Publications

• The glycyl radical enzyme TdcE can replace pyruvate formate-lyase in glucose fermentation.

  Sawers G., Hesslinger C., Muller N., Kaiser M.

  Mutants of Escherichia coli unable to synthesize a functional pyruvate formate-lyase (PFL) are severely impaired in their capacity to grow by glucose fermentation. In a functional complementation assay designed to isolate the pfl gene from Clostridium butyricum, we fortuitously identified a gene t ... >> More

  Mutants of Escherichia coli unable to synthesize a functional pyruvate formate-lyase (PFL) are severely impaired in their capacity to grow by glucose fermentation. In a functional complementation assay designed to isolate the pfl gene from Clostridium butyricum, we fortuitously identified a gene that did not encode a PFL but nonetheless was able to complement the phenotypic defects caused by an E. coli pfl mutation. The clostridial gene encoded a basic 14. 5-kDa protein (TcbC) which, based on amino acid similarity and analysis of immediately adjacent DNA sequences, was part of a transposase exhibiting extensive similarity to the product of the site-specific transposon Tn554 from Staphylococcus aureus. Our studies revealed that the clostridial TcbC protein activated the transcription of the E. coli tdcABCDEFG operon, which encodes an anaerobic L-threonine-degradative pathway. Normally, anaerobic synthesis of the pathway is optimal when E. coli grows in the absence of catabolite-repressing sugars and in the presence of L-threonine. Although anaerobic control of pathway synthesis was maintained, TcbC alleviated glucose repression. One of the products encoded by the tdc operon, TdcE, has recently been shown to be a 2-keto acid formate-lyase (C. Hesslinger, S. A. Fairhurst, and G. Sawers, Mol. Microbiol. 27:477-492, 1998) that can accept pyruvate as an enzyme substrate. Here we show that TdcE is directly responsible for the restoration of fermentative growth to pfl mutants. << Less

  J Bacteriol 180:3509-3516(1998) [PubMed] [EuropePMC]

• Novel keto acid formate-lyase and propionate kinase enzymes are components of an anaerobic pathway in Escherichia coli that degrades L-threonine to propionate.

  Hesslinger C., Fairhurst S.A., Sawers G.

  An immunological analysis of an Escherichia coli strain unable to synthesize the main pyruvate formate-lyase enzyme Pfl revealed the existence of a weak, cross-reacting 85 kDa polypeptide that exhibited the characteristic oxygen-dependent fragmentation typical of a glycyl radical enzyme. Polypepti ... >> More

  An immunological analysis of an Escherichia coli strain unable to synthesize the main pyruvate formate-lyase enzyme Pfl revealed the existence of a weak, cross-reacting 85 kDa polypeptide that exhibited the characteristic oxygen-dependent fragmentation typical of a glycyl radical enzyme. Polypeptide fragmentation of this cross-reacting species was shown to be dependent on Pfl activase. Cloning and sequence analysis of the gene encoding this protein revealed that it coded for a new enzyme, termed TdcE, which has 82% identity with Pfl. On the basis of RNA analyses, the tdcE gene was shown to be part of a large operon that included the tdcABC genes, encoding an anaerobic threonine dehydratase, tdcD, coding for a propionate kinase, tdcF, the function of which is unknown, and the tdcG gene, which encodes a L-serine dehydratase. Expression of the tdcABCDEFG operon was strongly catabolite repressed. Enzyme studies showed that TdcE has both pyruvate formate-lyase and 2-ketobutyrate formate-lyase activity, whereas the TdcD protein is a new propionate/acetate kinase. By monitoring culture supernatants from various mutants using 1H nuclear magnetic resonance (NMR), we followed the anaerobic conversion of L-threonine to propionate. These studies confirmed that 2-ketobutyrate, the product of threonine deamination, is converted in vivo by TdcE to propionyl-CoA. These studies also revealed that Pfl and an as yet unidentified thiamine pyrophosphate-dependent enzyme(s) can perform this reaction. Double null mutants deficient in phosphotransacetylase (Pta) and acetate kinase (AckA) or AckA and TdcD were unable to metabolize threonine to propionate, indicating that propionyl-CoA and propionyl-phosphate are intermediates in the pathway and that ATP is generated during the conversion of propionyl-P to propionate by AckA or TdcD. << Less

  Mol. Microbiol. 27:477-492(1998) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.