Large-scale purification and characterization of dihydrofolate reductase from a methotrexate-resistant strain of Lactobacillus casei

Abstract

Dihydrofolate reductase has been purified from a methotrexate-resistant strain of Lactobacillus casei NCB 6375. By careful attention to growth conditions, up to 2.5g of enzyme is obtained from a 400 litre culture. The purification procedure, involving polyethyleneimine treatment, DEAE-cellulose chromatography and affinity chromatography on methotrexate-aminohexyl-Sepharose, operates on the gram scale, with overall yields of 50-60%. Elution of the affinity column by reverse (upward) flow was used, as it led to recovery of the enzyme in a much smaller volume. The enzyme obtained appears to be more than 98% pure, as judged by gel electrophoresis, isoelectric focusing, and gel filtration. It has a mol.wt. ofapprox. 17900 and a turnover number of4s-(5OmM-triethanolamine/400mM-KCI, pH7.2, 25°C) with dihydrofolate and NADPH as substrates.The turnover number for folate is 0.02s-1. Michaelis constants for a variety of substrates have been measured by using a new fluorimetric assay (0.361uM-dihydrofolate; 0.78 uM-NADPH), and binding constants determined by using the quenching of protein fluorescence (dihydrofolate, 2.25 x 106M-1; NADPH, >108M-1). The pH/activity profile shows a single maximum at pH 7.3; at this pH, marked activation by 0.5M-NaCl is observed.Dihydrofolate reductase (tetrahydrofolate-NADP+ oxidoreductase, EC 1.5.1.3) is responsible for maintaining the intracellular pool of tetrahydrofolate by reducing dihydrofolate (arising either by biosynthesis de novo or by the action of thymidylate synthetase on 5,10-methylenetetrahydrofolate) to tetrahydrofolate (Blakley, 1969). This enzyme is of considerable pharmacological interest as the site of action of a group of powerful chemotherapeutic agents, the 'anti-folates', which includes methotrexate, trimethoprim and pyrimethamine (Blakley, 1969;Baker, 1967).We are undertaking a detailed study of the binding of substrates, coenzymes and inhibitors to the dihydrofolate reductase from a methotrexate-resistant strain of Lactobacillus casei, principally by highresolution n.m.r. (nuclear-magnetic-resonance) spec-* Present address:

Moritella Cold-Active Dihydrofolate Reductase: Are There Natural Limits to Optimization of Catalytic Efficiency at Low Temperature?

“…The apparent K m value for DHF of DHFR Mp (Table 2) is more than 50 times the value currently reported by several authors for the cognate enzyme in E. coli (27,29,36) and is also considerably higher than for L. casei (8) and T. maritima (41). Detailed examination of published data does not suggest that assay conditions influenced actual values to an extent in anyway comparable to the differences we observe.…”

A 1H n.m.r. study of the role of the glutamate moiety in the binding of methotrexate to Lactobacillus casei dihydrofolate reductase

“…On working up and crystallization from dimethylformamide-water, methotrexate Methotrexate dimethylester was prepared by esterifying methotrexate with methanol HCI by a modification of the method of Pfiffner et al (1947) as described previously (Wyeth et al, 1980). Dihydrofolate reductase was isolated and purified from L. casei as described previously (Dann et al, 1976). The enzyme was lyophilized twice from 2H20 solution to exchange all the exchangeable protons for deuterons, and was then examined as a 1 mM solution in a 50 mM potassium phosphate, 500 mM potassium chloride 2H20 buffer containing 1 mM dioxane as an internal reference.…”

Crystal Structure of Bacillus anthracis Dihydrofolate Reductase with the Dihydrophthalazine-Based Trimethoprim Derivative RAB1 Provides a Structural Explanation of Potency and Selectivity

“…This is directly related to TMP resistance and generally correlates with more favorable K m values for FAH 2 (3,12,39,45), thus making it harder to displace. However, exceptions that are TMP sensitive with a low K m for FAH 2 do exist, such as Lactobacillus casei (24). Previous work has also highlighted single amino acid changes relative to susceptible organisms to explain innate resistance.…”