Infection & immunity by brown, april 1977

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INFECTION AND IMMUNITY, Apr. 1977, p. 163-173

Vol. 16, No. 1

Copyright X 1977 American Society for Microbiology

Printed in U.S.A.

Polyol Metabolism by a Caries-Conducive Streptococcus: Purification and Properties of a Nicotinamide Adenine Dinucleotide-Dependent Mannitol-1-Phosphate Dehydrogenase
ALBERT T. BROWN* AND ROBERT D. BOWLES Department of Oral Biology, College of Dentistry,University of Kentucky, Lexington, Kentucky 40506 Received for publication 30 November 1976

The mannitol-1-phosphate dehydrogenase (M1PDH) (EC 1.1.1.17) from Streptococcus mutans strain FA-1 was purified to approximately a 425-fold increase in specific activity with a 29% recovery of total enzyme units, using a combination of (i) streptomycin sulfate and ammonium sulfate precipitation and (ii)diethylaminoethyl-cellulose (DE-52), agarose A 0.5M, and agarose-nicotinamide adenine dinucleotide (NAD) affinity column chromatography. Polyacrylamide gel electrophoresis of the purified enzyme preparation showed a single protein component that coincided with a band of M1PDH activity. The enzyme had a molecular weight of approximately 45,000 and was stable for long periods of time when stored at-80°C in the presence of ,3-mercaptoethanol. Its activity was not affected by mono- or divalent cations, and high concentrations of ethylenediaminetetraacetic acid were not inhibitory. The M1PDH catalyzed both the NADdependent oxidation of mannitol-1-phosphate and the reduced NAD (NADH)dependent reduction of fructose-6-phosphate. The forward reaction was highly specific for mannitol-1-phosphate andNAD, whereas the reverse reaction was highly specific for NADH and fructose-6-phosphate. The K,,, values for mannitol1-phosphate and NAD were 0.15 and 0.066 mM, respectively, and the K,, values for fructose-6-phosphate and NADH were 1.66 and 0.016 mM, respectively. The forward and reverse reactions catalyzed by the M1PDH from S. mutans appeared to be under cellular control. Both adenosine5'-triphosphate and fructose6-phosphate were negative effectors of the forward reaction, whereas adenosine 5'-diphosphate served as a negative effector of the reverse reaction catalyzed by the enzyme.

Oral strains of Streptococcus mutans are causative agents of multisurface dental caries in rodents and humans (5, 10, 11, 15, 16, 18, 21, 22). A nutritional characteristic that serves to distinguish S.mutans from other streptococcal components of the oral microflora is their ability to utilize either mannitol or sorbitol as a primary energy source (8, 9, 11, 14). We have shown that mannitol and sorbitol are phosphorylated to mannitol-1-phosphate and sorbitol-6phosphate, respectively, before their conversion to the glycolytic intermediate fructose-6phosphate by the action of mannitol-1-phosphatedehydrogenase (M1PDH) (EC 1.1.1.17) or sorbitol-6-phosphate dehydrogenase (S6PDH) (4). The M1PDH and S6PDH are separate and distinct enzymes, both of which exhibit specificity for the coenzyme nicotinamide adenine dinucleotide (NAD) for catalytic activity. Since recent interest has focused upon the use of polyalcohols as dietary sucrose substitutes, we

decided to investigate more thoroughly themetabolism of mannitol and sorbitol by oral strains of S. mutans. The purpose of this communication is to report the purification of M1PDH from S. mutans strain FA-1 and to describe some of its physical, kinetic, and regulatory properties.
MATERIALS AND METHODS Chemicals. Mannitol-1-phosphate was purchased as the barium salt from Sigma Chemical Co., St. Louis, Mo., and the barium was removedbefore use by treatment with Sigma Dowex 50X as previously described (4). All enzyme assay components were also purchased from Sigma. Diethylaminoethyl (DEAE)-cellulose (DE-52) was purchased from Reeve Angel, Clifton, N.J.; agarose A 0.5M from Bio-Rad, Richmond, Calif.; and agarose-NAD (AGNAD) from PBL Biochemicals, Milwaukee, Wis. All components of the S. mutans growth medium were purchased from...
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