Novel inhibitors of glutamyl-tRNA(Glu) reductase identified through cell-based screening of the heme/chlorophyll biosynthetic pathway.

The metabolite 5-aminolevulinic acid (ALA) is an early committed intermediate in the biosynthetic pathway of heme and chlorophyll formation. In plants, 5-aminolevulinic acid is synthesized via a two-step pathway in which glutamyl-tRNA(Glu) is reduced by glutamyl-tRNA(Glu) reductase (GluTR) to glutamate 1-semialdehyde, followed by transformation to 5-aminolevulinic acid catalyzed by glutamate 1-semialdehyde aminotransferase. Using an Escherichia coli cell-based high-throughput assay to screen small molecule libraries, we identified several chemical classes that specifically inhibit heme/chlorophyll biosynthesis at this point by demonstrating that the observed cell growth inhibition is reversed by supplementing the medium with 5-aminolevulinic acid. These compounds were further tested in vitro for inhibition of the purified enzymes GluTR and glutamate 1-semialdehyde aminotransferase as confirmation of the specificity and site of action. Several promising compounds were identified from the high-throughput screen that inhibit GluTR with an I(0.5) of less than 10 microM. Our results demonstrate the efficacy of cell-based high-throughput screening for identifying inhibitors of 5-aminolevulinic acid biosynthesis, thus representing the first report of exogenous inhibitors of this enzyme.

Design and synthesis of beta-carboxamido phosphonates as potent inhibitors of imidazole glycerol phosphate dehydratase.

We describe the synthesis and enzymatic activity of a library of beta-carboxamido phosphonates as inhibitors of imidazole glycerol phosphate dehydratase (IGPD). Biological results suggest the presence of an enzymatic interaction site not previously observed for other inhibitors of IGPD.

Stereoselective hydroxylation of norcamphor by cytochrome P450cam. Experimental verification of molecular dynamics simulations.

The stereoselectivity of cytochrome P450cam hydroxylation has been investigated with the enantiomerically pure substrate analog norcamphor. (1R)- and (1S)-norcamphor (> 92 enantiomeric excess) were characterized in the hydroxylation reaction with cytochrome P450cam with respect to the product profile, steady state kinetics, coupling efficiency, and free energy of substrate dissociation. The experimental results demonstrate regiospecificity that is enantiomer-specific and confirm our previously reported prediction that (1R)-norcamphor is hydroxylated preferentially at the 5-carbon and (1S)-norcamphor at the 6-carbon (Bass, M. B., and Ornstein, R. L. (1993) J. Comput. Chem. 14, 541-548); these simulation results are now compared with simulations involving a ferryl oxygen intermediate. Hydroxylation of (1R)-norcamphor was found at the 5-, 6-, and 3-carbons in a ratio of 65:30:5 (respectively), whereas (1S)-norcamphor was oxidized to produce a 28:62:10 ratio of the same products. With the exception of the regiospecificity, all of the reaction and physical parameters are similar for each enantiomer of norcamphor. These results show that the position of the carbonyl group on the hydrocarbon skeleton of norcamphor plays a role in determining the average orientation of this substrate in the active site and suggests that hydrogen bonding interactions can aid in directing the regiospecificity and stereospecificity of the hydroxylation reaction catalyzed by cytochrome P450cam.

Molecular recognition in cytochrome P-450: mechanism for the control of uncoupling reactions.

The pathway for utilization of pyridine nucleotide derived reducing equivalents in the cytochrome P-450 monooxygenase systems has three major branch points. The first is a partitioning between autoxidation of a ferrous, oxygenated heme adduct and input of the second reducing equivalent required for monooxygenase stoichiometry. The second is between dioxygen bond scission and release of two-electron-reduced O2 as hydrogen peroxide. The third is between substrate hydrogen abstraction initiated by a putative higher valent iron-oxo species and reduction of this intermediate by two additional electrons to produce water in an overall oxidase stoichiometry. For all substrates investigated, the direct release of superoxide at the first branch point never competes with second electron input. In order to elucidate the aspects of molecular recognition of a substrate-P-450 complex which affect these individual branch points in the catalytic cycle, we have measured the NADH-derived reducing equivalents recovered in hydroxylated substrate, hydrogen peroxide, and water for a series of active-site mutants designed to alter the coupling of ethylbenzene hydroxylation. We find that the reaction specificity at the second and third branch points is affected by site-directed mutations that alter the topology of the binding pocket. The increased commitment to catalysis observed for all mutants suggests that active-site hydration is important in the uncoupling to form hydrogen peroxide at the second branch point. The liberation of hydrogen peroxide does not correlate with the location of the mutation in the pocket, as expected if the two-electron-reduced dioxygen-bound intermediate is not directly participating in the substrate activation step.(ABSTRACT TRUNCATED AT 250 WORDS)

Engineering cytochrome P-450cam to increase the stereospecificity and coupling of aliphatic hydroxylation.

Site-directed mutants were constructed in cytochrome P-450cam to re-engineer the stereochemistry and coupling of ethylbenzene hydroxylation. The reaction with wild-type (WT) enzyme produces one regioisomer 1-phenylethanol with 5% reduced nicotinamide adenine deoxyribonucleic acid to product conversion of and a ratio of 73:27 for the R and S enantiomers respectively. Ethylbenzene was modeled into the active site of WT P-450cam in a rigid mode and oriented to optimize either pro-R or pro-S hydrogen abstraction. Residues T101, T185 and V247 make extensive contacts with the substrate in the static complexes and were therefore chosen for site-directed mutagenesis. Single mutants T101M, V247A and V247M are more stereospecific producing 89, 87 and 82% (R)-1-phenylethanol respectively. The coupling of the reaction is doubled for the single mutants T185L, T185F and V247M. In an effort to engineer increased stereospecificity and coupling into a single catalyst the T101M, T185F and V247M mutants were combined in a multiple mutant of P-450cam. This protein hydroxylates ethylbenzene resulting in an R:S ratio of 87:13 for the 1-phenylethanols and 13% coupling of reducing equivalents to product. The catalytic stereospecificity and stoichiometry with T101M--T185F--V247M does not represent a summation of the changes observed for the single mutants. A portion of the individual effects on substrate recognition produced by the single substitutions is either eliminated or degenerate within the triple mutant.

Ethylbenzene hydroxylation by cytochrome P450cam.

The metabolism of ethylbenzene by cytochrome P450cam was analyzed by experimental and theoretical methods. The present experiments indicate that ethylbenzene is hydroxylated almost exclusively at the secondary ethyl carbon with about a 2:1 ratio of R:S product. Several molecular dynamics trajectories were performed with different starting conformations of ethylbenzene in the active site of P450cam. The stereochemistry of hydroxylation predicted from the molecular dynamics simulations was found to be in good agreement with the observed products.