NOVEL KETO ACID FORMATE-LYASE AND PROPIONATE KINASE ENZYMES ARE COMPONENTS OF AN ANAEROBIC PATHWAY IN ESCHERICHIA-COLI THAT DEGRADES L-THREONINE TO PROPIONATE

An immunological analysis of an Escherichia coil strain unable to synt hesize the main pyruvate formate-lyase enzyme Pfl revealed the existen ce of a weak, crossreacting 85 kDa polypeptide that exhibited the char acteristic oxygen-dependent fragmentation typical of a glycyl radical enzyme. Polypeptide fragmentation of this cross-reacting species was s hown to be dependent on Pfl activase. Cloning and sequence analysis of the gene encoding this protein revealed that it coded for a new enzym e, termed TdcE, which has 82% identity with Pfl. On the basis of RNA a nalyses, the tdcE gene was shown to be part of a large operon that inc luded the fdcABC genes, encoding an anaerobic threonine dehydratase, t dcD, coding for a propionate kinase, tdcF, the function of which is un known, and the tdcG gene, which encodes a L-serine dehydratase. Expres sion of the tdcABCDEFG operon was strongly catabolite repressed. Enzym e studies showed that TdcE has both pyruvate formate-lyase and 2-ketob utyrate formate-lyase activity, whereas the TdcD protein is a new prop ionate/acetate kinase. By monitoring culture supernatants from various mutants using H-1 nuclear magnetic resonance (NMR), we followed the a naerobic conversion of L-threonine to propionate. These studies confir med that 2-ketobutyrate, the product of threonine deamination, is conv erted in vivo by TdcE to propionyl-CoA. These studies also revealed th at Pfl and an as yet unidentified thiamine pyrophosphate-dependent enz yme(s) can perform this reaction. Double null mutants deficient in pho sphotransacetylase (Pta) and acetate kinase (AckA) or AckA and TdcD we re unable to metabolize threonine to propionate, indicating that propi onyl-CoA and propionyl-phosphate are intermediates in the pathway and that ATP is generated during the conversion of propionyl-P to propiona te by AckA or TdcD.