Reconstitution of the Escherichia coli pyruvate dehydrogenase complex.

Reed, Lester J.; Pettit, Flora H.; Eley, Michael H.; Hamilton, Lynn; Collins, Jim H.; Oliver, Robert M.

doi:10.1073/pnas.72.8.3068

Cited by 73 publications

(39 citation statements)

References 15 publications

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“…K m values for pyruvate and NAD + were 47 µM and 66 µM, respectively (Fig. 5A and B), similar to those of other bacterial and mammalian PDH complexes (Bresters et al ., 1975; Reed et al ., 1975; Mareck et al ., 1988; Korotchkina et al ., 1995). We were not able to determine the K m value for CoA due to sigmoidal saturation curves and non‐linear Lineweaver‐Burk plots.…”

Section: Resultsmentioning

confidence: 99%

Mycobacterium tuberculosis appears to lack α‐ketoglutarate dehydrogenase and encodes pyruvate dehydrogenase in widely separated genes

Tian

Bryk

Shi

et al. 2005

Molecular Microbiology

100

107

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SummaryMycobacterium tuberculosis (Mtb) persists for prolonged periods in macrophages, where it must adapt to metabolic limitations and oxidative/nitrosative stress. However, little is known about Mtb's intermediary metabolism or antioxidant defences. We recently identified a peroxynitrite reductase-peroxidase complex in Mtb that included products of the genes sucB and lpd , which are annotated to encode the dihydrolipoamide succinyltransferase (E2) and lipoamide dehydrogenase (E3) components of a a a a -ketoglutarate dehydrogenase (KDH). However, we could detect no KDH activity in Mtb lysates, nor could we reconstitute KDH by combining the recombinant proteins SucA (annotated as the E1 component of KDH), SucB and Lpd. We therefore renamed the sucB product dihydrolipoamide acyltransferase (DlaT). Mtb lysates contained pyruvate dehydrogenase (PDH) activity, which was lost when the dlaT gene (formerly, sucB ) was disrupted. Purification of PDH from Mtb yielded AceE, annotated as an E1 component of PDH, along with DlaT and Lpd. Moreover, anti-DlaT antibody coimmunoprecipitated AceE. Finally, recombinant AceE, DlaT and Lpd, although encoded by genes that are widely separated on the chromosome, reconstituted PDH in vitro with K m values typical of bacterial PDH complexes. In sum, Mtb appears to lack KDH. Instead, DlaT and Lpd join with AceE to constitute PDH.

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Section: Resultsmentioning

confidence: 99%

Mycobacterium tuberculosis appears to lack α‐ketoglutarate dehydrogenase and encodes pyruvate dehydrogenase in widely separated genes

Tian

Bryk

Shi

et al. 2005

Molecular Microbiology

100

107

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“…This has similarly been disclosed for the PDH of Escherichia coli (23), as well as for components of ODH (6,8,18,37). The PDH possesses specific E1p and E2p proteins, and ODH possesses specific E1o and E2o proteins, whereas the dihydrolipoyl dehydrogenase component E3 is shared by the two multienzyme complexes (28,29). Thus, PDH and ODH complexes share one identical polypeptide plus very similar polypeptides, and they also have a similar overall quaternary structure (21, 23).…”

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confidence: 99%

The E2 Domain of OdhA of Corynebacterium glutamicum Has Succinyltransferase Activity Dependent on Lipoyl Residues of the Acetyltransferase AceF

et al. 2010

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Oxoglutarate dehydrogenase (ODH) and pyruvate dehydrogenase (PDH) complexes catalyze key reactions in central metabolism, and in Corynebacterium glutamicum there is indication of an unusual supercomplex consisting of AceE (E1), AceF (E2), and Lpd (E3) together with OdhA. OdhA is a fusion protein of additional E1 and E2 domains, and odhA orthologs are present in all Corynebacterineae, including, for instance, Mycobacterium tuberculosis. Here we show that deletion of any of the individual domains of OdhA in C. glutamicum resulted in loss of ODH activity, whereas PDH was still functional. On the other hand, deletion of AceF disabled both PDH activity and ODH activity as well, although isolated AceF protein had solely transacetylase activity and no transsuccinylase activity. Surprisingly, the isolated OdhA protein was inactive with 2-oxoglutarate as the substrate, but it gained transsuccinylase activity upon addition of dihydrolipoamide. Further enzymatic analysis of mutant proteins and mutant cells revealed that OdhA specifically catalyzes the E1 and E2 reaction to convert 2-oxoglutarate to succinyl-coenzyme A (CoA) but fully relies on the lipoyl residues provided by AceF involved in the reactions to convert pyruvate to acetyl-CoA. It therefore appears that in the putative supercomplex in C. glutamicum, in addition to dihydrolipoyl dehydrogenase E3, lipoyl domains are also shared, thus confirming the unique evolutionary position of bacteria such as C. glutamicum and M. tuberculosis.Pyruvate dehydrogenase (PDH) and 2-oxoglutarate dehydrogenase (ODH) activities catalyze key reactions in central metabolism. They exist as huge enzyme complexes of up to 11 MDa to convert a 2-oxoacid to an acyl-coenzyme A (CoA) derivative, which is acetyl-or succinyl-CoA, respectively (for reviews, see references 28 and 29 and references therein). The reaction requires distinct enzyme activities and involves the sequential actions of thiamine-pyrophosphate-dependent oxidative decarboxylation (E1, EC 1.2.4.2), with the concomitant transfer of the respective acyl group to a lipoamide residue. This is followed by the acyl group transfer to CoA, catalyzed by dihydrolipoyl transacylase activity (E2, EC 2.3.1.6), and, finally, the last step is dihydrolipoamide reoxidation to lipoamide by an FAD-dependent dihydrolipoyl dehydrogenase (E3, EC 1.8.1.4), thus enabling the initiation of a new catalytic cycle. As a result, the energy of the C 1 -C 2 bond of an ␣-oxoacid is preserved in acetyl-CoA and succinyl-CoA, respectively, and NADH.PDH and ODH are structurally closely related assemblies. Structural data for the three-dimensional organization of PDH of Bacillus stearothermophilus have culminated in the current view that the complex consists of an E2 core, to which E1 and E3 are flexibly tethered (20)(21)(22). This has similarly been disclosed for the PDH of Escherichia coli (23), as well as for components of ODH (6,8,18,37). The PDH possesses specific E1p and E2p proteins, and ODH possesses specific E1o and E2o proteins, whereas the dihydrolipoyl...

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“…This procedure mimics the evolution of naturally occurring bifunctional enzymes, which might have evolved from smaller proteins through gene fusion (22). It also imitates naturally occurring multienzyme systems, such as the pyruvate dehydrogenase complex and fatty acyl coenzyme A synthase complex (18,37). Fusion enzymes sometimes demonstrate their superiority over a mixture of individual native enzymes catalyzing the same multistep sequential reaction, but their effects have not been demonstrated definitively in kinetic parameters governing the reactions (5).…”

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confidence: 99%

Characterization of a Bifunctional Enzyme Fusion of Trehalose-6-Phosphate Synthetase and Trehalose-6-Phosphate Phosphatase of Escherichia coli

Seo

Koo

Lim

et al. 2000

Appl Environ Microbiol

100

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To test the effect of the physical proximity of two enzymes catalyzing sequential reactions, a bifunctional fusion enzyme, TPSP, was constructed by fusing the Escherichia coli genes for trehalose-6-phosphate (T6P) synthetase (TPS) and trehalose-6-phosphate phosphatase (TPP). TPSP catalyzes the sequential reaction in which T6P is formed and then dephosphorylated, leading to the synthesis of trehalose. The fused chimeric gene was overexpressed in E. coli and purified to near homogeneity; its molecular weight was 88,300, as expected. The K m values of the TPSP fusion enzyme for the sequential overall reaction from UDP-glucose and glucose 6-phosphate to trehalose were smaller than those of an equimolar mixture of TPS and TPP (TPS/TPP). However, the k cat values of TPSP were similar to those of TPS/TPP, resulting in a 3.5-to 4.0-fold increase in the catalytic efficiency (k cat /K m ). The K m and k cat values of TPSP and TPP for the phosphatase reaction from T6P to trehalose were quite similar. This suggests that the increased catalytic efficiency results from the proximity of TPS and TPP in the TPSP fusion enzyme. The thermal stability of the TPSP fusion enzyme was quite similar to that of the TPS/TPP mixture, suggesting that the structure of each enzyme moiety in TPSP is unperturbed by intramolecular constraint. These results clearly demonstrate that the bifunctional fusion enzyme TPSP catalyzing sequential reactions has kinetic advantages over a mixture of both enzymes (TPS and TPP). These results are also supported by the in vivo accumulation of up to 0.48 mg of trehalose per g of cells after isopropyl-␤-D-thiogalactopyranoside treatment of cells harboring the construct encoding TPSP.The nonreducing disaccharide trehalose [␣-D-glucopyranosyl-(131)-␣-D-glucopyranose] has high water-holding activities, which maintain the fluidity of membranes under dry conditions (25). It also stabilizes enzymes, foods, cosmetics, and pharmaceuticals at high temperatures (8,9,38). Due to its desirable physical and chemical characteristics, commercial production of trehalose is anticipated. Escherichia coli synthesizes trehalose when exposed to high osmolarity (12,20,39,41). In E. coli, trehalose is synthesized by two separate enzymes, trehalose-6-phosphate (T6P) synthetase (TPS) and trehalose-6-phosphate phosphatase (TPP), encoded by the genes otsA and otsB, respectively (15, 21). This is different from Saccharomyces cerevisiae, in which trehalose is synthesized by a large multisubunit complex with the catalytic activities of both TPS and TPP (6, 31, 43).Overexpression of TPS and TPP might be one way to produce trehalose. We assumed that the physical proximity of two enzymes catalyzing sequential reactions might increase the reaction rate by facilitating transfer of the reaction intermediate when they are present in a complex. A variety of techniques have been applied to better understand the proximity effect of enzymes catalyzing sequential reactions, including cross-linking and coimmobilization (23,32,34). In many of these cases,...

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Reconstitution of the Escherichia coli pyruvate dehydrogenase complex.

Abstract: MATERIALS AND METHODSEnzyme Preparations. The basic procedures for growing cells of E. coli (Crookes strain) and for purification and resolution of the pyruvate dehydrogenase complex were carried out as described (1,4

Cited by 73 publications

References 15 publications

Mycobacterium tuberculosis appears to lack α‐ketoglutarate dehydrogenase and encodes pyruvate dehydrogenase in widely separated genes

Mycobacterium tuberculosis appears to lack α‐ketoglutarate dehydrogenase and encodes pyruvate dehydrogenase in widely separated genes

The E2 Domain of OdhA of Corynebacterium glutamicum Has Succinyltransferase Activity Dependent on Lipoyl Residues of the Acetyltransferase AceF

Characterization of a Bifunctional Enzyme Fusion of Trehalose-6-Phosphate Synthetase and Trehalose-6-Phosphate Phosphatase of Escherichia coli

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