Dihydrofolate reductase from Daucus carota cell suspension cultures: purification, molecular and kinetic characterization

Abstract

The purification of dihydrofolate reductase (5, 6, 7, 8 tetrahydrofolate: NADP(+) oxidoreductase, E.C.: 1.5.1.3) from Daucus carota to apparent homogeneity, is described. The enzyme is a soluble protein with a molecular weight of 183 000±2 500, composed of identical subunits of 58 400±1 000. The enzyme is only weakly recognized by antibodies against human DHFR. The carrot DHFR is characterized by a pH optimum of 5.9, Km values for dihydrofolate and NADPH of 3.7 μM and 2.2 μM, respectively and a turnover number of 4 750 or 1 500 when referring to the 183 K form or the 58 K monomer, respectively. Molecular and kinetic properties are remarkably different from those reported for the soybean enzyme. Sensitivity to methotrexate is similar to that of bacterial and mammalian enzymes while sensitivity to trimethoprim and dihydrotriazine is intermediate between the two groups of organisms.

Mitochondria Are a Major Site for Folate and Thymidylate Synthesis in Plants

“…The DHFR activity was eluted with a molecular mass corresponding to ϳ140 kDa. Similar molecular masses were previously reported for DHFR originating from soybean seedling (41) and carrot cell suspension cultures (42). In protozoa, the apparent molecular mass of the enzyme was also estimated at 150 kDa by gel filtration (43).…”

On the use of PIPSA to Guide Target‐Selective Drug Design

“…For example, DHFRs from species that form the conserved salt bridge to the nitrogens of DHF by glutamate (human Glu 30) are separated from those using aspartate (E. coli Asp 27) ( Figure 2 and Figure 4). Furthermore, an interesting clustering can be found [28] Mus musculus Chordata W1 D3 N1 0.30 1.36 [29] Homo sapiens Chordata W1 D3 N1 0.11 2.5 [30] Bos taurus Chordata W1 D3 N1 2.3 33 [31] Sus scrofa Chordata W1 D3 N1 0.74 3.22 [29] Gallus gallus Chordata W1 D3 N1 0.15 1.8 [32] Thermotoga maritima Thermotogae W1 D1 N1 0.3 4.0 [33] Salmonella typhimurium Proteobacteria W1 D1 N1 0.4 n. d. [34] Escherichia coli Proteobacteria W1 D2 N4 0.27 1.05 [35] Saccharomyces cerevisiae Ascomycota W2 D4 N2 13 45 [36] Candida albicans Ascomycota W2 D4 N2 2.7 3.6 [5] Pneumocystis carinii Ascomycota W2 D4 N1 2.3 3.0 [37] Glycine max Magnoliophyta W2 D4 N1 35 415 [36] Daucus carota Magnoliophyta W2 D4 N1 3.7 2.2 [38] Neisseria gonorrhoeae Proteobacteria W2 D1 N1 2.6 10 [39] Mycobacterium tuberculosis Actinobacteria W2 D3 N1 4.5 4.2 [40] Heliothis virescens Arthropoda W3 D3 N1 7.5 6.7 [41] Lactobacillus casei Firmicutes W3 D1 N3 0.36 0.78 [42] Crithidia fasciculata Euglenozoa W4 D1 N1 1.1 2.7 [43] Leishmania major Euglenozoa W4 D1 N3 1.3 0.9 [44] [a] The clustering gives four clusters based on the PIPSA analysis with the human template structure (see Figures 2 and 4). Table 1 and middle panel of Figure 2; D1: triangles, D2: squares, D3: circles, D4: diamonds).…”

Proteolytic and partial sequencing studies of the bifunctional dihydrofolate reductase-thymidylate synthase from Daucus carota

“…The Mr of 183 000-185 000 was initially interpreted as an evidence of the presence of a homotrimer [ 1 ]. However the calculation of the Mr, using an average of the Stokes radius of 4.7 nm and a sedimentation coefficient of 6.6, gives a value of 124000 suggesting that, similarly to the situation reported in several protozoa, the native carrot protein is a dimer of identical subunits.…”

“…From these data aMr of 124000 was calculated according to Siegel and Monty [25] with a frictional coefficient of 1.42. This value is only slightly more than twice 977 that of the Mr 58 000 monomer [ 1 ] suggesting that carrot D H F R -T S occurs as a homodimer. This is not changed by KC1 up to 1 M.…”

“…The purification to homogeneity of the bifunctional DHFR-TS from carrot suspension cells was obtained by methotrexate-Sepharose affinity chromatography as described [1 ].…”