Site-directed mutagenesis study of the three catalytic residues of the fructosyltransferases of Lactobacillus reuteri 121
Bacterial fructosyltransferases (FTFs) are retaining-type glycosidases that belong to family 68 of glycoside hydrolases. Recently, the high-resolution 3D structure of the Bacillus subtilis levansucrase has been solved [Meng, G. and Futterer, K., Nat. Struct. Biol. 10 (2003) 935–941]. Based on this s...
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| Veröffentlicht in: | FEBS letters 2004-02, Vol.560 (1), p.131-133 |
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| creator | Ozimek, L.K van Hijum, S.A.F.T van Koningsveld, G.A van der Maarel, M.J.E.C van Geel-Schutten, G.H Dijkhuizen, L |
| description | Bacterial fructosyltransferases (FTFs) are retaining-type glycosidases that belong to family 68 of glycoside hydrolases. Recently, the high-resolution 3D structure of the
Bacillus subtilis levansucrase has been solved [Meng, G. and Futterer, K., Nat. Struct. Biol. 10 (2003) 935–941]. Based on this structure, the catalytic nucleophile, general acid/base catalyst, and transition state stabilizer were identified. However, a detailed characterization of site-directed mutants of the catalytic nucleophile has not been presented for any FTF enzyme. We have constructed site-directed mutants of the three putative catalytic residues of the
Lactobacillus reuteri 121 levansucrase and inulosucrase and characterized the mutant proteins. Changing the putative catalytic nucleophiles D272 (inulosucrase) and D249 (levansucrase) into their amido counterparts resulted in a 1.5–4×10
5 times reduction of total sucrase activity. |
| doi_str_mv | 10.1016/S0014-5793(04)00085-7 |
| format | Article |
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Bacillus subtilis levansucrase has been solved [Meng, G. and Futterer, K., Nat. Struct. Biol. 10 (2003) 935–941]. Based on this structure, the catalytic nucleophile, general acid/base catalyst, and transition state stabilizer were identified. However, a detailed characterization of site-directed mutants of the catalytic nucleophile has not been presented for any FTF enzyme. We have constructed site-directed mutants of the three putative catalytic residues of the
Lactobacillus reuteri 121 levansucrase and inulosucrase and characterized the mutant proteins. Changing the putative catalytic nucleophiles D272 (inulosucrase) and D249 (levansucrase) into their amido counterparts resulted in a 1.5–4×10
5 times reduction of total sucrase activity.</description><identifier>ISSN: 0014-5793</identifier><identifier>EISSN: 1873-3468</identifier><identifier>DOI: 10.1016/S0014-5793(04)00085-7</identifier><identifier>PMID: 14988011</identifier><language>eng</language><publisher>England: Elsevier B.V</publisher><subject>Amino Acid Motifs ; Amino Acid Sequence ; Amino Acid Substitution ; Bacillus subtilis ; Catalysis ; Catalytic residue ; Circular Dichroism ; Cloning, Molecular ; Conserved Sequence ; Escherichia coli - genetics ; Fructosyltransferase ; Gene Expression ; Genes, Bacterial ; Hexosyltransferases - chemistry ; Hexosyltransferases - genetics ; Hexosyltransferases - isolation & purification ; Hexosyltransferases - metabolism ; Inulosucrase ; Kinetics ; Lactobacillus - enzymology ; Lactobacillus - genetics ; Lactobacillus reuteri ; Levansucrase ; Molecular Sequence Data ; Mutagenesis ; Mutagenesis, Site-Directed ; Sequence Homology, Amino Acid ; Substrate Specificity</subject><ispartof>FEBS letters, 2004-02, Vol.560 (1), p.131-133</ispartof><rights>2004 Federation of European Biochemical Societies</rights><rights>FEBS Letters 560 (2004) 1873-3468 © 2015 Federation of European Biochemical Societies</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c5047-c846ad828111275abc14dc430813df303893809749d9d63cd9637043670667a33</citedby><cites>FETCH-LOGICAL-c5047-c846ad828111275abc14dc430813df303893809749d9d63cd9637043670667a33</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://onlinelibrary.wiley.com/doi/pdf/10.1016%2FS0014-5793%2804%2900085-7$$EPDF$$P50$$Gwiley$$H</linktopdf><linktohtml>$$Uhttps://www.sciencedirect.com/science/article/pii/S0014579304000857$$EHTML$$P50$$Gelsevier$$Hfree_for_read</linktohtml><link.rule.ids>314,776,780,1411,1427,3537,27901,27902,45550,45551,46384,46808,65534</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/14988011$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Ozimek, L.K</creatorcontrib><creatorcontrib>van Hijum, S.A.F.T</creatorcontrib><creatorcontrib>van Koningsveld, G.A</creatorcontrib><creatorcontrib>van der Maarel, M.J.E.C</creatorcontrib><creatorcontrib>van Geel-Schutten, G.H</creatorcontrib><creatorcontrib>Dijkhuizen, L</creatorcontrib><title>Site-directed mutagenesis study of the three catalytic residues of the fructosyltransferases of Lactobacillus reuteri 121</title><title>FEBS letters</title><addtitle>FEBS Lett</addtitle><description>Bacterial fructosyltransferases (FTFs) are retaining-type glycosidases that belong to family 68 of glycoside hydrolases. Recently, the high-resolution 3D structure of the
Bacillus subtilis levansucrase has been solved [Meng, G. and Futterer, K., Nat. Struct. Biol. 10 (2003) 935–941]. Based on this structure, the catalytic nucleophile, general acid/base catalyst, and transition state stabilizer were identified. However, a detailed characterization of site-directed mutants of the catalytic nucleophile has not been presented for any FTF enzyme. We have constructed site-directed mutants of the three putative catalytic residues of the
Lactobacillus reuteri 121 levansucrase and inulosucrase and characterized the mutant proteins. Changing the putative catalytic nucleophiles D272 (inulosucrase) and D249 (levansucrase) into their amido counterparts resulted in a 1.5–4×10
5 times reduction of total sucrase activity.</description><subject>Amino Acid Motifs</subject><subject>Amino Acid Sequence</subject><subject>Amino Acid Substitution</subject><subject>Bacillus subtilis</subject><subject>Catalysis</subject><subject>Catalytic residue</subject><subject>Circular Dichroism</subject><subject>Cloning, Molecular</subject><subject>Conserved Sequence</subject><subject>Escherichia coli - genetics</subject><subject>Fructosyltransferase</subject><subject>Gene Expression</subject><subject>Genes, Bacterial</subject><subject>Hexosyltransferases - chemistry</subject><subject>Hexosyltransferases - genetics</subject><subject>Hexosyltransferases - isolation & purification</subject><subject>Hexosyltransferases - metabolism</subject><subject>Inulosucrase</subject><subject>Kinetics</subject><subject>Lactobacillus - enzymology</subject><subject>Lactobacillus - genetics</subject><subject>Lactobacillus reuteri</subject><subject>Levansucrase</subject><subject>Molecular Sequence Data</subject><subject>Mutagenesis</subject><subject>Mutagenesis, Site-Directed</subject><subject>Sequence Homology, Amino Acid</subject><subject>Substrate Specificity</subject><issn>0014-5793</issn><issn>1873-3468</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2004</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNqNkU9P3DAQxa2KqmxpP0JRTogeAjNrJ3ZOFSD-SSv1AD1bXnsCrrIbsB1Qvj3ezdIe24Nlad7vzYzeMPYN4QQB69M7ABRlJRt-DOI7AKiqlB_YDJXkJRe12mOzP8g--xzj7wyhwuYT20fRKAWIMzbe-USl84FsIleshmQeaE3RxyKmwY1F3xbpkfILRIU1yXRj8rYIGXEDxXe9DYNNfRy7FMw6thRMnMSFyfWlsb7rhphtQ6LgC5zjF_axNV2kr7v_gP26ury_uCkXP69vL84Wpa1AyNIqURun5goR57IyS4vCWcFBIXctB64arqCRonGNq7l1Tc0lCF5LqGtpOD9gR1Pfp9A_542TXvloqevMmvohapRQVRnOYDWBNvQxBmr1U_ArE0aNoDeZ623mehOoBqG3mWuZfYe7AcNyRe6vaxdyBm4m4NV3NP5fV311eT7fKhsBxLa8mfVjakU5sRdPQUfraW1puqB2vf_Htm8YNqUl</recordid><startdate>20040227</startdate><enddate>20040227</enddate><creator>Ozimek, L.K</creator><creator>van Hijum, S.A.F.T</creator><creator>van Koningsveld, G.A</creator><creator>van der Maarel, M.J.E.C</creator><creator>van Geel-Schutten, G.H</creator><creator>Dijkhuizen, L</creator><general>Elsevier B.V</general><scope>6I.</scope><scope>AAFTH</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>C1K</scope></search><sort><creationdate>20040227</creationdate><title>Site-directed mutagenesis study of the three catalytic residues of the fructosyltransferases of Lactobacillus reuteri 121</title><author>Ozimek, L.K ; van Hijum, S.A.F.T ; van Koningsveld, G.A ; van der Maarel, M.J.E.C ; van Geel-Schutten, G.H ; Dijkhuizen, L</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c5047-c846ad828111275abc14dc430813df303893809749d9d63cd9637043670667a33</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2004</creationdate><topic>Amino Acid Motifs</topic><topic>Amino Acid Sequence</topic><topic>Amino Acid Substitution</topic><topic>Bacillus subtilis</topic><topic>Catalysis</topic><topic>Catalytic residue</topic><topic>Circular Dichroism</topic><topic>Cloning, Molecular</topic><topic>Conserved Sequence</topic><topic>Escherichia coli - genetics</topic><topic>Fructosyltransferase</topic><topic>Gene Expression</topic><topic>Genes, Bacterial</topic><topic>Hexosyltransferases - chemistry</topic><topic>Hexosyltransferases - genetics</topic><topic>Hexosyltransferases - isolation & purification</topic><topic>Hexosyltransferases - metabolism</topic><topic>Inulosucrase</topic><topic>Kinetics</topic><topic>Lactobacillus - enzymology</topic><topic>Lactobacillus - genetics</topic><topic>Lactobacillus reuteri</topic><topic>Levansucrase</topic><topic>Molecular Sequence Data</topic><topic>Mutagenesis</topic><topic>Mutagenesis, Site-Directed</topic><topic>Sequence Homology, Amino Acid</topic><topic>Substrate Specificity</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Ozimek, L.K</creatorcontrib><creatorcontrib>van Hijum, S.A.F.T</creatorcontrib><creatorcontrib>van Koningsveld, G.A</creatorcontrib><creatorcontrib>van der Maarel, M.J.E.C</creatorcontrib><creatorcontrib>van Geel-Schutten, G.H</creatorcontrib><creatorcontrib>Dijkhuizen, L</creatorcontrib><collection>ScienceDirect Open Access Titles</collection><collection>Elsevier:ScienceDirect:Open Access</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>Environmental Sciences and Pollution Management</collection><jtitle>FEBS letters</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Ozimek, L.K</au><au>van Hijum, S.A.F.T</au><au>van Koningsveld, G.A</au><au>van der Maarel, M.J.E.C</au><au>van Geel-Schutten, G.H</au><au>Dijkhuizen, L</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Site-directed mutagenesis study of the three catalytic residues of the fructosyltransferases of Lactobacillus reuteri 121</atitle><jtitle>FEBS letters</jtitle><addtitle>FEBS Lett</addtitle><date>2004-02-27</date><risdate>2004</risdate><volume>560</volume><issue>1</issue><spage>131</spage><epage>133</epage><pages>131-133</pages><issn>0014-5793</issn><eissn>1873-3468</eissn><abstract>Bacterial fructosyltransferases (FTFs) are retaining-type glycosidases that belong to family 68 of glycoside hydrolases. Recently, the high-resolution 3D structure of the
Bacillus subtilis levansucrase has been solved [Meng, G. and Futterer, K., Nat. Struct. Biol. 10 (2003) 935–941]. Based on this structure, the catalytic nucleophile, general acid/base catalyst, and transition state stabilizer were identified. However, a detailed characterization of site-directed mutants of the catalytic nucleophile has not been presented for any FTF enzyme. We have constructed site-directed mutants of the three putative catalytic residues of the
Lactobacillus reuteri 121 levansucrase and inulosucrase and characterized the mutant proteins. Changing the putative catalytic nucleophiles D272 (inulosucrase) and D249 (levansucrase) into their amido counterparts resulted in a 1.5–4×10
5 times reduction of total sucrase activity.</abstract><cop>England</cop><pub>Elsevier B.V</pub><pmid>14988011</pmid><doi>10.1016/S0014-5793(04)00085-7</doi><tpages>3</tpages><oa>free_for_read</oa></addata></record> |
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| subjects | Amino Acid Motifs Amino Acid Sequence Amino Acid Substitution Bacillus subtilis Catalysis Catalytic residue Circular Dichroism Cloning, Molecular Conserved Sequence Escherichia coli - genetics Fructosyltransferase Gene Expression Genes, Bacterial Hexosyltransferases - chemistry Hexosyltransferases - genetics Hexosyltransferases - isolation & purification Hexosyltransferases - metabolism Inulosucrase Kinetics Lactobacillus - enzymology Lactobacillus - genetics Lactobacillus reuteri Levansucrase Molecular Sequence Data Mutagenesis Mutagenesis, Site-Directed Sequence Homology, Amino Acid Substrate Specificity |
| title | Site-directed mutagenesis study of the three catalytic residues of the fructosyltransferases of Lactobacillus reuteri 121 |
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