Deflavination and reconstitution of flavoproteins

Hefti, Marco H.; Vervoort, Jacques; Berkel, Willem J.H. van

doi:10.1046/j.1432-1033.2003.03802.x

Cited by 119 publications

(111 citation statements)

References 235 publications

(255 reference statements)

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“…The prosthetic FMNs in the tetrameric enzyme may hold the subunits together (Van Berkel et al, 1988). During hydrophobic interaction chromatography in the presence of high concentrations of KBr and ammonium sulfate, the yellow flavin compound was eluted with decreasing pH, indicating a non-covalent association with the protein (Hefti et al, 2003). Azoreductases from Bacillus sp.…”

Section: Discussionmentioning

confidence: 99%

Biochemical and molecular characterization of an azoreductase from Staphylococcus aureus, a tetrameric NADPH-dependent flavoprotein

2005

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Azo dyes are a predominant class of colourants used in tattooing, cosmetics, foods and consumer products. A gene encoding NADPH-flavin azoreductase (Azo1) from the skin bacterium Staphylococcus aureus ATCC 25923 was identified and overexpressed in Escherichia coli. RT-PCR results demonstrated that the azo1 gene was constitutively expressed at the mRNA level in S. aureus. Azo1 was found to be a tetramer with a native molecular mass of 85 kDa containing four non-covalently bound FMN. Azo1 requires NADPH, but not NADH, as an electron donor for its activity. The enzyme was resolved to dimeric apoprotein by removing the flavin prosthetic groups using hydrophobic-interaction chromatography. The dimeric apoprotein was reconstituted on-column and in free stage with FMN, resulting in the formation of a fully functional native-like tetrameric enzyme. The enzyme cleaved the model azo dye 2-[4-(dimethylamino)phenylazo]benzoic acid (Methyl Red) into N,N-dimethyl-p-phenylenediamine and 2-aminobenzoic acid. The apparent K m values for NADPH and Methyl Red substrates were 0?074 and 0?057 mM, respectively. The apparent V max was 0?4 mM min "1 (mg protein) "1 . Azo1was also able to metabolize Orange II, Amaranth, Ponceau BS and Ponceau S azo dyes. Azo1 represents the first azoreductase to be identified and characterized from human skin microflora.

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Section: Discussionmentioning

confidence: 99%

Biochemical and molecular characterization of an azoreductase from Staphylococcus aureus, a tetrameric NADPH-dependent flavoprotein

2005

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“…All flavo-enzymes by definition contain a covalently or non-covalently bound flavin as a functional cofactor. Typically, they contain either flavin mononucleotide (FMN) or flavin adenine dinucleotide (FAD) having an isoalloxazine ring system, which enables them to catalyze one and two electron transfer reactions (Hefti et al, 2003). Recently, a strictly anaerobic azoreductase enzyme was also characterized from Clostridium perfringens by Morrison et al (2012).…”

Section: Structure and Function Of Dye-degrading Enzymesmentioning

confidence: 99%

Bacterial diversity and composition in major fresh produce growing soils affected by physiochemical properties and geographic locations

Ibekwe

Yang

et al. 2016

Science of The Total Environment

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Azo dyes and their intermediate degradation products are common contaminants of soil and groundwater in developing countries where textile and leather dye products are produced. The toxicity of azo dyes is primarily associated with their molecular structure, substitution groups and reactivity. To avoid contamination of natural resources and to minimize risk to human health, this wastewater requires treatment in an environmentally safe manner. This manuscript critically reviews biological treatment systems and the role of bacterial reductive and oxidative enzymes/processes in the bioremediation of dye-polluted wastewaters. Many studies have shown that a variety of culturable bacteria have efficient enzymatic systems that can carry out complete mineralization of dye chemicals and their metabolites (aromatic compounds) over a wide range of environmental conditions. Complete mineralization of azo dyes generally involves a two-step process requiring initial anaerobic treatment for decolorization, followed by an oxidative process that results in degradation of the toxic intermediates that are formed during the first step. Molecular studies have revealed that the first reductive process can be carried out by two classes of enzymes involving flavin-dependent and flavin-free azoreductases under anaerobic or low oxygen conditions. The second step that is carried out by oxidative enzymes that primarily involves broad specificity peroxidases, laccases and tyrosinases. This review focuses, in particular, on the characterization of these enzymes with respect to their enzyme kinetics and the environmental conditions that are necessary for bioreactor systems to treat azo dyes contained in wastewater.

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“…4 Hence, inhibition of MAO B is one of the strategies for the treatment of the latter illness. 10 Most MAO B inhibitors that are in clinical use nowadays are irreversible. 10,11 Scheme 1.…”

Section: Introductionmentioning

confidence: 99%

“…10 Most MAO B inhibitors that are in clinical use nowadays are irreversible. 10,11 Scheme 1. Atom numbering of the flavin moiety, without which MAO enzymes are catalytically inactive.…”

Section: Introductionmentioning

confidence: 99%

Computational Study of the pK_aValues of Potential Catalytic Residues in the Active Site of Monoamine Oxidase B

Borštnar

Repič

Kamerlin

et al. 2012

J. Chem. Theory Comput.

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Monoamine oxidase (MAO), which exists in two isozymic forms, MAO A and MAO B, is an important flavoenzyme responsible for the metabolism of amine neurotransmitters such as dopamine, serotonin and norepinephrine. Despite extensive research effort, neither the catalytic nor the inhibition mechanisms of MAO have been completely understood. There has also been dispute with regard to the protonation state of the substrate upon entering the active site, as well as the identity of residues that are important for the initial deprotonation of irreversible acetylenic inhibitors, in accordance with the recently proposed mechanism. Therefore, in order to investigate features essential for the modes of action of MAO, we have calculated pK a values of three relevant tyrosine residues in the MAO B active site, with and without dopamine bound as the substrate (as well as the pK a of the dopamine itself in the active site). The calculated pK a values for Tyr188, Tyr398 and Tyr435 in the complex are found to be shifted upwards to 13.0, 13.7 and 14.7, respectively, relative to 10.1 in aqueous solution, ruling out the likelihood that they are viable proton acceptors. The altered tyrosine pK a values could be rationalized as an interplay of two opposing effects: insertion of positively charged bulky dopamine that lowers tyrosine pK a values, and subsequent removal of water molecules from the active site that elevates tyrosine pK a values, in which the latter prevails. Additionally, the pK a value of the bound dopamine (8.8) is practically unchanged compared to the corresponding value in aqueous solution (8.9), as would be expected from a charged amine placed in a hydrophobic active site consisting of aromatic moieties. We also observed potentially favorable cation-π interactions between -NH 3 + group on dopamine and aromatic moieties, which provide stabilizing effect to the charged fragment. Thus, we offer here theoretical evidence that the amine is most likely to be present in the active site in its protonated form, which is similar to the conclusion from experimental studies of MAO A (Jones et al. J. Neural Trans. 2007, 114, 707-712). However, the free energy cost of 3 transferring the proton from the substrate to the bulk solvent is only 1.9 kcal mol -1 , leaving open the possibility that the amine enters the chemical step in its neutral form. In conjunction with additional experimental and computational work, the data presented here should lead towards a deeper understanding of mechanisms of the catalytic activity and irreversible inhibition of MAO B, which can allow for the design of novel and improved MAO B inhibitors.

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Deflavination and reconstitution of flavoproteins

Cited by 119 publications

References 235 publications

Biochemical and molecular characterization of an azoreductase from Staphylococcus aureus, a tetrameric NADPH-dependent flavoprotein

Biochemical and molecular characterization of an azoreductase from Staphylococcus aureus, a tetrameric NADPH-dependent flavoprotein

Bacterial diversity and composition in major fresh produce growing soils affected by physiochemical properties and geographic locations

Computational Study of the pK_aValues of Potential Catalytic Residues in the Active Site of Monoamine Oxidase B

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Deflavination and reconstitution of flavoproteins

Cited by 119 publications

References 235 publications

Biochemical and molecular characterization of an azoreductase from Staphylococcus aureus, a tetrameric NADPH-dependent flavoprotein

Biochemical and molecular characterization of an azoreductase from Staphylococcus aureus, a tetrameric NADPH-dependent flavoprotein

Bacterial diversity and composition in major fresh produce growing soils affected by physiochemical properties and geographic locations

Computational Study of the pKaValues of Potential Catalytic Residues in the Active Site of Monoamine Oxidase B

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Computational Study of the pK_aValues of Potential Catalytic Residues in the Active Site of Monoamine Oxidase B