Structures of ceftazidime and its transition-state analogue in complex with AmpC beta-lactamase: implications for resistance mutations and inhibitor design.

Third-generation cephalosporins are widely used beta-lactam antibiotics that resist hydrolysis by beta-lactamases. Recently, mutant beta-lactamases that rapidly inactivate these drugs have emerged. To investigate why third-generation cephalosporins are relatively stable to wild-type class C beta-lactamases and how mutant enzymes might overcome this, the structures of the class C beta-lactamase AmpC in complex with the third-generation cephalosporin ceftazidime and with a transition-state analogue of ceftazidime were determined by X-ray crystallography to 2.0 and 2.3 A resolution, respectively. Comparison of the acyl-enzyme structures of ceftazidime and loracarbef, a beta-lactam substrate, reveals that the conformation of ceftazidime in the active site differs from that of substrates. Comparison of the structures of the acyl-enzyme intermediate and the transition-state analogue suggests that ceftazidime blocks formation of the tetrahedral transition state, explaining why it is an inhibitor of AmpC. Ceftazidime cannot adopt a conformation competent for catalysis due to steric clashes that would occur with conserved residues Val211 and Tyr221. The X-ray crystal structure of the mutant beta-lactamase GC1, which has improved activity against third-generation cephalosporins, suggests that a tandem tripeptide insertion in the Omega loop, which contains Val211, has caused a shift of this residue and also of Tyr221 that would allow ceftazidime and other third-generation cephalosporins to adopt a more catalytically competent conformation. These structural differences may explain the extended spectrum activity of GC1 against this class of cephalosporins. In addition, the complexed structure of the transition-state analogue inhibitor (K(i) 20 nM) with AmpC reveals potential opportunities for further inhibitor design.

The role for an invariant aspartic acid in hypoxanthine phosphoribosyltransferases is examined using saturation mutagenesis, functional analysis, and X-ray crystallography.

The role of an invariant aspartic acid (Asp137) in hypoxanthine phosphoribosyltransferases (HPRTs) was examined by site-directed and saturation mutagenesis, functional analysis, and X-ray crystallography using the HPRT from Trypanosoma cruzi. Alanine substitution (D137A) resulted in a 30-fold decrease of k(cat), suggesting that Asp137 participates in catalysis. Saturation mutagenesis was used to generate a library of mutant HPRTs with random substitutions at position 137, and active enzymes were identified by complementation of a bacterial purine auxotroph. Functional analyses of the mutants, including determination of steady-state kinetic parameters and pH-rate dependence, indicate that glutamic acid or glutamine can replace the wild-type aspartate. However, the catalytic efficiency and pH-rate profile for the structural isosteric mutant, D137N, were similar to the D137A mutant. Crystal structures of four of the mutant enzymes were determined in ternary complex with substrate ligands. Structures of the D137E and D137Q mutants reveal potential hydrogen bonds, utilizing several bound water molecules in addition to protein atoms, that position these side chains within hydrogen bond distance of the bound purine analogue, similar in position to the aspartate in the wild-type structure. The crystal structure of the D137N mutant demonstrates that the Asn137 side chain does not form interactions with the purine substrate but instead forms novel interactions that cause the side chain to adopt a nonfunctional rotamer. The results from these structural and functional analyses demonstrate that HPRTs do not require a general base at position 137 for catalysis. Instead, hydrogen bonding sufficiently stabilizes the developing partial positive charge at the N7-atom of the purine substrate in the transition-state to promote catalysis.

Efficient identification of inhibitors targeting the closed active site conformation of the HPRT from Trypanosoma cruzi.

BACKGROUND: Currently, only two drugs are recommended for treatment of infection with Trypanosoma cruzi, the etiologic agent of Chagas' disease. These compounds kill the trypomastigote forms of the parasite circulating in the bloodstream, but are relatively ineffective against the intracellular stage of the parasite life cycle. Neither drug is approved by the FDA for use in the US. The hypoxanthine phosphoribosyltransferase (HPRT) from T. cruzi is a possible new target for antiparasite chemotherapy. The crystal structure of the HPRT in a conformation approximating the transition state reveals a closed active site that provides a well-defined target for computational structure-based drug discovery. RESULTS: A flexible ligand docking program incorporating a desolvation correction was used to screen the Available Chemicals Directory for inhibitors targeted to the closed conformation of the trypanosomal HPRT. Of 22 potential inhibitors identified, acquired and tested, 16 yielded K(i)'s between 0.5 and 17 microM versus the substrate phosphoribosylpyrophosphate. Surprisingly, three of eight compounds tested were effective in inhibiting the growth of parasites in infected mammalian cells. CONCLUSIONS: This structure-based docking method provided a remarkably efficient path for the identification of inhibitors targeting the closed conformation of the trypanosomal HPRT. The inhibition constants of the lead inhibitors identified are unusually favorable, and the trypanostatic activity of three of the compounds in cell culture suggests that they may provide useful starting points for drug design for the treatment of Chagas' disease.

Ternary complex structure of human HGPRTase, PRPP, Mg2+, and the inhibitor HPP reveals the involvement of the flexible loop in substrate binding.

Site-directed mutagenesis was used to replace Lys68 of the human hypoxanthine phosphoribosyltransferase (HGPRTase) with alanine to exploit this less reactive form of the enzyme to gain additional insights into the structure activity relationship of HGPRTase. Although this substitution resulted in only a minimal (one- to threefold) increase in the Km values for binding pyrophosphate or phosphoribosylpyrophosphate, the catalytic efficiencies (k(cat)/Km) of the forward and reverse reactions were more severely reduced (6- to 30-fold), and the mutant enzyme showed positive cooperativity in binding of alpha-D-5-phosphoribosyl-1-pyrophosphate (PRPP) and nucleotide. The K68A form of the human HGPRTase was cocrystallized with 7-hydroxy [4,3-d] pyrazolo pyrimidine (HPP) and Mg PRPP, and the refined structure reported. The PRPP molecule built into the [(Fo - Fc)phi(calc)] electron density shows atomic interactions between the Mg PRPP and enzyme residues in the pyrophosphate binding domain as well as in a long flexible loop (residues Leu101 to Gly111) that closes over the active site. Loop closure reveals the functional roles for the conserved SY dipeptide of the loop as well as the molecular basis for one form of gouty arthritis (S103R). In addition, the closed loop conformation provides structural information relevant to the mechanism of catalysis in human HGPRTase.

Limited proteolysis of a trypanosomal hypoxanthine phosphoribosyltransferase yields crystals that diffract X-rays to near atomic resolution.

Two crystal forms of the hypoxanthine phosphoribosyltransferase from Trypanosoma cruzi were grown and characterized. Proteolytic modification at the C-terminus of the recombinant enzyme yielded monoclinic crystals that diffract X-rays to higher resolution than the original, trigonal crystal form. Data from the monoclinic crystal form enabled determination of the crystal structure for the trypanosomal HPRT to 1.4 A resolution.

Approaching the transition state in the crystal structure of a phosphoribosyltransferase.

Hypoxanthine phosphoribosyltransferase (HPRT) salvages 6-oxopurine bases in the nucleotide metabolic pathway. The 1.8 A crystal structure of an asymmetric dimer of the HPRT from the protozoan parasite Trypanosoma cruzi was determined in a ternary complex with the primary substrate phosphoribosylpyrophosphate (PRPP) and an analogue of the substrate hypoxanthine, revealing both open and closed active site conformations. The ligands are positioned for in-line nucleophilic attack at the PRPP ribose C1' by two metal ions which straddle the pyrophosphate leaving group. The structure provides the first evidence for the involvement of two metal ions in the HPRT-catalyzed reaction, and structural details further suggest the mechanism may proceed via SN2-type chemistry. The closed conformation reveals the structural roles for invariant flexible loop residues Ser103 and Tyr104 and supports a role for the loop in the liberation of pyrophosphate. The pre-transition state structure is valuable for understanding the enzyme mechanism, as well as providing a foundation for antiparasite drug design efforts against T. cruzi, which causes Chagas' disease in humans. Additionally, the structure illuminates the molecular basis of three inherited mutations in the human HPRT leading to Lesch-Nyhan syndrome (D193N) or gout (S103R or S109L), as the homologous residues in the trypanosomal enzyme contribute to the previously unrecognized Mg2+ ion binding site and to the formation of the closed flexible loop, respectively.

A 1.4 A crystal structure for the hypoxanthine phosphoribosyltransferase of Trypanosoma cruzi.

The hypoxanthine phosphoribosyltransferase (HPRT) from Trypanosoma cruzi, etiologic agent of Chagas' disease, was cocrystallized with the inosine analogue Formycin B (FmB) and the structure determined to 1.4 A resolution. This is the highest resolution structure yet reported for a phosphoribosyltransferase (PRT), and the asymmetric unit of the crystal contains a dimer of closely associated, nearly identical subunits. A conserved nonproline cis peptide in one active-site loop exposes the main-chain nitrogen to the enzyme active site, while the adjacent lysine side chain interacts with the other subunit of the dimer, thereby providing a possible mechanism for communication between the subunits and their active sites. The three-dimensional coordinates for the invariant Ser103-Tyr104 dipeptide are reported here for the first time. These are the only highly conserved residues in a second active-site loop, termed the long flexible loop, which is predicted to close over the active site of HPRTs to protect a labile transition state [Eads et al. (1994) Cell 78, 325-334]. This structure represents a major step forward in efforts to design/discover potent selective inhibitors of the HPRT of T. cruzi.

Hypoxanthine phosphoribosyltransferase from Trypanosoma cruzi as a target for structure-based inhibitor design: crystallization and inhibition studies with purine analogs.

The hypoxanthine phosphoribosyltransferase (HPRT) from Trypanosoma cruzi is a potential target for enzyme structure-based inhibitor design, based on previous studies which indicate that these parasites lack the metabolic enzymes required for de novo synthesis of purine nucleotides. By using a bacterial complement selection system, 59 purine analogs were assayed for their interaction with the HPRTs from T. cruzi and Homo sapiens. Eight compounds were identified from the bacterial assay to have an affinity for the trypanosomal enzyme. Inhibition constants for four of these compounds against purified recombinant trypanosomal and human HPRTs were determined and compared. The results confirm that the recombinant system can be used to identify compounds which have affinity for the trypanosomal HPRT. Furthermore, the results provide evidence for the importance of chemical modifications at positions 6 and 8 of the purine ring in the binding of these compounds to the HPRTs. An accurate three-dimensional structure of the trypanosomal enzyme will greatly enhance our understanding of the interactions between HPRTs and these compounds. Toward this end, crystallization conditions for the trypanosomal HPRT and preliminary analysis of X-ray diffraction data to a resolution of 2 A is reported. These results represent significant progress toward a structure-based approach to the design of inhibitors of the HPRT of trypanosomes with the long-range goal of developing new drugs for the treatment of Chagas' disease.

Substitution of lysine for arginine at position 199 of a hypoxanthine phosphoribosyltransferase interferes with binding of the primary substrate to the active site.

Lysine was substituted for a conserved arginine at position 199 of the schistosomal hypoxanthine phosphoribosyltransferase (HPRT). This resulted in a > or = 35-fold increase in the K(M) for binding phosphoribosyl-pyrophosphate (PRPP). The possible functional role of R199 in tertiary structure, as well as in the binding of PRPP, is interpreted in the context of the reported three dimensional structure for the human HPRT.

Crystal structure of the hypoxanthine-guanine-xanthine phosphoribosyltransferase from the protozoan parasite Tritrichomonas foetus.

The crystal structure of the hypoxanthine-guanine-xanthine phosphoribosyltransferase (HGXPRTase) from Tritrichomonas foetus has been determined and refined against X-ray data to 1.9 A resolution. T. foetus HGXPRTase crystallizes as an asymmetric dimer, with GMP bound to only one of the two molecules that form the asymmetric unit. Each molecule of HGXPRTase is formed by two lobes joined by a short "hinge" region, and the GMP binds in a cavity between the two lobes. A comparison of the two molecules in the asymmetric unit shows that the hinge region is flexible and that ligand binding affects the relative positions of the two lobes. The binding of GMP brings the two lobes closer together, rotating one lobe by about 5 degrees relative to the other. T. foetus appears to depend on HGXPRTase for its supply of GMP, making this enzyme a target for antiparasite drug design. A comparison of the structures of T. foetus HGXPRTase and human HGPRTase reveals that, while these enzymes retain a similar polypeptide fold, there are substantial differences between the active sites of these two homologs. These differences suggest that it will be possible to find compounds that selectively inhibit the parasite enzyme.