scholarly journals Segmental structure and protein domains in the pyruvate dehydrogenase multienzyme complex of Escherichia coli. Genetic reconstruction in vitro and 1H-n.m.r. spectroscopy

1987 ◽  
Vol 247 (3) ◽  
pp. 641-649 ◽  
Author(s):  
S E Radford ◽  
E D Laue ◽  
R N Perham ◽  
J S Miles ◽  
J R Guest
Keyword(s):  
Escherichia Coli ◽  
Pyruvate Dehydrogenase ◽  
Inner Core ◽  
Polypeptide Chain ◽  
Enzyme Complex ◽  
Multienzyme Complex ◽  
Wild Type ◽  
Dihydrolipoamide Dehydrogenase ◽  
Core Domain

A deletion in vitro can be made in the aceEF-lpd operon encoding the pyruvate dehydrogenase multienzyme complex of Escherichia coli, which causes deletion of two of the three homologous lipoyl domains that comprise the N-terminal half of each dihydrolipoamide acetyltransferase (E2p) polypeptide chain. An active complex is still formed and 1H-n.m.r. spectroscopy of this modified complex revealed that many of the unusually sharp resonances previously attributed to conformationally mobile segments in the wild-type E2p polypeptide chains had correspondingly disappeared. A further deletion was engineered in the long (alanine + proline)-rich segment of polypeptide chain that linked the one remaining lipoyl domain to the C-terminal half of the E2p chain. 1H-n.m.r. spectroscopy of the resulting enzyme complex, which was also active, revealed a further corresponding loss in the unusually sharp resonances observed in the spectrum. These experiments strongly support the view that the sharp resonances derive, principally at least, from the three long (alanine + proline)-rich sequences which separate the three lipoyl domains and link them to the C-terminal half of the E2p chain. Closer examination of the 400 MHz 1H-n.m.r. spectra of the wild-type and restructured complexes, and of the products of limited proteolysis, revealed another sharp but smaller resonance. This was tentatively attributed to another, but smaller, (alanine + proline)-rich sequence that separates the dihydrolipoamide dehydrogenase-binding domain from the inner core domain in the C-terminal half of the E2p chain. If this sequence is also conformationally flexible, it may explain previous fluorescence data which suggest that dihydrolipoamide dehydrogenase bound to the enzyme complex is quite mobile. The acetyltransferase active site in the E2p chain was shown to reside in the inner core domain, between residues 370 and 629.

The pyruvate dehydrogenase multi-enzyme complex of Escherichia coli : genetic reconstruction and functional analysis of the lipoyl domains

1986 ◽  
Vol 317 (1540) ◽  
pp. 391-404 ◽  
Keyword(s):  
Escherichia Coli ◽  
Pyruvate Dehydrogenase ◽  
Active Site ◽  
Inner Core ◽  
Polypeptide Chain ◽  
Pyruvate Dehydrogenase Complex ◽  
Site Directed Mutagenesis ◽  
Enzyme Complex ◽  
Site Selective

The dihydrolipoamide acetyltransferase (E2p) component of the pyruvate dehydrogenase complex of Escherichia coli contains three highly homologous lipoyl domains ( ca . 100 residues) that are tandemly repeated to form the N-terminal half of the polypeptide chain. These lipoyl domains are linked to a much larger ( ca . 300 residues) subunit-binding domain that aggregates to form the octahedral inner core of the complex and also contains the acetyltransferase active site. Selective in vitro deletions in the E2p gene ( aceF )have allowed the creation of truncated E2p chains in which one or more of the lipoyl domains has been excised. Site-directed mutagenesis has been used to change individual residues. The effects of these deletions and mutations on the assembly, catalytic activity and active-site coupling in the complex are assessed.

scholarly journals Evidence for two lipoic acid residues per lipoate acetyltransferase chain in the pyruvate dehydrogenase multienzyme complex of Escherichia coli

1976 ◽  
Vol 159 (3) ◽  
pp. 677-682 ◽  
Author(s):  
M J Danson ◽  
R N Perham
Keyword(s):  
Escherichia Coli ◽  
Lipoic Acid ◽  
Pyruvate Dehydrogenase ◽  
Acid Content ◽  
Polypeptide Chain ◽  
Multienzyme Complex ◽  
Covalently Bound

The reaction of two maleimides, N-ethylmaleimide and bis-(N-maleimidomethyl) ether, with the pyruvate dehydrogenase multienzyme complex of Escherichia coli in the presence of the substrate, pyruvate, was examined. In both cases, the reaction was demonstrated to be almost exclusively with the lipoate acetyltransferase component, and evidence is presented to show that the most likely sites of reaction are the lipoic acid residues covalently bound to this component. With both reagents the stoicheiometry of the reaction was measured: 2 mol of reagent reacted with each polypeptide chain of lipoate acetyltransferase, implying that each chain bears two functionally active lipolic acid residues. This observation can be reconciled with previous determinations of the lipoic acid content of the complex by allowing for the variability of the subunit polypeptide-chain ratio that can be demonstrated for this multimeric enzyme.

scholarly journals Domain structure and1H-n.m.r. spectroscopy of the pyruvate dehydrogenase complex of Bacillus stearothermophilus

1984 ◽  
Vol 217 (1) ◽  
pp. 219-227 ◽  
Author(s):  
L C Packman ◽  
R N Perham ◽  
G C K Roberts
Keyword(s):  
Pyruvate Dehydrogenase ◽  
Active Sites ◽  
Bacillus Stearothermophilus ◽  
Inner Core ◽  
Polypeptide Chain ◽  
Pyruvate Dehydrogenase Complex ◽  
Terminal Region ◽  
Enzyme Complex ◽  
Dehydrogenase Complex ◽  
Lipoyl Domain

The pyruvate dehydrogenase complex of Bacillus stearothermophilus was treated with Staphylococcus aureus V8 proteinase, causing cleavage of the dihydrolipoamide acetyltransferase polypeptide chain (apparent Mr 57 000), inhibition of the enzymic activity and disassembly of the complex. Fragments of the dihydrolipoamide acetyltransferase chains with apparent Mr 28 000, which contained the acetyltransferase activity, remained assembled as a particle ascribed the role of an inner core of the complex. The lipoic acid residue of each dihydrolipoamide acetyltransferase chain was found as part of a small but stable domain that, unlike free lipoamide, was able still to function as a substrate for reductive acetylation by pyruvate in the presence of intact enzyme complex or isolated pyruvate dehydrogenase (lipoamide) component. The lipoyl domain was acidic and had an apparent Mr of 6500 (by sedimentation equilibrium), 7800 (by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis) and 10 000 and 20 400 (by gel filtration in the presence and in the absence respectively of 6M-guanidinium chloride). 1H-n.m.r. spectroscopy of the dihydrolipoamide acetyltransferase inner core demonstrated that it did not contain the segments of highly mobile polypeptide chain found in the pyruvate dehydrogenase complex. 1H-n.m.r. spectroscopy of the lipoyl domain demonstrated that it had a stable and defined tertiary structure. From these and other experiments, a model of the dihydrolipoamide acetyltransferase chain is proposed in which the small, folded, lipoyl domain comprises the N-terminal region, and the large, folded, core-forming domain that contains the acetyltransferase active site comprises the C-terminal region. These two regions are separated by a third segment of the chain, which includes a substantial region of polypeptide chain that enjoys high conformational mobility and facilitates movement of the lipoyl domain between the various active sites in the enzyme complex.

scholarly journals Intramolecular coupling of active sites in the pyruvate dehydrogenase multienzyme complex of Escherichia coli

1978 ◽  
Vol 175 (1) ◽  
pp. 193-198 ◽  
Author(s):  
M J D Danson ◽  
E A Hooper ◽  
R N Perham
Keyword(s):  
Escherichia Coli ◽  
Lipoic Acid ◽  
Pyruvate Dehydrogenase ◽  
Active Sites ◽  
Pyruvate Decarboxylase ◽  
Enzyme Complex ◽  
Multienzyme Complex ◽  
Thiol Groups ◽  
Specific Reaction ◽  
Good Agreement

The intramolecular passage of substrate between the component enzymes of the pyruvate dehydrogenase multienzyme complex of Escherichia coli was examined. A series of partly reassembled complexes, varying only in their E1 (pyruvate decarboxylase, EC 1.2.4.1) content, was incubated with pyruvate in the absence of CoA, conditions under which the lipoic acid residues covalently bound to the E2 (lipoate acetyltransferase, EC2.3.1.12) chains of the complex become reductively acetylated, and the reaction then ceases. The fraction of E2 chains thus acetylated was estimated by specific reaction of the thiol groups in the acetyl-lipoic acid moieties with N-ethyl[2,3-14C]maleimide. The simplest interpretation of the results was that a single E1 dimer is capable of catalysing the rapid acetylation of 8-12 E2 chains, in good agreement with the results of Bates, Danson, Hale, Hooper & Perham [(1977) Nature (London) 268, 313-316]. This novel functional connexion of active sites must be brought about by transacetylation reactions between lipoic acid residues of neighbouring E2 chains in the enzyme complex. There was also a slow transacylation process between the rapidly acetylated lipoic acid residues and those that did not react in the initial, faster phase. This interaction was not investigated in detail, since it is too slow to be of kinetic significance in the normal enzymic reaction.

scholarly journals Further Studies with Lipoamide Dehydrogenase Mutants of Escherichia coli k12

Microbiology ◽  
2000 ◽  
Vol 81 (1) ◽  
pp. 237-245 ◽  
Author(s):  
J. R. Guest ◽  
I. T. Creaghan
Keyword(s):  
Escherichia Coli ◽  
Pyruvate Dehydrogenase ◽  
Recombination Frequency ◽  
Regulatory Element ◽  
Multienzyme Complex ◽  
Wild Type ◽  
Mutant Site ◽  
Lipoamide Dehydrogenase ◽  
Distal Gene ◽  
P1 Transduction

The immunological properties of ten lipoamide dehydrogenase mutants of Escherichia coli were investigated with antiserum raised against purified lipoamide dehydrogenase. Seven mutants were CRM+ (cross-reacting material present) as they contained lipoamide dehydrogenase proteins exhibiting either complete or partial immunological identity with the wild-type protein. This indicates that at least seven of the mutations affect the lipoamide dehydrogenase structural gene (lpd). The remaining three mutants (CRM-) contained no detectable cross-reacting protein. None of the lpd mutations were sensitive to any of three different amber-suppressors. Genetic analysis by P1-transduction showed that all the lpd mutant sites were clustered very near the distal gene (aceF) of the ace region which specifies the dehydrogenase (aceE) and transacetylase (aceF) components of the pyruvate dehydrogenase multienzyme complex. Calculations based on the recombination frequency between an aceF mutant and the nearest lpd mutant site support the conclusion that apart from the possible presence of a regulatory element, the aceF and lpd genes are contiguous.

scholarly journals Differential Suppression of priA2::kan Phenotypes in Escherichia coli K-12 by Mutations in priA, lexA, and dnaC

Genetics ◽  
1996 ◽  
Vol 143 (1) ◽  
pp. 5-13 ◽  
Author(s):  
Steven J Sandler ◽  
Hardeep S Samra ◽  
Alvin J Clark
Keyword(s):  
Escherichia Coli ◽  
Dna Replication ◽  
Mutant Allele ◽  
Wild Type ◽  
Suppressor Mutations ◽  
Dnab Helicase ◽  
K 12 ◽  
Extragenic Suppressors ◽  
Assembly Activity

Abstract First identified as an essential component of the ϕX174 in vitro DNA replication system, PriA has ATPase, helicase, translocase, and primosome-assembly activities. priA1::kan strains of Escherichia coli are sensitive to UV irradiation, deficient in homologous recombination following transduction, and filamentous. priA2::kan strains have eightfold higher levels of uninduced SOS expression than wild type. We show that (1) priA1::kan strains have eightfold higher levels of uninduced SOS expression, (2) priA2::kan strains are UVS and Rec−, (3) lexA3 suppresses the high basal levels of SOS expression of a priA2::kan strain, and (4) plasmid-encoded priA300 (K230R), a mutant allele retaining only the primosome-assembly activity of priA+, restores both UVR and Rec+ phenotypes to a priA2::kan strain. Finally, we have isolated 17 independent UVR Rec+ revertants of priA2::kan strains that carry extragenic suppressors. All 17 map in the C-terminal half of the dnaC gene. DnaC loads the DnaB helicase onto DNA as a prelude for primosome assembly and DNA replication. We conclude that priA's primosome-assembly activity is essential for DNA repair and recombination and that the dnaC suppressor mutations allow these processes to occur in the absence of priA.

scholarly journals Enhancement of the Efficiency of Secretion of Heterologous Lipase in Escherichia coli by Directed Evolution of the ABC Transporter System

2005 ◽  
Vol 71 (7) ◽  
pp. 3468-3474 ◽  
Author(s):  
Gyeong Tae Eom ◽  
Jae Kwang Song ◽  
Jung Hoon Ahn ◽  
Yeon Soo Seo ◽  
Joon Shick Rhee
Keyword(s):  
Escherichia Coli ◽  
Directed Evolution ◽  
Abc Transporter ◽  
Microtiter Plate ◽  
Single Amino Acid ◽  
Wild Type ◽  
E Coli ◽  
Single Amino Acid Change ◽  
Secretion Efficiency

ABSTRACT The ABC transporter (TliDEF) from Pseudomonas fluorescens SIK W1, which mediated the secretion of a thermostable lipase (TliA) into the extracellular space in Escherichia coli, was engineered using directed evolution (error-prone PCR) to improve its secretion efficiency. TliD mutants with increased secretion efficiency were identified by coexpressing the mutated tliD library with the wild-type tliA lipase in E. coli and by screening the library with a tributyrin-emulsified indicator plate assay and a microtiter plate-based assay. Four selected mutants from one round of error-prone PCR mutagenesis, T6, T8, T24, and T35, showed 3.2-, 2.6-, 2.9-, and 3.0-fold increases in the level of secretion of TliA lipase, respectively, but had almost the same level of expression of TliD in the membrane as the strain with the wild-type TliDEF transporter. These results indicated that the improved secretion of TliA lipase was mediated by the transporter mutations. Each mutant had a single amino acid change in the predicted cytoplasmic regions in the membrane domain of TliD, implying that the corresponding region of TliD was important for the improved and successful secretion of the target protein. We therefore concluded that the efficiency of secretion of a heterologous protein in E. coli can be enhanced by in vitro engineering of the ABC transporter.

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