Formate is the hydrogen donor for the anaerobic ribonucleotide reductase from Escherichia coli.

During anaerobic growth Escherichia coli uses a specific ribonucleoside-triphosphate reductase (class III enzyme) for the production of deoxyribonucleoside triphosphates. In its active form, the enzyme contains an iron-sulfur center and an oxygen-sensitive glycyl radical (Gly-681). The radical is generated in the inactive protein from S-adenosylmethionine by an auxiliary enzyme system present in E. coli. By modification of the previous purification procedure, we now prepared a glycyl radical-containing reductase, active in the absence of the auxiliary reducing enzyme system. This reductase uses formate as hydrogen donor in the reaction. During catalysis, formate is stoichiometrically oxidized to CO2, and isotope from [3H]formate appears in water. Thus E. coli uses completely different hydrogen donors for the reduction of ribonucleotides during anaerobic and aerobic growth. The aerobic class I reductase employs redox-active thiols from thioredoxin or glutaredoxin to this purpose. The present results strengthen speculations that class III enzymes arose early during the evolution of DNA.

The three-dimensional structure of NAD(P)H:quinone reductase, a flavoprotein involved in cancer chemoprotection and chemotherapy: mechanism of the two-electron reduction.

Quinone reductase [NAD(P)H:(quinone acceptor) oxidoreductase, EC 1.6.99.2], also called DT diaphorase, is a homodimeric FAD-containing enzyme that catalyzes obligatory NAD(P)H-dependent two-electron reductions of quinones and protects cells against the toxic and neoplastic effects of free radicals and reactive oxygen species arising from one-electron reductions. These two-electron reductions participate in the reductive bioactivation of cancer chemotherapeutic agents such as mitomycin C in tumor cells. Thus, surprisingly, the same enzymatic reaction that protects normal cells activates cytotoxic drugs used in cancer chemotherapy. The 2.1-A crystal structure of rat liver quinone reductase reveals that the folding of a portion of each monomer is similar to that of flavodoxin, a bacterial FMN-containing protein. Two additional portions of the polypeptide chains are involved in dimerization and in formation of the two identical catalytic sites to which both monomers contribute. The crystallographic structures of two FAD-containing enzyme complexes (one containing NADP+, the other containing duroquinone) suggest that direct hydride transfers from NAD(P)H to FAD and from FADH2 to the quinone [which occupies the site vacated by NAD(P)H] provide a simple rationale for the obligatory two-electron reductions involving a ping-pong mechanism.

Animal model for the therapy of acquired immunodeficiency syndrome with reverse transcriptase inhibitors.

The reverse transcriptase (RT) of the human immunodeficiency virus type 1 (HIV-1) is the major target for antiretroviral therapy of the acquired immunodeficiency syndrome (AIDS). While some inhibitors exhibit activity against most retroviral RTs, others are specific for the HIV-1 enzyme. To develop an animal model for the therapy of the HIV-1 infection with RT inhibitors, the RT of the simian immunodeficiency virus (SIV) was replaced by the RT of HIV-1. Macaques infected with this SIV/HIV-1 hybrid virus developed AIDS-like symptoms and pathology. The HIV-1-specific RT inhibitor LY300046.HCl, but not zidovudine [3'-azido-3'-deoxythymidine (AZT)] delayed the appearance of plasma antigenemia in macaques infected with a high dose of the chimeric virus. Infection of macaques with the chimeric virus seems to be a valuable model to study the in vivo efficacy of new RT inhibitors, the emergence and reversal of drug resistance, the therapy of infections with drug-resistant viruses, and the efficacy of combination therapy.

Cloning of an organic solvent-resistance gene in Escherichia coli: the unexpected role of alkylhydroperoxide reductase.

Although bacterial strain able to grow in the presence of organic solvents have been isolated, little is known about the mechanism of their resistance. In the present study, 1,2,3,4-tetrahydronaphthalene (tetralin), a solvent with potential applications in industrial biocatalysis, was used to select a resistant mutant of Escherichia coli. The resultant mutant strain was tested for resistance to a wide range of solvents of varying hydrophobicities and was found to be resistant not only to tetralin itself but also to cyclohexane, propylbenzene, and 1,2-dihydronaphthalene. A recombinant library from mutant DNA was used to clone the resistance gene. The sequence of the cloned locus was determined and found to match the sequence of the previously described alkylhydroperoxide reductase operon ahpCF. The mutation was localized to a substitution of valine for glycine at position 142 in the coding region of ahpC, which is the gene encoding the catalytic subunit of the enzyme. The ahpC mutant was found to have an activity that was three times that of the wild type in reducing tetralin hydroperoxide to 1,2,3,4-tetrahydro-1-naphthol. We conclude that the toxicity of such solvents as tetralin is caused by the formation of toxic hydroperoxides in the cell. The ahpC mutation increases the activity of the enzyme toward hydrophobic hydroperoxides, thereby conferring resistance. The ahpC mutant was sensitive to the more hydrophilic solvents xylene and toluene, suggesting that there are additional mechanisms of solvent toxicity. Mutants resistant to a mixture of xylene and tetralin were isolated from the ahpC mutant but not from the wild-type strain.

Adaptation of Spodoptera exigua larvae to plant proteinase inhibitors by induction of gut proteinase activity insensitive to inhibition.

Tobacco plants were transformed with a cDNA clone of chymotrypsin/trypsin-specific potato proteinase inhibitor II (PI2) under the control of a constitutive promoter. Although considerable levels of transgene expression could be demonstrated, the growth of Spodoptera exigua larvae fed with detached leaves of PI2-expressing plants was not affected. Analysis of the composition of tryptic gut activity demonstrated that only 18% of the proteinase activity of insects reared on these transgenic plants was sensitive to inhibition by PI2, whereas 78% was sensitive in insects reared on control plants. Larvae had compensated for this loss of tryptic activity by a 2.5-fold induction of new activity that was insensitive to inhibition by PI2. PI2-insensitive proteolytic activity was also induced in response to endogenous proteinase inhibitors of tobacco; therefore, induction of such proteinase activity may represent the mechanism by which insects that feed on plants overcome plant proteinase inhibitor defense.

Human immunodeficiency virus reverse transcriptase substrate-induced conformational changes and the mechanism of inhibition by nonnucleoside inhibitors.

A combination of transient kinetic and equilibrium titration methods has been used to show that both primer/template and nucleotide binding to human immunodeficiency virus type 1 (HIV-1) reverse transcriptase are two-step processes. In both cases, after initial formation of relatively weakly bound states, isomerization reactions lead to tightly bound states. In the case of deoxynucleotide binding to the reverse transcriptase-primer/template complex, the second step in the interaction is rate-limiting in the overall reaction during processive polymerization. Discrimination against incorrect nucleotides occurs both in the initial weak binding and in the second step but is purely kinetic in the second step (as opposed to thermodynamic in the first step). Nonnucleoside inhibitors have a relatively small effect on nucleotide-binding steps (overall affinity is reduced by a factor of ca. 10), while the affinity of the primer/template duplex is increased by at least a factor of 10. The major effect of nonnucleoside inhibitors is on the chemical step (nucleotide transfer).

Inhibitors of human heart chymase based on a peptide library.

We have synthesized two sets of noncleavable peptide-inhibitor libraries to map the S and S' subsites of human heart chymase. Human heart chymase is a chymotrypsin-like enzyme that converts angiotensin I to angiotensin II. The first library consists of peptides with 3-fluorobenzylpyruvamides in the P1 position. (Amino acid residues of substrates numbered P1, P2, etc., are toward the N-terminal direction, and P'1, P'2, etc., are toward the C-terminal direction from the scissile bond.) The P'1 and P'2 positions were varied to contain each one of the 20 naturally occurring amino acids and P'3 was kept constant as an arginine. The second library consists of peptides with phenylalanine keto-amides at P1, glycine in P'1, and benzyloxycarbonyl (Z)-isoleucine in P4. The P2 and P3 positions were varied to contain each of the naturally occurring amino acids, except for cysteine and methionine. The peptides of both libraries are attached to a solid support (pins). The peptides are evaluated by immersing the pins in a solution of the target enzyme and evaluating the amount of enzyme absorbed. The pins with the best inhibitors will absorb most enzyme. The libraries select the best and worst inhibitors within each group of peptides and provide an approximate ranking of the remaining peptides according to Ki. Through this library, we determined that Z-Ile-Glu-Pro-Phe-CO2Me and (F)-Phe-CO-Glu-Asp-ArgOMe should be the best inhibitors of chymase in this collection of peptide inhibitors. We synthesized the peptides and found Ki values were 1 nM and 1 microM, respectively. The corresponding Ki values for chymotrypsin were 10 nM and 100 microM. The use of libraries of inhibitors has advantages over the classical method of synthesis of potential inhibitors in solution: the libraries are reusable, the same libraries can be used with a variety of different serine proteases, and the method allows the screening of hundreds of compounds in short periods of time.

A potassium channel beta subunit related to the aldo-keto reductase superfamily is encoded by the Drosophila hyperkinetic locus.

Genetic and physiological studies of the Drosophila Hyperkinetic (Hk) mutant revealed defects in the function or regulation of K+ channels encoded by the Shaker (Sh) locus. The Hk polypeptide, determined from analysis of cDNA clones, is a homologue of mammalian K+ channel beta subunits (Kv beta). Coexpression of Hk with Sh in Xenopus oocytes increases current amplitudes and changes the voltage dependence and kinetics of activation and inactivation, consistent with predicted functions of Hk in vivo. Sequence alignments show that Hk, together with mammalian Kv beta, represents an additional branch of the aldo-keto reductase superfamily. These results are relevant to understanding the function and evolutionary origin of Kv beta.

Suppression of the breakthrough of human immunodeficiency virus type 1 (HIV-1) in cell culture by thiocarboxanilide derivatives when used individually or in combination with other HIV-1-specific inhibitors (i.e., TSAO derivatives).

Five structurally related thiophene and furane analogues of the oxathiin carboxanilide derivative NSC 615985 (UC84) (designated UC10, UC68, UC81, UC42, and UC16) were identified as potent inhibitors of HIV-1 replication in cell culture and HIV-1 reverse transcriptase activity. These compounds were markedly active against a series of mutant HIV-1 strains, containing the Leu-100-->Ile, Val-106-->Ala, Glu-138-->Lys, or Tyr-181-->Cys mutations in their reverse transcriptase. However, the thiocarboxanilide derivatives selected for mutations at amino acid positions 100 (Leu-->Ile), 101 (Lys-->Ile/Glu), 103 (Lys-->Thr/Asp) and 141 (Gly-->Glu) in the HIV-1 reverse transcriptase. The compounds completely suppressed HIV-1 replication and prevented the emergence of resistant virus strains when used at 1.3-6.6 microM--that is, 10- to 25-fold lower than the concentration required for nevirapine and bis(heteroaryl)piperazine (BHAP) U90152 to do so. If UC42 was combined with the [2',5'-bis-O-(tert-butyldimethylsilyl)-3'-spiro-5"-(4"-amino-1",2"- oxathiole-2",2"-dioxide)]-beta-D-pentofuranosyl (TSAO) derivative of N3-methylthymine (TSAO-m3T), virus breakthrough could be prevented for a much longer time, and at much lower concentrations, than if the compounds were used individually. Virus breakthrough could be suppressed for even longer, and at lower drug concentrations, if BHAP was added to the combination of UC42 with TSAO-m3T, which points to the feasibility of two- or three-drug combinations in preventing virus breakthrough and resistance development.

DNA synthesis and topoisomerase inhibitors increase transduction by adeno-associated virus vectors.

Viral vectors based on adeno-associated virus (AAV) preferentially transduce cells in S phase of the cell cycle. We recently found that DNA-damaging agents increased the transduction of nondividing cells. However, the optimal concentrations were toxic to cells. Here we show that the transduction of normal human fibroblasts by AAV vectors is increased by prior exposure to DNA synthesis inhibitors, such as aphidicolin or hydroxyurea, and topoisomerase inhibitors, such as etoposide or camptothecin. Transduction efficiencies could be increased > 300-fold in stationary cultures at concentrations that did not affect cell viability or proliferative potential. Both S-phase and non-S-phase cells were affected, suggesting that cellular functions other than replicative DNA synthesis may be involved. Applying these methods to gene transfer protocols should improve prospects for gene therapy by AAV vectors.

Crystallization and preliminary x-ray studies of NADPH-cytochrome P450 reductase.

NADPH-cytochrome P450 reductase (CPR; NADPH:ferrihemoprotein reductase, EC 1.6.2.4) catalyzes the transfer of electrons to all known microsomal cytochromes P450. CPR is unique in that it is one of only two mammalian enzymes known to contain both flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN), the other being the various isoforms of nitric oxide synthase. Similarities in amino acid sequence and in functional domain arrangement with other key flavoproteins, including nitric oxide synthase, make CPR an excellent prototype for studies of interactions between two flavin cofactors. We have obtained diffraction-quality crystals of rat liver CPR, expressed in Escherichia coli and solubilized by limited proteolysis with trypsin. The crystals were grown in Hepes buffer (pH 7.0), containing polyethylene glycol 4500 and NaCl. The crystals belong to the orthorhombic space group P2(1)2(1)2(1), with unit cell dimensions a = 103.3 A, b = 116.1 A, and c = 120.4 A. If we assume that there are two molecules of the 72-kDa CPR polypeptide per asymmetric unit, the calculated value of Vm is 2.54 A3/Da.

Protein structure-based design of potent orally bioavailable, nonpeptide inhibitors of human immunodeficiency virus protease.

A class of potent nonpeptidic inhibitors of human immunodeficiency virus protease has been designed by using the three-dimensional structure of the enzyme as a guide. By employing iterative protein cocrystal structure analysis, design, and synthesis the binding affinity of the lead compound was incrementally improved by over four orders of magnitude. An inversion in inhibitor binding mode was observed crystallographically, providing information critical for subsequent design and highlighting the utility of structural feedback in inhibitor optimization. These inhibitors are selective for the viral protease enzyme, possess good antiviral activity, and are orally available in three species.

Ras farnesyltransferase inhibitors suppress the phenotype resulting from an activated ras mutation in Caenorhabditis elegans.

Attachment of Ras protein to the membrane, which requires farnesylation at its C terminus, is essential for its biological activity. A promising pharmacological approach of antagonizing oncogenic Ras activity is to develop inhibitors of farnesyltransferase. We use Caenorhabditis elegans vulval differentiation, which is controlled by a Ras-mediated signal transduction pathway, as a model system to test previously identified farnesyltransferase inhibitors. We show here that two farnesyltransferase inhibitors, manumycin and gliotoxin, suppress the Multivulva phenotype resulting from an activated let-60 ras mutation, but not the Multivulva phenotype resulting from mutations in the lin-1 gene or the lin-15 gene, which act downstream and upstream of let-60 ras, respectively, in the signaling pathway. These results are consistent with the idea that the suppression of the Multivulva phenotype of let-60 ras by the two inhibitors is specific for Ras protein and that the mutant Ras protein might be more sensitive than wild-type Ras to the farnesyltransferase inhibitors. This work suggests that C. elegans vulval development could be a simple and effective in vivo system for evaluation of farnesyltransferase inhibitors against Ras-activated tumors.