Pyruvate formate-lyase (inactive form) and pyruvate formate-lyase activating enzyme of Escherichia coli: Isolation and structural properties

Conradt, Harald S.; Hohmann-Berger, Marita; Hohmann, Hans‐Peter; Blaschkowski, Hans P.; Knappe, J

doi:10.1016/0003-9861(84)90054-7

Cited by 102 publications

(105 citation statements)

References 20 publications

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“…Growth of bacteria and enzyme preparations E. coli K12 cells, transformed with plasmid p29 [6], were grown anaerobically with glucose (10 g/L) and chloramphenicol (30 mg/L) in mineral medium as described previously [7]. (Pyruvate formate-lyase constitutes about 20% of the soluble proteins in these cells.)…”

Section: Materidrmentioning

confidence: 99%

“…Enzyme activity was measured with the coupled optical assay [7]. Active sites were chemically quantified from [14C]acetyl charging (I nmol is bound to a protein equivalent of 35 u [3]).…”

Section: Materidrmentioning

confidence: 99%

“…With a dissociable cofactor ruled out [7], we expect that the unpaired electron resides on a standard amino acid residue of the polypeptide chain. This assumption has recently been confirmed by expressing the plasmid-borne pfl gene of E. coli in Pseudomonus putidn cells (which do not harbor the pfl system in their chromosome).…”

Section: Isotopic Substitutionsmentioning

confidence: 99%

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The free radical of pyruvate formate‐lyase

Unkrig

Neugebauer

Knappe

1989

European Journal of Biochemistry

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The first-derivative EPR spectrum of the active form of Escherichiu coli pyruvate formate-lyase shows an asymmetric doublet with partially resolved hyperfine splittings (g = 2.0037). Isotope substitution studies demonstrated couplings of a carbon-centered unpaired electron to a solvent-exchangeable proton (a = 1.5 mT) and to further hydrogen nuclei (a = 0.36 and 0.57 mT). By selective incorporation of unlabelled tyrosine into 2H-labelled enzyme protein, a tyrosyl radical structure has been ruled out. Circumstantial evidence indicates that the organic free radical, which also displays an ultraviolet absorption signal at 365 nm, is located on a standard amino acid residue of the polypeptide chain. EPR signal quantification found a stoichiometry of I spin per active site.The formate analogue hypophosphite has been characterized as a specific k,,, inhibitor of pyruvate formatelyase which destroys the enzyme radical. Protein-linked 1 -hydroxyethylphosphonate was previously described as the dead-end product after reaction of the analogue with the intermediary acetyl-enzyme form of the catalytic cycle [W. Plaga et al. (1988) Eur. J. Biochem. 178,. EPR spectroscopy of this system has now identified the corresponding a-phosphoryl radical as a reaction intermediate [g = 2.0032; u(P) = 2.72 mT, a(3H) = 1.96 mT]; it showed a half-life of about 20 min at 0°C. This finding proves that the enzyme radical is a hydrogen-atomtransferring coenzymic element.Pyruvate formate-lyase is the key enzyme of the anaerobic glucose degradation route in Escherichia coli cells [l]. It shows unique mechanisms of enzyme regulation and catalysis. Transcription of the structural gene (~f l )is highly controlled by anaerobic induction [2]. At the post-translational level, pyruvate formate-lyase is interconverted by specific converter enzymes between a fully inactive form (Ei) and an active form (E,) that contains a persistent organic free radical [3]. Pyruvate is nonoxidatively processed to acetyl-CoA by a two-step reaction pattern with an intermediary acetyl-enzyme:(2) Addressing structure/function relationships, we have recently mapped the covalent catalytic SH-group as the Cys-419 residue of the polypeptide chain [4]. These studies also showed that the acetyl-enzyme intermediate will react with the formate analogue hypophosphite to produce l-hydroxyethylphosphonate, linked as a thioester to Cys-418. The C-P bond-forming process, which resembles pyruvate synthesis from formate (reversal of Eqn l), has now been investigated by EPR spectroscopy. We have identified a substrate-based radical intermediate, thus demonstrating for the first time a functional role of the free radical of pyruvate formate-lyase. Our findings substantiate the notion that the reversible C-C bond cleavage/synthesis in the first half-reaction of the catalytic cycle occurs by a radical mechanism.We also report in this paper the EPR and ultraviolet spectroscopic characterization of the enzyme radical, its quantification, and several isotope substitution experiments for approachi...

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Section: Materidrmentioning

confidence: 99%

“…Enzyme activity was measured with the coupled optical assay [7]. Active sites were chemically quantified from [14C]acetyl charging (I nmol is bound to a protein equivalent of 35 u [3]).…”

Section: Materidrmentioning

confidence: 99%

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The free radical of pyruvate formate‐lyase

Unkrig

Neugebauer

Knappe

1989

European Journal of Biochemistry

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“…Induction of pfl gene expression under anaerobic conditions has been shown to involve both the FNR and the ArcA/ArcB regulatory proteins, which regulate gene expression in response to O 2 and various metabolites, respectively [3,4]. In addition to this transcriptional regulation, PFL requires post-translational activation by the pyruvate formate-lyase activating enzyme (PFL-AE) [5]. PFL-AE is constitutively expressed under both aerobic and anaerobic conditions [6], however in vitro studies have demonstrated that it is catalytically active only under anaerobic reducing conditions [7,8].…”

Section: Introductionmentioning

confidence: 99%

Inactivation of E. coli pyruvate formate-lyase: Role of AdhE and small molecules

Nnyepi

Peng

Broderick

2007

Archives of Biochemistry and Biophysics

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E. coli AdhE has been reported to harbor three distinct enzymatic activities: alcohol dehydrogenase, acetaldehyde-CoA dehydrogenase, and pyruvate formate-lyase (PFL) deactivase. Herein we report on the cloning, expression, and purification of E. coli AdhE, and the re-investigation of its purported enzymatic activities. While both the alcohol dehydrogenase and acetaldehyde-CoA dehydrogenase activities were readily detectible, we were unable to obtain any evidence for catalytic deactivation of PFL by AdhE, regardless of whether the reported cofactors for deactivation (Fe(II), NAD, and CoA) were present. Our results demonstrate that AdhE is not a PFL deactivating enzyme. We have also examined the potential for deactivation of active PFL by small-molecule thiols. Both β-mercaptoethanol and dithiothreitol deactivate PFL efficiently, with the former providing quite rapid deactivation. PFL deactivated by these thiols can be reactivated, suggesting that this deactivation is non-destructive transfer of an H atom equivalent to quench the glycyl radical.

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“…Pyruvate formate-lyase (PFL; EC 2.3.1.54) is a key enzyme in fermentative growth and is responsible for converting pyruvate to acetyl coenzyme A and formate, which serve as substrates for the production of acetate, ethanol, hydrogen gas, and carbon dioxide (14). The activity of PFL is regulated by a reversible enzymatic interconversion of an active form to an inactive form in response to oxygen (6). Smith and Neidhardt (27) determined that the anaerobic steady-state level of PFL is 10 times higher than the aerobic level.…”

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confidence: 99%

Transcription of pfl is regulated by anaerobiosis, catabolite repression, pyruvate, and oxrA: pfl::Mu dA operon fusions of Salmonella typhimurium

1989

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Pyruvate formate-lyase (EC 2.3.1.54), a key enzyme in the anaerobic metabolism of Salmonella typhiunuium, catalyzes the conversion of pyruvate to acetyl coenzyme A and formate. pfl::Mu dA operon fusions were isolated for the study of transcriptional regulation. pfl was transcribed both aerobically and anaerobically, but the activity increased about sixfold under anaerobic conditions. The addition of pyruvate, formate, and acetate in nutrient broth did not have any effect on the anaerobic expression of pfl. However, the addition of pyruvate to minimal glucose medium increased the anaerobic expression of pfl. The expression of pfl varied in different growth media. Anaerobic expression of pfl was lower when the culture was grown in minimal glucose medium than when it was grown in nutrient broth. When Casamino Acids (Difco Laboratories, Detroit, Mich.) were added to minimal glucose medium, the expression ofpfl increased proportionally with the amount of Casamino Acids added. The transcription of pfl was positively controlled by the oxrA gene product and was affected by both the cya and crp mutations. However, mutations in genes affecting the cyclic AMP-cyclic AMP receptor protein complex or oxrA could not completely abolish the anaerobic derepression ofpfl. In merodiploid strains, pfl::Mu dA/F' pjt, the j-galactosidase activities were decreased. The mutations gyrA, oxrC, and oxrE, which affected anaerobic metabolism, did not affect anaerobic expression of pfl.

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Pyruvate formate-lyase (inactive form) and pyruvate formate-lyase activating enzyme of Escherichia coli: Isolation and structural properties

Cited by 102 publications

References 20 publications

The free radical of pyruvate formate‐lyase

The free radical of pyruvate formate‐lyase

Inactivation of E. coli pyruvate formate-lyase: Role of AdhE and small molecules

Transcription of pfl is regulated by anaerobiosis, catabolite repression, pyruvate, and oxrA: pfl::Mu dA operon fusions of Salmonella typhimurium

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