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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Veröffentlicht in:The Journal of biological chemistry 2002-11, Vol.277 (45), p.42926-42936
Hauptverfasser: Hoffmann, Nils, Amara, Amro A, Beermann, Br Bernd, Qi, Qingsheng, Hinz, Hans-Jurgen, Rehm, Bernd H A
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container_end_page 42936
container_issue 45
container_start_page 42926
container_title The Journal of biological chemistry
container_volume 277
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% α−helix, 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.
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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% α−helix, 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 &amp; 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 &amp; 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% α−helix, 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. 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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% α−helix, 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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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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