Alpha-pyridine nucleotides as substrates for a plasmid-specified dihydrofolate reductase

Area of Science:

• Biochemistry
• Enzymology
• Molecular Biology

Background:

• Alpha epimers of pyridine nucleotides are generally inactive in dehydrogenase reactions.
• Dihydrofolate reductase (DHFR) is crucial for folate metabolism, with NADP+ as a cofactor.
• The R67 plasmid-encoded DHFR confers trimethoprim resistance in bacteria.

Purpose of the Study:

• To investigate the substrate specificity of the R67 plasmid-encoded dihydrofolate reductase concerning pyridine nucleotide epimers.
• To compare the catalytic activity and binding kinetics of the R67 DHFR with beta-NADPH and alpha-NADPH.
• To explore the structural basis for cofactor binding in the R67 DHFR.

Main Methods:

• Enzyme kinetics assays using purified R67 dihydrofolate reductase.
• Characterization of substrate utilization with beta-NADPH and alpha-NADPH.
• Analysis of competitive inhibition using NADP+ analogues.
• Testing the activity of pyridine nucleotide analogues.

Main Results:

• The R67 DHFR utilized both alpha-NADPH and alpha-NADH, unlike bacterial and mammalian DHFRs which exclusively use beta-NADPH and beta-NADH.
• The Michaelis constant (Km) for alpha-NADPH was 16 microM, fourfold higher than for beta-NADPH (4 microM).
• The maximal velocity (Vmax) with alpha-NADPH was 70% of that observed with beta-NADPH.
• Alpha-NADP+ and beta-NADP+ acted as competitive inhibitors for the R67 enzyme.
• The R67 DHFR uniquely utilized thio-NADPH, unlike other tested enzymes.

Conclusions:

• The R67 plasmid-derived dihydrofolate reductase possesses a unique pyridine nucleotide binding site.
• This distinct binding site accommodates alpha-epimers of NAD(P)H, differing from other DHFRs and dehydrogenases.
• The structural divergence in the cofactor binding pocket may explain the altered substrate specificity.