Studies on pyridine nucleotide-dependent processes in Haemophilus influenzae

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1989
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Virginia Polytechnic Institute and State University
Abstract

Haemophilus influenzae and related species have a unique requirement for externally-provided NAD; therefore, several pyridine nucleotide-requiring enzymes become important for the survival of these pathogens.

Haemophilus influenzae ATP:NMN adenylyltransferase was partially purified 15-fold with a 27% yield using dye affinity chromatography. Affinity chromatography was also used to purify NAD kinase from Haemophilus influenzae, 18-fold with a 32% yield. Substrate specificity studies of these enzymes demonstrated the enzymes to function with 3-acetylpyridine analogs of their respective substrates.

A membrane-bound NMN glycohydrolase was demonstrated in Haemophilus influenzae. The enzyme functions with 3-acetylpyridine mononucleotide as a substrate, and is inhibited effectively by 3-aminopyridine mononucleotide. The possible involvement of this enzyme in the transport of NMN into the cytoplasm is discussed

Growth inhibition studies demonstrated that 3-aminopyridine mononucleotide is a potent inhibitor of growth of the organism and could inhibit growth by inhibiting the transport of NMN. The previously reported inhibition of growth by the 3-aminopyridine adenine dinucleotide was attributed to the formation of the mononucleotide through the reaction catalyzed by the Haemophilus influenzae periplasmic nucleotide pyrophosphatase.

A cytosolic lactate dehydrogenase, specific for D(-)-lactate was purified to electrophoretic homogeneity 2100-fold with a 14% yield. The purified enzyme was demonstrated to be a tetramer of M, = 135,000. It catalyzes essentially the reduction of pyruvate with very low activity observed for the oxidation of D(-)-lactate. An optimum pH of 7.2 was determined for the reduction of pyruvate with NADH as the coenzyme. Several NADH analogs, altered either in the pyridine or purine moiety, functioned as coenzymes. Coenzyme-competitive inhibition by adenosine derivatives demonstrated important interactions of the pyrophosphate region of the coenzyme in binding with the enzyme. Several structural analogs of NADH and pyruvate were evaluated as selective inhibitors of the enzyme. Chemical modification of the purified D-lactate dehydrogenase was effectively achieved by micromolar concentrations of several N-alkylmaleimides. Positive chain length effects in the inactivation by maleimides indicated the presence of a hydrophobic region close to the sulfhydryl groups being modified. The product of the reaction catalyzed by D-lactate dehydrogenase, D(-)-lactate, provides the substrate for a membrane-bound D-lactate oxidase. The D-lactate oxidase converts D(-)-lactate back to pyruvate and transfers electrons to the respiratory chain. No cytosolic L(+)-lactate dehydrogenase was found in Haemophilus influenzae; however, the organism possesses an L-lactate oxidase associated with the cell membrane. The L-lactate oxidase is also part of the respiratory chain, and utilizes exogenous L(+)-lactate to give pyruvate for the organism to use as a carbon source.

Haemophilus influenzae and related species have a unique requirement for externally-provided NAD; therefore, several pyridine nucleotide-requiring enzymes become important for the survival of these pathogens.

Haemophilus influenzae ATP:NMN adenylyltransferase was partially purified 15-fold with a 27% yield using dye affinity chromatography. Affinity chromatography was also used to purify NAD kinase from Haemophilus influenzae, 18-fold with a 32% yield. Substrate specificity studies of these enzymes demonstrated the enzymes to function with 3-acetylpyridine analogs of their respective substrates.

A membrane-bound NMN glycohydrolase was demonstrated in Haemophilus influenzae. The enzyme functions with 3-acetylpyridine mononucleotide as a substrate, and is inhibited effectively by 3-aminopyridine mononucleotide. The possible involvement of this enzyme in the transport of NMN into the cytoplasm is discussed.

Growth inhibition studies demonstrated that 3-aminopyridine mononucleotide is a potent inhibitor of growth of the organism and could inhibit growth by inhibiting the transport of NMN. The previously reported inhibition of growth by the 3-aminopyridine adenine dinucleotide was attributed to the formation of the mononucleotide through the reaction catalyzed by the Haemophilus influenzae periplasmic nucleotide pyrophosphatase.

A cytosolic lactate dehydrogenase, specific for D(-)-lactate was purified to electrophoretic homogeneity 2100-fold with a 14% yield. The purified enzyme was demonstrated to be a tetramer of M, = 135,000. It catalyzes essentially the reduction of pyruvate with very low activity observed for the oxidation of D(-)-lactate. An optimum pH of 7.2 was determined for the reduction of pyruvate with NADH as the coenzyme. Several NADH analogs, altered either in the pyridine or purine moiety, functioned as coenzymes. Coenzyme-competitive inhibition by adenosine derivatives demonstrated important interactions of the pyrophosphate region of the coenzyme in binding with the enzyme. Several structural analogs of NADH and pyruvate were evaluated as selective inhibitors of the enzyme. Chemical modification of the purified D-lactate dehydrogenase was effectively achieved by micromolar concentrations of several N-alkylmaleimides. Positive chain length effects in the inactivation by maleimides indicated the presence of a hydrophobic region close to the sulfhydryl groups being modified. The product of the reaction catalyzed by D-lactate dehydrogenase, D(-)-lactate, provides the substrate for a membrane-bound D-lactate oxidase. The D-lactate oxidase converts D(-)-lactate back to pyruvate and transfers electrons to the respiratory chain. No cytosolic L(+)-lactate dehydrogenase was found in Haemophilus influenzae; however, the organism possesses an L-lactate oxidase associated with the cell membrane. The L-lactate oxidase is also part of the respiratory chain, and utilizes exogenous L(+)-lactate to give pyruvate for the organism to use as a carbon source.

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