Screening, Molecular Cloning, and Biochemical Characterization of an Alcohol Dehydrogenase from Pichia pastoris Useful for the Kinetic Resolution of a Racemic β-Hydroxy-β-trifluoromethyl Ketone
The stereoselective synthesis of chiral 1,3‐diols with the aid of biocatalysts is an attractive tool in organic chemistry. Besides the reduction of diketones, an alternative approach consists of the stereoselective reduction of β‐hydroxy ketones (aldols). Thus, we screened for an alcohol dehydrogena...
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Veröffentlicht in: | Chembiochem : a European journal of chemical biology 2016-07, Vol.17 (14), p.1349-1358 |
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creator | Bulut, Dalia Duangdee, Nongnaphat Gröger, Harald Berkessel, Albrecht Hummel, Werner |
description | The stereoselective synthesis of chiral 1,3‐diols with the aid of biocatalysts is an attractive tool in organic chemistry. Besides the reduction of diketones, an alternative approach consists of the stereoselective reduction of β‐hydroxy ketones (aldols). Thus, we screened for an alcohol dehydrogenase (ADH) that would selectively reduce a β‐hydroxy‐β‐trifluoromethyl ketone. One potential starting material for this process is readily available by aldol addition of acetone to 2,2,2‐trifluoroacetophenone. Over 200 strains were screened, and only a few yeast strains showed stereoselective reduction activities. The enzyme responsible for the reduction of the β‐hydroxy‐β‐trifluoromethyl ketone was identified after purification and subsequent MALDI‐TOF mass spectrometric analysis. As a result, a new NADP+‐dependent ADH from Pichia pastoris (PPADH) was identified and confirmed to be capable of stereospecific and diastereoselective reduction of the β‐hydroxy‐β‐trifluoromethyl ketone to its corresponding 1,3‐diol. The gene encoding PPADH was cloned and heterologously expressed in Escherichia coli BL21(DE3). To determine the influence of an N‐ or C‐terminal His‐tag fusion, three different recombinant plasmids were constructed. Interestingly, the variant with the N‐terminal His‐tag showed the highest activity; consequently, this variant was purified and characterized. Kinetic parameters and the dependency of activity on pH and temperature were determined. PPADH shows a substrate preference for the reduction of linear and branched aliphatic aldehydes. Surprisingly, the enzyme shows no comparable activity towards ketones other than the β‐hydroxy‐β‐trifluoromethyl ketone.
Find the needle in the haystack! A new alcohol dehydrogenase—PPADH from P. pastoris—was identified. It is able to distinguish between the two enantiomers of the racemic β‐hydroxy‐β‐trifluoromethyl ketone 1 by efficiently reducing the S form to the corresponding stereochemically uniform 1,3‐diol (S,S)‐2. |
doi_str_mv | 10.1002/cbic.201600101 |
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Find the needle in the haystack! A new alcohol dehydrogenase—PPADH from P. pastoris—was identified. It is able to distinguish between the two enantiomers of the racemic β‐hydroxy‐β‐trifluoromethyl ketone 1 by efficiently reducing the S form to the corresponding stereochemically uniform 1,3‐diol (S,S)‐2.</description><identifier>ISSN: 1439-4227</identifier><identifier>EISSN: 1439-7633</identifier><identifier>DOI: 10.1002/cbic.201600101</identifier><identifier>PMID: 27123855</identifier><language>eng</language><publisher>Germany: Blackwell Publishing Ltd</publisher><subject>Alcohol Dehydrogenase - genetics ; Alcohol Dehydrogenase - metabolism ; alcohol dehydrogenases ; asymmetric reduction and oxidation ; biocatalysis ; Cloning, Molecular - methods ; diols ; enantioselectivity ; Escherichia coli ; Hydrogen-Ion Concentration ; Ketones - chemistry ; Ketones - isolation & purification ; kinetic resolution ; Kinetics ; Oxidoreductases ; Pichia - enzymology ; Pichia pastoris ; Stereoisomerism ; Substrate Specificity ; Temperature</subject><ispartof>Chembiochem : a European journal of chemical biology, 2016-07, Vol.17 (14), p.1349-1358</ispartof><rights>2016 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim</rights><rights>2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.</rights><lds50>peer_reviewed</lds50><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c4861-69831f0f6c155dcb354c53fac326914d878c3eaecbf271d4b81bcae29baca2e43</citedby><cites>FETCH-LOGICAL-c4861-69831f0f6c155dcb354c53fac326914d878c3eaecbf271d4b81bcae29baca2e43</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://onlinelibrary.wiley.com/doi/pdf/10.1002%2Fcbic.201600101$$EPDF$$P50$$Gwiley$$H</linktopdf><linktohtml>$$Uhttps://onlinelibrary.wiley.com/doi/full/10.1002%2Fcbic.201600101$$EHTML$$P50$$Gwiley$$H</linktohtml><link.rule.ids>314,780,784,1417,27924,27925,45574,45575</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/27123855$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Bulut, Dalia</creatorcontrib><creatorcontrib>Duangdee, Nongnaphat</creatorcontrib><creatorcontrib>Gröger, Harald</creatorcontrib><creatorcontrib>Berkessel, Albrecht</creatorcontrib><creatorcontrib>Hummel, Werner</creatorcontrib><title>Screening, Molecular Cloning, and Biochemical Characterization of an Alcohol Dehydrogenase from Pichia pastoris Useful for the Kinetic Resolution of a Racemic β-Hydroxy-β-trifluoromethyl Ketone</title><title>Chembiochem : a European journal of chemical biology</title><addtitle>ChemBioChem</addtitle><description>The stereoselective synthesis of chiral 1,3‐diols with the aid of biocatalysts is an attractive tool in organic chemistry. Besides the reduction of diketones, an alternative approach consists of the stereoselective reduction of β‐hydroxy ketones (aldols). Thus, we screened for an alcohol dehydrogenase (ADH) that would selectively reduce a β‐hydroxy‐β‐trifluoromethyl ketone. One potential starting material for this process is readily available by aldol addition of acetone to 2,2,2‐trifluoroacetophenone. Over 200 strains were screened, and only a few yeast strains showed stereoselective reduction activities. The enzyme responsible for the reduction of the β‐hydroxy‐β‐trifluoromethyl ketone was identified after purification and subsequent MALDI‐TOF mass spectrometric analysis. As a result, a new NADP+‐dependent ADH from Pichia pastoris (PPADH) was identified and confirmed to be capable of stereospecific and diastereoselective reduction of the β‐hydroxy‐β‐trifluoromethyl ketone to its corresponding 1,3‐diol. The gene encoding PPADH was cloned and heterologously expressed in Escherichia coli BL21(DE3). To determine the influence of an N‐ or C‐terminal His‐tag fusion, three different recombinant plasmids were constructed. Interestingly, the variant with the N‐terminal His‐tag showed the highest activity; consequently, this variant was purified and characterized. Kinetic parameters and the dependency of activity on pH and temperature were determined. PPADH shows a substrate preference for the reduction of linear and branched aliphatic aldehydes. Surprisingly, the enzyme shows no comparable activity towards ketones other than the β‐hydroxy‐β‐trifluoromethyl ketone.
Find the needle in the haystack! A new alcohol dehydrogenase—PPADH from P. pastoris—was identified. It is able to distinguish between the two enantiomers of the racemic β‐hydroxy‐β‐trifluoromethyl ketone 1 by efficiently reducing the S form to the corresponding stereochemically uniform 1,3‐diol (S,S)‐2.</description><subject>Alcohol Dehydrogenase - genetics</subject><subject>Alcohol Dehydrogenase - metabolism</subject><subject>alcohol dehydrogenases</subject><subject>asymmetric reduction and oxidation</subject><subject>biocatalysis</subject><subject>Cloning, Molecular - methods</subject><subject>diols</subject><subject>enantioselectivity</subject><subject>Escherichia coli</subject><subject>Hydrogen-Ion Concentration</subject><subject>Ketones - chemistry</subject><subject>Ketones - isolation & purification</subject><subject>kinetic resolution</subject><subject>Kinetics</subject><subject>Oxidoreductases</subject><subject>Pichia - enzymology</subject><subject>Pichia pastoris</subject><subject>Stereoisomerism</subject><subject>Substrate Specificity</subject><subject>Temperature</subject><issn>1439-4227</issn><issn>1439-7633</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2016</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNqNkctu1DAUhiMEoqWwZYm8ZEEGO06cZNmm0FYtt14EO8s5OW4MTjzYiWh4LF6BPc9ERhlG7GDlI-v7Px_5j6KnjK4YpclLqA2sEsoEpYyye9E-S3kZ54Lz-9s5TZJ8L3oUwmdKaSk4exjtJTlLeJFl-9HPK_CIvelvX5A3ziKMVnlSWbdcqb4hR8ZBi50BZUnVKq9gQG--q8G4njg9M-TQgmudJcfYTo13t9irgER715H3BlqjyFqFwXkTyE1APVqinSdDi-Tc9DgYIJcYnB13SnKpYPMk-fUjPt0o76Z4HgdvtB3d7MWhnSw5x8H1-Dh6oJUN-GR7HkQ3r19dV6fxxbuTs-rwIoa0ECwWZcGZploAy7IGap6lkHGtgCeiZGlT5AVwVAi1nv-nSeuC1aAwKWsFKsGUH0TPF-_au68jhkF2JgBaq3p0Y5CsoIVIS8r-C81yMW8kZnS1oOBdCB61XHvTKT9JRuWmY7npWO46ngPPtu6x7rDZ4X9KnYFyAb4Zi9M_dLI6Oqv-lsdL1oQB73ZZ5b9IkfM8kx_fnkhapdXV9YdPMuG_AbRYx8I</recordid><startdate>20160715</startdate><enddate>20160715</enddate><creator>Bulut, Dalia</creator><creator>Duangdee, Nongnaphat</creator><creator>Gröger, Harald</creator><creator>Berkessel, Albrecht</creator><creator>Hummel, Werner</creator><general>Blackwell Publishing Ltd</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>7X8</scope><scope>7QO</scope><scope>8FD</scope><scope>FR3</scope><scope>P64</scope></search><sort><creationdate>20160715</creationdate><title>Screening, Molecular Cloning, and Biochemical Characterization of an Alcohol Dehydrogenase from Pichia pastoris Useful for the Kinetic Resolution of a Racemic β-Hydroxy-β-trifluoromethyl Ketone</title><author>Bulut, Dalia ; Duangdee, Nongnaphat ; Gröger, Harald ; Berkessel, Albrecht ; Hummel, Werner</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c4861-69831f0f6c155dcb354c53fac326914d878c3eaecbf271d4b81bcae29baca2e43</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2016</creationdate><topic>Alcohol Dehydrogenase - genetics</topic><topic>Alcohol Dehydrogenase - metabolism</topic><topic>alcohol dehydrogenases</topic><topic>asymmetric reduction and oxidation</topic><topic>biocatalysis</topic><topic>Cloning, Molecular - methods</topic><topic>diols</topic><topic>enantioselectivity</topic><topic>Escherichia coli</topic><topic>Hydrogen-Ion Concentration</topic><topic>Ketones - chemistry</topic><topic>Ketones - isolation & purification</topic><topic>kinetic resolution</topic><topic>Kinetics</topic><topic>Oxidoreductases</topic><topic>Pichia - enzymology</topic><topic>Pichia pastoris</topic><topic>Stereoisomerism</topic><topic>Substrate Specificity</topic><topic>Temperature</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Bulut, Dalia</creatorcontrib><creatorcontrib>Duangdee, Nongnaphat</creatorcontrib><creatorcontrib>Gröger, Harald</creatorcontrib><creatorcontrib>Berkessel, Albrecht</creatorcontrib><creatorcontrib>Hummel, Werner</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>MEDLINE - Academic</collection><collection>Biotechnology Research Abstracts</collection><collection>Technology Research Database</collection><collection>Engineering Research Database</collection><collection>Biotechnology and BioEngineering Abstracts</collection><jtitle>Chembiochem : a European journal of chemical biology</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Bulut, Dalia</au><au>Duangdee, Nongnaphat</au><au>Gröger, Harald</au><au>Berkessel, Albrecht</au><au>Hummel, Werner</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Screening, Molecular Cloning, and Biochemical Characterization of an Alcohol Dehydrogenase from Pichia pastoris Useful for the Kinetic Resolution of a Racemic β-Hydroxy-β-trifluoromethyl Ketone</atitle><jtitle>Chembiochem : a European journal of chemical biology</jtitle><addtitle>ChemBioChem</addtitle><date>2016-07-15</date><risdate>2016</risdate><volume>17</volume><issue>14</issue><spage>1349</spage><epage>1358</epage><pages>1349-1358</pages><issn>1439-4227</issn><eissn>1439-7633</eissn><abstract>The stereoselective synthesis of chiral 1,3‐diols with the aid of biocatalysts is an attractive tool in organic chemistry. Besides the reduction of diketones, an alternative approach consists of the stereoselective reduction of β‐hydroxy ketones (aldols). Thus, we screened for an alcohol dehydrogenase (ADH) that would selectively reduce a β‐hydroxy‐β‐trifluoromethyl ketone. One potential starting material for this process is readily available by aldol addition of acetone to 2,2,2‐trifluoroacetophenone. Over 200 strains were screened, and only a few yeast strains showed stereoselective reduction activities. The enzyme responsible for the reduction of the β‐hydroxy‐β‐trifluoromethyl ketone was identified after purification and subsequent MALDI‐TOF mass spectrometric analysis. As a result, a new NADP+‐dependent ADH from Pichia pastoris (PPADH) was identified and confirmed to be capable of stereospecific and diastereoselective reduction of the β‐hydroxy‐β‐trifluoromethyl ketone to its corresponding 1,3‐diol. The gene encoding PPADH was cloned and heterologously expressed in Escherichia coli BL21(DE3). To determine the influence of an N‐ or C‐terminal His‐tag fusion, three different recombinant plasmids were constructed. Interestingly, the variant with the N‐terminal His‐tag showed the highest activity; consequently, this variant was purified and characterized. Kinetic parameters and the dependency of activity on pH and temperature were determined. PPADH shows a substrate preference for the reduction of linear and branched aliphatic aldehydes. Surprisingly, the enzyme shows no comparable activity towards ketones other than the β‐hydroxy‐β‐trifluoromethyl ketone.
Find the needle in the haystack! A new alcohol dehydrogenase—PPADH from P. pastoris—was identified. It is able to distinguish between the two enantiomers of the racemic β‐hydroxy‐β‐trifluoromethyl ketone 1 by efficiently reducing the S form to the corresponding stereochemically uniform 1,3‐diol (S,S)‐2.</abstract><cop>Germany</cop><pub>Blackwell Publishing Ltd</pub><pmid>27123855</pmid><doi>10.1002/cbic.201600101</doi><tpages>10</tpages></addata></record> |
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subjects | Alcohol Dehydrogenase - genetics Alcohol Dehydrogenase - metabolism alcohol dehydrogenases asymmetric reduction and oxidation biocatalysis Cloning, Molecular - methods diols enantioselectivity Escherichia coli Hydrogen-Ion Concentration Ketones - chemistry Ketones - isolation & purification kinetic resolution Kinetics Oxidoreductases Pichia - enzymology Pichia pastoris Stereoisomerism Substrate Specificity Temperature |
title | Screening, Molecular Cloning, and Biochemical Characterization of an Alcohol Dehydrogenase from Pichia pastoris Useful for the Kinetic Resolution of a Racemic β-Hydroxy-β-trifluoromethyl Ketone |
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