Identifying and Quantitating FAD and FMN in Simple and in Iron-Sulfur-Containing Flavoproteins

Aliverti, Alessandro; Curti, Bruno; Vanoni, Maria A.

doi:10.1385/1-59259-266-x:9

Cited by 136 publications

(171 citation statements)

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“…In addition, the unknown flavin had absorption maxima at 376 and 450 nm and a spectrum nearly identical to that of a FAD standard and differed from that of FMN or riboflavin standards that both have absorption maxima at 375 and 447 nm (data not shown). The absorption maxima of FAD at 450 nm and FMN at 447 nm are consistent with previous studies and can be used to distinguish between the two molecules (34). Together these data indicate that NWMN2274 is an FAD-binding protein.…”

Section: Identification Of Nwmn2274 As a Putative Electron Donor Tosupporting

confidence: 89%

“…All samples were at 30 M in a buffer of 50 mM Tris-HCl (pH 8.0), 100 mM NaCl. To heatdenature and remove NWMN2274 from the flavin molecule, the protein solution was boiled for 10 min and centrifuged at 21,000 ϫ g for 3 min, and the supernatant was centrifuged through a Nanosep centrifugal device (PALL Life Sciences) with a molecular mass cut-off of 3 kDa as described previously (34).…”

Section: Methodsmentioning

confidence: 99%

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IruO Is a Reductase for Heme Degradation by IsdI and IsdG Proteins in Staphylococcus aureus

Loutet¹,

Kobylarz

Chau

et al. 2013

Journal of Biological Chemistry

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Section: Identification Of Nwmn2274 As a Putative Electron Donor Tosupporting

confidence: 89%

Section: Methodsmentioning

confidence: 99%

IruO Is a Reductase for Heme Degradation by IsdI and IsdG Proteins in Staphylococcus aureus

Loutet¹,

Kobylarz

Chau

et al. 2013

Journal of Biological Chemistry

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“…The flavin cofactor of FdR_A, however, remained bound to the precipitated protein. To remove the prosthetic group of FdR_A, the protein was diluted in water and denatured by incubation at 100°C for 10 min in the dark (26). The resulting flavin-containing supernatant was concentrated by evaporation and afterwards diluted with methanol to a final concentration of 10 M. Five l of the flavin solution were analyzed by LC-MS (Agilent 1100 series, Bruker HCT plus Ion Trap mass spectrometer).…”

Section: Characterization Of S Cellulosum So Ce56 Ferredoxins and Fementioning

confidence: 99%

Genome Mining in Sorangium cellulosum So ce56

Ewen

Hannemann

Khatri

et al. 2009

Journal of Biological Chemistry

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Myxobacteria, especially members of the genus Sorangium, are known for their biotechnological potential as producers of pharmaceutically valuable secondary metabolites. The biosynthesis of several of those myxobacterial compounds includes cytochrome P450 activity. Although class I cytochrome P450 enzymes occur wide-spread in bacteria and rely on ferredoxins and ferredoxin reductases as essential electron mediators, the study of these proteins is often neglected. Therefore, we decided to search in the Sorangium cellulosum So ce56 genome for putative interaction partners of cytochromes P450. In this work we report the investigation of eight myxobacterial ferredoxins and two ferredoxin reductases with respect to their activity in cytochrome P450 systems. Intriguingly, we found not only one, but two ferredoxins whose ability to sustain an endogenous So ce56 cytochrome P450 was demonstrated by CYP260A1-dependent conversion of nootkatone. Moreover, we could demonstrate that the two ferredoxins were able to receive electrons from both ferredoxin reductases. These findings indicate that S. cellulosum can alternate between different electron transport pathways to sustain cytochrome P450 activity. The cytochrome P450 (CYP)2 enzymes constitute a superfamily of external monooxygenases. The catalytic versatility of the family members explains their involvement in such diverse biological processes as biosynthesis of steroid hormones, carbon source assimilation, and metabolism of xenobiotics. In addition, cytochrome P450 enzymes have been reported to be involved in the biosynthesis of many pharmaceutically interesting secondary metabolites from a variety of microorganisms (1-4). Cytochromes P450 are usually dependent on an external electron donor. With respect to their electron transport system they can be divided into several classes, with class I (the mitochondrial/bacterial cytochrome P450 systems) being the predominant form in prokaryotes (5). In this system the electrons required for the enzymatic reaction originate from NAD(P)H and are delivered to the cytochrome P450 via a ferredoxin reductase and a ferredoxin. In a number of examples, the heterologous reconstitution of the electron transfer chain has been shown to be ineffective, if possible at all (5). Thus, it is desirable to identify the natural redox partners, especially if genomic sequence information is available. However, even then the identification of the correct interaction partners remains challenging because the encoding genes are frequently located at genomic loci distant to the cytochrome P450 genes (6, 7 To fulfill the role as electron mediator, the ferredoxin component of any given cytochrome P450 system has to be reduced. This reduction is achieved by a ferredoxin reductase, which in turn takes up electrons from NAD(P)H. The ferredoxin reductase is often the least characterized constituent of the cytochrome P450 system because these flavoproteins may be unstable (i.e. easily lose their cofactor) and usually show a relatively low level of expression (10...

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“…The flavin cofactor in PcpD was identified by denaturation of the protein and HPLC analysis of the released flavin as described by Aliverti et al (21). The cofactor was found to be flavin mononucleotide (FMN), which is consistent with the evolutionary relationship between PcpD and phthalate dioxygenase reductases, which also contain FMN.…”

mentioning

confidence: 52%

Sequestration of a highly reactive intermediate in an evolving pathway for degradation of pentachlorophenol

Yadid

Rudolph

Hlouchová

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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Microbes in contaminated environments often evolve new metabolic pathways for detoxification or degradation of pollutants. In some cases, intermediates in newly evolved pathways are more toxic than the initial compound. The initial step in the degradation of pentachlorophenol by Sphingobium chlorophenolicum generates a particularly reactive intermediate; tetrachlorobenzoquinone (TCBQ) is a potent alkylating agent that reacts with cellular thiols at a diffusion-controlled rate. TCBQ reductase (PcpD), an FMN-and NADH-dependent reductase, catalyzes the reduction of TCBQ to tetrachlorohydroquinone. In the presence of PcpD, TCBQ formed by pentachlorophenol hydroxylase (PcpB) is sequestered until it is reduced to the less toxic tetrachlorohydroquinone, protecting the bacterium from the toxic effects of TCBQ and maintaining flux through the pathway. The toxicity of TCBQ may have exerted selective pressure to maintain slow turnover of PcpB (0.02 s −1 ) so that a transient interaction between PcpB and PcpD can occur before TCBQ is released from the active site of PcpB.biodegradation | molecular evolution | channeling | quinone reductase

show abstract

Identifying and Quantitating FAD and FMN in Simple and in Iron-Sulfur-Containing Flavoproteins

Cited by 136 publications

References 14 publications

IruO Is a Reductase for Heme Degradation by IsdI and IsdG Proteins in Staphylococcus aureus

IruO Is a Reductase for Heme Degradation by IsdI and IsdG Proteins in Staphylococcus aureus

Genome Mining in Sorangium cellulosum So ce56

Sequestration of a highly reactive intermediate in an evolving pathway for degradation of pentachlorophenol

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