Identification of Key Amino Acid Residues in the Assembly of Enzymes into the Pyruvate Dehydrogenase Complex ofBacillus stearothermophilus:  A Kinetic and Thermodynamic Analysis†

Biochemistry ◽  
2002 ◽  
Vol 41 (33) ◽  
pp. 10446-10453 ◽  
Author(s):  
Hyo-Il Jung ◽  
Alan Cooper ◽  
Richard N. Perham
Keyword(s):  
Amino Acid ◽  
Thermodynamic Analysis ◽  
Pyruvate Dehydrogenase ◽  
Pyruvate Dehydrogenase Complex ◽  
Amino Acid Residues ◽  
Ofbacillus Stearothermophilus ◽  
Kinetic And Thermodynamic Analysis ◽  
Dehydrogenase Complex

scholarly journals Isolation and characterization of lipoylated and unlipoylated domains of the E2p subunit of the pyruvate dehydrogenase complex of Escherichia coli

1990 ◽  
Vol 271 (1) ◽  
pp. 139-145 ◽  
Author(s):  
S T Ali ◽  
J R Guest
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Pyruvate Dehydrogenase ◽  
Pyruvate Dehydrogenase Complex ◽  
Amino Acid Residues ◽  
E Coli ◽  
Isolation And Characterization ◽  
Lysine Residues ◽  
Dehydrogenase Complex ◽  
Lipoyl Domain

The dihydrolipoamide acetyltransferase subunit (E2p) of the pyruvate dehydrogenase complex of Escherichia coli has three highly conserved and tandemly repeated lipoyl domains, each containing approx. 80 amino acid residues. These domains are covalently modified with lipoyl groups bound in amide linkage to the N6-amino groups of specific lysine residues, and the cofactors perform essential roles in the formation and transfer of acetyl groups by the dehydrogenase (E1p) and acetyltransferase (E2p) subunits. A subgene encoding a hybrid lipoyl domain was previously shown to generate two products when overexpressed, whereas a mutant subgene, in which the lipoyl-lysine codon is replaced by a glutamine codon, expresses only one product. A method has been devised for purifying the three types of independently folded domain from crude extracts of E. coli, based on their pH-(and heat-)stabilities. The domains were characterized by: amino acid and N-terminal sequence analysis, lipoic acid content, acetylation by E1p, tryptic peptide analysis and immunochemical activity. This has shown that the two forms of domain expressed from the parental subgene are lipoylated (L203) and unlipoylated (U203) derivatives of the hybrid lipoyl domain, whereas the mutant subgene produces a single unlipoylatable domain (204) containing the Lys-244----Gln substitution.

scholarly journals Amino acid sequence analysis of the lipoyl and peripheral subunit-binding domains in the lipoate acetyltransferase component of the pyruvate dehydrogenase complex from Bacillus stearothermophilus

1988 ◽  
Vol 252 (1) ◽  
pp. 79-86 ◽  
Author(s):  
L C Packman ◽  
A Borges ◽  
R N Perham
Keyword(s):  
Amino Acid ◽  
Sequence Analysis ◽  
Pyruvate Dehydrogenase ◽  
Bacillus Stearothermophilus ◽  
Pyruvate Dehydrogenase Complex ◽  
Limited Proteolysis ◽  
Binding Domain ◽  
British Library ◽  
Amino Acid Residues ◽  
Lipoyl Domain

The pyruvate dehydrogenase multienzyme complex from Bacillus stearothermophilus comprises a structural core, composed of 60 dihydrolipoamide acetyltransferase (E2p) subunits, which binds multiple copies of pyruvate decarboxylase (E1p) and dihydrolipoamide dehydrogenase (E3) subunits. After limited proteolysis with chymotrypsin, the N-terminal lipoyl domain of E2p was excised, purified and sequenced. The residual complex, which remained assembled, was then digested with trypsin under mild conditions. This treatment promoted complete disassembly of the complex and the various components were separated by gel filtration and h.p.l.c. A folded fragment of E2p containing about 50 amino acid residues was identified as being responsible for binding the E3 subunits, although, unlike the corresponding region of the E2p or E2o chains of the pyruvate dehydrogenase or 2-oxoglutarate dehydrogenase complexes from Escherichia coli, the fragment also bound E1p molecules. Further peptide purification and sequence analysis allowed the determination of the first 211 amino acid residues of the B. stearothermophilus E2p chain, thus providing the complete primary structure of the lipoyl domain, the E1p/E3-binding domain and the regions of polypeptide chain, probably highly flexible in nature, that link the domains to each other and to the inner-core (E2p-binding) domain. Several of the proteolytically sensitive sites were also identified. The sequence of the B. stearothermophilus E2p chain shows close homology with the sequences of the E2p and E2o chains from E. coli, although significant differences in structure are apparent. Detailed evidence for the sequence of the peptides obtained by limited proteolysis and further chemical and enzymic cleavages have been deposited as Supplementary Publication SUP 50142 (11 pages) at the British Library Lending Division, Boston Spa, Wetherby, West Yorkshire LS23 6BQ, U.K., from whom copies may be obtained as indicated in Biochem. J. (1988) 249, 5.

scholarly journals Pyruvate:Quinone Oxidoreductase in Corynebacterium glutamicum: Molecular Analysis of the pqo Gene, Significance of the Enzyme, and Phylogenetic Aspects

2006 ◽  
Vol 188 (4) ◽  
pp. 1341-1350 ◽  
Author(s):  
Mark E. Schreiner ◽  
Christian Riedel ◽  
Jiři Holátko ◽  
Miroslav Pátek ◽  
Bernhard J. Eikmanns
Keyword(s):  
Amino Acid ◽  
Corynebacterium Glutamicum ◽  
Pyruvate Dehydrogenase ◽  
Acid Production ◽  
Pyruvate Dehydrogenase Complex ◽  
Wide Distribution ◽  
Oxidative Decarboxylation ◽  
Amino Acid Production ◽  
Acetohydroxy Acid Synthase ◽  
Dehydrogenase Complex

ABSTRACT Corynebacterium glutamicum recently has been shown to possess pyruvate:quinone oxidoreductase (PQO), catalyzing the oxidative decarboxylation of pyruvate to acetate and CO2 with a quinone as the electron acceptor. Here, we analyze the expression of the C. glutamicum pqo gene, investigate the relevance of the PQO enzyme for growth and amino acid production, and perform phylogenetic studies. Expression analyses revealed that transcription of pqo is initiated 45 bp upstream of the translational start site and that it is organized in an operon together with genes encoding a putative metal-activated pyridoxal enzyme and a putative activator protein. Inactivation of the chromosomal pqo gene led to the absence of PQO activity; however, growth and amino acid production were not affected under either condition tested. Introduction of plasmid-bound pqo into a pyruvate dehydrogenase complex-negative C. glutamicum strain partially relieved the growth phenotype of this mutant, indicating that high PQO activity can compensate for the function of the pyruvate dehydrogenase complex. To investigate the distribution of PQO enzymes in prokaryotes and to clarify the relationship between PQO, pyruvate oxidase (POX), and acetohydroxy acid synthase enzymes, we compiled and analyzed the phylogeny of respective proteins deposited in public databases. The analyses revealed a wide distribution of PQOs among prokaryotes, corroborated the hypothesis of a common ancestry of the three enzymes, and led us to propose that the POX enzymes of Lactobacillales were derived from a PQO.

scholarly journals Amino acid sequences around the sites of phosphorylation in the pig heart pyruvate dehydrogenase complex

1979 ◽  
Vol 181 (2) ◽  
pp. 419-426 ◽  
Author(s):  
P H Sugden ◽  
A L Kerbey ◽  
P J Randle ◽  
C A Waller ◽  
K B M Reid
Keyword(s):  
Amino Acid ◽  
Pyruvate Dehydrogenase ◽  
Pyruvate Dehydrogenase Complex ◽  
Amino Acid Sequences ◽  
Phosphorylation Sites ◽  
Complete Inactivation ◽  
Total P ◽  
Dehydrogenase Complex

1. When pig heart pyruvate dehydrogenase complex was phosphorylated to completion with [gamma-32P]ATP by its intrinsic kinase, three phosphorylation sites were observed. The amino acid sequences around these sites were: sequence 1, Tyr-Gly-Met-Gly-Thr-Ser(P)-Val-Glu-Arg; and sequence 2, Tyr-His-Gly-His-Ser(P)-Met-Ser-Asp-Pro-Gly-Val-Ser(P)-Tyr-Arg. 2. When phosphorylated to inactivation by repetitive additions of limiting quantities of [gamma-32P]ATP, phosphate was incorporated mainly (more than 90%) into Ser-5 of sequence 2. Phosphorylation of this site thus results in activation of pyruvate dehydrogenase. 3. If Ser-5 is phosphorylated with ATP and the enzyme then incubated with [gamma-32P]ATP, phosphorylation of the remaining sites occurred. Ser-12 of sequence 2 is phosphorylated about twice as rapidly as Ser-6 of sequence 1. 4. Incubation of pyruvate dehydrogenase with excess [gamma-32P]ATP with termination of phosphorylation at about 50% complete inactivation showed that Ser-5 of sequence 2 was phosphorylated most rapidly, but also that Ser-12 of sequence 2 was significantly (15% of total) phosphorylated. Ser-6 sequence 1 contained about 1% total P. 5. These results suggest that addition of limiting amounts of ATP produces primarily phosphorylation of Ser-5 of sequence 2 (inactivating site). This also occurs during incubation with excess ATP before complete inactivation occurs, but a greater occupancy of other sites also occurs during this treatment.

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