The Pden_5119 protein oxidizes NADH with oxygen under mediation by the bound flavin mononucleotide (FMN) and may be involved in the maintenance of the cellular redox pool. In biochemical characterization, the curve of the pH-rate dependence was bell-shaped with pKa1 = 6.6 and pKa2 = 9.2 at 2 μM FMN while it contained only a descending limb pKa of 9.7 at 50 μM FMN. The enzyme was found to undergo inactivation by reagents reactive with histidine, lysine, tyrosine, and arginine. In the first three cases, FMN exerted a protective effect against the inactivation. X-ray structural analysis coupled with site-directed mutagenesis identified three amino acid residues important to the catalysis. Structural and kinetic data suggest that His-117 plays a role in the binding and positioning of the isoalloxazine ring of FMN, Lys-82 fixes the nicotinamide ring of NADH to support the proS-hydride transfer, and Arg-116 with its positive charge promotes the reaction between dioxygen and reduced flavin.

• Keywords
• FMN, NADH, Paracoccus denitrificans, dioxygen reduction,
• MeSH
• Flavin Mononucleotide chemistry   MeSH
• Flavins chemistry   MeSH
• Catalysis MeSH
• Kinetics MeSH
• NAD metabolism   MeSH
• Oxidation-Reduction MeSH
• Paracoccus denitrificans * metabolism   MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Flavin Mononucleotide MeSH
• Flavins MeSH
• NAD MeSH
• NADH oxidase MeSH Browser

Paracoccus denitrificans ArsH is encoded by two identical genes located in two distinct putative arsenic resistance (ars) operons. Escherichia coli-produced recombinant N-His6-ArsH was characterized both structurally and kinetically. The X-ray structure of ArsH revealed a flavodoxin-like domain and motifs for the binding of flavin mononucleotide (FMN) and reduced nicotinamide adenine dinucleotide phosphate (NADPH). The protein catalyzed FMN reduction by NADPH via ternary complex mechanism. At a fixed saturating FMN concentration, it acted as an NADPH-dependent organoarsenic reductase displaying ping-pong kinetics. A 1:1 enzymatic reaction of phenylarsonic acid with the reduced form of FMN (FMNH2) and formation of phenylarsonous acid were observed. Growth experiments with P. denitrificans and E. coli revealed increased toxicity of phenylarsonic acid to cells expressing arsH, which may be related to in vivo conversion of pentavalent As to more toxic trivalent form. ArsH expression was upregulated not only by arsenite, but also by redox-active agents paraquat, tert-butyl hydroperoxide and diamide. A crucial role is played by the homodimeric transcriptional repressor ArsR, which was shown in in vitro experiments to monomerize and release from the DNA-target site. Collectively, our results establish ArsH as responsible for enhancement of organo-As(V) toxicity and demonstrate redox control of ars operon.

• Keywords
• FMN, NADPH, Paracoccus denitrificans, organoarsenicals, redox-responsive repressor,
• Publication type
• Journal Article MeSH

Chromium pollution is increasing incessantly due to continuing industrialization. Of various oxidation states, Cr(6+) is very toxic due to its carcinogenic and mutagenic nature. It also has deleterious effects on different microorganisms as well as on plants. Many species of bacteria thriving in the Cr(6+)-contaminated environments have evolved novel strategies to cope with Cr(6+) toxicity. Generally, decreased uptake or exclusion of Cr(6+) compounds through the membranes, biosorption, and the upregulation of genes associated with oxidative stress response are some of the resistance mechanisms in bacterial cells to overcome the Cr(6+) stress. In addition, bacterial Cr(6+) reduction into Cr(3+) is also a mechanism of specific significance as it transforms toxic and mobile chromium derivatives into reduced species which are innocuous and immobile. Ecologically, the bacterial trait of reductive immobilization of Cr(6+) derivatives is of great advantage in bioremediation. The present review is an effort to underline the bacterial resistance and reducing mechanisms to Cr(6+) compounds with recent development in order to garner a broad perspective.

• MeSH
• Bacteria metabolism   MeSH
• Biodegradation, Environmental MeSH
• Chromium metabolism   toxicity   MeSH
• Oxidation-Reduction MeSH
• Oxidative Stress physiology   MeSH
• Publication type
• Journal Article MeSH
• Review MeSH
• Names of Substances
• Chromium MeSH
• chromium hexavalent ion MeSH Browser

FerB from Paracoccus denitrificans is a soluble cytoplasmic flavoprotein that accepts redox equivalents from NADH or NADPH and transfers them to various acceptors such as quinones, ferric complexes and chromate. The crystal structure and small-angle X-ray scattering measurements in solution reported here reveal a head-to-tail dimer with two flavin mononucleotide groups bound at the opposite sides of the subunit interface. The dimers tend to self-associate to a tetrameric form at higher protein concentrations. Amino acid residues important for the binding of FMN and NADH and for the catalytic activity are identified and verified by site-directed mutagenesis. In particular, we show that Glu77 anchors a conserved water molecule in close proximity to the O2 of FMN, with the probable role of facilitating flavin reduction. Hydride transfer is shown to occur from the 4-pro-S position of NADH to the solvent-accessible si side of the flavin ring. When using deuterated NADH, this process exhibits a kinetic isotope effect of about 6 just as does the NADH-dependent quinone reductase activity of FerB; the first, reductive half-reaction of flavin cofactor is thus rate-limiting. Replacing the bulky Arg95 in the vicinity of the active site with alanine substantially enhances the activity towards external flavins that obeys the standard bi-bi ping-pong reaction mechanism. The new evidence for a cryptic flavin reductase activity of FerB justifies the previous inclusion of this enzyme in the protein family of NADPH-dependent FMN reductases.

• MeSH
• Amino Acids chemistry   genetics   metabolism   MeSH
• Bacterial Proteins chemistry   genetics   metabolism   MeSH
• Biocatalysis MeSH
• X-Ray Diffraction MeSH
• Flavin Mononucleotide chemistry   metabolism   MeSH
• Flavins chemistry   metabolism   MeSH
• Flavoproteins chemistry   genetics   metabolism   MeSH
• Catalytic Domain genetics   MeSH
• Kinetics MeSH
• Crystallography, X-Ray MeSH
• Scattering, Small Angle MeSH
• Models, Molecular MeSH
• Molecular Sequence Data MeSH
• Protein Multimerization MeSH
• Mutagenesis, Site-Directed MeSH
• NADH, NADPH Oxidoreductases chemistry   classification   metabolism   MeSH
• NADP chemistry   metabolism   MeSH
• Oxidation-Reduction MeSH
• Paracoccus denitrificans enzymology   genetics   MeSH
• Amino Acid Sequence MeSH
• Sequence Homology, Amino Acid MeSH
• Protein Structure, Tertiary * MeSH
• Protein Binding MeSH
• Binding Sites genetics   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Amino Acids MeSH
• Bacterial Proteins MeSH
• Flavin Mononucleotide MeSH
• Flavins MeSH
• Flavoproteins MeSH
• NADH, NADPH Oxidoreductases MeSH
• NADP MeSH

The flavin-dependent enzyme FerB from Paracoccus denitrificans reduces a broad range of compounds, including ferric complexes, chromate and most notably quinones, at the expense of the reduced nicotinamide adenine dinucleotide cofactors NADH or NADPH. Recombinant unmodified and SeMet-substituted FerB were crystallized under similar conditions by the hanging-drop vapour-diffusion method with microseeding using PEG 4000 as the precipitant. FerB crystallized in several different crystal forms, some of which diffracted to approximately 1.8 A resolution. The crystals of native FerB belonged to space group P2(1), with unit-cell parameters a = 61.6, b = 110.1, c = 65.2 A, beta = 118.2 degrees and four protein molecules in the asymmetric unit, whilst the SeMet-substituted form crystallized in space group P2(1)2(1)2, with unit-cell parameters a = 61.2, b = 89.2, c = 71.5 A and two protein molecules in the asymmetric unit. Structure determination by the three-wavelength MAD/MRSAD method is now in progress.

• MeSH
• Crystallization MeSH
• Crystallography, X-Ray MeSH
• NADH Dehydrogenase chemistry   MeSH
• Paracoccus denitrificans enzymology   MeSH
• Soil Microbiology MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• NADH Dehydrogenase MeSH