FMN Reductase Research Articles

CYP116B5-SOX: An artificial peroxygenase for drug metabolites production and bioremediation.

CYP116B5 is a class VII P450 in which the heme domain is linked to a FMN and 2Fe2S-binding reductase. Our laboratory has proved that the CYP116B5 heme domain (CYP116B5-hd) is capable of catalyzing the oxidation of substrates using H2O2. Recently, the Molecular Lego approach was applied to join the heme domain of CYP116B5 to sarcosine oxidase (SOX), which provides H2O2 in-situ by the sarcosine oxidation. In this work, the chimeric self-sufficient fusion enzyme CYP116B5-SOX was heterologously expressed, purified, and characterized for its functionality by absorbance and fluorescence spectroscopy. Differential scanning calorimetry (DSC) experiments revealed a TM of 48.4 ± 0.04 and 58.3 ± 0.02°C and a enthalpy value of 175,500 ± 1850 and 120,500 ± 1350 cal mol-1 for the CYP116B5 and SOX domains respectively. The fusion enzyme showed an outstanding chemical stability in presence of up to 200 mM sarcosine or 5 mM H2O2 (4.4 ± 0.8 and 11.0 ± 2.6% heme leakage respectively). Thanks to the in-situ H2O2 generation, an improved kcat/KM for the p-nitrophenol conversion was observed (kcat of 20.1 ± 0.6 min-1 and KM of 0.23 ± 0.03 mM), corresponding to 4 times the kcat/KM of the CYP116B5-hd. The aim of this work is the development of an engineered biocatalyst to be exploited in bioremediation. In order to tackle this challenge, an E. coli strain expressing CYP116B5-SOX was employed to exploit this biocatalyst for the oxidation of the wastewater contaminating-drug tamoxifen. Data show a 12-fold increase in tamoxifen N-oxide production-herein detected for the first time as CYP116B5 metabolite-compared to the direct H2O2 supply, equal to the 25% of the total drug conversion.

Decolorization and detoxification of Brilliant Crocein GR by a newly enriched thermophilic consortium

The environmental pollution caused by azo dyes at high temperatures has become an urgent problem. However, little attention has been paid to decolorizing azo dyes by thermophilic consortiums. In this study, a thermophilic bacterial consortium (BCGR-T) mainly composed of two genera, namely, Caldibacillus (70.90%) and Aeribacillus (17.63%) was first enriched, which can decolorize Brilliant Crocein GR (BCGR) at high temperatures (50–75 °C), pH values of 6∼8, dye concentrations (100–400 mg/L) and salinities (1–5%, w/v). The enzyme activity results showed that the azoreductase activity was nearly 8.8 times that of the control (p < 0.01), and the intracellular lignin peroxidase was also highly expressed with enzyme activity of 5.64 U (min−1 mg−1 protein) (p < 0.05), indicated that both azoreductase and intracellular lignin peroxidase played an important part in the decolorization process. Furthermore, seven new intermediate metabolic products, including aniline, phthalic acid, 2-carboxy benzaldehyde, phenylacetic acid, benzoic acid, toluene, and 4-methyl-hexanoic acid, were identified. In addition, functional genes related with the azo dye decolorization, such as those encoding the azoreductase, laccase, FMN reductase, NADPH-/NADH-quinone oxidoreductases and NADPH-/NADH dehydrogenases, catechol dioxygenase, homogentisate 1,2-dioxygenase, protocatechuate 3,4-dioxygenase, gentisate 1,2-dioxygenase, azobenzene reductase, naphthalene 1,2-dioxygenase, benzoate/toluate 1,2-dioxygenase, and anthranilate 1,2-dioxygenase and so on were found in the metagenome of the consortium BCGR-T. Finally, a new decolorization pathway of the thermophilic consortium BCGR-T was proposed. In addition, the phototoxicity of BCGR decreased after decolorization. Overall, the thermophilic consortium BCGR-T could be a promising candidate in the treatment of high concentration azo dye wastewater at high temperatures.

Metabolic engineering of Paracoccus denitrificans for dual degradation of sulfamethoxazole and ammonia nitrogen.

Sulfamethoxazole (SMX), as one of the most widely used sulfonamide antibiotics, has been frequently detected in the aqueous environment, posing potential risks to the environment and human health. Although microbial degradation methods have been widely applied, some issues remain, including low degradation efficiency and poor environmental adaptability. In this regard, constructing efficient degrading bacteria by metabolic engineering is an ideal solution to these challenges. In this study, we used Paracoccus denitrificans DYTN-1, a superior nitrogen removal environment strain, as chassis to construct an SMX degradation pathway, obtaining a new bacteria for simultaneous degradation of SMX and removal of ammonia nitrogen. In doing this, we first identified and characterized four native promoters of P. denitrificans DYTN-1 with gradient strength to control the expression of the SMX degradation pathway. After degradation pathway expression level optimization and FMN reductase optimization, SMX degradation efficiency was significantly improved. The constructed P. d-pIAB4-PCS-sutR strain exhibited superior co-degradation of SMX and ammonia nitrogen contaminants with degradation rates of 44% and 71%, respectively. This study could pave the way for SMX degradation engineered strain design and evolution of environmental bioremediation. IMPORTANCE The abuse of sulfamethoxazole (SMX) had led to an increased accumulation in the environment, resulting in the disruption of the structure of microbial communities, further disrupting the bio-degradation process of other pollutants, such as ammonia nitrogen. To solve this challenge, we first identified and characterized four native promoters of Paracoccus denitrificans DYTN-1 with gradient strength to control the expression of the SMX degradation pathway. Then SMX degradation efficiency was significantly improved with degradation pathway expression level optimization and FMN reductase optimization. Finally, the superior nitrogen removal environment strain, P. denitrificans DYTN-1, obtained an SMX degradation function. This pioneering study of metabolic engineering to enhance the SMX degradation in microorganisms could pave the way for designing the engineered strains of SMX and nitrogen co-degradation and the environmental bioremediation.

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.

Engineering the Biosynthesis of prFMN Promotes the Conversion between Styrene/CO2 and Cinnamic Acid Catalyzed by the Ferulic Acid Decarboxylase Fdc1

Enzymatic decarboxylation and carboxylation are emerging as prospective processes to produce high-value compounds under mild conditions. Ferulic acid decarboxylase Fdc1 catalyzes broad substrate tolerance against α, β-unsaturated carboxylic acids, and provides green routes for carbon dioxide fixation with the reversible carboxylation, while the activity of the enzyme is limited by the indispensable cofactor prenylated flavin (prFMN), which is unstable and is rarely detected in nature. In this study, a prFMN efficient synthesis route was built using six exogenous enzymes introduced into E. coli cells, leading to the construction of a powerful cell catalyst named SC-6. Based on the metabolic analysis, the results indicated that the reduction of FMN to FMNH2 was the bottleneck in prFMN synthesis pathway, and introducing FMN reductase increased the production of prFMN 3.8-fold compared with the common flavin prenyltransferase UbiX overexpression strain. Using SC-6 cell catalyst, the decarboxylation activity of Fdc1 increased more than 20 times with cinnamic acid and 4-acetoxycinnamic acid as substrates. Furthermore, the reversible carboxylation reaction was carried out, and the cell catalyst presented 20 times carbon dioxide fixation activity using styrene to produce cinnamic acid. Finally, the maximum yield of cinnamic acid catalyzed by SC-6 achieved 833.68 ± 34.51 mM·mg−1 in two hours. The constructed prFMN pathway in vivo provides fundamentals for efficient decarboxylation and carbon fixation reactions catalyzed by prFMN-dependent enzymes.

Investigating Structural Features Utilized by Two‐component Flavin‐dependent Systems

Many enzymes exist in different oligomeric states which serves to regulate enzyme activity, integrate different pathways, expose new active sites at the interface of the interacting subunit, and enhance protein stability. Two‐component FMN‐dependent systems expressed in bacteria during times of limited sulfur availability regulate enzyme activity through the coordination of oligomeric changes of the reductase with reduced flavin transfer to the monooxygenase. The alkanesulfonate monooxygenase system is ubiquitous in bacteria, the reductase (SsuE) and the monooxygenase (SsuD) catalyze the desulfonation of alkanesulfonates to the corresponding aldehyde and sulfite. Pseudomonas aeruginosa has a more complex mechanism for acquiring sulfur. In addition to the SsuE/SsuD system, the organism also encodes a flavin‐dependent reductase (MsuE) and two monooxygenases (MsuC/MsuD) that together convert methanesulfinate to formaldehyde and sulfite. In the alkanesulfonate monooxygenase systems, the FMN reductases exist in different oligomeric states. SsuE exists as a tetramer (dimer of dimers) but can shift to a dimer in the presence of flavin and/or substrate. Coordinated structural dynamics of the FMN reductases and monooxygenase enables efficient synchronization of labile flavin transfer and catalysis. However, the unique structural features that integrates the oligomeric state of the reductase with the mode of flavin transfer to the monooxygenase has not been fully explored.Protein‐protein interactions between the FMN reductases and monooxygenases prevent the unproductive oxidation of reduced flavin. Studies were performed to investigate the interaction sites and oligomeric alterations critical for the transfer of reduced flavin from the reductase to the partner monooxygenase. SsuE and MsuE share 30% amino acid identity and SsuD and MsuD have 60% amino acid sequence identity, implying that although the enzymes utilize different substrates, they share similar structural strategies to regulate flavin transfer. Results from fluorescence spectroscopy show a high binding affinity between SsuE/SsuD and MsuE/MsuD. The high binding affinity observed ensures the safe transfer of reduced flavin from the reductase to the monooxygenase. Protein‐protein interactions sites were identified using hydrogen‐deuterium exchange with mass spectrometry (HDX‐MS) to evaluate flavin transfer in the alkanesulfonate monooxygenase systems (SsuE/SsuD and MsuE/MsuD). The interaction sites in SsuE are located at the dimer‐dimer interface and are close to the active site, while the monooxygenase interactions sites are conserved across SsuD enzymes from different bacterial sources. Comparable protein‐protein interaction sites observed in the alkanesulfonate monooxygenase system (SsuE/SsuD) were identified in the methanesulfinate monooxygenase system (MsuE/MsuD). Taken together, oligomeric changes of the flavin reductase regulates the transfer of flavin and the dimer‐dimer interface houses the sites for protein‐protein interactions with the monooxygenase facilitating catalysis.

Enhancement effects of dissolved organic matter leached from sewage sludge on microbial reduction and immobilization of Cr(VI) by Geobacter sulfurreducens

Sewage sludge has a high concentration of dissolved organic matter (DOM) which contains compounds that can serve as electron donors or shuttles for metal reduction by dissimilatory metal reducing bacteria (DMRB). In this study, Cr(VI) removal by G. sulfurreducens, a common DMRB present in anaerobic soils, was examined in the presence or absence of sludge DOM. Two different types of sludge DOM were tested; composted sludge DOM (C-DOM) and anaerobically digested sludge DOM (A-DOM). Both sludge DOMs enhanced Cr(VI) reduction by G. sulfurreducens, but C-DOM was more effective likely because it had higher concentrations of humic substances that served as electron shuttles. Transcriptomic studies indicated that G. sulfurreducens utilizes several different mechanisms to tolerate chromium including extracellular Cr(VI) reduction and immobilization by outer membrane c-type cytochromes and electrically conductive pili, intracellular Cr(VI) reduction by triheme cytochromes and NAD(P)H FMN reductase proteins, and chromium efflux by several P-type ATPase and RND transporter proteins. Microscopy experiments also showed that Cr(III) crystals formed on the surface of the cells, indicating that extracellular Cr(VI) reduction and adsorption was involved in the chromium removal process. These results help provide insight into the potential use of sewage sludge as an additive to enhance the bioremediation of chromium contaminated soils.

Molecular dynamics study of in silico mutations in the auto-inhibitory loop of human endothelial nitric oxide synthase FMN sub-domain.

Structural flexibility of the peptide linker connecting two domains is essential for the functioning of multi-domain complex. Nitric oxide synthase (NOS) isoforms contain the oxygenase and the reductase domains connected by calmodulin binding linker (CBL) region. Additionally, the endothelial NOS (eNOS) isoform contain an auto-inhibitory loop (AI) in the FMN reductase sub-domain which represses the inter-domain electron transfer process. Binding of Ca2+-Calmodulin complex on the CBL region relieves the AI loop repression and facilitates electron transfer from FMN in the reductase domain to the heme in the oxygenase domain. Few experimental studies have reported that in vitro mutation of Serine-615 (S615D) and Serine-633 (S633D) in the FMN reductase sub-domain to aspartic acid increased NO production and increased Ca2+ sensitivity. To understand the role of AI loop in eNOS repression and activation in serine mutants (S615D and S633D), we modelled the FMN reductase sub-domain of human eNOS protein with and without the CBL region. Molecular dynamics simulations performed indicated that the mutant protein AI loop structure was stabilized by salt bridge formed between D615 and R602. It was also found that mutation increased the flexibility of C-terminal residues of eNOS CBL region. The hinge-like movement of the AI loop allowed rotation of the FMN sub-domain clockwise which may favour electron-transfer in the mutant protein. This study provides insight on mutation (S615D and S633D) induced changes in AI loop and increased flexibility of CBL region which may lead to the protein activation and may also facilitate Calmodulin binding at physiological Ca2+ concentration. Graphical Abstract Mutation of amino acid residues contribute to structural changes at molecular level leading to alteration in protein dynamics and its function. Serine-615 and Serine-633 in the auto-inhibitory loop of human eNOS reductase model was mutated to aspartic acid in silico and molecular dynamics simulations of the protein showed that steric hindrance due to mutation altered the auto-inhibitory loop rearrangement and the FMN sub-domain movement favouring electron transfer.

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.

Biocatalytic in Vitro and in Vivo FMN Prenylation and (De)carboxylase Activation

Reversible UbiD-like (de)carboxylases represent a large family of mostly uncharacterized enzymes, which require the recently discovered prenylated FMN (prFMN) cofactor for activity. Functional characterization of novel UbiDs is hampered by a lack of robust protocols for prFMN generation and UbiD activation. Here, we report two systems for in vitro and in vivo FMN prenylation and UbiD activation under aerobic conditions. The in vitro one-pot prFMN cascade includes five enzymes: FMN prenyltransferase (UbiX), prenol kinase, polyphosphate kinase, formate dehydrogenase, and FMN reductase, which use prenol, polyphosphate, formate, ATP, NAD+, and FMN as substrates and cofactors. Under aerobic conditions, this cascade produced prFMN from FMN with over 98% conversion and activated purified ferulic acid decarboxylase Fdc1 from Aspergillus niger and protocatechuic acid decarboxylase ENC0058 from Enterobacter cloaceae. The in vivo system for FMN prenylation and UbiD activation is based on the coexpression of Fdc1 and UbiX in Escherichia coli cells under aerobic conditions in the presence of prenol. The in vitro and in vivo FMN prenylation cascades will facilitate functional characterization of novel UbiDs and their applications.

Toward Bioluminescent Materials by Plasma Treatment of Microfibrous Nonwovens, Followed by Immobilization of One or Both Enzyme(s) (Luciferase and FMN Reductase) Involved in Luminescent Bacteria.

Bioluminescent living organisms emit light through a specific biocatalyzed reaction involving a luciferin substrate and a luciferase enzyme. The present work investigated the possibility of creating optimal luminescence by immobilization of one or both the enzymes Luciferase (Luc) and FMN reductase (Red) involved in a bioluminescent bacterial system onto a plasma-activated microfibrous PET nonwoven. Parameters affecting the catalytic activity and efficiency of the bacterial system in aqueous medium were determined by luminescence intensity measurements using a luminometer. Two types of plasma, air atmospheric plasma (ATMP) and cold remote plasma (CRPNO) treatment, were used to activate the PET nonwoven. Further, one or both enzyme(s) were immobilized using a physical adsorption technique, without or with the use of natural biopolymers (gelatin and starch) and bovine serum albumin-BSA protein, to improve enzyme stability and activity. Coimmobilization of both Red and Luc enzymes on the CRPNO plasma-activated nonwoven in the presence of BSA led to the maximum luminescence. As high as 60,000 RLU equivalent to that of an LED light used for calibration was observed and showed stable intensity up to 6 min. Fiber surface analysis was tested using wettability tests (water contact angle and capillary uptake), while scanning electron microscopy, atomic force microscopy, and electron spectroscopy for chemical analysis showed changes in fiber surface morphology and chemical functional groups. A considerable increase in "N" atom content after coimmobilization of enzymes in the presence of BSA was detected. This study is the first successful attempt to use a biomimetic strategy for immobilization of enzymes involved in bacterial luminescence on a plasma-activated microfibrous nonwoven in an attempt to attain bioluminescent materials.

Putative Allergens Identified in Mango (Mangifera indica Linn) Leaf and Fruit with Transcriptome Analysis

Some allergens have previously been identified in mango (Mangifera indica Linn), including profilins, Bet v 1-like proteins and chitinase. In this paper, we identified the deepest investigation of mango potential allergens using high-throughput Illumina sequencing. RNA-Seq generated 11,751,123 contigs that were assembled into 99,328 unigenes with 16,848 unigenes of &gt;1000 bp. A total of 230,242 unigenes were annotated using public protein databases, with a cut-off E-value above 10−5, of which 27,295, 46,030, 24,227 and 14,023 unigenes were assigned to gene ontology terms, Nr, Swiss-Prot and clusters of orthologous groups, respectively. A total of 66 potential allergen genes were identified, and their relative expressions were evaluated using Illumina RNA-Seq technology. Allergens mainly belonged to pollen allergen, pathogenesis-related protein Bet v I family and NADPH-dependent FMN reductase. We selected c61327.graph_c0, highly expressed in fruit and annotated as Pollen Ole e 1, was used as a template to obtain homologous protein structure in the RCSB PDB bank (PDB: 4z8w). We over-expressed and purified c61327.graph_c0, which could bind to human IgE by immunoblotting analysis. The epitope (74-SFRQEVKTEKHGEFKVHLPFSVSEHV-99) was speculated to confer allergic reactions. Therefore, this study provided a comprehensive systemic view of the transcriptome between mango leaf and fruit allergens endowed with biological activities, which will be useful for further genomic research studies and breeding of lower allergenic mango cultivars. Keywords: Mangifera indica Linn; transcriptome analysis; allergens; protein structure

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.

Not as easy as π: An insertional residue does not explain the π-helix gain-of-function in two-component FMN reductases.

The π-helix located at the tetramer interface of two-component FMN-dependent reductases contributes to the structural divergence from canonical FMN-bound reductases within the NADPH:FMN reductase family. The π-helix in the SsuE FMN-dependent reductase of the alkanesulfonate monooxygenase system has been proposed to be generated by the insertion of a Tyr residue in the conserved α4-helix. Variants of Tyr118 were generated, and their X-ray crystal structures determined, to evaluate how these alterations affect the structural integrity of the π-helix. The structure of the Y118A SsuE π-helix was converted to an α-helix, similar to the FMN-bound members of the NADPH:FMN reductase family. Although the π-helix was altered, the FMN binding region remained unchanged. Conversely, deletion of Tyr118 disrupted the secondary structural properties of the π-helix, generating a random coil region in the middle of helix 4. Both the Y118A and Δ118 SsuE SsuE variants crystallize as a dimer. The MsuE FMN reductase involved in the desulfonation of methanesulfonates is structurally similar to SsuE, but the π-helix contains a His insertional residue. Exchanging the π-helix insertional residue of each enzyme did not result in equivalent kinetic properties. Structure-based sequence analysis further demonstrated the presence of a similar Tyr residue in an FMN-bound reductase in the NADPH:FMN reductase family that is not sufficient to generate a π-helix. Results from the structural and functional studies of the FMN-dependent reductases suggest that the insertional residue alone is not solely responsible for generating the π-helix, and additional structural adaptions occur to provide the altered gain of function.

Functional Evaluation of the π-Helix in the NAD(P)H:FMN Reductase of the Alkanesulfonate Monooxygenase System.

A subgroup of enzymes in the NAD(P)H:FMN reductase family is comprised of flavin reductases from two-component monooxygenase systems. The diverging structural feature in these FMN reductases is a π-helix centrally located at the tetramer interface that is generated by the insertion of an amino acid in a conserved α4 helix. The Tyr insertional residue of SsuE makes specific contacts across the dimer interface that may assist in the altered mechanistic properties of this enzyme. The Y118F SsuE variant maintained the π-π stacking interactions at the tetramer interface and had kinetic parameters similar to those of wild-type SsuE. Substitution of the π-helical residue (Tyr118) to Ala or Ser transformed the enzymes into flavin-bound SsuE variants that could no longer support flavin reductase and desulfonation activities. These variants existed as dimers and could form protein-protein interactions with SsuD even though flavin transfer was not sustained. The ΔY118 SsuE variant was flavin-free as purified and did not undergo the tetramer to dimer oligomeric shift with the addition of flavin. The absence of desulfonation activity can be attributed to the inability of ΔY118 SsuE to promote flavin transfer and undergo the requisite oligomeric changes to support desulfonation. Results from these studies provide insights into the role of the SsuE π-helix in promoting flavin transfer and oligomeric changes that support protein-protein interactions with SsuD.

2-DE Compared with iTRAQ-based Proteomic Analysis of the Functional Regulation of Proteins in Rhodococcus sp. BAP-1 Response to Fluoranthene

Although the degradation pathways of Polycyclic aromatic hydrocarbons (PAHs) have been extensively studied in many bacteria, the variations in the expression levels of the key functional regulation of proteins during catabolism are still not quantitatively understood. In this study, we compared two proteomic methods, that one is two-dimensional gel electrophoresis (2-DE), a traditional widely used way and the other is isobaric tags for relative and absolute quantization (iTRAQ), an innovative approach, in order to analyze the functional regulation at the protein level in high effective fluoranthene-degrading bacteria named Rhodococcus sp. BAP-1. The number of differentially expressed proteins identified using iTRAQ is much larger than employing 2-DE. Response to fluoranthene, the key over expressed proteins in BAP-1 were NADPH-dependent FMN reductase, 30S ribosomal protein S2, S-ribosylhomocysteinase, etc.; the significant down-regulated proteins were cytochrome ubiquinol oxidase subunit, NAD(P) transhydrogenase subunit alpha, 5-methyltetrahydropteroyltriglutamate-homocysteine methyltransferase, et al.

FMNH2-dependent monooxygenases initiate catabolism of sulfonamides in Microbacterium sp. strain BR1 subsisting on sulfonamide antibiotics

We report a cluster of genes encoding two monooxygenases (SadA and SadB) and one FMN reductase (SadC) that enable Microbacterium sp. strain BR1 and other Actinomycetes to inactivate sulfonamide antibiotics. Our results show that SadA and SadC are responsible for the initial attack of sulfonamide molecules resulting in the release of 4-aminophenol. The latter is further transformed into 1,2,4-trihydroxybenzene by SadB and SadC prior to mineralization and concomitant production of biomass. As the degradation products lack antibiotic activity, the presence of SadA will result in an alleviated bacteriostatic effect of sulfonamides. In addition to the relief from antibiotic stress this bacterium gains access to an additional carbon source when this gene cluster is expressed. As degradation of sulfonamides was also observed when Microbacterium sp. strain BR1 was grown on artificial urine medium, colonization with such strains may impede common sulfonamide treatment during co-infections with pathogens of the urinary tract. This case of biodegradation exemplifies the evolving catabolic capacity of bacteria, given that sulfonamide bacteriostatic are purely of synthetic origin. The wide distribution of this cluster in Actinomycetes and the presence of traA encoding a relaxase in its vicinity suggest that this cluster is mobile and that is rather alarming.

Identification of Arsenic Resistance Genes from Marine Sediment Metagenome.

A metagenomic library of sea sediment metagenome containing 245,000 recombinant clones representing~2.45Gb of sea sediment microbial DNA was constructed. Two unique arsenic resistance clones, A7 and A12, were identified by selection on sodium arsenite containing medium. Clone A7 showed a six-fold higher resistance to arsenate [As(V)], a three-fold higher resistance to arsenite [As(III)] and significantly increased resistance to antimony [Sb(III)], while clone A12 showed increased resistance only to sodium arsenite and not to the other two metalloids. The clones harbored inserts of 8.848Kb and 6.771Kb, respectively. Both the clones possess A+T rich nucleotide sequence with similarity to sequences from marine psychrophilic bacteria. Sequence and transposon-mutagenesis based analysis revealed the presence of a putative arsenate reductase (ArsC), a putative arsenite efflux pump (ArsB/ACR) and a putative NADPH-dependent FMN reductase (ArsH) in both the clones and also a putative transcriptional regulatory protein (ArsR) in pA7. The increased resistance of clone A7 to As(V), As(III) and Sb(III) indicates functional expression of ArsC and ArsB proteins from pA7. The absence of increased As(V) resistance in clone A12 may be due tothe expression of a possible inactive ArsC, as conserved Arg60 residue in this protein was replaced by Glu60, while the absence of Sb(III) resistance may be due to the presence of an ACR3p-type arsenite pump, which is known to lack antimony transport ability.

The mitochondrial outer membrane protein mitoNEET is a redox enzyme catalyzing electron transfer from FMNH2 to oxygen or ubiquinone

Increasing evidence suggests that mitoNEET, a target of the type II diabetes drug pioglitazone, is a key regulator of energy metabolism in mitochondria. MitoNEET is anchored to the mitochondrial outer membrane via its N-terminal α helix domain and hosts a redox-active [2Fe-2S] cluster in its C-terminal cytosolic region. The mechanism by which mitoNEET regulates energy metabolism in mitochondria, however, is not fully understood. Previous studies have shown that mitoNEET specifically interacts with the reduced flavin mononucleotide (FMNH2) and that FMNH2 can quickly reduce the mitoNEET [2Fe-2S] clusters. Here we report that the reduced mitoNEET [2Fe-2S] clusters can be readily oxidized by oxygen. In the presence of FMN, NADH, and flavin reductase, which reduces FMN to FMNH2 using NADH as the electron donor, mitoNEET mediates oxidation of NADH with a concomitant reduction of oxygen. Ubiquinone-2, an analog of ubiquinone-10, can also oxidize the reduced mitoNEET [2Fe-2S] clusters under anaerobic or aerobic conditions. Compared with oxygen, ubiquinone-2 is more efficient in oxidizing the mitoNEET [2Fe-2S] clusters, suggesting that ubiquinone could be an intrinsic electron acceptor of the reduced mitoNEET [2Fe-2S] clusters in mitochondria. Pioglitazone or its analog NL-1 appears to inhibit the electron transfer activity of mitoNEET by forming a unique complex with mitoNEET and FMNH2 The results suggest that mitoNEET is a redox enzyme that may promote oxidation of NADH to facilitate enhanced glycolysis in the cytosol and that pioglitazone may regulate energy metabolism in mitochondria by inhibiting the electron transfer activity of mitoNEET.

Comparative genomics and metabolomics analyses of the adaptation mechanism in Ketogulonicigenium vulgare-Bacillus thuringiensis consortium

Adaptive evolution by serial subcultivation of co-cultured Bacillus thuringiensis and Ketogulonicigenium vulgare significantly enhanced the productivity of 2-keto-L-gulonic acid in two-step vitamin C production. The adaptation mechanism in K. vulgare-B. thuringiensis consortium was investigated in this study based on comparative genomics and metabolomics studies. It was found that the growth, anti-oxidation, transcription and regulation were significantly enhanced in the adapted consortium. The mutation of the genes, which encode amidohydrolase in adapted K. vulgare (K150) and amino acid permease in adapted B. thuringiensis (B150), resulted in the increase of some amino acids levels in each species, and further enhanced the metabolic exchange and growth ability of the two species. Besides, the mutation of the gene encoding spore germination protein enhanced the metabolic levels of tricarboxylic acid cycle, and decreased the sporulation in B150, which induced its growth. The mutation of the genes, which encode NADPH nitroreductase in K150 and NADPH-dependent FMN reductase in B150, may enhance the ability of anti-oxidation. Overall, the long-term adaptation of K. vulgare and B. thuringiensis influenced the global regulation and made them more inseparable in metabolite exchange. Our work will provide ideas for the molecular design and optimization in microbial consortium.