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.

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.

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.

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.

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.

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.

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.

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.

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.

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.