Lactate dehydrogenase from Streptococcus mutans: purification, characterization, and crossed antigenicity with lactate dehydrogenases from Lactobacillus casei, Actinomyces viscosus, and Streptococcus sanguis

Abstract

A cytoplasmic fructose-1,6-diphosphate-dependent lactate dehydrogenase (LDH; EC 1.1.1.27) from Streptococcus mutans OMZ175 was purified to homogeneity as judged by sodium dodecyl sulfate-gel electrophoresis. The purification consisted of ammonium sulfate precipitation of the cytoplasmic fraction, DEAE-Sephacel and Blue-Sepharose CL.6B chromatography, and Sephacryl S200 gel permeation. The catalytic activity of the purified enzyme required the presence of fructose-1,6-diphosphate with a broad optimum between pH 5 and 6.2. The concentration of fructose-1,6-diphosphate required for half-maximal velocity was around 0.02 mM and was affected by the pyruvate concentration. The enzyme seemed to have at least two binding sites for the activator which interact in a cooperative manner. Increasing concentrations of fructose-1,6-diphosphate up to 2 mM enhanced the relative affinity of the enzyme for pyruvate and modified the pyruvate saturation curve from sigmoidal to hyperbolic. The enzyme activity showed also a sigmoidal response to NADH, exhibiting two binding sites for the cofactor with a Hill coefficient of about 1.9. The molecular weight of the native enzyme was 150,000 as determined by gel permeation on Sephacryl S200. Monomers (38,000 daltons) and dimers (85,000 daltons) were observed by sodium dodecyl sulfate-gel electrophoresis; the latter form was dissociated after reduction with 2-mercaptoethanol, and the enzyme could be considered a tetramer. Antibodies obtained against the purified S. mutans OMZ175 LDH cross-reacted with the sodium dodecyl sulfate-dissociated forms of LDHs from different S. mutans serotypes, Streptococcus sanguis OMZ9, Lactobacillus casei ATCC 4646, and Actinomyces viscosus NY 1. A competitive enzyme-linked immunosorbent assay allowed us to detect a very close relationship between the native states of L-LDHs from S. mutans serotypes and S. sanguis. Cross-reactions were also observed with the LDHs from A. viscosus and L. casei, the latter being the least related. A very weak immunological relationship was obtained between the L-LDH from S. mutans OMZ175 and the D-LDH from Lactobacillus leichmannii, whereas no cross-reaction could be detected with mammal LDHs.

Immunological study of lactate dehydrogenase from Streptococcus mutans and evidence of common antigenic domains with lactate dehydrogenases from lactic bacteria

“…A significant degree of similarity exists between their active centers, at least for the Lactobacillus casei (14) and the Streptococcus cremoris (7) enzymes, when respectively compared with vertebrate enzymes. Immunological crossreactions between extragenera lactic bacterial LDHs revealed antigenic relationships (21), confirming the structural conservation of these molecules.…”

“…Organisms, culture conditions, and enzyme purification. S. mutans OMZ 175 (serotype f, 3, 19), S. sanguis OMZ 9, A. viscosus NY.1, and L. casei ATCC 4646 were previously described (20,21). Bacteria were prepared from exponentialphase cultures grown in brain heart infusion broth (Difco Laboratories, Detroit, Mich.).…”

Influence of Environmental Conditions on Hydrogen Peroxide Formation by Streptococcus gordonii

“…1). In this case, inhibition of LDH would most likely occur due to decreases in the levels of glycolytic intermediates and, in particular, fructose-1,6-bis-phosphate, which is an allosteric activator of LDH (30).…”

Effects of the natural compound, oxyresveratrol, on the growth of Streptococcus mutans, and on biofilm formation, acid production, and virulence gene expression

“…Glycolysis is another important sucrose metabolism pathway in S. mutans. In this process, LDH catalyzes the conversion of pyruvate to lactic acid, which in turn reduces the pH of the environmental medium (34). Our results showed that the unit bacterial count activity of LDH was markedly increased at an oxyresveratrol concentration of 62.5 lg ml À1 and showed smaller increases at 125 and 250 lg ml À1 of oxyresveratrol: however, all were still significantly higher than the unit bacterial count activity of LDH in the control group.…”