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.

FIG. 1

Interconversion of different forms of PFL in E. coli (19). act, PFL activase; deact, PFL deactivase; ●, the free radical of active PFL.

FIG. 2

Specificities of anti-PFL antibodies and analysis of PFL cleavage by Western blotting. (A) Western blot analysis of L. lactis MG1363 (lane 1) and the pfl mutant strain MGKAS13 (lane 2). (B) Western blot analysis of extracts of MG1363. Lane 1, anaerobic growth and extraction; lane 2, anaerobic growth and extraction with subsequent exposure to air for 5 min; lane 3, anaerobic growth and aerobic extraction.

FIG. 3

Western blot of protein extracts from L. lactis MG1363 grown in defined MS10 medium (A) and complex M17 broth (B). Aerobic (lane 1) and anaerobic (lane 2) growth on glucose is shown. Aerobic (lane 3) and anaerobic (lane 4) growth on galactose is also shown. Protein extracts were prepared aerobically, which led to oxygenolytic cleavage of activated PFL subunits.

FIG. 4

PFL Western blot analysis of L. lactis MG1363 (lane 1) and the adhE mutant strain MGKAS15 (lane 2) grown aerobically in complex medium. Protein extraction was carried out aerobically.