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Genetic reconstruction and characterization of the recombinant transacylase (E2b) component of bovine branched-chain alpha-keto acid dehydrogenase complex. Implication of histidine 391 as an active site residue.

Identifieur interne : 004999 ( Main/Curation ); précédent : 004998; suivant : 004A00

Genetic reconstruction and characterization of the recombinant transacylase (E2b) component of bovine branched-chain alpha-keto acid dehydrogenase complex. Implication of histidine 391 as an active site residue.

Auteurs : T A Griffin ; D T Chuang

Source :

RBID : pubmed:2198287

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Abstract

Genetically altered transacylase (E2b) proteins of the bovine branched-chain alpha-keto acid dehydrogenase complex were overexpressed in Escherichia coli and characterized. Deletion by PstI or Bal31 digestion of the amino-terminal region of the inner-core domain (residues 175-421) beyond residue 209 resulted in a complete loss of transacylase activity. The enzyme assay was carried out using [1-14C]isovaleryl-CoA and exogenous dihydrolipoamide as substrates. The removal of 4 residues (Thr-Ile-Pro-Ile) (residues 175-178) from the amino terminus of the inner-core domain significantly reduced the level of transacylase activity. The results establish that the segment between residues 175 and 209 is an integral part of the active site of E2b. The residue His-391 in the recombinant inner-core domain (E2b delta 167) was changed to Asn or Gln by site-directed mutagenesis. The wild-type and the two mutant inner-core domains were assembled into 24-mers as determined by gel filtration. However, both Asn and Gln mutations were accompanied by a complete loss of the enzymatic activity. Titration of the natural branched-chain alpha-keto dehydrogenase complex from pH 8 to 6 sharply reduced transacylase activity. The above data support the hypothesis that a conserved histidine residue in E2 acts as a general base for the transacylation reaction by analogy with E. coli chloramphenicol acetyltransferases.

PubMed: 2198287

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T A Griffin
<affiliation>
<nlm:affiliation>Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas 75235.</nlm:affiliation>
<wicri:noCountry code="subField">Dallas 75235</wicri:noCountry>
</affiliation>

Le document en format XML

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<title xml:lang="en">Genetic reconstruction and characterization of the recombinant transacylase (E2b) component of bovine branched-chain alpha-keto acid dehydrogenase complex. Implication of histidine 391 as an active site residue.</title>
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<name sortKey="Griffin, T A" sort="Griffin, T A" uniqKey="Griffin T" first="T A" last="Griffin">T A Griffin</name>
<affiliation>
<nlm:affiliation>Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas 75235.</nlm:affiliation>
<wicri:noCountry code="subField">Dallas 75235</wicri:noCountry>
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<name sortKey="Chuang, D T" sort="Chuang, D T" uniqKey="Chuang D" first="D T" last="Chuang">D T Chuang</name>
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<title xml:lang="en">Genetic reconstruction and characterization of the recombinant transacylase (E2b) component of bovine branched-chain alpha-keto acid dehydrogenase complex. Implication of histidine 391 as an active site residue.</title>
<author>
<name sortKey="Griffin, T A" sort="Griffin, T A" uniqKey="Griffin T" first="T A" last="Griffin">T A Griffin</name>
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<term>3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide)</term>
<term>Acyltransferases (genetics)</term>
<term>Acyltransferases (isolation & purification)</term>
<term>Acyltransferases (metabolism)</term>
<term>Amino Acid Sequence</term>
<term>Animals</term>
<term>Base Sequence</term>
<term>Binding Sites</term>
<term>Blotting, Western</term>
<term>Cattle</term>
<term>Chromosome Deletion</term>
<term>Cloning, Molecular</term>
<term>Escherichia coli (genetics)</term>
<term>Genetic Vectors</term>
<term>Histidine</term>
<term>Hydrogen-Ion Concentration</term>
<term>Ketone Oxidoreductases (genetics)</term>
<term>Ketone Oxidoreductases (metabolism)</term>
<term>Kinetics</term>
<term>Molecular Sequence Data</term>
<term>Molecular Weight</term>
<term>Multienzyme Complexes (genetics)</term>
<term>Multienzyme Complexes (metabolism)</term>
<term>Mutation</term>
<term>Oligonucleotide Probes</term>
<term>Recombinant Proteins (isolation & purification)</term>
<term>Recombinant Proteins (metabolism)</term>
<term>Restriction Mapping</term>
<term>Sequence Homology, Nucleic Acid</term>
</keywords>
<keywords scheme="KwdFr" xml:lang="fr">
<term>3-Methyl-2-oxobutanoate dehydrogenase (lipoamide)</term>
<term>Acyltransferases (génétique)</term>
<term>Acyltransferases (isolement et purification)</term>
<term>Acyltransferases (métabolisme)</term>
<term>Animaux</term>
<term>Bovins</term>
<term>Cartographie de restriction</term>
<term>Cetone oxidoreductases (génétique)</term>
<term>Cetone oxidoreductases (métabolisme)</term>
<term>Cinétique</term>
<term>Clonage moléculaire</term>
<term>Complexes multienzymatiques (génétique)</term>
<term>Complexes multienzymatiques (métabolisme)</term>
<term>Concentration en ions d'hydrogène</term>
<term>Données de séquences moléculaires</term>
<term>Délétion de segment de chromosome</term>
<term>Escherichia coli (génétique)</term>
<term>Histidine</term>
<term>Masse moléculaire</term>
<term>Mutation</term>
<term>Protéines recombinantes (isolement et purification)</term>
<term>Protéines recombinantes (métabolisme)</term>
<term>Similitude de séquences d'acides nucléiques</term>
<term>Sites de fixation</term>
<term>Sondes oligonucléotidiques</term>
<term>Séquence d'acides aminés</term>
<term>Séquence nucléotidique</term>
<term>Technique de Western</term>
<term>Vecteurs génétiques</term>
</keywords>
<keywords scheme="MESH" type="chemical" qualifier="genetics" xml:lang="en">
<term>Acyltransferases</term>
<term>Ketone Oxidoreductases</term>
<term>Multienzyme Complexes</term>
</keywords>
<keywords scheme="MESH" type="chemical" qualifier="isolation & purification" xml:lang="en">
<term>Acyltransferases</term>
<term>Recombinant Proteins</term>
</keywords>
<keywords scheme="MESH" type="chemical" qualifier="metabolism" xml:lang="en">
<term>Acyltransferases</term>
<term>Ketone Oxidoreductases</term>
<term>Multienzyme Complexes</term>
<term>Recombinant Proteins</term>
</keywords>
<keywords scheme="MESH" type="chemical" xml:lang="en">
<term>3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide)</term>
<term>Histidine</term>
<term>Oligonucleotide Probes</term>
</keywords>
<keywords scheme="MESH" qualifier="genetics" xml:lang="en">
<term>Escherichia coli</term>
</keywords>
<keywords scheme="MESH" qualifier="génétique" xml:lang="fr">
<term>Acyltransferases</term>
<term>Cetone oxidoreductases</term>
<term>Complexes multienzymatiques</term>
<term>Escherichia coli</term>
</keywords>
<keywords scheme="MESH" qualifier="isolement et purification" xml:lang="fr">
<term>Acyltransferases</term>
<term>Protéines recombinantes</term>
</keywords>
<keywords scheme="MESH" qualifier="métabolisme" xml:lang="fr">
<term>Acyltransferases</term>
<term>Cetone oxidoreductases</term>
<term>Complexes multienzymatiques</term>
<term>Protéines recombinantes</term>
</keywords>
<keywords scheme="MESH" xml:lang="en">
<term>Amino Acid Sequence</term>
<term>Animals</term>
<term>Base Sequence</term>
<term>Binding Sites</term>
<term>Blotting, Western</term>
<term>Cattle</term>
<term>Chromosome Deletion</term>
<term>Cloning, Molecular</term>
<term>Genetic Vectors</term>
<term>Hydrogen-Ion Concentration</term>
<term>Kinetics</term>
<term>Molecular Sequence Data</term>
<term>Molecular Weight</term>
<term>Mutation</term>
<term>Restriction Mapping</term>
<term>Sequence Homology, Nucleic Acid</term>
</keywords>
<keywords scheme="MESH" xml:lang="fr">
<term>3-Methyl-2-oxobutanoate dehydrogenase (lipoamide)</term>
<term>Animaux</term>
<term>Bovins</term>
<term>Cartographie de restriction</term>
<term>Cinétique</term>
<term>Clonage moléculaire</term>
<term>Concentration en ions d'hydrogène</term>
<term>Données de séquences moléculaires</term>
<term>Délétion de segment de chromosome</term>
<term>Histidine</term>
<term>Masse moléculaire</term>
<term>Mutation</term>
<term>Similitude de séquences d'acides nucléiques</term>
<term>Sites de fixation</term>
<term>Sondes oligonucléotidiques</term>
<term>Séquence d'acides aminés</term>
<term>Séquence nucléotidique</term>
<term>Technique de Western</term>
<term>Vecteurs génétiques</term>
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<front>
<div type="abstract" xml:lang="en">Genetically altered transacylase (E2b) proteins of the bovine branched-chain alpha-keto acid dehydrogenase complex were overexpressed in Escherichia coli and characterized. Deletion by PstI or Bal31 digestion of the amino-terminal region of the inner-core domain (residues 175-421) beyond residue 209 resulted in a complete loss of transacylase activity. The enzyme assay was carried out using [1-14C]isovaleryl-CoA and exogenous dihydrolipoamide as substrates. The removal of 4 residues (Thr-Ile-Pro-Ile) (residues 175-178) from the amino terminus of the inner-core domain significantly reduced the level of transacylase activity. The results establish that the segment between residues 175 and 209 is an integral part of the active site of E2b. The residue His-391 in the recombinant inner-core domain (E2b delta 167) was changed to Asn or Gln by site-directed mutagenesis. The wild-type and the two mutant inner-core domains were assembled into 24-mers as determined by gel filtration. However, both Asn and Gln mutations were accompanied by a complete loss of the enzymatic activity. Titration of the natural branched-chain alpha-keto dehydrogenase complex from pH 8 to 6 sharply reduced transacylase activity. The above data support the hypothesis that a conserved histidine residue in E2 acts as a general base for the transacylation reaction by analogy with E. coli chloramphenicol acetyltransferases.</div>
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