Pyruvate formate-lyase (PFL) is an important enzyme in the metabolic pathway of lactic acid bacteria (LAB) and is held responsible for the regulation of the shift between homolactic acid to mixed acid fermentation. PFL catalysis the reversible reaction of acetyl-CoA and formate into pyruvate and CoA. A glycyl radical, who is regenerated within the reaction, is involved; therefore, PFL works only under strictly anaerobic conditions. For its activation, the C-terminal domain has to bind to the iron-sulfur cluster containing pyruvate formate lyase activating enzyme (PFLae) so that the catalytic Gly734 of PFL can bind to the active site of PFLae for forming a glycyl radical. An exposure of the activated PFL to air leads to an oxygenolytic cleavage of the C-terminus. An earlier proposed inactivation by the bifunctional alcohole dehydrogenase (AdhE) for preventing this irreversible oxgenolytic cleavage was reported controversially and can be doubted. For PFL of some organisms, transcriptional and post-transcriptional regulations depending on growth and environment conditions were reported in literature. In particular, for PFL of L. lactis and S. mutans, an inhibition by triose phosphate (GAP, DHAP) was described but no potential binding sites were stated. Based on our docking experiments for finding potential binding sites, we propose an allosteric mechanism of inhibition. by triose phosphate.
This report is organised as follows: After summarizing the literature describing the role of PFL and PFLae (section 2 and 3), we structurally compare PFL of different organisms and describe in this report potential allosteric binding sites close to the C-terminus of PFL using homology modelling and computational docking experiments(section 4). A binding of triose phosphate to those sites may prevent a PFL-PFLae complex formation by inhibiting the movement of the C-terminus. The proposed sites are not strictly conserved among E. coli and LAB, and in some cases a binding of triose phosphate is either not possible or not likely. This reveals differences between the LAB which is in agreement with differences in the locations of the genes pflA (PFLae) and pflB (PFL) presented in section 5. An analysis of the gene locations revealed that in the organisms E. coli, E. faecalis and L. plantarum the two genes, pflA and pflB, are laying close to each other whereas in L. lactis, S. pyogenes and S. mutans the genes can be found at distinct locations leading to the conclusion that differences in the transcriptional and post-transcriptional regulation of PFL and PFLae can be expected for the different LAB and E. coli. At the end of the report, we present briefly a literature summary of PFL in respect to metabolism and growth rate (section 6), before we discuss the possibility of PFL inactivation by AdhE (section 7). Finally, we want to address the last section (section 8) to the experimentalist of SysMO-LAB2 suggesting some experiments for analysing the role and regulation of PFL and PFLae in LAB in more details. Here, any input and discussion regarding such experiments is highly welcome.
Created: 1st Mar 2012 at 18:32
Last updated: 12th May 2016 at 17:55