A multifunctional polyketide-peptide synthetase essential for albicidin biosynthesis in Xanthomonas albilineans

Albicidins, a family of potent antibiotics and phytotoxins produced by the sugarcane leaf scald pathogen Xanthomonas albilineans, inhibit DNA replication in bacteria and plastids. A gene located by Tn5-tagging was confirmed by complementation to participate in albicidin biosynthesis. The gene (xabB) encodes a large protein (predicted Mr 525695), with a modular architecture indicative of a multifunctional polyketide synthase (PKS) linked to a non-ribosomal peptide synthetase (NRPS). At 4801 amino acids in length, XabB is the largest reported PKS–NRPS. Twelve catalytic domains in this multifunctional enzyme are arranged in the order N terminus–acyl-CoA ligase (AL)–acyl carrier protein (ACP)–ß-ketoacyl synthase (KS)–ß-ketoacyl reductase (KR)–ACP–ACP–KS–peptidyl carrier protein (PCP)–condensation (C)–adenylation–PCP–C. The modular architecture of XabB indicates likely steps in albicidin biosynthesis and approaches to enhance antibiotic yield. The novel pattern of domains, in comparison with known PKS–NRPS enzymes for antibiotic production, also contributes to the knowledge base for rational design of enzymes producing novel antibiotics.

Metabolic activation of heterocyclic amines and other procarcinogens in Salmonella typhimurium umu tester strains expressing human cytochrome P4501A1, 1A2, 1B1, 2C9, 2D6, 2E1, and 3A4 and human NADPH-P450 reductase and bacterial O-acetyltransferase

We investigated roles of different forms of cytochrome P450 (P450 or CYP) in the metabolic activation of heterocyclic amines (HCAs) and other procarcinogens to genotoxic metabolite(s) in the newly developed umu tester strains Salmonella typhimurium (S. typhimurium) OY1002/1A1, OY1002/1A2, OY1002/1B1, OY1002/2C9, OY1002/2D6, OY1002/2E1 and OY 1002/3A4. which express respective human P450 enzymes and NADPH-cytochrome P350 reductase (reductase) and bacterial O-acetyltransferase (O-AT). These strains were established by introducing two plasmids into S. typhimurium TA 1535, one carrying both P450 and the reductase cDNA in a bicistronic construct under control of an IPTG-inducible double me promoter and the other, pOA 102, carrying O-AT and umuClacZ fusion genes. Expression levels of CYP were found to range between 35 to 550 nmol/l cell culture in the strains tested. O-AT activities in different strains ranged from 52 to 135 nmol isoniazid acetylated/min/mg protein. All HCAs tested, and 2-aminoanthracene and 2-aminofluorene exhibited high genotoxicity in the OY1002/1A2 strain, and genotoxicity of 2-amino-3-methylimidazo [4,5-f]quinoline was detected in both the OY1002/1A1 and OY1002/1A2 strains. 1-Amino-1,4-dimethyl-5H-pyrido[4.3-b]-indole and 3-amino-1-methyl-5H-pyrido[4,3-b]-indole were activated in the OY1002/1A1, OY1002/1B1, OY1002/1A2, and OY1002/3A4 strains. Aflatoxin B-1 exhibited genotoxicity in the OY1002/1A2, OY1002/1A1, and OY1002/3A4 strains. beta -Naphthylamine and benzo[a]pyrene did not exhibit genotoxicity in any of the strains. These results suggest that CYP1A2 is the major cytochrom P450 enzyme involved in bioactivation of HCAs. (C) 2001 Elsevier Science B.V. All rights reserved.

Site-Directed Mutagenesis of Dimethyl Sulfoxide Reductase from Rhodobacter capsulatus: Characterization of a Y114 → F Mutant

A system for expressing site-directed mutants of the molybdenum enzyme dimethyl sulfoxide reductase from Rhodobacter capsulatus in the natural host was constructed. This system was used to Generate and express dimethyl sulfoxide reductase with a Y114F mutation. The Y114F mutant had an increased k(cat) and increased K-m toward both dimethyl sulfoxide and trimethylamine N-oxide compared to the native enzyme, and the value of k(cat)/K-m was lower for both substrates in the mutant enzyme. The Y114F mutant, as isolated, was able to oxidize dimethyl sulfide with phenazine ethosulfate as the electron acceptor but with a lower k(cat) than that of the native enzyme. The pH optimum of dimethyl sulfide: acceptor oxidoreductase activity in the Y114F mutant was shown to be shifted by +1 pH unit compared to the native enzyme. The Y114F mutant did not form a pink complex with dimethyl sulfide, which is characteristic of the native enzyme. The mutant enzyme showed a large increase in the K-d for DMS. Direct electrochemistry showed that the Mo(V)/Mo(IV) couple was unaffected by the Y114F mutant, but the midpoint potential of the Mo(VI)/Mo(V) couple was raised by about 50 mV. These data confirm that the Y114 residue plays a critical role in oxidation-reduction processes at the molybdenum active site and in oxygen atom transfer associated with sulfoxide reduction.

The first non-turnover voltammetric response from a molybdenum enzyme: direct electroscopy of dimethylsulfoxide reductase from Rhodobacter capsulatus

The first direct voltammetric response from a molybdenum enzyme under non-turnover conditions is reported. Cyclic voltammetry of dimethylsulfoxide reductase from Rhodobacter capsulatus reveals a reversible Mo-VI/V response at + 161 mV followed by a reversible Mo-V/IV response at -102 mV versus NHE at pH 8. The higher potential couple exhibits a pH dependence consistent with protonation upon reduction to the Mo-V state and we have determined the pK(a) for this semi-reduced species to be 9.0. The lower potential couple is pH independent within the range 5 < pH < 10. The optical spectrum of the Mo chromophore has been investigated with spectroelectrochemistry. At high potential, in its resting state, the enzyme exhibits a spectrum characteristic of the Mo-VI form. This changes significantly following bulk electrolysis (-400 mV versus NHE) at an optically transparent, indium-doped tin oxide working electrode, where a single visible electronic maximum at 632 nm is observed, which is comparable with spectra reported previously for the dithionite-reduced enzyme. This two-electron process is chemically reversible by reoxidizing the enzyme at the electrode in the absence of mediators or promoters. The activity of the enzyme has been established by observation of a catalytic current in the presence of DMSO at pH 8, where a sigmoidal (steady state) voltammogram is seen. Electronic supplementary material to this paper (Fig. S 1) can be obtained by using the Springer Link server located at http://dx.doi.org/10.1007/s00775-002-0374-y.

Molecular analysis of dimethyl sulphide dehydrogenase from Rhodovulum sulfidophilum: its place in the dimethyl sulphoxide reductase family of microbial molybdopterin-containing enzymes

Dimethyl sulphide dehydrogenase catalyses the oxidation of dimethyl sulphide to dimethyl sulphoxide (DMSO) during photoautotrophic growth of Rhodovulum sulfidophilum . Dimethyl sulphide dehydrogenase was shown to contain bis (molybdopterin guanine dinucleotide)Mo, the form of the pterin molybdenum cofactor unique to enzymes of the DMSO reductase family. Sequence analysis of the ddh gene cluster showed that the ddhA gene encodes a polypeptide with highest sequence similarity to the molybdop-terin-containing subunits of selenate reductase, ethylbenzene dehydrogenase. These polypeptides form a distinct clade within the DMSO reductase family. Further sequence analysis of the ddh gene cluster identified three genes, ddhB , ddhD and ddhC . DdhB showed sequence homology to NarH, suggesting that it contains multiple iron-sulphur clusters. Analysis of the N-terminal signal sequence of DdhA suggests that it is secreted via the Tat secretory system in complex with DdhB, whereas DdhC is probably secreted via a Sec-dependent mechanism. Analysis of a ddhA mutant showed that dimethyl sulphide dehydrogenase was essential for photolithotrophic growth of Rv. sulfidophilum on dimethyl sulphide but not for chemo-trophic growth on the same substrate. Mutational analysis showed that cytochrome c (2) mediated photosynthetic electron transfer from dimethyl sulphide dehydrogenase to the photochemical reaction centre, although this cytochrome was not essential for photoheterotrophic growth of the bacterium.

Control of dimethylsulfoxide reductase expression in Rhodobacter capsulatus: the role of carbon metabolites and the response regulators DorR and RegA

Regulation of the expression of dimethylsulfoxide (DMSO) reductase was investigated in the purple phototrophic bacterium Rhodobacter capsulatus. Under phototrophic, anaerobic conditions with malate as carbon source, DMSO caused an approximately 150-fold induction of DMSO reductase activity. The response regulator DorR was required for DMSO-dependent induction and also appeared to slightly repress DMSO reductase expression in the absence of substrate. Likewise, when pyruvate replaced malate as carbon source there was an induction of DMSO reductase activity in cells grown at low light intensity (16 W m(-2)) and again this induction was dependent on DorR. The level of DMSO reductase activity in aerobically grown cells was elevated when pyruvate replaced malate as carbon source. One possible explanation for this is that acetyl phosphate, produced from pyruvate, may activate expression of DMSO reductase by direct phosphorylation of DorR, leading to low levels of induction of dor gene expression in the absence of DMSO. A mutant lacking the global response regulator of photosynthesis gene expression, RegA, exhibited high levels of DMSO reductase in the absence of DMSO, when grown phototrophically with malate as carbon source. This suggests that phosphorylated RegA acts as a repressor of dor operon expression under these conditions. It has been proposed elsewhere that RegA-dependent expression is negatively regulated by the cytochrome cbb(3) oxidase. A cco mutant lacking cytochrome cbb(3) exhibited significantly higher levels of Phi[dorA::lacZ] activity in the presence of DMSO compared to wild-type cells and this is consistent with the above model. Pyruvate restored DMSO reductase expression in the regA mutant to the same pattern as found in wild-type cells. These data suggest that R. capsulatus contains a regulator of DMSO respiration that is distinct from DorR and RegA, is activated in the presence of pyruvate, and acts as a negative regulator of DMSO reductase expression.

The DMSO reductase family of microbial molybdenum enzymes; Molecular properties and role in the dissimilatory reduction of toxic elements

The dimethylsulfoxide (DMSO) reductase family of molybdenum enzymes is a large and diverse group that is found in bacteria and archaea. These enzymes are characterised by a bis(molybdopterin guanine dinucleotide)Mo form of the molybdenum cofactor, and they are particularly important in anaerobic respiration including the dissimilatory reduction of certain toxic oxoanions. The structural and phylogenetic relationship between the proteins of this family is discussed. High-resolution crystal structures of enzymes of the DMSO reductase family have revealed a high degree of similarity in tertiary structure. However, there is considerable variation in the structure of the molybdenum active site and it seems likely that these subtle but important differences lead to the great diversity of function seen in this family of enzymes. This diversity of catalytic capability is associated with several distinct pathways of electron transport.

Dizygotic twinning is not associated with methylenetetrahydrofolate reductase haplotypes

Background: Folate metabolism is critical to embryonic development, influencing neural tube defects (NTD) and recurrent early pregnancy loss. Polymorphisms in 5,10-methylenetetrahydrofolate reductase (MTHFR) have been associated with dizygotic (DZ) twinning through pregnancy loss. Methods: The C677T and A1298C polymorphisms in MTHFR were genotyped in 258 Australasian families (1016 individuals) and 118 Dutch families (462 individuals) of mothers of DZ twins and a population sample of 462 adolescent twin families (1861 individuals). Haplotypes were constructed from the alleles, and transmission of the MTHFR haplotypes to mothers of DZ twins and from parents to twins in the adolescent twin families analysed. Results: The C677T and A1298C were common in all three populations (frequencies > 0.29). There was strong linkage disequilibrium (D'=1) between the variants, showing that specific combinations of alleles (haplotypes) were transmitted together. Three haplotypes accounted for nearly all the variation. There was no evidence of any association between MTHFR genotype and twinning in mothers of twins, or of the loss of specific MTHFR genotypes during twin pregnancies. Conclusions: It is concluded that variation in twinning frequency is not associated with MTHFR genotype.

The critical role of tryptophan-116 in the catalytic cycle of dimethylsulfoxide reductase from Rhodobacter capsulatus

In dimethylsulfoxide reductase of Rhodobacter capsulatus tryptophan-116 forms a hydrogen bond with a single oxo ligand bound to the molybdenum ion. Mutation of this residue to phenylalanine affected the UV/visible spectrum of the purified Mo-VI form of dimethylsulfoxide reductase resulting in the loss of the characteristic transition at 720 nm. Results of steady-state kinetic analysis and electrochemical studies suggest that tryptophan 116 plays a critical role in stabilizing the hexacoordinate monooxo Mo-VI form of the enzyme and prevents the formation of a dioxo pentacoordinate Mo-VI species, generated as a consequence of the dissociation of one of the dithiolene ligands of the molybdopterin cofactor from the Mo ion. (C) 2004 Published by Elsevier B.V. on behalf of the Federation of European Biochemical Societies.

Crystallization of the C-terminal domain of the mouse brain cytosolic long-chain acyl-CoA thioesterase

The mammalian long-chain acyl-CoA thioesterase, the enzyme that catalyses the hydrolysis of acyl-CoAs to free fatty acids, contains two fused 4HBT (4-hydroxybenzoyl-CoA thioesterase) motifs. The C-terminal domain of the mouse long-chain acyl-CoA thioesterase (Acot7) has been expressed in bacteria and crystallized. The crystals were obtained by vapour diffusion using PEG 2000 MME as precipitant at pH 7.0 and 290 K. The crystals have the symmetry of space group R32 ( unit-cell parameters a = b = 136.83, c = 99.82 angstrom, gamma = 120 degrees). Two molecules are expected in the asymmetric unit. The crystals diffract to 2.4 angstrom resolution using the laboratory X-ray source and are suitable for crystal structure determination.

Direct electrochemistry of human and rat NADPH cytochrome P450 reductase

The diflavo-protein NADPH cytochrome P450 reductase (CPR) is the key electron transfer partner for all drug metabolizing cytochrome P450 enzymes in humans. The protein delivers, consecutively, two electrons to the heme active site of the P450 in a carefully orchestrated process which ultimately leads to the generation of a high valent oxo-heme moiety. Despite its central role in P450 function, no direct electrochemical investigation of the purified protein has been reported. Here we report the first voltammetric study of purified human CPR where responses from both the FMN and FAD cofactors have been identified using both cyclic and square wave voltammetry. For human CPR redox responses at -2 and -278 mV (with a ratio of 1e(-):3e(-)) vs NHE were seen at pH 7.9 while the potentials for rat CPR at pH 8.0 were -20 and -254 mV. All redox responses exhibit a pH dependence of approximately -59 mV/pH unit consistent with proton coupled electron transfer reactions of equal stoichiometry. (c) 2006 Elsevier B.V. All rights reserved.