Biochemical Characterization of the Pseudomonas putida 3-Hydroxyacyl ACP:CoA Transacylase, Which Diverts Intermediates of Fatty Acid de Novo Biosynthesis
The 3-hydroxyacyl ACP:CoA transacylase (PhaG) was recently identified in various Pseudomonas species and catalyzes the diversion of ACP thioester intermediates of fatty acid de novo biosynthesis toward the respective CoA thioesters, which serve as precursors for polyester and rhamnolipid biosynthesi...
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creator | Hoffmann, Nils Amara, Amro A Beermann, Br Bernd Qi, Qingsheng Hinz, Hans-Jurgen Rehm, Bernd H A |
description | The 3-hydroxyacyl ACP:CoA transacylase (PhaG) was recently identified in various Pseudomonas species and catalyzes the diversion of ACP thioester intermediates of fatty acid de novo biosynthesis toward the respective CoA thioesters, which serve as precursors for polyester and rhamnolipid biosynthesis.
PhaG from Pseudomonas putida was overproduced in Escherichia coli as a C-terminal hexahistidine-tagged (His 6 ) fusion protein in high yield. The His 6 -PhaG was purified to homogeneity by refolding of PhaG obtained from inclusion bodies, and a new enzyme assay was established.
Kinetic analysis of the 3-hydroxyacyl transfer to ACP, catalyzed by His 6 -PhaG, gave K 0.5 values of 28 μ m (ACP) and 65 μ m (3-hydroxyacyl-CoA) considering V
max values of 11.7 milliunits/mg and 12.4 milliunits/mg, respectively. A Hill coefficient of 1.38 (ACP) and 1.32 (3-hydroxyacyl-CoA)
indicated a positive substrate cooperativity. Subcellular localization studies showed that PhaG is not attached to polyester
granules and resides in the cytosol. Gel filtration chromatography analysis in combination with light scattering analysis
indicated substrate-induced dimerization of the transacylase. A threading model of PhaG was developed based on the homology
to an epoxide hydrolase (1cqz). In addition, the alignment with the α/β-hydrolase fold region indicated that PhaG belongs
to α/β-hydrolase superfamily. Accordingly, CD analysis suggested a secondary structure composition of 29% αâhelix, 22% β-sheet,
18% β-turn, and 31% random coil. Site-specific mutagenesis of seven highly conserved amino acid residues (Asp-60, Ser-102,
His-177, Asp-182, His-192, Asp-223, His-251) was used to validate the protein model and to investigate organization of the
transacylase active site. Only the D182(A/E) mutation was permissive with about 30% specific activity of the wild type enzyme.
Furthermore, this mutation caused a change in substrate specificity, indicating a functional role in substrate binding. The
serine-specific agent phenylmethylsulfonyl fluoride (PMSF) or the histidine-specific agent diethylpyrocarbonate (DEPC) caused
inhibition of 3-hydroxyacyl transfer to holo-ACP, and the S102(A/T) or H251(A/R) PhaG mutant was incapable of catalyzing 3-hydroxyacyl
transfer, suggesting that these residues are part of a catalytic triad. |
doi_str_mv | 10.1074/jbc.M207821200 |
format | Article |
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PhaG from Pseudomonas putida was overproduced in Escherichia coli as a C-terminal hexahistidine-tagged (His 6 ) fusion protein in high yield. The His 6 -PhaG was purified to homogeneity by refolding of PhaG obtained from inclusion bodies, and a new enzyme assay was established.
Kinetic analysis of the 3-hydroxyacyl transfer to ACP, catalyzed by His 6 -PhaG, gave K 0.5 values of 28 μ m (ACP) and 65 μ m (3-hydroxyacyl-CoA) considering V
max values of 11.7 milliunits/mg and 12.4 milliunits/mg, respectively. A Hill coefficient of 1.38 (ACP) and 1.32 (3-hydroxyacyl-CoA)
indicated a positive substrate cooperativity. Subcellular localization studies showed that PhaG is not attached to polyester
granules and resides in the cytosol. Gel filtration chromatography analysis in combination with light scattering analysis
indicated substrate-induced dimerization of the transacylase. A threading model of PhaG was developed based on the homology
to an epoxide hydrolase (1cqz). In addition, the alignment with the α/β-hydrolase fold region indicated that PhaG belongs
to α/β-hydrolase superfamily. Accordingly, CD analysis suggested a secondary structure composition of 29% αâhelix, 22% β-sheet,
18% β-turn, and 31% random coil. Site-specific mutagenesis of seven highly conserved amino acid residues (Asp-60, Ser-102,
His-177, Asp-182, His-192, Asp-223, His-251) was used to validate the protein model and to investigate organization of the
transacylase active site. Only the D182(A/E) mutation was permissive with about 30% specific activity of the wild type enzyme.
Furthermore, this mutation caused a change in substrate specificity, indicating a functional role in substrate binding. The
serine-specific agent phenylmethylsulfonyl fluoride (PMSF) or the histidine-specific agent diethylpyrocarbonate (DEPC) caused
inhibition of 3-hydroxyacyl transfer to holo-ACP, and the S102(A/T) or H251(A/R) PhaG mutant was incapable of catalyzing 3-hydroxyacyl
transfer, suggesting that these residues are part of a catalytic triad.</description><identifier>ISSN: 0021-9258</identifier><identifier>EISSN: 1083-351X</identifier><identifier>DOI: 10.1074/jbc.M207821200</identifier><identifier>PMID: 12200450</identifier><language>eng</language><publisher>United States: American Society for Biochemistry and Molecular Biology</publisher><subject>Acyltransferases - chemistry ; Acyltransferases - genetics ; Acyltransferases - isolation & purification ; Acyltransferases - metabolism ; Amino Acid Sequence ; Amino Acid Substitution ; Animals ; Chromatography, Gel ; Circular Dichroism ; Epoxide Hydrolases - chemistry ; Fatty Acids - biosynthesis ; Kinetics ; Mice ; Molecular Sequence Data ; Mutagenesis, Site-Directed ; Protein Conformation ; Pseudomonas putida - enzymology ; Recombinant Proteins - chemistry ; Recombinant Proteins - isolation & purification ; Recombinant Proteins - metabolism ; Sequence Alignment ; Subcellular Fractions - enzymology</subject><ispartof>The Journal of biological chemistry, 2002-11, Vol.277 (45), p.42926-42936</ispartof><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c391t-a9fe99e62b06bf9ef0e4230cee02106ef7c23e275ed502216689e4361ae4eca93</citedby><cites>FETCH-LOGICAL-c391t-a9fe99e62b06bf9ef0e4230cee02106ef7c23e275ed502216689e4361ae4eca93</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><link.rule.ids>314,776,780,27903,27904</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/12200450$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Hoffmann, Nils</creatorcontrib><creatorcontrib>Amara, Amro A</creatorcontrib><creatorcontrib>Beermann, Br Bernd</creatorcontrib><creatorcontrib>Qi, Qingsheng</creatorcontrib><creatorcontrib>Hinz, Hans-Jurgen</creatorcontrib><creatorcontrib>Rehm, Bernd H A</creatorcontrib><title>Biochemical Characterization of the Pseudomonas putida 3-Hydroxyacyl ACP:CoA Transacylase, Which Diverts Intermediates of Fatty Acid de Novo Biosynthesis</title><title>The Journal of biological chemistry</title><addtitle>J Biol Chem</addtitle><description>The 3-hydroxyacyl ACP:CoA transacylase (PhaG) was recently identified in various Pseudomonas species and catalyzes the diversion of ACP thioester intermediates of fatty acid de novo biosynthesis toward the respective CoA thioesters, which serve as precursors for polyester and rhamnolipid biosynthesis.
PhaG from Pseudomonas putida was overproduced in Escherichia coli as a C-terminal hexahistidine-tagged (His 6 ) fusion protein in high yield. The His 6 -PhaG was purified to homogeneity by refolding of PhaG obtained from inclusion bodies, and a new enzyme assay was established.
Kinetic analysis of the 3-hydroxyacyl transfer to ACP, catalyzed by His 6 -PhaG, gave K 0.5 values of 28 μ m (ACP) and 65 μ m (3-hydroxyacyl-CoA) considering V
max values of 11.7 milliunits/mg and 12.4 milliunits/mg, respectively. A Hill coefficient of 1.38 (ACP) and 1.32 (3-hydroxyacyl-CoA)
indicated a positive substrate cooperativity. Subcellular localization studies showed that PhaG is not attached to polyester
granules and resides in the cytosol. Gel filtration chromatography analysis in combination with light scattering analysis
indicated substrate-induced dimerization of the transacylase. A threading model of PhaG was developed based on the homology
to an epoxide hydrolase (1cqz). In addition, the alignment with the α/β-hydrolase fold region indicated that PhaG belongs
to α/β-hydrolase superfamily. Accordingly, CD analysis suggested a secondary structure composition of 29% αâhelix, 22% β-sheet,
18% β-turn, and 31% random coil. Site-specific mutagenesis of seven highly conserved amino acid residues (Asp-60, Ser-102,
His-177, Asp-182, His-192, Asp-223, His-251) was used to validate the protein model and to investigate organization of the
transacylase active site. Only the D182(A/E) mutation was permissive with about 30% specific activity of the wild type enzyme.
Furthermore, this mutation caused a change in substrate specificity, indicating a functional role in substrate binding. The
serine-specific agent phenylmethylsulfonyl fluoride (PMSF) or the histidine-specific agent diethylpyrocarbonate (DEPC) caused
inhibition of 3-hydroxyacyl transfer to holo-ACP, and the S102(A/T) or H251(A/R) PhaG mutant was incapable of catalyzing 3-hydroxyacyl
transfer, suggesting that these residues are part of a catalytic triad.</description><subject>Acyltransferases - chemistry</subject><subject>Acyltransferases - genetics</subject><subject>Acyltransferases - isolation & purification</subject><subject>Acyltransferases - metabolism</subject><subject>Amino Acid Sequence</subject><subject>Amino Acid Substitution</subject><subject>Animals</subject><subject>Chromatography, Gel</subject><subject>Circular Dichroism</subject><subject>Epoxide Hydrolases - chemistry</subject><subject>Fatty Acids - biosynthesis</subject><subject>Kinetics</subject><subject>Mice</subject><subject>Molecular Sequence Data</subject><subject>Mutagenesis, Site-Directed</subject><subject>Protein Conformation</subject><subject>Pseudomonas putida - enzymology</subject><subject>Recombinant Proteins - chemistry</subject><subject>Recombinant Proteins - isolation & purification</subject><subject>Recombinant Proteins - metabolism</subject><subject>Sequence Alignment</subject><subject>Subcellular Fractions - enzymology</subject><issn>0021-9258</issn><issn>1083-351X</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2002</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNpFkblu3DAQhokgRrw-2pQBiyBVtOahi-k2SnwAvgobSUdQ1CiiIYlrkrIjv4nf1lzsAp5mgME3_xw_Qp8pWVJSpCcPtV5eMVKUjDJCPqAFJSVPeEb_fkQLQhhNBMvKfXTg_QOJkQr6Ce1TFuE0Iwv0-tNY3cFgtOpx1SmndABnXlQwdsS2xaEDfOthauxgR-XxegqmUZgn53Pj7P9Z6bnHq-r2R2VX-M6p0W8qysN3_KczusO_zBO44PHFGIUHaIwK4DfKpyqEGa-0aXAD-No-WRyX8fMYR3rjj9Beq3oPx7t8iO5Pf99V58nlzdlFtbpMNBc0JEq0IATkrCZ53QpoCaSMEw0Qjyc5tIVmHFiRQZMRxmielwJSnlMFKWgl-CH6ttVdO_s4gQ9yMF5D36sR7OQlLXNCKS0juNyC2lnvHbRy7cyg3CwpkRszZDRDvpsRG77slKc6Hv6O774fga9boDP_umfjQNZbNyQrCplmMmWC5fwNXpGTTw</recordid><startdate>20021108</startdate><enddate>20021108</enddate><creator>Hoffmann, Nils</creator><creator>Amara, Amro A</creator><creator>Beermann, Br Bernd</creator><creator>Qi, Qingsheng</creator><creator>Hinz, Hans-Jurgen</creator><creator>Rehm, Bernd H A</creator><general>American Society for Biochemistry and Molecular Biology</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>7QL</scope><scope>C1K</scope></search><sort><creationdate>20021108</creationdate><title>Biochemical Characterization of the Pseudomonas putida 3-Hydroxyacyl ACP:CoA Transacylase, Which Diverts Intermediates of Fatty Acid de Novo Biosynthesis</title><author>Hoffmann, Nils ; Amara, Amro A ; Beermann, Br Bernd ; Qi, Qingsheng ; Hinz, Hans-Jurgen ; Rehm, Bernd H A</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c391t-a9fe99e62b06bf9ef0e4230cee02106ef7c23e275ed502216689e4361ae4eca93</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2002</creationdate><topic>Acyltransferases - chemistry</topic><topic>Acyltransferases - genetics</topic><topic>Acyltransferases - isolation & purification</topic><topic>Acyltransferases - metabolism</topic><topic>Amino Acid Sequence</topic><topic>Amino Acid Substitution</topic><topic>Animals</topic><topic>Chromatography, Gel</topic><topic>Circular Dichroism</topic><topic>Epoxide Hydrolases - chemistry</topic><topic>Fatty Acids - biosynthesis</topic><topic>Kinetics</topic><topic>Mice</topic><topic>Molecular Sequence Data</topic><topic>Mutagenesis, Site-Directed</topic><topic>Protein Conformation</topic><topic>Pseudomonas putida - enzymology</topic><topic>Recombinant Proteins - chemistry</topic><topic>Recombinant Proteins - isolation & purification</topic><topic>Recombinant Proteins - metabolism</topic><topic>Sequence Alignment</topic><topic>Subcellular Fractions - enzymology</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Hoffmann, Nils</creatorcontrib><creatorcontrib>Amara, Amro A</creatorcontrib><creatorcontrib>Beermann, Br Bernd</creatorcontrib><creatorcontrib>Qi, Qingsheng</creatorcontrib><creatorcontrib>Hinz, Hans-Jurgen</creatorcontrib><creatorcontrib>Rehm, Bernd H A</creatorcontrib><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>The Journal of biological chemistry</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Hoffmann, Nils</au><au>Amara, Amro A</au><au>Beermann, Br Bernd</au><au>Qi, Qingsheng</au><au>Hinz, Hans-Jurgen</au><au>Rehm, Bernd H A</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Biochemical Characterization of the Pseudomonas putida 3-Hydroxyacyl ACP:CoA Transacylase, Which Diverts Intermediates of Fatty Acid de Novo Biosynthesis</atitle><jtitle>The Journal of biological chemistry</jtitle><addtitle>J Biol Chem</addtitle><date>2002-11-08</date><risdate>2002</risdate><volume>277</volume><issue>45</issue><spage>42926</spage><epage>42936</epage><pages>42926-42936</pages><issn>0021-9258</issn><eissn>1083-351X</eissn><abstract>The 3-hydroxyacyl ACP:CoA transacylase (PhaG) was recently identified in various Pseudomonas species and catalyzes the diversion of ACP thioester intermediates of fatty acid de novo biosynthesis toward the respective CoA thioesters, which serve as precursors for polyester and rhamnolipid biosynthesis.
PhaG from Pseudomonas putida was overproduced in Escherichia coli as a C-terminal hexahistidine-tagged (His 6 ) fusion protein in high yield. The His 6 -PhaG was purified to homogeneity by refolding of PhaG obtained from inclusion bodies, and a new enzyme assay was established.
Kinetic analysis of the 3-hydroxyacyl transfer to ACP, catalyzed by His 6 -PhaG, gave K 0.5 values of 28 μ m (ACP) and 65 μ m (3-hydroxyacyl-CoA) considering V
max values of 11.7 milliunits/mg and 12.4 milliunits/mg, respectively. A Hill coefficient of 1.38 (ACP) and 1.32 (3-hydroxyacyl-CoA)
indicated a positive substrate cooperativity. Subcellular localization studies showed that PhaG is not attached to polyester
granules and resides in the cytosol. Gel filtration chromatography analysis in combination with light scattering analysis
indicated substrate-induced dimerization of the transacylase. A threading model of PhaG was developed based on the homology
to an epoxide hydrolase (1cqz). In addition, the alignment with the α/β-hydrolase fold region indicated that PhaG belongs
to α/β-hydrolase superfamily. Accordingly, CD analysis suggested a secondary structure composition of 29% αâhelix, 22% β-sheet,
18% β-turn, and 31% random coil. Site-specific mutagenesis of seven highly conserved amino acid residues (Asp-60, Ser-102,
His-177, Asp-182, His-192, Asp-223, His-251) was used to validate the protein model and to investigate organization of the
transacylase active site. Only the D182(A/E) mutation was permissive with about 30% specific activity of the wild type enzyme.
Furthermore, this mutation caused a change in substrate specificity, indicating a functional role in substrate binding. The
serine-specific agent phenylmethylsulfonyl fluoride (PMSF) or the histidine-specific agent diethylpyrocarbonate (DEPC) caused
inhibition of 3-hydroxyacyl transfer to holo-ACP, and the S102(A/T) or H251(A/R) PhaG mutant was incapable of catalyzing 3-hydroxyacyl
transfer, suggesting that these residues are part of a catalytic triad.</abstract><cop>United States</cop><pub>American Society for Biochemistry and Molecular Biology</pub><pmid>12200450</pmid><doi>10.1074/jbc.M207821200</doi><tpages>11</tpages><oa>free_for_read</oa></addata></record> |
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source | MEDLINE; EZB-FREE-00999 freely available EZB journals; Alma/SFX Local Collection |
subjects | Acyltransferases - chemistry Acyltransferases - genetics Acyltransferases - isolation & purification Acyltransferases - metabolism Amino Acid Sequence Amino Acid Substitution Animals Chromatography, Gel Circular Dichroism Epoxide Hydrolases - chemistry Fatty Acids - biosynthesis Kinetics Mice Molecular Sequence Data Mutagenesis, Site-Directed Protein Conformation Pseudomonas putida - enzymology Recombinant Proteins - chemistry Recombinant Proteins - isolation & purification Recombinant Proteins - metabolism Sequence Alignment Subcellular Fractions - enzymology |
title | Biochemical Characterization of the Pseudomonas putida 3-Hydroxyacyl ACP:CoA Transacylase, Which Diverts Intermediates of Fatty Acid de Novo Biosynthesis |
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