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Saturation mutagenesis reveals the importance of residues αR145 and αF146 of penicillin acylase in the synthesis of β-lactam antibiotics
Penicillin acylase (PA) from Escherichia coli can catalyze the coupling of an acyl group to penicillin- and cephalosporin-derived β-lactam nuclei, a conversion that can be used for the industrial synthesis of β-lactam antibiotics. The modest synthetic properties of the wild-type enzyme make it desir...
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| Published in: | Journal of biotechnology 2008, Vol.133 (1), p.18-26 |
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| container_end_page | 26 |
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| container_title | Journal of biotechnology |
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| creator | Jager, Simon A.W. Shapovalova, Irina V. Jekel, Peter A. Alkema, Wynand B.L. Švedas, Vytas K. Janssen, Dick B. |
| description | Penicillin acylase (PA) from
Escherichia coli can catalyze the coupling of an acyl group to penicillin- and cephalosporin-derived β-lactam nuclei, a conversion that can be used for the industrial synthesis of β-lactam antibiotics. The modest synthetic properties of the wild-type enzyme make it desirable to engineer improved mutants. Analysis of the crystal structure of PA has shown that residues αR145 and αF146 undergo extensive repositioning upon binding of large ligands to the active site, suggesting that these residues may be good targets for mutagenesis aimed at improving the catalytic performance of PA. Therefore, site-saturation mutagenesis was performed on both positions and a complete set of all 38 variants was subjected to rapid HPLC screening for improved ampicillin synthesis. Not less than 33 mutants showed improved synthesis, indicating the importance of the mutated residues in PA-catalyzed acyl transfer kinetics. In several mutants at low substrate concentrations, the maximum level of ampicillin production was increased up to 1.5-fold, and the ratio of the synthetic rate over the hydrolytic rate was increased 5–15-fold. Moreover, due to increased tendency of the acyl–enzyme intermediate to react with β-lactam nucleophile instead of water, mutants αR145G, αR145S and αR145L demonstrated an enhanced synthetic yield over wild-type PA at high substrate concentrations. This was accompanied by an increased conversion of 6-APA to ampicillin as well as a decreased undesirable hydrolysis of the acyl donor. Therefore, these mutants are interesting candidates for the enzymatic production of semi-synthetic β-lactam antibiotics. |
| doi_str_mv | 10.1016/j.jbiotec.2007.08.039 |
| format | article |
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Escherichia coli can catalyze the coupling of an acyl group to penicillin- and cephalosporin-derived β-lactam nuclei, a conversion that can be used for the industrial synthesis of β-lactam antibiotics. The modest synthetic properties of the wild-type enzyme make it desirable to engineer improved mutants. Analysis of the crystal structure of PA has shown that residues αR145 and αF146 undergo extensive repositioning upon binding of large ligands to the active site, suggesting that these residues may be good targets for mutagenesis aimed at improving the catalytic performance of PA. Therefore, site-saturation mutagenesis was performed on both positions and a complete set of all 38 variants was subjected to rapid HPLC screening for improved ampicillin synthesis. Not less than 33 mutants showed improved synthesis, indicating the importance of the mutated residues in PA-catalyzed acyl transfer kinetics. In several mutants at low substrate concentrations, the maximum level of ampicillin production was increased up to 1.5-fold, and the ratio of the synthetic rate over the hydrolytic rate was increased 5–15-fold. Moreover, due to increased tendency of the acyl–enzyme intermediate to react with β-lactam nucleophile instead of water, mutants αR145G, αR145S and αR145L demonstrated an enhanced synthetic yield over wild-type PA at high substrate concentrations. This was accompanied by an increased conversion of 6-APA to ampicillin as well as a decreased undesirable hydrolysis of the acyl donor. Therefore, these mutants are interesting candidates for the enzymatic production of semi-synthetic β-lactam antibiotics.</description><identifier>ISSN: 0168-1656</identifier><identifier>EISSN: 1873-4863</identifier><identifier>DOI: 10.1016/j.jbiotec.2007.08.039</identifier><identifier>CODEN: JBITD4</identifier><language>eng ; rus</language><publisher>Lausanne: Elsevier B.V</publisher><subject>Ampicillin ; Antibiotic synthesis ; Directed evolution ; Escherichia coli ; Mutagenesis ; Penicillin acylase ; Protein engineering</subject><ispartof>Journal of biotechnology, 2008, Vol.133 (1), p.18-26</ispartof><rights>2007 Elsevier B.V.</rights><rights>2008 INIST-CNRS</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c441t-50e3a5acc8def297a8f4b9b6c40644ed240cafae9ef8dd2ea390b81bf0e3f9ce3</citedby><cites>FETCH-LOGICAL-c441t-50e3a5acc8def297a8f4b9b6c40644ed240cafae9ef8dd2ea390b81bf0e3f9ce3</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><link.rule.ids>315,786,790,4043,27956,27957,27958</link.rule.ids><backlink>$$Uhttp://pascal-francis.inist.fr/vibad/index.php?action=getRecordDetail&idt=19874084$$DView record in Pascal Francis$$Hfree_for_read</backlink></links><search><creatorcontrib>Jager, Simon A.W.</creatorcontrib><creatorcontrib>Shapovalova, Irina V.</creatorcontrib><creatorcontrib>Jekel, Peter A.</creatorcontrib><creatorcontrib>Alkema, Wynand B.L.</creatorcontrib><creatorcontrib>Švedas, Vytas K.</creatorcontrib><creatorcontrib>Janssen, Dick B.</creatorcontrib><title>Saturation mutagenesis reveals the importance of residues αR145 and αF146 of penicillin acylase in the synthesis of β-lactam antibiotics</title><title>Journal of biotechnology</title><description>Penicillin acylase (PA) from
Escherichia coli can catalyze the coupling of an acyl group to penicillin- and cephalosporin-derived β-lactam nuclei, a conversion that can be used for the industrial synthesis of β-lactam antibiotics. The modest synthetic properties of the wild-type enzyme make it desirable to engineer improved mutants. Analysis of the crystal structure of PA has shown that residues αR145 and αF146 undergo extensive repositioning upon binding of large ligands to the active site, suggesting that these residues may be good targets for mutagenesis aimed at improving the catalytic performance of PA. Therefore, site-saturation mutagenesis was performed on both positions and a complete set of all 38 variants was subjected to rapid HPLC screening for improved ampicillin synthesis. Not less than 33 mutants showed improved synthesis, indicating the importance of the mutated residues in PA-catalyzed acyl transfer kinetics. In several mutants at low substrate concentrations, the maximum level of ampicillin production was increased up to 1.5-fold, and the ratio of the synthetic rate over the hydrolytic rate was increased 5–15-fold. Moreover, due to increased tendency of the acyl–enzyme intermediate to react with β-lactam nucleophile instead of water, mutants αR145G, αR145S and αR145L demonstrated an enhanced synthetic yield over wild-type PA at high substrate concentrations. This was accompanied by an increased conversion of 6-APA to ampicillin as well as a decreased undesirable hydrolysis of the acyl donor. Therefore, these mutants are interesting candidates for the enzymatic production of semi-synthetic β-lactam antibiotics.</description><subject>Ampicillin</subject><subject>Antibiotic synthesis</subject><subject>Directed evolution</subject><subject>Escherichia coli</subject><subject>Mutagenesis</subject><subject>Penicillin acylase</subject><subject>Protein engineering</subject><issn>0168-1656</issn><issn>1873-4863</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2008</creationdate><recordtype>article</recordtype><recordid>eNqFkUGO1DAQRS0EEs3AERDZwC6hnDhpZ4XQiAGkkZAYZm1VnPLgVuI0tnukPgOngYPMmajQLbFkVZbq_V_lX0K8lFBJkN3bXbUb_JLJVjXAtgJdQdM_Ehupt02pdNc8FhvmdCm7tnsqnqW0AwDVt3Ijft5gPkTMfgnFfMh4R4GST0Wke8IpFfk7FX7eLzFjsFQsjjvJjwdKxcOvr1K1BYaRn1dSdWt3T8FbP00-FGiPEyaWh78u6Ri4rN6MPfwuJ7QZZ5Znv27vbXounjieSS_O9ULcXn34dvmpvP7y8fPl--vSKiVz2QI12KK1eiRX91vUTg390FkFnVI01gosOqSenB7HmrDpYdBycKxzvaXmQrw5-e7j8oN_ks3sk6VpwkDLIZkatKpbpRlsT6CNS0qRnNlHP2M8Gglmjd7szDl6s0ZvQBuOnnWvzwMwWZxc5Ox8-ifu9VbxDOZenTiHi8G7yMztTQ2yAeCrqa5h4t2JIM7j3lM0yXriS4w-ks1mXPx_dvkDXdirYA</recordid><startdate>2008</startdate><enddate>2008</enddate><creator>Jager, Simon A.W.</creator><creator>Shapovalova, Irina V.</creator><creator>Jekel, Peter A.</creator><creator>Alkema, Wynand B.L.</creator><creator>Švedas, Vytas K.</creator><creator>Janssen, Dick B.</creator><general>Elsevier B.V</general><general>[New York, NY]: Elsevier</general><general>Elsevier</general><scope>FBQ</scope><scope>IQODW</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>7QL</scope><scope>7QO</scope><scope>8FD</scope><scope>C1K</scope><scope>FR3</scope><scope>P64</scope></search><sort><creationdate>2008</creationdate><title>Saturation mutagenesis reveals the importance of residues αR145 and αF146 of penicillin acylase in the synthesis of β-lactam antibiotics</title><author>Jager, Simon A.W. ; Shapovalova, Irina V. ; Jekel, Peter A. ; Alkema, Wynand B.L. ; Švedas, Vytas K. ; Janssen, Dick B.</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c441t-50e3a5acc8def297a8f4b9b6c40644ed240cafae9ef8dd2ea390b81bf0e3f9ce3</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng ; rus</language><creationdate>2008</creationdate><topic>Ampicillin</topic><topic>Antibiotic synthesis</topic><topic>Directed evolution</topic><topic>Escherichia coli</topic><topic>Mutagenesis</topic><topic>Penicillin acylase</topic><topic>Protein engineering</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Jager, Simon A.W.</creatorcontrib><creatorcontrib>Shapovalova, Irina V.</creatorcontrib><creatorcontrib>Jekel, Peter A.</creatorcontrib><creatorcontrib>Alkema, Wynand B.L.</creatorcontrib><creatorcontrib>Švedas, Vytas K.</creatorcontrib><creatorcontrib>Janssen, Dick B.</creatorcontrib><collection>AGRIS</collection><collection>Pascal-Francis</collection><collection>CrossRef</collection><collection>Bacteriology Abstracts (Microbiology B)</collection><collection>Biotechnology Research Abstracts</collection><collection>Technology Research Database</collection><collection>Environmental Sciences and Pollution Management</collection><collection>Engineering Research Database</collection><collection>Biotechnology and BioEngineering Abstracts</collection><jtitle>Journal of biotechnology</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Jager, Simon A.W.</au><au>Shapovalova, Irina V.</au><au>Jekel, Peter A.</au><au>Alkema, Wynand B.L.</au><au>Švedas, Vytas K.</au><au>Janssen, Dick B.</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Saturation mutagenesis reveals the importance of residues αR145 and αF146 of penicillin acylase in the synthesis of β-lactam antibiotics</atitle><jtitle>Journal of biotechnology</jtitle><date>2008</date><risdate>2008</risdate><volume>133</volume><issue>1</issue><spage>18</spage><epage>26</epage><pages>18-26</pages><issn>0168-1656</issn><eissn>1873-4863</eissn><coden>JBITD4</coden><notes>http://dx.doi.org/10.1016/j.jbiotec.2007.08.039</notes><notes>ObjectType-Article-1</notes><notes>SourceType-Scholarly Journals-1</notes><notes>ObjectType-Feature-2</notes><notes>content type line 23</notes><abstract>Penicillin acylase (PA) from
Escherichia coli can catalyze the coupling of an acyl group to penicillin- and cephalosporin-derived β-lactam nuclei, a conversion that can be used for the industrial synthesis of β-lactam antibiotics. The modest synthetic properties of the wild-type enzyme make it desirable to engineer improved mutants. Analysis of the crystal structure of PA has shown that residues αR145 and αF146 undergo extensive repositioning upon binding of large ligands to the active site, suggesting that these residues may be good targets for mutagenesis aimed at improving the catalytic performance of PA. Therefore, site-saturation mutagenesis was performed on both positions and a complete set of all 38 variants was subjected to rapid HPLC screening for improved ampicillin synthesis. Not less than 33 mutants showed improved synthesis, indicating the importance of the mutated residues in PA-catalyzed acyl transfer kinetics. In several mutants at low substrate concentrations, the maximum level of ampicillin production was increased up to 1.5-fold, and the ratio of the synthetic rate over the hydrolytic rate was increased 5–15-fold. Moreover, due to increased tendency of the acyl–enzyme intermediate to react with β-lactam nucleophile instead of water, mutants αR145G, αR145S and αR145L demonstrated an enhanced synthetic yield over wild-type PA at high substrate concentrations. This was accompanied by an increased conversion of 6-APA to ampicillin as well as a decreased undesirable hydrolysis of the acyl donor. Therefore, these mutants are interesting candidates for the enzymatic production of semi-synthetic β-lactam antibiotics.</abstract><cop>Lausanne</cop><cop>Amsterdam</cop><cop>New York, NY</cop><pub>Elsevier B.V</pub><doi>10.1016/j.jbiotec.2007.08.039</doi><tpages>9</tpages><oa>free_for_read</oa></addata></record> |
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| language | eng ; rus |
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| source | ScienceDirect Freedom Collection 2022-2024 |
| subjects | Ampicillin Antibiotic synthesis Directed evolution Escherichia coli Mutagenesis Penicillin acylase Protein engineering |
| title | Saturation mutagenesis reveals the importance of residues αR145 and αF146 of penicillin acylase in the synthesis of β-lactam antibiotics |
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