Characterization of mutationally altered dihydropteroate synthase and its ability to form a sulfonamide-containing dihydrofolate analog

Abstract

Among spontaneous mutants of Escherichia coli selected for resistance against sulfonamides, thermosensitive strains were found. These were shown to possess a changed dihydropteroate synthase (EC 2.5.1.15), which had a substantially higher Km value for its normal substrate, p-aminobenzoic acid, and an about 150fold higher Km for sulfonamides. The mutationally changed dihydropteroate synthase was found to be thermosensitive by in vitro assays. The thermosensitivity was used as an enzyme marker to demonstrate the complex formation between 2-amino-4-hydroxy-6-pyrophosphorylmethyl pteridine and sulfonamides by partially purified dihydropteroate synthase. The formation of folate from 2-amino-4hydroxy-6-pyrophosphorylmethyl pteridine and p-aminobenzoylglutamic acid by dihydropteroate synthase was found to be very sensitive to inhibition by sulfonamides and very inefficient with the mutationally changed enzyme.The formation of dihydropteroate from paminobenzoic acid (AB) and reduced 2-amino-4hydroxy-6-hydroxymethyl pteridine (pteridine-CH20H) has for a long time been known to be the target of sulfonamide action (4). Part of the inhibitory effect of sulfonamides on bacterial growth has been ascribed to the incorporation of the drug into a pteridine complex to give a sulfonamide-containing H2-pteroate (7,8-dihydropteroic acid) analog (3,4). These investigations, however, have not linked the analog formation to the activity of dihydropteroate synthase (EC 2.5.1.15).In a previous study on plasmid-mediated sulfonamide resistance, the occurrence of a plasmid-borne, drug-resistant dihydropteroate synthase was described (15). Spontaneous mutants of Escherichia coli were isolated that carried temperature sensitivity lesions. These were allegedly located in the chromosomal dihydropteroate synthase gene and were used as tools for the identification of extrachromosomal synthase activity.Experiments are described here which demonstrate the temperature sensitivity of isolated mutant dihydropteroate synthase activities. Partially purified enzyme from these mutants catalyzed the formation of a sulfonamide-containing complex from 2-amino-4-hydroxy-6-pyro-phosphoryl-methyl pteridine (H2-pteridine-CH20-PP) and [35S]sulfathiazole, as did the wild-type enzyme, but the formation of sulfonamide com-plex was temperature sensitive with mutant enzyme.The temperature-sensitive mutants were originally isolated as sulfonamide-resistant strains of E. coli C (15). The temperature sensitivity was interpreted to be a consequence of the mutational change in the enzyme to sulfonamide resistance. In concurrence with this interpretation, dihydropteroate synthase isolated from the mutants showed a Km value about 150-fold higher for sulfathiazole than that isolated from the wild type.The efficiency of the enzymatic formation of dihydrofolate from H2-pteridine-CH20-PP and N-(p-aminobenzoyl)-L-[ U-14C]glutamate (ABG) was also seen to decrease drastically as a consequence of mutational change in the dihydropteroate synthase. MATERIALS AND METHODSBacteria. The E....

Mechanistic link between uptake of sulfonamides and bacteriostatic effect: Model development and application to experimental data from two soil microorganisms

“…(S2)). Regarding the high variability of enzyme tests, these values are in excellent agreement with the few reported enzyme affinities for E. coli of 0.03 mg/L for SMX [32,33] and 0.69 mg/L for STZ [33]. Figure 4 shows the regression lines between 1/EC50 and AAF for Ps.…”

“…The CAF values are specific for the possible effect of each sulfonamide in a microbial species, and provide the missing link between the SA concentration in the medium and the observed effects. This conclusion is corroborated by other studies, which found significantly different DHPS enzyme affinity constants for STZ and SMX [32,33]. The model developed here enables the estimation of the CAF for a specific sulfonamide from a single EC50 value.…”

Plasmid-borne sulfonamide resistance determinants studied by restriction enzyme analysis

“…The tubes were centrifuged in an Eppendorf centrifuge, and the supematant was collected. The protein content was estimated by the method of Bradford (4), and dihydropteroate synthase activity was assayed as described before (21 containing the corresponding resista ments from RI, R100, and pGS02 tested for fragment orientation with results. Sulfonamide resistance genes from p pGSOS.…”

Genetic dominance governs the evolution and spread of mobile genetic elements in bacteria

“…Palmer and Kishony recently described that the fraction of PDP that is converted to DHP (the correct product) or to dihydropterin-sulfonamide (the incorrect one) depends on the ratio of drug to drug-competing substrate (sulfonamide to PABA ratio), weighted by binding affinity of the DHPS enzyme to each substrate ( 31 ). Resistance mutations in folP decrease the binding affinity to sulfonamides drastically ( 47, 48 ). As a consequence, in this scenario the degree of dominance of mutations is going to depend on the rate of synthesis of DHP by the mutant allele, compared to the rate of synthesis of dihydropterin-sulfonamide by the wild type allele (in other words what fraction of PDP is used in the correct or the incorrect reaction).…”

“…Therefore, the higher the binding affinity of the mutant FolP to PABA the higher coefficient of dominance of the mutation will be. In summary, DHPS resistant alleles will range from mildly dominant (point mutations in folP with reduced affinity to PABA ( 47 )) to strongly dominant (mobile resistant alleles of DHPS, such as sul1 and sul2 , with high binding affinity to PABA ( 48 )).…”