Synthesis and Posttranslational Regulation of Pyruvate Formate-Lyase in Lactococcus lactis

Abstract

The enzyme pyruvate formate-lyase (PFL) from Lactococcus lactis was produced in Escherichia coli and purified to obtain anti-PFL antibodies that were shown to be specific for L. lactis PFL. It was demonstrated that activated L. lactis PFL was sensitive to oxygen, as in E. coli, resulting in the cleavage of the PFL polypeptide. The PFL protein level and its in vivo activity and regulation were shown by Western blotting, enzyme-linked immunosorbent assay, and metabolite measurement to be dependent on the growth conditions. The PFL level during anaerobic growth on the slowly fermentable sugar galactose was higher than that on glucose. This shows that variation in the PFL protein level may play an important role in the regulation of metabolic shift from homolactic to mixed-acid product formation, observed during growth on glucose and galactose, respectively. During anaerobic growth in defined medium, complete activation of PFL was observed. Strikingly, although no formate was produced during aerobic growth of L. lactis, PFL protein was indeed detected under these conditions, in which the enzyme is dispensable due to the irreversible inactivation of PFL by oxygen. In contrast, no oxygenolytic cleavage was detected during aerobic growth in complex medium. This observation may be the result of either an effective PFL deactivase activity or the lack of PFL activation. In E. coli, the PFL deactivase activity resides in the multifunctional alcohol dehydrogenase ADHE. It was shown that in L. lactis, ADHE does not participate in the protection of PFL against oxygen under the conditions analyzed. Our results provide evidence for major differences in the mechanisms of posttranslational regulation of PFL activity in E. coli and L. lactis.

Pyruvate Formate-lyase and a Novel Route of Eukaryotic ATP Synthesis in Chlamydomonas Mitochondria

“…Alternatively, the 3-kDa difference might be explained by differential activation and radical-induced cleavage at the C-terminal end. However, it is not currently known whether C. reinhardtii PFL may be subject to oxygenolytic cleavage as its counterparts in bacteria (2,14,15). In our mitochondrial proteome analysis, one peptide that matches with the C-terminal sequence of PFL was identified.…”

“…This peptide stems from (i) a protein of high molecular mass (ϳ60 -75 kDa) and (ii) a region of the PFL that is C-terminal to the glycine residue to be activated into a glycyl radical. In E. coli, PFL is a homodimer that is post-translationally activated by introduction of a radical on the ultimate Gly residue of only one of the two monomers (2,15). Whether only one or both PFL subunits are activated in C. reinhardtii is yet unknown.…”

“…The radical-containing protein is extremely sensitive to oxygen: exposure of activated E. coli PFL to air results into its cleavage in two polypeptides of ϳ82 and 3 kDa (2,13). Fragmentation of activated PFL by oxygen has also been reported in the ruminal bacterium Streptococcus bovis (14) and in the lactic acid bacterium Lactococcus lactis (15). Oxygenolytic cleavage of PFL appears to be a drastic means to inactivate an enzyme that would produce less energy than its aerobic counterpart, pyruvate dehydrogenase.…”

“…Quenching of the radical present on activated PFL by ADHE (16,17) occurs via a yet unknown mechanism. However, ADHE does not always exhibit the PFL deactivase activity, as shown in L. lactis (15).…”

Metabolic Behavior of Lactococcus lactis MG1363 in Microaerobic Continuous Cultivation at a Low Dilution Rate

“…In this light, it is surprising that, to our knowledge, no investigations have been carried out on the microaerobic physiology of starter cultures. A number of studies on the effect of oxygen in fully aerated cultures have been published, but little light has been shed on the window ranging from anaerobic to fully aerobic conditions of growth (4,7,8,20).Interest in the microaerobic physiology of LAB is furthermore spurred by reports on the extreme sensitivity of pyruvate formate-lyase (PFL) to oxygen (1, 25) and the negative effect of oxygen on pfl gene expression (2,19). Similarly, it has recently been reported that vigorous aeration reduces the transcription of the adhE gene, which encodes alcohol dehydrogenase (ADH) in Lactococcus lactis (3).…”