protein Conformation</topic><topic>Protein Engineering - methods</topic><topic>Recombinant Proteins - chemistry</topic><topic>Recombinant Proteins - genetics</topic><topic>Recombinant Proteins - metabolism</topic><topic>Sequence Alignment - methods</topic><topic>Sequence Homology, Amino Acid</topic><topic>three-dimensional structure</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Lehmann, Martin</creatorcontrib><creatorcontrib>Kostrewa, Dirk</creatorcontrib><creatorcontrib>Wyss, Markus</creatorcontrib><creatorcontrib>Brugger, Roland</creatorcontrib><creatorcontrib>D'Arcy, Allan</creatorcontrib><creatorcontrib>Pasamontes, Luis</creatorcontrib><creatorcontrib>van Loon, Adolphus P.G.M.</creatorcontrib><collection>Istex</collection><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>Bacteriology Abstracts (Microbiology B)</collection><collection>Biotechnology Research Abstracts</collection><collection>Calcium & Calcified Tissue Abstracts</collection><collection>Neurosciences Abstracts</collection><collection>Nucleic Acids Abstracts</collection><collection>Virology and AIDS Abstracts</collection><collection>Technology Research Database</collection><collection>Environmental Sciences and Pollution Management</collection><collection>Engineering Research Database</collection><collection>AIDS and Cancer Research Abstracts</collection><collection>ProQuest Health & Medical Complete (Alumni)</collection><collection>Algology Mycology and Protozoology Abstracts (Microbiology C)</collection><collection>Biotechnology and BioEngineering Abstracts</collection><collection>MEDLINE - Academic</collection><jtitle>Protein engineering</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Lehmann, Martin</au><au>Kostrewa, Dirk</au><au>Wyss, Markus</au><au>Brugger, Roland</au><au>D'Arcy, Allan</au><au>Pasamontes, Luis</au><au>van Loon, Adolphus P.G.M.</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>From DNA sequence to improved functionality: using protein sequence comparisons to rapidly design a thermostable consensus phytase</atitle><jtitle>Protein engineering</jtitle><stitle>Protein Eng</stitle><addtitle>Protein Eng</addtitle><date>2000-01</date><risdate>2000</risdate><volume>13</volume><issue>1</issue><spage>49</spage><epage>57</epage><pages>49-57</pages><issn>0269-2139</issn><issn>1741-0126</issn><eissn>1460-213X</eissn><eissn>1741-0134</eissn><abstract>Naturally-occurring phytases having the required level of thermostability for application in animal feeding have not been found in nature thus far. We decided to de novo construct consensus phytases using primary protein sequence comparisons. A consensus enzyme based on 13 fungal phytase sequences had normal catalytic properties, but showed an unexpected 15–22°C increase in unfolding temperature compared with each of its parents. As a first step towards understanding the molecular basis of increased heat resistance, the crystal structure of consensus phytase was determined and compared with that of Aspergillus niger phytase. Aspergillus niger phytase unfolds at much lower temperatures. In most cases, consensus residues were indeed expected, based on comparisons of both three-dimensional structures, to contribute more to phytase stabilization than non-consensus amino acids. For some consensus amino acids, predicted by structural comparisons to destabilize the protein, mutational analysis was performed. Interestingly, these consensus residues in fact increased the unfolding temperature of the consensus phytase. In summary, for fungal phytases apparently an unexpected direct link between protein sequence conservation and protein stability exists.</abstract><cop>England</cop><pub>Oxford University Press</pub><pmid>10679530</pmid><doi>10.1093/protein/13.1.49</doi><tpages>9</tpages><oa>free_for_read</oa></addata></record> |
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| source | MEDLINE; Oxford University Press Journals All Titles (1996-Current); EZB-FREE-00999 freely available EZB journals |
| subjects | 6-Phytase - chemistry 6-Phytase - genetics 6-Phytase - metabolism Amino Acid Sequence animal feed pelleting Aspergillus niger Aspergillus niger - enzymology consensus protein design Crystallography, X-Ray Enzyme Stability Fungal Proteins - chemistry Fungal Proteins - genetics Fungal Proteins - metabolism increased heat stability Models, Molecular Molecular Sequence Data Mutation phytase phytase family Protein Conformation Protein Engineering - methods Recombinant Proteins - chemistry Recombinant Proteins - genetics Recombinant Proteins - metabolism Sequence Alignment - methods Sequence Homology, Amino Acid three-dimensional structure |
| title | From DNA sequence to improved functionality: using protein sequence comparisons to rapidly design a thermostable consensus phytase |
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