Sulfonamides were amongst the first clinically useful antibacterial agents to be discovered. The identification of sulfanilamide as the active component of the dye Prontosil rubrum led to the synthesis of clinically useful analogues. Today sulfamethoxazole (in combination with trimethoprim), is used to treat urinary tract infections caused by bacteria such as Escherichia coli and is also a first-line treatment for pneumonia caused by the fungus Pneumocystis carinii, a common condition in AIDS patients. The site of action is the de novo folate biosynthesis enzyme dihydropteroate synthase (DHPS) where sulfonamides act as analogues of one of the substrates, para-aminobenzoic acid (pABA). We report here the crystal structure of E.coli DHPS at 2.0 A resolution refined to an R-factor of 0.185. The single domain of 282 residues forms an eight-stranded alpha/beta-barrel. The 7,8-dihydropterin pyrophosphate (DHPPP) substrate binds in a deep cleft in the barrel, whilst sulfanilamide binds closer to the surface. The DHPPP ligand site is highly conserved amongst prokaryotic and eukaryotic DHPSs.

These structures show how two drugs interact with a fungal DHFR. A comparison of the three-dimensional structure of this relatively large DHFR with bacterial or mammalian enzyme-inhibitor complexes determined previously highlights some additional secondary structure elements in this particular enzyme species. These comparisons provide further insight into the principles governing DHFR-inhibitor interaction, in which the volume of the active site appears to determine the strength of inhibitor binding.

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The X-ray crystal structure of 7,8-dihydro-6-hydroxymethylpterinpyrophosphokinase (PPPK) in a ternary complex with ATP and a pterin analogue has been solved to 2.0 A î resolution, giving, for the first time, detailed information of the PPPK/ATP intermolecular interactions and the accompanying conformational change. The first 100 residues of the 158 residue peptide contain a L LK KL LL LK KL L motif present in several other proteins including nucleoside diphosphate kinase. Comparative sequence examination of a wide range of prokaryotic and lower eukaryotic species confirms the conservation of the PPPK active site, indicating the value of this de novo folate biosynthesis pathway enzyme as a potential target for the development of novel broad-spectrum anti-infective agents.z 1999 Federation of European Biochemical Societies.

Infrared difference spectra show that at least 4 conformations coexist for the ester carbonyl group of the stable acylenzyme species formed between the antibiotic aztreonam and the class C β-lactamase from Citrobacter freundii. A novel method for the assignment of the bands that arise from the ester carbonyl group has been employed. This has made use of the finding that the infrared absorption intensity of aliphatic esters is surprisingly constant, so a direct comparison with simple model esters has been possible. This has allowed a clear distinction to be made between ester and amide (protein) absorptions.The polarity of the conformer environment varies from hexane-like to strongly hydrogen-bonded. We assume that the conformer with the lowest frequency (1690 cm -1 ) and hence the strongest hydrogenbonding is the singular conformer observed in the X-ray crystallographic structure, since a good interaction via two hydrogen bonds with the oxyanion hole is seen. Molecular dynamics simulation by the method of locally enhanced sampling revealed that the motion of the ester carbonyl of the acylenzyme species in and out of the oxyanion hole is facile. The simulation revealed two pathways for this motion that would go through intermediates that first break one or the other of the two hydrogen bonds to the oxyanion hole, prior to departure of the carbonyl moiety out of the active site. It is likely that such motion for the acyl-enzyme species might also occur with more typical β-lactam substrates for β-lactamases, but their detection in the more rapid time scale may prove a challenge. 2The question as to how well x-ray crystallographic structures represent the dynamic state of protein-ligand complexes in solution has been much debated. The question has been partly answered by comparison of crystallographic and NMR structures of stable complexes, such as trypsin-inhibitor complexes (1). Studies such as these have shown a general agreement between the techniques, although structures deduced from NMR data perhaps show mobility/dispersion more graphically. Loop motion is often dramatically portrayed. While there are now many reports in the literature concerning multiple conformations of amino acid side chains and larger scale conformational changes in proteins, there is very little in the literature concerning ligand conformational multiplicity (2-6). One well-studied case is that of dihydrofolate reductase with methotrexate bound (7,8 Conformational flexibility in the course of catalysis (9-17) and inhibition (18,19) has been documented for β-lactamases as well. It is critical that structural information be put in the context of the documented conformational changes to arrive at the details of the catalytic processes by these enzymes.Catalysis by serine-dependent β-lactamases proceeds via a two-step mechanism that involves acylation of the active site serine and the deacylation of the acyl-enzyme species (20). Binding of the substrate at the Michaelis complex stage involves sequestration of the β-lactam carbonyl in ...