Genome mining in Sorangium cellulosum So ce56: identification and characterization of the homologous electron transfer proteins of a myxobacterial cytochrome P450.

Myxobacteria, especially members of the genus Sorangium, are known for their biotechnological potential as producers of pharmaceutically valuable secondary metabolites. The biosynthesis of several of those myxobacterial compounds includes cytochrome P450 activity. Although class I cytochrome P450 enzymes occur wide-spread in bacteria and rely on ferredoxins and ferredoxin reductases as essential electron mediators, the study of these proteins is often neglected. Therefore, we decided to search in the Sorangium cellulosum So ce56 genome for putative interaction partners of cytochromes P450. In this work we report the investigation of eight myxobacterial ferredoxins and two ferredoxin reductases with respect to their activity in cytochrome P450 systems. Intriguingly, we found not only one, but two ferredoxins whose ability to sustain an endogenous So ce56 cytochrome P450 was demonstrated by CYP260A1-dependent conversion of nootkatone. Moreover, we could demonstrate that the two ferredoxins were able to receive electrons from both ferredoxin reductases. These findings indicate that S. cellulosum can alternate between different electron transport pathways to sustain cytochrome P450 activity.

The reduced [2Fe-2S] clusters in adrenodoxin and Arthrospira platensis ferredoxin share spin density with protein nitrogens, probed using 2D ESEEM.

We have used X-band ESEEM to study the reduced [2Fe-2S] cluster in adrenodoxin and Arthrospira platensis ferredoxin. By use of a 2D approach (HYSCORE), we have shown that the cluster is involved in weak magnetic interactions with several nitrogens in each protein. Despite substantial differences in the shape and orientational dependence of individual cross-peaks, the major spectral features in both proteins are attributable to two peptide nitrogens (N1 and N2) with similar hyperfine couplings approximately 1.1 and approximately 0.70 MHz. The couplings determined represent a small fraction (0.0003-0.0005) of the unpaired spin density of the reduced cluster transferred to these nitrogens over H-bond bridges or the covalent bonds of cysteine ligands. Simulation of the HYSCORE spectra has allowed us to estimate the orientation of the nuclear quadrupole tensors of N1 and N2 in the g-tensor coordinate system. The most likely candidates for the role of N1 and N2 have been identified in the protein environment by comparing magnetic-resonance data with crystallographic structures of the oxidized proteins. A possible influence of redox-linked structural changes on ESEEM data is analyzed using available structures for related proteins in two redox states.

Mechanism of inhibition of P-glycoprotein mediated efflux by vitamin E TPGS: influence on ATPase activity and membrane fluidity.

Efflux pump (e.g., P-gp, MRP1, and BCRP) inhibition has been recognized as a strategy to overcome multi-drug resistance and improve drug bioavailability. Besides small-molecule inhibitors, surfactants such as Tween 80, Cremophor EL, several Pluronics, and Vitamin E TPGS (TPGS 1000) are known to modulate efflux pump activity. Competitive inhibition of substrate binding, alteration of membrane fluidity, and inhibition of efflux pump ATPase have been proposed as possible mechanisms. Focusing on TPGS 1000, the aim of our study was to unravel the inhibitory mechanism by comparing the results of inhibition experiments in a Caco-2 transport assay with data from electron spin resonance (ESR) and from ATPase activity studies. ESR results, on Caco-2 cells using 5-doxyl stearic acid (5-SA) as a spin probe, ruled out cell membrane fluidization as a major contributor; change of membrane fluidity was only observed at surfactant concentrations 100 times higher than those needed to achieve full efflux inhibition. Concurrently, TPGS 1000 inhibited substrate induced ATPase activity without inducing significant ATPase activity on its own. By investigating TPGS analogues that varied by their PEG chain length, and/or possessed a modified hydrophobic core, transport studies revealed that modulation of ATPase activity correlated with inhibitory potential for P-gp mediated efflux. Hence, these results indicate that ATPase inhibition is an essential factor in the inhibitory mechanism of TPGS 1000 on cellular efflux pumps.

Spectroscopic and biochemical studies on protein variants of quinaldine 4-oxidase: Role of E736 in catalysis and effects of serine ligands on the FeSI and FeSII clusters.

Quinaldine 4-oxidase (Qox), which catalyzes the hydroxylation of quinaldine to 1H-4-oxoquinaldine, is a heterotrimeric (LMS)2 molybdo-iron/sulfur flavoprotein belonging to the xanthine oxidase family. Variants of Qox were generated by site-directed mutagenesis. Replacement in the large subunit at E736, which is presumed to be located close to the molybdenum, by aspartate (QoxLE736D) resulted in a marked decrease in kcat app for quinaldine, while Km app was largely unaffected. Although a minor reduction of the glutamine substituted variant QoxLE736Q by quinaldine occurred, its activity was below detection, indicating that the carboxylate group of E736 is crucial for catalysis. Replacement of cysteine ligands C40, C45, or C60 (FeSII) and of the C120 or C154 ligands to FeSI in the small subunit of Qox by serine led to decreased iron contents of the protein preparations. Substitutions C40S and C45S (Fe1 of FeSII) suppressed the characteristic FeSII EPR signals and significantly reduced catalytic activity. In QoxSC154S (Fe1 of FeSI), the g-factor components of FeSI were drastically changed. In contrast, Qox proteins with substitutions of C48 and C60 (Fe2 of FeSII), and of the C120 ligand at Fe2 of FeSI, retained considerable activity and showed less pronounced changes in their EPR parameters. Taken together, the properties of the Qox variants suggest that Fe1 of both FeSI and FeSII are the reducible iron sites, whereas the Fe2 ions remain in the ferric state. The location of the reducible iron sites of FeSI and FeSII appears to be conserved in enzymes of the xanthine oxidase family.

Postirradiation electron transfer vs differential radical decay in X-irradiated DNA and its mixtures with additives. Electron spin resonance spectroscopy in LiBr glass at low temperatures.

Free radical formation in DNA and in colyophilized mixtures of DNA with the additives mitoxantrone and riboflavin was monitored after X-ray irradiation in frozen aqueous glasses (7 M LiBr/D2O) at 77 K by electron spin resonance (ESR) spectroscopy. Specifically, the postirradiation time course at 77 K of the respective free radical intensity residing on DNA or on the additive was probed in order to test the hypothesis of electron transfer from DNA, e.g., to mitoxantrone after irradiation under these conditions (e.g., Messer, A.; Carpenter, K.; Forzley, K.; Buchanan, J.; Yang, S.; Razskazovskii, Y.; Cai, Z.; Sevilla, M. D. J. Phys. Chem. B 2000, 104, 1128). For both additives, different additive loadings and irradiation doses were employed. The observed relative change in contributions of DNA and of additive radical components to the experimental spectra with time could be ascribed, for both additives, unequivocally to independent, differential fading of component radicals. Transfer from DNA to the additive, e.g., by electron tunneling as proposed before could be ruled out to occur by a detailed, quantitative analysis of the experimental spectra using reconstruction techniques. Additional studies were performed with the nucleotides TMP and dCMP and its mixtures with mitoxantrone in order to describe the time course in systems which are expected to behave independently; the results supported the conclusions arrived at from the analysis of the DNA/additive system. A model was proposed to describe the postirradiation radical fading mechanisms which involve liberation of radiation-induced matrix-trapped defects with time. It was assumed that these defects are ESR-mute and react with radicals by net radical destruction. Some experimental observations are presented concerning influence of temperature and of the matrix on the fading processes. These seem to argue in favor of such a model although a detailed, quantitative description is still not possible.

Dioxygenases without requirement for cofactors and their chemical model reaction: compulsory order ternary complex mechanism of 1H-3-hydroxy-4-oxoquinaldine 2,4-dioxygenase involving general base catalysis by histidine 251 and single-electron oxidation of the substrate dianion.

1H-3-Hydroxy-4-oxoquinaldine 2,4-dioxygenase (Hod) is a cofactor-less dioxygenase belonging to the alpha/beta hydrolase fold family, catalyzing the cleavage of 1H-3-hydroxy-4-oxoquinaldine (I) and 1H-3-hydroxy-4-oxoquinoline (II) to N-acetyl- and N-formylanthranilate, respectively, and carbon monoxide. Bisubstrate steady-state kinetics and product inhibition patterns of HodC, the C69A protein variant of Hod, suggested a compulsory-order ternary-complex mechanism, in which binding of the organic substrate precedes dioxygen binding, and carbon monoxide is released first. The specificity constants, k(cat)/K(m,A) and k(cat)/K(m,O)()2, were 1.4 x 10(8) and 3.0 x 10(5) M(-1) s(-1) with I and 1.2 x 10(5) and 0.41 x 10(5) M(-1) s(-1) with II, respectively. Whereas HodC catalyzes formation of the dianion of its organic substrate prior to dioxygen binding, HodC-H251A does not, suggesting that H251, which aligns with the histidine of the catalytic triad of the alpha/beta hydrolases, acts as general base in catalysis. Investigation of base-catalyzed dioxygenolysis of I by electron paramagnetic resonance (EPR) spectroscopy revealed formation of a resonance-stabilized radical upon exposure to dioxygen. Since in D(2)O spectral properties are not affected, exchangeable protons are not involved, confirming that the dianion is the reactive intermediate that undergoes single-electron oxidation. We suggest that in the ternary complex of the enzyme, direct single-electron transfer from the substrate dianion to dioxygen may occur, resulting in a radical pair. Based on the estimated spin distribution within the radical anion (observed in the model reaction of I), radical recombination may produce a C4- or C2-hydroperoxy(di)anion. Subsequent intramolecular attack would result in the 2,4-endoperoxy (di)anion that may collapse to the reaction products.

Gene cluster of Arthrobacter ilicis Ru61a involved in the degradation of quinaldine to anthranilate: characterization and functional expression of the quinaldine 4-oxidase qoxLMS genes.

A genetic analysis of the anthranilate pathway of quinaldine degradation was performed. A 23-kb region of DNA from Arthrobacter ilicis Rü61a was cloned into the cosmid pVK100. Although Escherichia coli clones containing the recombinant cosmid did not transform quinaldine, cosmids harboring the 23-kb region, or a 10.8-kb stretch of this region, conferred to Pseudomonas putida KT2440 the ability to cometabolically convert quinaldine to anthranilate. The 10.8-kb fragment thus contains the genes coding for quinaldine 4-oxidase (Qox), 1H-4-oxoquinaldine 3-monooxygenase, 1H-3-hydroxy-4-oxoquinaldine 2,4-dioxygenase, and N-acetylanthranilate amidase. The qoxLMS genes coding for the molybdopterin cytosine dinucleotide-(MCD-), FeSI-, FeSII-, and FAD-containing Qox were inserted into the expression vector pJB653, generating pKP1. Qox is the first MCD-containing enzyme to be synthesized in a catalytically fully competent form by a heterologous host, P. putida KT2440 pKP1; the catalytic properties and the UV-visible and EPR spectra of Qox purified from P. putida KT2440 pKP1 were essentially like those of wild-type Qox. This provides a starting point for the construction of protein variants of Qox by site-directed mutagenesis. Downstream of the qoxLMS genes, a putative gene whose deduced amino acid sequence showed 37% similarity to the cofactor-inserting chaperone XdhC was located. Additional open reading frames identified on the 23-kb segment may encode further enzymes (a glutamyl tRNA synthetase, an esterase, two short-chain dehydrogenases/reductases, an ATPase belonging to the AAA family, a 2-hydroxyhepta-2,4-diene-1,7-dioate isomerase/5-oxopent-3-ene-1,2,5-tricarboxylate decarboxylase-like protein, and an enzyme of the mandelate racemase group) and hypothetical proteins involved in transcriptional regulation, and metabolite transport.

Functional expression of the quinoline 2-oxidoreductase genes (qorMSL) in Pseudomonas putida KT2440 pUF1 and in P. putida 86-1 deltaqor pUF1 and analysis of the Qor proteins.

The availability of a system for the functional expression of genes coding for molybdenum hydroxylases is a prerequisite for the construction of enzyme variants by mutagenesis. For the expression cloning of quinoline 2-oxidoreductase (Qor) from Pseudomonas putida 86--that contains the molybdopterin cytosine dinucleotide molybdenum cofactor (Mo-MCD), two distinct [2Fe-2S] clusters and FAD--the qorMSL genes were inserted into the broad host range vector, pJB653, generating pUF1. P. putida KT2440 and P. putida 86-1 deltaqor were used as recipients for pUF1. Whereas Qor from the wild-type strain showed a specific activity of 19-23 U x mg(-1), the specific activity of Qor purified from P. putida KT2440 pUF1 was only 0.8-2.5 U x mg(-1), and its apparent k(cat) (quinoline) was about ninefold lower than that of wild-type Qor. The apparent Km values for quinoline were similar for both proteins. UV/visible and EPR spectroscopy indicated the presence of the full set of [2Fe-2S] clusters and FAD in Qor from P. putida KT2440 pUF1, however, the very low intensity of the Mo(V)-rapid signal, that occurs in the presence of quinoline, as well as metal analysis indicated a deficiency of the molybdenum center. In contrast, the metal content, and the spectroscopic and catalytic properties of Qor produced by P. putida 86-1 deltaqor pUF1 were essentially like those of wild-type Qor. Release of CMP upon acidic hydrolysis of the Qor proteins suggested the presence of the MCD form of the pyranopterin cofactor; the CMP contents of the three enzymes were similar.

Hydrogenases in the "active" state: determination of g-matrix axes and electron spin distribution at the active site by 1H ENDOR spectroscopy.

Hydrons and electrons are substrates for the enzyme hydrogenase, but cannot be observed in X-ray crystal structures. High-resolution 1H electron nuclear double resonance (ENDOR) spectroscopy offers a means to detect the distribution of protons and unpaired electrons. ENDOR spectra were recorded from frozen solutions of the nickel-iron hydrogenases of Desulfovibrio gigas and Desulfomicrobium baculatum, in the "active" state ("Ni-C" EPR signal) and analyzed by orientationally selective simulation methods. The experimental spectra were fitted using a structural model of the nickel-iron centre based on crystallographic results, allowing for differences in electron spin distribution as well as the spatial orientation of the g-matrix ( g-tensor), and anisotropic and isotropic hyperfine couplings of the protons nearest to the nickel ion. ENDOR signals, detected after complete deuterium exchange, were assigned to six protons of the cysteines bound to nickel. The assignment took advantage of the substitution of a selenium for a sulfur ligand, which occurs naturally between the [NiFeSe] and [NiFe] hydrogenases from Dm. baculatum and D. gigas, respectively, and was found to affect just two signals. The four signals with the largest hyperfine couplings, including isotropic contributions from 4.5 to 13.5 MHz, were assigned to the beta-methylene protons of the two terminal cysteine ligands, one of which is substituted by seleno-cysteine in [NiFeSe] hydrogenase. The electron spin is delocalized onto the nickel (50%) and its sulfur ligands, with a higher proportion on the terminal than the bridging ligands. The g-matrix was found to align with the active site in such a way that the g1- g2 plane is nearly coplanar (18.3 degrees) with the plane defined by nickel and three sulfur atoms, and the g2 axis deviates by 22.9 degrees from the vector between nickel and iron. Significantly for the reaction of the enzyme, direct evidence for the binding of hydrons at the active site was obtained by the detection of H/D-exchangeable ENDOR signals.

Bacterial Degradation of Quinoline and Derivatives-Pathways and Their Biocatalysts.

A series of interesting enzymes were discovered during investigations on the degradation of quinoline by microorganisms. These include the molybdenum-containing hydroxylases that catalyze the transformation 1→2 and the unusual 2,4-dioxygenases that catalyze the reaction 3→4. The application of the hydroxylases may even be interesting in industry, because several quinoline derivatives are used as pharmaceuticals or agrochemicals.