Oligomeric Changes Regulate Flavin Transfer in Two-Component FMN Reductases Involved in Sulfur Metabolism.

The FMN reductases (SsuE and MsuE of the alkanesulfonate monooxygenase systems) supply reduced flavin to their partner monooxygenases for the desulfonation of alkanesulfonates. Flavin reductases that comprise two-component systems must be able to regulate both flavin reduction and transfer. One mechanism to control these distinct processes is through changes in the oligomeric state of the enzymes. Despite their similar overall structures, SsuE and MsuE showed clear differences in their oligomeric states in the presence of substrates. The oligomeric state of SsuE was converted from a tetramer to a dimer/tetramer equilibrium in the presence of FMN or NADPH in analytical ultracentrifugation studies. Conversely, MsuE shifted from a dimer to a single tetrameric state with FMN, and the NADPH substrate did not induce a similar oligomeric shift. There was a fast tetramer to dimer equilibrium shift occurring at the dimer/dimer interface in H/D-X investigations with apo SsuE. Formation of the SsuE/FMN complex slowed the tetramer/dimer conversion, leading to a slower exchange along the dimer/dimer interface. The oligomeric shift of the MsuE/FMN complex from a dimer to a distinct tetramer showed a decrease in H/D-X in the region around the π-helices at the dimer/dimer interface. Both SsuE and MsuE showed a comparable and significant increase in the melting temperature with the addition of FMN, indicating the conformers formed by each FMN-bound enzyme had increased stability. A mechanism that supports the different structural shifts is rationalized by the different roles these enzymes play in providing reduced flavin to single or multiple monooxygenase enzymes.

Assay of Fe(III) Chelate Reductase Activity in Arabidopsis thaliana Root.

A sensitive FerroZine assay is used to measure the membrane-bound ferric-chelate reductase activity in the Arabidopsis thaliana roots. In Arabidopsis, FRO2 (FERRIC CHELATE REDUCTASE 2) encodes the Fe(III) chelate reductase and its expression is induced by iron deficiency. As FRO2 reduces Fe(III) to soluble Fe(II), the resulting Fe(II) forms a purple-colored complex with the dye FerroZine. The concentration of the Fe(II)-FerroZine is directly proportional to the absorbance at 562 nm.

Iron metabolism in the social amoeba Dictyostelium discoideum: A role for ferric chelate reductases.

Iron is the most abundant transition metal in all living organisms and is essential for several cellular activities, including respiration, oxygen transport, energy production and regulation of gene expression. Iron starvation is used by professional phagocytes, from Dictyostelium to macrophages, as a form of defense mechanism against intracellular pathogens. Previously, we showed that Dictyostelium cells express the proton-driven iron transporter Nramp1 (Natural Resistance-Associated Macrophage Protein 1) and the homolog NrampB (Nramp2) in membranes of macropinosomes and phagosomes or of the contractile vacuole network, respectively. The Nramp-driven transport of iron across membranes is selective for ferrous ions. Since iron is mostly present as ferric ions in growth media and in engulfed bacteria, we have looked for proteins with ferric reductase activity. The Dictyostelium genome does not encode for classical STEAP (Six-Transmembrane Epithelial Antigen of Prostate) ferric reductases, but harbors three genes encoding putative ferric chelate reductase belonging to the Cytochrome b561 family containing a N terminus DOMON domain (DOpamine ß-MONooxygenase N-terminal domain). We have cloned the three genes, naming them fr1A, fr1B and fr1C. fr1A and fr1B are mainly expressed in the vegetative stage while fr1C is highly expressed in the post aggregative stage. All three reductases are localized in the endoplasmic reticulum, but Fr1A is also found in endolysosomal vesicles, in the Golgi and, to a much lower degree, in the plasma membrane, whereas Fr1C is homogeneously distributed in the plasma membrane and in macropinosomal and phagosomal membranes. To gain insight in the function of the three genes we generated KO mutants, but gene disruption was successful only for two of them (fr1A and fr1C), being very likely lethal for fr1B. fr1A- shows a slight delay in the aggregation stage of development, while fr1C- gives rise to large multi-tipped streams during aggregation and displays a strong delay in fruiting body formation. The two single mutants display altered cell growth under conditions of ferric ions overloading and, in the ability to reduce Fe3+, confirming a role of these putative ferric reductases in iron reduction and transport from endo-lysosomal vesicles to the cytosol.

MSMEG_3955 from Mycobacterium smegmatis is a FMN bounded homotrimeric NAD(P)H:Flavin mononucleotide (FMN) oxidoreductase.

BACKGROUND: Tuberculosis (TB) remains an important public health problem since it is the major cause of elevated morbidity and mortality globally. Previous works have shown that Mycobacterium tuberculosis (Mtb); the prime causative agent of the deadly disease has dormancy survival regulator (DosR) regulon, a two-component regulatory system which controls the transcription of more than 50 genes. However, the structure and detailed functions of these DosR regulated genes are largely undetermined. Out of many DosR regulon genes, Rv3131 gets up regulated in hypoxic conditions and was believed to encode for a nitroreductase flavoprotein. The utilization of mycobacteria-specific model systems has greatly added to our understanding of the molecular mechanisms involved in the life cycle and pathogenesis of Mtb. RESULTS: In this study the non-pathogenic mycobacterial model organism Mycobacterium smegmatis (Msmeg) was used to reveal the structure and function of MSMEG_3955; which is a homologue of Rv3131 from Mtb. Using chromatography and spectroscopy techniques it was revealed that cofactor flavin mononucleotide (FMN) was bound to flavoprotein MSMEG_3955. Consistent with the homology modelling predictions, Circular Dichroism (CD) analysis indicated that the MSMEG_3955 is composed of 39.3% α-helix and 24.9% ß-pleated sheets. In contrast to the current notions, the enzymatic assays performed in the present study revealed that MSMEG_3955 was not capable of reducing nitro substrates but showed NADPH dependent FMN oxidoreductase activity. Also, gel permeation chromatography, dynamic light scattering and native acidic gels showed that MSMEG_3955 exists as a homotrimer. Furthermore, the presence of NADPH dependent FMN oxidoreductase and homotrimeric existence could be an alternative function of the protein to help the bacteria survive in dormant state or may be involved in other biochemical pathways. CONCLUSION: MSMEG_3955 is a FMN bound flavoprotein, which exits as a trimer under in vitro conditions. There is no disulphide linkages in between the three protomers of the homotrimer MSMEG_3955. It has a NADPH dependent FMN oxidoreductase activity.

Fission yeast RNA-binding proteins Puf2 and Puf4 are involved in repression of ferrireductase Frp1 expression in response to iron.

This study identifies a post-transcriptional mechanism of iron uptake regulation by Puf2 and Puf4 of the Pumilio and FBF (Puf) family of RNA-binding proteins in Schizosaccharomyces pombe. Cells expressing Puf2 and Puf4 stimulate decay of the frp1+ mRNA encoding a key enzyme of the reductive iron uptake pathway. Results consistently showed that frp1+ mRNA is stabilized in puf2Δ puf4Δ mutant cells under iron-replete conditions. As a result, puf2Δ puf4Δ cells exhibit an increased sensitivity to iron accompanied by enhanced ferrireductase activity. A pool of GFP-frp1+ 3'UTR RNAs was generated using a reporter gene containing the 3' untranslated region (UTR) of frp1+ that was under the control of a regulatable promoter. Results showed that Puf2 and Puf4 accelerate the destabilization of mRNAs containing the frp1+ 3'UTR which harbors two Pumilio response elements (PREs). Binding studies revealed that the PUM-homology RNA-binding domain of Puf2 and Puf4 expressed in Escherichia coli specifically interacts with PREs in the frp1+ 3'UTR. Using RNA immunoprecipitation in combination with reverse transcription qPCR assays, results showed that Puf2 and Puf4 interact preferentially with frp1+ mRNA under basal and iron-replete conditions, thereby contributing to inhibit Frp1 production and protecting cells against toxic levels of iron.

CYB561A3 is the key lysosomal iron reductase required for Burkitt B-cell growth and survival.

Epstein-Barr virus (EBV) causes endemic Burkitt lymphoma, the leading childhood cancer in sub-Saharan Africa. Burkitt cells retain aspects of germinal center B-cell physiology with MYC-driven B-cell hyperproliferation; however, little is presently known about their iron metabolism. CRISPR/Cas9 analysis highlighted the little-studied ferrireductase CYB561A3 as critical for Burkitt proliferation but not for that of the closely related EBV-transformed lymphoblastoid cells or nearly all other Cancer Dependency Map cell lines. Burkitt CYB561A3 knockout induced profound iron starvation, despite ferritinophagy ad plasma membrane transferrin upregulation. Elevated concentrations of ascorbic acid, a key CYB561 family electron donor, or the labile iron source ferrous citrate rescued Burkitt CYB561A3 deficiency. CYB561A3 knockout caused catastrophic lysosomal and mitochondrial damage and impaired mitochondrial respiration. Conversely, lymphoblastoid B cells with the transforming EBV latency III program were instead dependent on the STEAP3 ferrireductase. These results highlight CYB561A3 as an attractive therapeutic Burkitt lymphoma target.

Production of Tyrian purple indigoid dye from tryptophan in Escherichia coli.

Tyrian purple, mainly composed of 6,6'-dibromoindigo (6BrIG), is an ancient dye extracted from sea snails and was recently demonstrated as a biocompatible semiconductor material. However, its synthesis remains limited due to uncharacterized biosynthetic pathways and the difficulty of regiospecific bromination. Here, we introduce an effective 6BrIG production strategy in Escherichia coli using tryptophan 6-halogenase SttH, tryptophanase TnaA and flavin-containing monooxygenase MaFMO. Since tryptophan halogenases are expressed in highly insoluble forms in E. coli, a flavin reductase (Fre) that regenerates FADH2 for the halogenase reaction was used as an N-terminal soluble tag of SttH. A consecutive two-cell reaction system was designed to overproduce regiospecifically brominated precursors of 6BrIG by spatiotemporal separation of bromination and bromotryptophan degradation. These approaches led to 315.0 mg l-1 6BrIG production from tryptophan and successful synthesis of regiospecifically dihalogenated indigos. Furthermore, it was demonstrated that 6BrIG overproducing cells can be directly used as a bacterial dye.

NfoR: Chromate Reductase or Flavin Mononucleotide Reductase?

Soil bacteria can detoxify Cr(VI) ions by reduction. Within the last 2 decades, numerous reports of chromate reductase enzymes have been published. These reports describe catalytic reduction of chromate ions by specific enzymes. These enzymes each have sequence similarity to known redox-active flavoproteins. We investigated the enzyme NfoR from Staphylococcus aureus, which was reported to be upregulated in chromate-rich soils and to have chromate reductase activity (H. Han, Z. Ling, T. Zhou, R. Xu, et al., Sci Rep 7:15481, 2017, https://doi.org/10.1038/s41598-017-15588-y). We show that NfoR has structural similarity to known flavin mononucleotide (FMN) reductases and reduces FMN as a substrate. NfoR binds FMN with a dissociation constant of 0.4 µM. The enzyme then binds NADPH with a dissociation constant of 140 µM and reduces the flavin at a rate of 1,350 s-1 Turnover of the enzyme is apparently limited by the rate of product release that occurs, with a net rate constant of 0.45 s-1 The rate of product release limits the rate of observed chromate reduction, so the net rate of chromate reduction by NfoR is orders of magnitude lower than when this process occurs in solution. We propose that NfoR is an FMN reductase and that the criterion required to define chromate reduction as enzymatic has not been met. That NfoR expression is increased in the presence of chromate suggests that the survival adaption was to increase the net rate of chromate reduction by facile, adventitious redox processes.IMPORTANCE Chromate is a toxic by-product of multiple industrial processes. Chromate reduction is an important biological activity that ameliorates Cr(VI) toxicity. Numerous researchers have identified chromate reductase activity by observing chromate reduction. However, all identified chromate reductase enzymes have flavin as a cofactor or use a flavin as a substrate. We show here that NfoR, an enzyme claimed to be a chromate reductase, is in fact an FMN reductase. In addition, we show that reduction of a flavin is a viable way to transfer electrons to chromate but that it is unlikely to be the native function of enzymes. We propose that upregulation of a redox-active flavoprotein is a viable means to detoxify chromate that relies on adventitious reduction that is not catalyzed.

VuuB and IutB reduce ferric-vulnibactin in Vibrio vulnificus M2799.

Vibrio vulnificus, a pathogenic bacterium that causes serious infections in humans, requires iron for growth. Clinical isolate, V. vulnificus M2799, secretes a catecholate siderophore, namely, vulnibactin, to capture iron (III) from the environment. Growth experiments using a deletion mutant indicated that VuuB, a member of the FAD-containing siderophore-interacting protein family, plays a crucial role in Fe3+-vulnibactin reduction. IutB, a member of the ferric-siderophore reductase family, stands a substitute for VuuB in its absence. It remained unclear why V. vulnificus M2799 has two proteins with relevant functions. Here we biochemically characterized VuuB and IutB using purified recombinant proteins. Purified VuuB, a flavoprotein, catalyzed the reduction of Fe3+-nitrilotriacetic acid as its electron acceptor, in the presence of NADH as its electron donor and FAD as its cofactor. IutB catalyzed the reduction of Fe3+-nitrilotriacetic acid, in the presence of NADH, NADPH, or reduced glutathione as its electron donor. The optimal pH values and temperatures of VuuB and IutB were 7.0 and 37 °C, and 8.5 and 45 °C, respectively. On analyzing their ferric-chelate reductase activities, both VuuB and IutB were found to catalyze the reduction of Fe3+-aerobactin, Fe3+-vibriobactin, and Fe3+-vulnibactin. When the biologically relevant substrate, Fe3+-vulnibactin, was used, the levels of ferric-chelate reductase activities were similar between VuuB and IutB. Finally, the mRNA levels were quantified by qRT-PCR in M2799 cells cultivated under low-iron conditions. The number of vuuB mRNA was 8.5 times greater than that of iutB. The expression ratio correlated with the growth of their mutants in the presence of vulnibactin.

ArsH protects Pseudomonas putida from oxidative damage caused by exposure to arsenic.

The two As resistance arsRBC operons of Pseudomonas putida KT2440 are followed by a downstream gene called arsH that encodes an NADPH-dependent flavin mononucleotide reductase. In this work, we show that the arsH1 and (to a lesser extent) arsH2 genes of P. putida KT2440 strengthened its tolerance to both inorganic As(V) and As(III) and relieved the oxidative stress undergone by cells exposed to either oxyanion. Furthermore, overexpression of arsH1 and arsH2 endowed P. putida with a high tolerance to the oxidative stress caused by diamide (a drainer of metabolic NADPH) in the absence of any arsenic. To examine whether the activity of ArsH was linked to a direct action on the arsenic compounds tested, arsH1 and arsH2 genes were expressed in Escherichia coli, which has an endogenous arsRBC operon but lacks an arsH ortholog. The resulting clones both deployed a lower production of reactive oxygen species (ROS) when exposed to As salts and had a superior endurance to physiological redox insults. These results suggest that besides the claimed direct action on organoarsenicals, ArsH contributes to relieve toxicity of As species by mediating reduction of ROS produced in vivo upon exposure to the oxyanion, e.g. by generating FMNH2 to fuel ROS-quenching activities.

Expression, biochemical characterization, and mutation of a water forming NADH: FMN oxidoreductase from Lactobacillus rhamnosus.

Enzyme-catalyzed cofactor regeneration is a significant approach to avoid large quantities consumption of oxidized cofactor, which is vital in a variety of bioconversion reactions. NADH: FMN oxidoreductase is an ideal regenerating enzyme because innocuous molecular oxygen is required as an oxidant. But the by-product H2O2 limits its further applications at the industrial scale. Here, novel NADH: FMN oxidoreductase (LrFOR) from Lactobacillus rhamnosus comprised of 1146 bp with a predicted molecular weight of 42 kDa was cloned and overexpressed in Escherichia coli BL21 (DE3). Enzyme assay shows that the purified recombinant LrFOR has both the NADPH and NADH oxidation activity. Biochemical characterizations suggested that LrFOR exhibits the specific activity of 39.8 U·mg-1 with the optimal pH and temperature of 5.6 and 35 °C and produces H2O instead of potentially harmful peroxide. To further study its catalytic function, a critical Thr29 residue and its six mutants were investigated. Mutants T29G, T29A, and T29D show notable enhancement in activities compared with the wild type. Molecular docking of NADH into wild type and its mutants reveal that a small size or electronegative of residue in position29 could shorten the distance of NADH and FMN, promoting the electrons transfer and resulting in the increased activity. This work reveals the pivotal role of position 29 in the catalytic function of LrFOR and provides effective catalysts in NAD+ regeneration.

Creating Flavin Reductase Variants with Thermostable and Solvent-Tolerant Properties by Rational-Design Engineering.

We have employed computational approaches-FireProt and FRESCO-to predict thermostable variants of the reductase component (C1 ) of (4-hydroxyphenyl)acetate 3-hydroxylase. With the additional aid of experimental results, two C1 variants, A166L and A58P, were identified as thermotolerant enzymes, with thermostability improvements of 2.6-5.6 °C and increased catalytic efficiency of 2- to 3.5-fold. After heat treatment at 45 °C, both of the thermostable C1 variants remain active and generate reduced flavin mononucleotide (FMNH- ) for reactions catalyzed by bacterial luciferase and by the monooxygenase C2 more efficiently than the wild type (WT). In addition to thermotolerance, the A166L and A58P variants also exhibited solvent tolerance. Molecular dynamics (MD) simulations (6 ns) at 300-500 K indicated that mutation of A166 to L and of A58 to P resulted in structural changes with increased stabilization of hydrophobic interactions, and thus in improved thermostability. Our findings demonstrated that improvements in the thermostability of C1 enzyme can lead to broad-spectrum uses of C1 as a redox biocatalyst for future industrial applications.

Distinct associations of the Saccharomyces cerevisiae Rad9 protein link Mac1-regulated transcription to DNA repair.

While it is known that ScRad9 DNA damage checkpoint protein is recruited to damaged DNA by recognizing specific histone modifications, here we report a different way of Rad9 recruitment on chromatin under non DNA damaging conditions. We found Rad9 to bind directly with the copper-modulated transcriptional activator Mac1, suppressing both its DNA binding and transactivation functions. Rad9 was recruited to active Mac1-target promoters (CTR1, FRE1) and along CTR1 coding region following the association pattern of RNA polymerase (Pol) II. Hir1 histone chaperone also interacted directly with Rad9 and was partly required for its localization throughout CTR1 gene. Moreover, Mac1-dependent transcriptional initiation was necessary and sufficient for Rad9 recruitment to the heterologous ACT1 coding region. In addition to Rad9, Rad53 kinase also localized to CTR1 coding region in a Rad9-dependent manner. Our data provide an example of a yeast DNA-binding transcriptional activator that interacts directly with a DNA damage checkpoint protein in vivo and is functionally restrained by this protein, suggesting a new role for Rad9 in connecting factors of the transcription machinery with the DNA repair pathway under unchallenged conditions.

Reduction-dependent siderophore assimilation in a model pennate diatom.

Iron uptake by diatoms is a biochemical process with global biogeochemical implications. In large regions of the surface ocean diatoms are both responsible for the majority of primary production and frequently experiencing iron limitation of growth. The strategies used by these phytoplankton to extract iron from seawater constrain carbon flux into higher trophic levels and sequestration into sediments. In this study we use reverse genetic techniques to target putative iron-acquisition genes in the model pennate diatom Phaeodactylum tricornutum We describe components of a reduction-dependent siderophore acquisition pathway that relies on a bacterial-derived receptor protein and provides a viable alternative to inorganic iron uptake under certain conditions. This form of iron uptake entails a close association between diatoms and siderophore-producing organisms during low-iron conditions. Homologs of these proteins are found distributed across diatom lineages, suggesting the significance of siderophore utilization by diatoms in the marine environment. Evaluation of specific proteins enables us to confirm independent iron-acquisition pathways in diatoms and characterize their preferred substrates. These findings refine our mechanistic understanding of the multiple iron-uptake systems used by diatoms and help us better predict the influence of iron speciation on taxa-specific iron bioavailability.

Redox-induced structural changes in the di-iron and di-manganese forms of Bacillus anthracis ribonucleotide reductase subunit NrdF suggest a mechanism for gating of radical access.

Class Ib ribonucleotide reductases (RNR) utilize a di-nuclear manganese or iron cofactor for reduction of superoxide or molecular oxygen, respectively. This generates a stable tyrosyl radical (Y·) in the R2 subunit (NrdF), which is further used for ribonucleotide reduction in the R1 subunit of RNR. Here, we report high-resolution crystal structures of Bacillus anthracis NrdF in the metal-free form (1.51 Å) and in complex with manganese (MnII/MnII, 1.30 Å). We also report three structures of the protein in complex with iron, either prepared anaerobically (FeII/FeII form, 1.32 Å), or prepared aerobically in the photo-reduced FeII/FeII form (1.63 Å) and with the partially oxidized metallo-cofactor (1.46 Å). The structures reveal significant conformational dynamics, likely to be associated with the generation, stabilization, and transfer of the radical to the R1 subunit. Based on observed redox-dependent structural changes, we propose that the passage for the superoxide, linking the FMN cofactor of NrdI and the metal site in NrdF, is closed upon metal oxidation, blocking access to the metal and radical sites. In addition, we describe the structural mechanics likely to be involved in this process.

Nonsense-mediated mRNA decay of the ferric and cupric reductase mRNAs FRE1 and FRE2 in Saccharomyces cerevisiae.

The nonsense-mediated mRNA decay (NMD) pathway regulates mRNAs that aberrantly terminate translation. This includes aberrant mRNAs and functional natural mRNAs. Natural mRNA degradation by NMD is triggered by mRNA features and environmental cues. Saccharomyces cerevisiae encodes multiple proteins with ferric and cupric reductase activity. Here, we examined the regulation by NMD of two mRNAs, FRE1 and FRE2, encoding ferric and cupric reductases in S. cerevisiae. We found that FRE2 mRNAs are regulated by NMD under noninducing conditions and that the FRE2 3'-UTR contributes to the degradation of the mRNAs by NMD. Conversely, FRE1 mRNAs are not regulated by NMD under comparable conditions. These findings suggest that regulation of functionally related mRNAs by NMD can be differential and conditional.

Interactions between iron and α-synuclein pathology in Parkinson's disease.

Both iron deposition and α-synuclein aggregation are neuropathological hallmarks of Parkinson's disease (PD). We aimed to summarize the extensive interactions between these two factors. The direct structural links between iron and α-synuclein suggest that structural reorganization provokes α-synuclein conformational change. Iron post-transcriptionally regulates α-synuclein synthesis in the presence of iron-responsive element. Increased oxidative/nitrative stress induced by iron is believed to be involved in the post-translational modulation of α-synuclein. Iron modulates proteolytic pathways and therefore participates in the regulation of α-synuclein levels. Meanwhile, the recycling of iron through ferritin degradation suggests a link from the aspects of the degradation signaling pathway. Finally, α-synuclein might regulate iron metabolism through its ferrireductase activity. A prominent role of α-synuclein in iron homeostasis is involved in the uptake of transferrin-Fe. These findings suggest that intracellular iron and α-synuclein are closely related to each other, contributing to the vulnerability of dopaminergic neurons or even to a vicious cycle of toxicity in the pathology of PD.

Improved Efficiency of the Desulfurization of Oil Sulfur Compounds in Escherichia coli Using a Combination of Desensitization Engineering and DszC Overexpression.

The 4S pathway of biodesulfurization, which can specifically desulfurize aromatic S-heterocyclic compounds without destroying their combustion value, is a low-cost and environmentally friendly technology that is complementary to hydrodesulfurization. The four Dsz enzymes convert the model compound dibenzothiophene (DBT) into the sulfur-free compound 2-hydroxybiphenyl (HBP). Of these four enzymes, DszC, the first enzyme in the 4S pathway, is the most severely affected by the feedback inhibition caused by HBP. This study is the first attempt to directly modify DszC to decrease its inhibition by HBP, with the results showing that the modified protein is insensitive to HBP. On the basis of the principle that the final HBP product could show a blue color with Gibbs reagent, a high-throughput screening method for its rapid detection was established. The screening method and the combinatorial mutagenesis generated the mutant AKWC (A101K/W327C) of DszC. After the IC50 was calculated, the feedback inhibition of the AKWC mutant was observed to have been substantially reduced. Interestingly, the substrate inhibition of DszC had also been reduced as a result of directed evolution. Finally, the recombinant BL21(DE3)/BADC*+C* (C* represents AKWC) strain exhibited a specific conversion rate of 214.84 µmolHBP/gDCW/h, which was 13.8-fold greater than that of the wild-type strain. Desensitization engineering and the overexpression of the desensitized DszC protein resulted in the elimination of the feedback inhibition bottleneck in the 4S pathway, which is practical and effective progress toward the production of sulfur-free fuel oil. The results of this study demonstrate the utility of desensitization of feedback inhibition regulation in metabolic pathways by protein engineering.

Functional and mechanistic characterization of an atypical flavin reductase encoded by the pden_5119 gene in Paracoccus denitrificans.

Pden_5119, annotated as an NADPH-dependent FMN reductase, shows homology to proteins assisting in utilization of alkanesulfonates in other bacteria. Here, we report that inactivation of the pden_5119 gene increased susceptibility to oxidative stress, decreased growth rate and increased growth yield; growth on lower alkanesulfonates as sulfur sources was not specifically influenced. Pden_5119 transcript rose in response to oxidative stressors, respiratory chain inhibitors and terminal oxidase downregulation. Kinetic analysis of a fusion protein suggested a sequential mechanism in which FMN binds first, followed by NADH. The affinity of flavin toward the protein decreased only slightly upon reduction. The observed strong viscosity dependence of kcat demonstrated that reduced FMN formed tends to remain bound to the enzyme where it can be re-oxidized by oxygen or, less efficiently, by various artificial electron acceptors. Stopped flow data were consistent with the enzyme-FMN complex being a functional oxidase that conducts the reduction of oxygen by NADH. Hydrogen peroxide was identified as the main product. As shown by isotope effects, hydride transfer occurs from the pro-S C4 position of the nicotinamide ring and partially limits the overall turnover rate. Collectively, our results point to a role for the Pden_5119 protein in maintaining the cellular redox state.

Arginine-95 is important for recruiting superoxide to the active site of the FerB flavoenzyme of Paracoccus denitrificans.

Ferric reductase B (FerB) is a flavin mononucleotide (FMN)-containing NAD(P)H:acceptor oxidoreductase structurally close to the Gluconacetobacter hansenii chromate reductase (ChrR). The crystal structure of ChrR was previously determined with a chloride bound proximal to FMN in the vicinity of Arg101, and the authors suggested that the anionic electron acceptors, chromate and uranyl tricarbonate, bind similarly. Here, we identify the corresponding arginine residue in FerB (Arg95) as being important for the reaction of FerB with superoxide. Four mutants at position 95 were prepared and found kinetically to have impaired capacity for superoxide binding. Stopped-flow data for the flavin cofactor showed that the oxidative step is rate limiting for catalytic turnover. The findings are consistent with a role for FerB as a superoxide scavenging contributor.