The Escherichia coli gene coding for dihydropteroate synthase (DHPS) has been cloned and sequenced. The protein has 282 amino acids and a compositional molecular mass of 30,314 daltons. Increased expression of the enzyme was realized by using a T7 expression system. The enzyme was purified and crystallized. A temperature-sensitive mutant was isolated and found to express a DHPS with a lower specific activity and lower affinities for para-aminobenzoic acid and sulfathiazole. The allele had a point mutation that changed a phenylalanine codon to a leucine codon, and the mutation was in a codon that is conserved among published DHPS sequences.Dihydropteroate synthase (DHPS) (EC 2.5.1.15) catalyzes the condensation of para-aminobenzoic acid (pAB) with 7,8-dihydro-6-hydroxymethylpterin-pyrophosphate, forming 7,8-dihydropteroate (39, 44). This intermediary metabolite is subsequently converted to tetrahydrofolic acid, essential for the syntheses of purines, thymidylate, glycine, methionine, pantothenic acid, and N-formylmethionyl-tRNA. Sulfonamides are pAB analogs that are recognized by DHPS as alternate substrates (4,7,45,57). In the presence of sulfonamides, DHPS forms a sulfa-pterin adduct that is metabolically inert and diffuses from the cell (40). Folate cofactor depletion results in growth inhibition and in the appropriate environment, cell death (55).DHPS activity was first identified in crude cell extracts of several organisms by Shiota and coworkers (44, 45) and was identified in Escherichia coli by Brown et al. (8). The kinetic characteristics of DHPS have been studied by using partially purified extracts. Recently, a purification procedure for DHPS was published which showed that this enzyme constituted less than 0.01% of all the proteins in a cell (54). DHPS was purified to homogeneity, the sequence of the first 28 amino acids was determined, and the protein was shown to be a homodimer of two 30-kDa subunits. However, the purification procedure yielded less than 2 mg of purified protein from 1 kg of starting material, emphasizing the need to clone and overexpress this important chemotherapeutic target.Recently, the chromosomal gene that codes for DHPS in Streptococcus pneumoniae was cloned, sequenced, and shown to code for a protein of 34 kDa (28). A similar sequence was also identified in a Bacillus subtilis folic acid biosynthetic operon (48). Two other genes (sulI and sulII) that code for plasmid-borne sulfonamide-resistant DHPSs have also been sequenced (38, 52). However, there is no information about the E. coli DHPS gene. In this communication, we report the cloning, sequencing, and enhanced expression of the E. coli DHPS gene, designated folP. We also report conditions that allow crystallization of this enzyme.