Reappraisal of the Regulation of Lactococcal l -Lactate Dehydrogenase

Abstract

Lactococcal lactate dehydrogenases (LDHs) are coregulated at the substrate level by at least two mechanisms: the fructose-1,6-biphosphate/phosphate ratio and the NADH/NAD ratio. Among the Lactococcus lactis species, there are strains that are predominantly regulated by the first mechanism (e.g., strain 65.1) or by the second mechanism (e.g., strain NCDO 2118). A more complete model of the kinetics of the regulation of lactococcal LDH is discussed.Lactococci are known for their homolactic metabolism, whereby more than 90% of the sugars present are converted into lactic acid. However, under certain conditions, the metabolism may shift to the production of mixed acids (acetate, ethanol, and formate). From the 1960s onward, the view that the control of this shift was modulated mainly by the intracellular concentration of fructose-1,6-biphosphate (FBP) activating both L-lactate dehydrogenase (L-LDH; EC 1.1.1.27) and pyruvate kinase (EC 2.7.1.40) was held (3,9,18,19). Inorganic phosphate (P i ) was recognized as a severe inhibitor of both enzymes. Apparently, both FBP and P i were seen to compete for the same allosteric site of LDH (9).Recently, this metabolic model was questioned by Garrigues et al. (5), who showed that the sugar metabolism of strain NCDO 2118 was instead regulated by the NADH/NAD ratio. A high ratio inhibited glyceraldehyde 3-phosphate dehydrogenase (GAPDH; EC 1.2.1.12) and increased LDH activity. Many, but not all, researchers took up this view without reflection, creating a confusing situation. Currently, metabolic flux models based on enzyme kinetics are applied as predictive tools in metabolic engineering (see, e.g., reference 8), illustrating the importance of expressing kinetic characteristics adequately. Therefore, we undertook an investigation regarding the nature of the regulation of LDH activity among several Lactococcus lactis strains, including those frequently used in metabolic flux studies. We also examined the effects of ATP, ADP, AMP, and phosphoenolpyruvate (PEP), since Jonas et al. (9) observed strong competitive inhibition by ATP of LDH activity in L. lactis strain US3 (ϭNCIMB701197).L. lactis strains (listed in Table 1) were each grown anaerobically at 30°C in pH-controlled batch cultures on glucose (10 g/liter) with SD3 medium (17). The external pH was kept at 6.5, which corresponds to an internal pH of 7.2 (12). The cultures were harvested in the late exponential phase, centrifuged at 4°C for 10 min at 5,000 ϫ g, washed, and resuspended in triethanolamine buffer (50 mM triethanolamine [pH 7.2], 5 mM MgCl 2 ⅐ 6H 2 O). Cell extracts (CE) were prepared by using glass beads (17). LDH activity was measured spectrophotometrically by monitoring the oxidation of NADH (340 nm; ε ϭ 6,220 M Ϫ1 cm Ϫ1 ) at 30°C. One milliliter of the standard assay mixture consisted of triethanolamine buffer (50 mM; pH 7.2), NADH (0.3 mM), FBP (10 mM), and CE (approximately 160 mg of protein/liter). The reaction was initiated by the addition of pyruvate (initial concentration, 10 mM). One unit of LDH was de...

The Pool of ADP and ATP Regulates Anaerobic Product Formation in Resting Cells of Lactococcus lactis

“…The concentrations of P i and FBP vary inversely (7,37,52), and for decades these compounds have been recognized as effectors of pyruvate kinase and LDH (10,11). A recent study showed that the extents of inhibition by P i and activation by FBP of LDH vary in different lactococcal strains commonly used in research (54). Since P i and FBP apparently compete for the same allosteric site (27), the inhibition of LDH by P i is most likely overcome by the presence of the high concentrations of FBP found in the present study and other studies (11,16).…”

Engineering of a Xylose Metabolic Pathway in Corynebacterium glutamicum

“…These intermediates are subsequently eventually converted into lactic and succinic acids under anaerobic conditions by the enzymes LDH and succinate dehydrogenase, respectively. LDH is known to be allosterically regulated in lactococci, as it is activated by intracellular fructose-1,6-bisphosphate (49). In addition, lower amounts of ldh transcripts on xylose than on glucose have been observed with other genera, for example, Rhizopus oryzae (42).…”

Comparative study of Trichoderma gene expression in interactions with tomato plants using high-density oligonucleotide microarrays

“…Taking the T87 data together, it is difficult to understand the role played by the downregulation of genes involved in amino acid metabolism, although the turn-off of glutamine synthetase and L-amino acid oxidase could indicate a sufficient availability of nitrogen. In bacteria, lactate dehydrogenases can be regulated by the fructose 1,6-bisphosphate/phosphate ratio (van Niel et al, 2004). The downregulation of lactate dehydrogenase in T87 could be due to the accumulation of fructose 6-phosphate obtained via the activation of Gln-6P deaminase observed in the T87-tomato interaction.…”