Preparation of the lactate oxidase apoenzyme and studies on the binding of flavin mononucleotide to the apoenzyme.
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ABSTRACT:
SUBMITTER: Choong YS
PROVIDER: S-EPMC1165184 | biostudies-other | 1975 Jan
REPOSITORIES: biostudies-other
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Project description: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.
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Project description:The flavin mononucleotide (FMN) riboswitch is an emerging target for the development of novel RNA-targeting antibiotics. We previously discovered an FMN derivative, 5FDQD, that protects mice against diarrhea-causing Clostridium difficile bacteria. Here, we present the structure-based drug design strategy that led to the discovery of this fluoro-phenyl derivative with antibacterial properties. This approach involved the following stages: (1) structural analysis of all available free and bound FMN riboswitch structures; (2) design, synthesis, and purification of derivatives; (3) in vitro testing for productive binding using two chemical probing methods; (4) in vitro transcription termination assays; and (5) resolution of the crystal structures of the FMN riboswitch in complex with the most mature candidates. In the process, we delineated principles for productive binding to this riboswitch, thereby demonstrating the effectiveness of a coordinated structure-guided approach to designing drugs against RNA.
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Project description:Riboswitches are structural RNA elements that control gene expression. These naturally occurring RNA sensors are of continued interest as antibiotic targets, molecular sensors, and functional elements of synthetic circuits. Here, we describe affinity-based profiling of the flavin mononucleotide (FMN) riboswitch to characterize ligand binding and structural folding. We designed and synthesized photoreactive ligands and used them for photoaffinity labeling. We showed selective labeling of the FMN riboswitch and used this covalent interaction to quantitatively measure ligand binding, which we demonstrate with the naturally occurring antibiotic roseoflavin. We measured conditional riboswitch folding as a function of temperature and cation concentration. Furthermore, combining photoaffinity labeling with reverse transcription revealed ligand binding sites within the aptamer domain with single-nucleotide resolution. The photoaffinity probe was applied to cellular extracts of Bacillus subtilis to demonstrate conditional folding of the endogenous low-abundant ribD FMN riboswitch in biologically derived samples using quantitative PCR. Lastly, binding of the riboswitch-targeting antibiotic roseoflavin to the FMN riboswitch was measured in live bacteria using the photoaffinity probe.
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Project description:The versatility in design of redox flow batteries makes them apt to efficiently store energy in large-scale applications at low cost. The discovery of inexpensive organic electroactive materials for use in aqueous flow battery electrolytes is highly attractive, but is thus far limited. Here we report on a flow battery using an aqueous electrolyte based on the sodium salt of flavin mononucleotide. Flavins are highly versatile electroactive molecules, which catalyse a multitude of redox reactions in biological systems. We use nicotinamide (vitamin B3) as a hydrotropic agent to enhance the water solubility of flavin mononucleotide. A redox flow battery using flavin mononucleotide negative and ferrocyanide positive electrolytes in strong base shows stable cycling performance, with over 99% capacity retention over the course of 100 cycles. We hypothesize that this is enabled due to the oxidized and reduced forms of FMN-Na being stabilized by resonance structures.
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Project description:Two high-resolution structures of a double mutant of bacterial cholesterol oxidase in the presence or absence of a ligand, glycerol, are presented, showing the trajectory of glycerol as it binds in a Michaelis complex-like position in the active site. A group of three aromatic residues forces the oxidized isoalloxazine moiety to bend along the N5-N10 axis as a response to the binding of glycerol in the active site. Movement of these aromatic residues is only observed in the glycerol-bound structure, indicating that some tuning of the FAD redox potential is caused by the formation of the Michaelis complex during regular catalysis. This structural study suggests a possible mechanism of substrate-assisted flavin activation, improves our understanding of the interplay between the enzyme, its flavin cofactor and its substrate, and is of use to the future design of effective cholesterol oxidase inhibitors.
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Project description:The biosynthesis of several protein cofactors is subject to feedback regulation by riboswitches. Flavin mononucleotide (FMN)-specific riboswitches, also known as RFN elements, direct expression of bacterial genes involved in the biosynthesis and transport of riboflavin (vitamin B(2)) and related compounds. Here we present the crystal structures of the Fusobacterium nucleatum riboswitch bound to FMN, riboflavin and antibiotic roseoflavin. The FMN riboswitch structure, centred on an FMN-bound six-stem junction, does not fold by collinear stacking of adjacent helices, typical for folding of large RNAs. Rather, it adopts a butterfly-like scaffold, stapled together by opposingly directed but nearly identically folded peripheral domains. FMN is positioned asymmetrically within the junctional site and is specifically bound to RNA through interactions with the isoalloxazine ring chromophore and direct and Mg(2+)-mediated contacts with the phosphate moiety. Our structural data, complemented by binding and footprinting experiments, imply a largely pre-folded tertiary RNA architecture and FMN recognition mediated by conformational transitions within the junctional binding pocket. The inherent plasticity of the FMN-binding pocket and the availability of large openings make the riboswitch an attractive target for structure-based design of FMN-like antimicrobial compounds. Our studies also explain the effects of spontaneous and antibiotic-induced deregulatory mutations and provided molecular insights into FMN-based control of gene expression in normal and riboflavin-overproducing bacterial strains.
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Project description:Two arginine residues, Arg-181 and Arg-268, are conserved throughout the known family of FMN-containing enzymes that catalyze the oxidation of alpha-hydroxyacids. In the lactate oxidase from Aerococcus viridans, these residues have been changed to lysine in two single mutations and in a double mutant form. In addition, Arg-181 has been replaced by methionine to determine the effect of removing the positive charge on the residue. The effects of these replacements on the kinetic and thermodynamic properties are reported. With all mutant forms, there are only small effects on the reactivity of the reduced flavin with oxygen. On the other hand, the efficiency of reduction of the oxidized flavin by l-lactate is greatly reduced, particularly with the R268K mutant forms. The results demonstrate the importance of the two arginine residues in the binding of substrate and its interaction with the flavin, and are consistent with a previous hypothesis that they also play a role of charge neutralization in the transition state of substrate dehydrogenation. The replacement of Arg-268 by lysine also results in a slow conversion of the 8-CH(3)- substituent of FMN to yield 8-formyl-FMN, still tightly bound to the enzyme, and with significantly different physical and chemical properties from those of the FMN-enzyme.
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