Crystal structures of Escherichia coli and Lactobacillus casei dihydrofolate reductase refined at 1.7 A resolution. I. General features and binding of methotrexate.

Analysis of a series of mutants of an Escherichia coli alanine transfer RNA shows that substitution of a single G-U base pair in the acceptor helix eliminates aminoacylation with alanine in vivo and in vitro. Introduction of that base pair into the analogous position of a cysteine and a phenylalanine transfer RNA confers upon each the ability to be aminoacylated with alanine. Thus, as little as a single base pair can direct an amino acid to a specific transfer RNA.

A simple structural feature is a major determinant of the identity of a transfer RNA

Trimethoprim (TMP) and sulfonamides (SULs) are synthetic antibacterial agents. The first SUL compounds were used in 1932, whereas TMP is a relatively new compound first used in 1962 in England (69). Since 1968, TMP has been used in combination with SULs because the combination of TMPSULs was supposed to be synergistic in vitro (18). However, clinical experience suggests that TMP-SUL combinations have no clear synergism in vivo (2, 16, 112). In the 1970s, TMP alone came into use, first for the prophylaxis of urinary tract infections and later for the treatment of acute urinary tract infections as well (81). TMP and SULs share both a wide antibacterial spectrum including common urinary tract pathogens (Escherichia coli and other members of the family Enterobacteriaceae), respiratory tract pathogens (Streptococcus pneumoniae, Haemophilus influenzae, and in combination, Moraxella catarrhalis), skin pathogens (Staphylococcus aureus), as well as certain enteric pathogens (E. coli and Shigella spp.). Because of the wide range of clinical indications, TMP-SUL combinations have been used extensively everywhere in the world. In addition, both compounds are relatively inexpensive, a fact that allows for the use of these drugs outside of developed countries. Today, the most important fear is the development of bacterial resistance to TMP and SULs. To counter bacterial resistance it is essential to understand the molecular background of resistance mechanisms. Analysis of TMP and SUL resistance determinants in clinical bacteria has already revealed new recombination mechanisms that have an impact on spread of the resistance in general. As synthetic antimicrobial agents, TMP and SULs are also examples of agents that bacteria have not met previously, and to which they can develop resistance; this excludes resistance mechanisms related to antibiotic-producing organisms. In this minireview, we describe the current knowledge of TMP and SUL resistance in major bacterial pathogens and review the TMP and SUL resistance mechanisms.

Trimethoprim and sulfonamide resistance.

The solution structure of the DNA binding domain of HIV-1 integrase (residues 220-270) has been determined by multidimensional NMR spectroscopy. The protein is a dimer in solution, and each subunit is composed of a five-stranded P-barrel with a topology very similar to that of the SH3 domain. The dimer is formed by a stacked @-interface comprising strands 2, 3, and 4, with the two triple-stranded antiparallel P-sheets, one from each subunit, oriented antiparallel to each other. One surface of the dimer, bounded by the loop between strands Pl and P2, forms a saddle-shaped groove with dimensions of approximately 24 x 23 x 12 8, in cross section. Lys264, which has been shown from mutational data to be involved in DNA binding, protrudes from this surface, implicating the saddle- shaped groove as the potential DNA binding site. The integrase protein of the human immunodeficiency virus (HIV) mediates a key step in the life cycle of the virus, namely the integration of a DNA copy of the viral genome into a host chromosome (see Goff, 1992; Vink & Plasterk, 1993; for reviews). HIV-1’ integrase is composed of three functional domains (Bushman et al., 1993; Engelman et al., 1993; van Gent et al., 1993; Vink et al., 1993): a small N-terminal domain that contains a His2Cys2 zinc binding motif, a central catalytic domain whose crystal structure has recently been solved (Dyda et al., 1994), and a C-terminal DNA binding domain. While the catalytic core domain can carry out a simple polynucleotidyl transfer termed disintegra- tion (Bushman et al., 1993), all three domains are required for the 3’ processing and DNA strand transfer activities that accomplish integration of the viral genome (Drelich et al., 1992; Schauer & Billich, 1992; Vink et al., 1993). Conse- quently, the N- and C-terminal domains provide additional potentially useful targets for rational drug design aimed at inhibiting HIV integration into the host genome. The function of the N-terminal domain is at present unknown, and in the case of the related Rous Sarcoma virus integrase at least, its integrity appears not to be essential for

/pdf/solution-structure-of-the-dna-binding-domain-of-hiv-1-4dghbmhwru.pdf

Solution structure of the DNA binding domain of HIV-1 integrase.

The crystal structure of dihydrofolate reductase (EC 1.5.1.3) from Escherichia coli has been solved as the binary complex with NADP+ (the holoenzyme) and as the ternary complex with NADP+ and folate. The Bragg law resolutions of the structures are 2.4 and 2.5 A, respectively. The new crystal forms are nonisomorphous with each other and with the methotrexate binary complex reported earlier [Bolin, J. T., Filman, D. J., Matthews, D. A., Hamlin, R. C., & Kraut, J. (1982) J. Biol. Chem. 257, 13650-13662]. In general, NADP+ and folate binding conform to predictions, but the nicotinamide moiety of NADP+ is disordered in the holoenzyme and ordered in the ternary complex. A mobile loop (residues 16-20) involved in binding the nicotinamide is also disordered in the holoenzyme. We report a detailed analysis of the binding interactions for both ligands, paying special attention to several apparently strained interactions that may favor the transition state for hydride transfer. Hypothetical models are presented for the binding of 7,8-dihydrofolate in the Michaelis complex and for the transition-state complex.

Crystal structures of Escherichia coli dihydrofolate reductase: the NADP+ holoenzyme and the folate.NADP+ ternary complex. Substrate binding and a model for the transition state.

Dihydrofolate reductase has been purified 40-fold to apparent homogeneity from a trimethoprim-resistant strain of Escherichia coli (RT 500) using a procedure that includes methotrexate affinity column chromatography. Determinations of the molecular weight of the enzyme based on its amino acid composition, sedimentation velocity, and sodium dodecyl sulfate gel electrophoresis gave values of 17680, 17470 and 18300, respectively. An aggregated form of the enzyme with a low specific activity can be separated from the monomer by gel filtration; treatment of the aggregate with mercaptoethanol or dithiothreitol results in an increase in enzymic activity and a regeneration of the monomer. Also, multiple molecular forms of the monomer have been detected by polyacrylamide gel electrophoresis. The unresolved enzyme exhibits two pH optima (pH 4.5 and pH 7.0) with dihydrofolate as a substrate. Highest activities are observed in buffers containing large organic cations. In 100 mM imidazolium chloride (pH 7), the specific activity is 47 mumol of dihydrofolate reduced per min per mg at 30 degrees. Folic acid also serves as a substrate with a single pH optimum of pH 4.5. At this pH the Km for folate is 16 muM, and the Vmax is 1/1000 of the rate observed with dihydrofolate as the substrate. Monovalent cations (Na+, K+, Rb+, and Cs+) inhibit dihydrofolate reductase; at a given ionic strength the degree of inhibition is a function of the ionic radius of the cation. Divalent cations are more potent inhibitors; the I50 of BaCl2 is 250 muM, as compared to 125 mM for KCl. Anions neither inhibit nor activate the enzyme.

Purification and properties of Escherichia coli dihydrofolate reductase.

Crystalline R67 dihydrofolate reductase (DHFR) is a dimeric molecule with two identical 78 amino acid subunits, each folded into a beta-barrel conformation. The outer surfaces of the three longest beta strands in each protomer together form a third beta barrel having six strands at the subunit interface. A unique feature of the enzyme structure is that while the intersubunit beta barrel is quite regular over most of its surface, an 8-A "gap" runs the full length of the barrel, disrupting potential hydrogen bonds between beta-strand D in subunit I and the adjacent corresponding strand of subunit II. It is proposed that this deep groove is the NADPH binding site and that the association between protein and cofactor is modulated by hydrogen-bonding interactions along one face of this antiparallel beta-barrel structure. A hypothetical model is proposed for the R67 DHFR-NADPH-folate ternary complex that is consistent with both the known reaction stereoselectivity and the weak binding of 2,4-diamino inhibitors to the plasmid-specified reductase. Geometrical comparison of this model with an experimentally determined structure for chicken DHFR suggests that chromosomal and type II R-plasmid specified enzymes may have independently evolved similar catalytic machinery for substrate reduction.

Crystal structure of a novel trimethoprim-resistant dihydrofolate reductase specified in Escherichia coli by R-plasmid R67.

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Purification and properties of Escherichia coli dihydrofolate reductase.

Crystal structure of a novel trimethoprim-resistant dihydrofolate reductase specified in Escherichia coli by R-plasmid R67.