Kinetics and specificity of reductive acylation of lipoyl domains from 2-oxo acid dehydrogenase multienzyme complexes

Graham, Lloyd D.; Packman, Leonard C.; Perham, Richard N.

doi:10.1021/bi00430a023

Cited by 81 publications

(83 citation statements)

References 46 publications

(53 reference statements)

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“…Integration of the ethanol production genes into the chromosomal pfl gene may further contribute to this problem by reducing the level of pyruvate formate-lyase activity. Although pyruvate dehydrogenase has a K m for pyruvate that is equal to that of pyruvate decarboxylase, pyruvate dehydrogenase is expressed at low levels during fermentation and is allosterically inhibited by the high levels of NADH present during fermentation (14,20). In CSLϩX medium, a portion of cellular pyruvate was converted to acetyl-CoA by KO11 during the first 24 h, as shown by the accumulation of acetate as a fermentation product.…”

Section: Discussionmentioning

confidence: 99%

“…At least three sites of allosteric regulation may contribute to the increase in growth. Both pyruvate dehydrogenase (14,20) and citrate synthase (17,48,49) are allosterically inhibited by NADH. Oxidation of NADH from glycolysis during reduction of acetaldehyde from added pyruvate or added acetaldehyde tends to decrease the NADH/NAD ϩ ratio.…”

Section: Discussionmentioning

confidence: 99%

“…The ratio of NAD(P)H to NAD(P) ϩ has been shown to alter cellular patterns of metabolic flux (14). NAD(P)H is an allosteric inhibitor of many enzymes, including pyruvate dehydrogenase (20), phosphotransacetylase (41), malate dehydrogenase (38), and citrate synthase (17,48). In KO11, addition of acetaldehyde or pyruvate (metabolized to acetaldehyde by recombinant pyruvate decarboxylase) would be expected to decrease the level of NAD(P)H and the NAD(P)H/NAD(P) ϩ ratio by increasing the pool of acetaldehyde available for reduction to ethanol.…”

Section: Vol 68 2002 Flux Through Citrate Synthase Limits Growth 1075mentioning

confidence: 99%

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Flux through Citrate Synthase Limits the Growth of EthanologenicEscherichia coliKO11 during Xylose Fermentation

Underwood

Buszko

Shanmugam

et al. 2002

Appl Environ Microbiol

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Previous studies have shown that high levels of complex nutrients (Luria broth or 5% corn steep liquor) were necessary for rapid ethanol production by the ethanologenic strain Escherichia coli KO11. Although this strain is prototrophic, cell density and ethanol production remained low in mineral salts media (10% xylose) unless complex nutrients were added. The basis for this nutrient requirement was identified as a regulatory problem created by metabolic engineering of an ethanol pathway. Cells must partition pyruvate between competing needs for biosynthesis and regeneration of NAD ؉ . Expression of low-K m Zymomonas mobilis pdc (pyruvate decarboxylase) in KO11 reduced the flow of pyruvate carbon into native fermentation pathways as desired, but it also restricted the flow of carbon skeletons into the 2-ketoglutarate arm of the tricarboxylic acid pathway (biosynthesis). In mineral salts medium containing 1% corn steep liquor and 10% xylose, the detrimental effect of metabolic engineering was substantially reduced by addition of pyruvate. A similar benefit was also observed when acetaldehyde, 2-ketoglutarate, or glutamate was added. In E. coli, citrate synthase links the cellular abundance of NADH to the supply of 2-ketoglutarate for glutamate biosynthesis. This enzyme is allosterically regulated and inhibited by high NADH concentrations. In addition, citrate synthase catalyzes the first committed step in 2-ketoglutarate synthesis. Oxidation of NADH by added acetaldehyde (or pyruvate) would be expected to increase the activity of E. coli citrate synthase and direct more carbon into 2-ketoglutarate, and this may explain the stimulation of growth. This hypothesis was tested, in part, by cloning the Bacillus subtilis citZ gene encoding an NADH-insensitive citrate synthase. Expression of recombinant citZ in KO11 was accompanied by increases in cell growth and ethanol production, which substantially reduced the need for complex nutrients.The engineering of metabolic pathways for production of industrial chemicals from renewable carbohydrates offers an alternative to petroleum-based processes and chemical synthesis (3,12,22). Genetically altering metabolic pathways, however, often causes unanticipated changes which may limit utility. Undesirable changes, such as reduced growth, decreased glycolytic flux, and low volumetric productivity, are generally attributed to a lack of ATP (19, 50), creation of futile cycles (10,11,37), changes in intracellular metabolite pools, or a metabolic imbalance (2,7,9,13,28,51,53). Often, these detrimental effects are masked by abundant complex nutrients in laboratory media and are apparent only in more minimal media (7,10,11,31).Salmonella enterica serovar Typhimurium was engineered for succinate production by increasing expression of pyc encoding pyruvate carboxylase (50). Although succinate production increased, the growth rate declined by 18% and the glycolytic flux decreased by 40%. Similar results were reported for an analogous construction in Escherichia coli (19). Donnelly and cow...

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Vol 68 2002 Flux Through Citrate Synthase Limits Growth 1075mentioning

confidence: 99%

See 1 more Smart Citation

Flux through Citrate Synthase Limits the Growth of EthanologenicEscherichia coliKO11 during Xylose Fermentation

Underwood

Buszko

Shanmugam

et al. 2002

Appl Environ Microbiol

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“…Not just is k,,,/K,,, raised in value by a factor of lo4, but the lipoyl domain confers specificity on the pendant lipoyl group for reductive acylation only by the E l component of the parent 2-oxo acid dehydrogenase complex (Graham et al, 1989). This offers a most effective means of substrate channeling (Perham, 1991), but the structural basis remains unclear.…”

Section: Comparison Of Loops Close In Space To the Modified Lysine Hamentioning

confidence: 99%

Prediction of the three‐dimensional structures of the biotinylated domain from yeast pyruvate carboxylase and of the lipoylated H‐protein from the pea leaf glycine cleavage system: A new automated method for the prediction of protein tertiary structure

Brocklehurst

Perham

1993

Protein Science

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A new, automated, knowledge‐based method for the construction of three‐dimensional models of proteins is described. Geometric restraints on target structures are calculated from a consideration of homologous template structures and the wider knowledge base of unrelated protein structures. Three‐dimensional structures are calculated from initial partly folded states by high‐temperature molecular dynamics simulations followed by slow cooling of the system (simulated annealing) using nonphysical potentials. Three‐dimensional models for the biotinylated domain from the pyruvate carboxylase of yeast and the lipoylated H‐protein from the glycine cleavage system of pea leaf were constructed, based on the known structures of two lipoylated domains of 2‐oxo acid dehydrogenase multienzyme complexes. Despite their weak sequence similarity, the three proteins are predicted to have similar three‐dimensional structures, representative of a new protein module. Implications for the mechanisms of posttranslational modification of these proteins and their catalytic function are discussed.

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“…The lipoyl domains were dialysed against 50 mM potassium phosphate pH 7.0. The purity and lipoylation (see also more, the three-dimensional structure of the lipoyl domain is required for promoting reductive acylation of its pendant lipoyl Berg et al, 1994) of the lipoyl domains was analysed by denaturing and non-denaturing PAGE (Schägger and von Jagow, group by the E1 component (Graham et al, 1989), although this does not seem to happen just simply by enhancement of the 1987), with resolving gel 16.5% T, 3 % C and stacking gel 4%…”

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confidence: 99%

Kinetics and specificity of reductive acylation of wild‐type and mutated lipoyl domains of 2‐oxo‐acid dehydrogenase complexes from Azotobacter vinelandii

Berg

Westphal

Bosma

et al. 1998

European Journal of Biochemistry

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The kinetics and specificity of reductive acylation of lipoyl domains derived from Azotobacter vinelandii 2-oxo-acid dehydrogenase complexes, catalysed by A. vinelandii and Escherichia coli complexes, have been investigated. With the wild-type pyruvate dehydrogenase complex from A. vinelandii the rate of reductive acetylation and deacetylation was studied by rapid mixing methods. The rate of reductive acetylation, 126 s Ϫ1 , corresponds well with the turnover rate derived from steady-state measurements. Deacetylation was rapid and specific for coenzyme A. No deacetylation was observed with reduced or oxidised lipoamide or with dithiothreitol. The rate of reductive acetylation of complex-bound lipoyl domains by pyruvate dehydrogenase (E1p) is at least 60 times higher than of free lipoyl domains under comparable conditions. This gain in catalytic rate indicates a large diffusion limitation of lipoyl domains when attached via the flexible linker segments to the complex, and illustrates the efficiency of substrate channeling in the multienzyme complex.The 2-oxo-acid dehydrogenases exhibit specificity for lipoyl domains in the reductive acylation reaction. The A. vinelandii lipoyl domain derived from the pyruvate dehydrogenase complex is a good substrate for A. vinelandii E1p, but not for A. vinelandii 2-oxoglutarate dehydrogenase (E1o), and vice versa. The A. vinelandii lipoyl domain of the pyruvate dehydrogenase complex is also, although at a lower rate, reductively acetylated by E. coli E1p and reductively succinylated by E. coli E1o. Likewise, the A. vinelandii lipoyl domain derived from the 2-oxoglutarate dehydrogenase complex is recognised by E. coli E1o, but not by E. coli E1p. This suggests that common determinants of the lipoyl domains exist that are responsible for recognition by the E1 components. On the basis of the observed specificity and lipoyl domain sequences and structures, an exposed loop of the A. vinelandii 2-oxoglutarate dehydrogenase complex lipoyl domain was subjected to mutagenesis. Although the reductive acylation experiments of mutants of the lipoyl domain indicate the importance of this loop for recognition, it is probably not the single determinant for specificity.Keywords : multienzyme complex; lipoyl domain; recognition; mutagenesis; Azotobacter vinelandii.The 2-oxo-acid dehydrogenase multienzyme complexes ca-nase complex (OGDHC) converts 2-oxoglutarate into succinylCoA. The complexes effectuate these reactions by combining talyse the oxidative decarboxylation of 2-oxo acids to the correthe activities of three enzymes : a substrate-specific 2-oxo-acid sponding acyl-CoA derivatives, accompanied with the reduction dehydrogenase [pyruvate dehydrogenase (E1p) or 2-oxoglutarate of NAD ϩ [for recent reviews see Mattevi et al. (1992a), Berg dehydrogenase (E1o)], an acyltransferase [acetyltransferase and de Kok (1997b)]. In the gram-negative bacteria Azotobacter (E2p) or succinyltransferase (E2o)], and a common lipoamide vinelandii and Escherichia coli two similar complexes are predehydrogenase...

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Kinetics and specificity of reductive acylation of lipoyl domains from 2-oxo acid dehydrogenase multienzyme complexes

Cited by 81 publications

References 46 publications

Flux through Citrate Synthase Limits the Growth of EthanologenicEscherichia coliKO11 during Xylose Fermentation

Flux through Citrate Synthase Limits the Growth of EthanologenicEscherichia coliKO11 during Xylose Fermentation

Prediction of the three‐dimensional structures of the biotinylated domain from yeast pyruvate carboxylase and of the lipoylated H‐protein from the pea leaf glycine cleavage system: A new automated method for the prediction of protein tertiary structure

Kinetics and specificity of reductive acylation of wild‐type and mutated lipoyl domains of 2‐oxo‐acid dehydrogenase complexes from Azotobacter vinelandii

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