Natural Flavins:Occurrence, Role, and Noncanonical Chemistry

Flavoproteins are of key importance to all life on earth for both primary and secondary metabolism. Most flavin-dependent enzymes utilize flavin mononucleotide (FMN) or flavin adenine dinucleotide (FAD) as redox cofactor for single-electron and hydride transfer as well as oxidation and oxygenation chemistry at the C4a-locus. Over the last decades, several naturally occurring modified flavins, like 8-formylFAD, F420, and prenylFMN, and covalently bound flavins have been discovered, and were found to further expand the toolbox of flavin chemistry, showcasing extraordinary redox potentials and unprecedented chemistry. Recently, also several examples of “exotic” flavin chemistry, such as N5-oxygenation, have been identified in enzymes that utilize the standard flavins FMN and FAD. It shows that nature has been extremely inventive in exploiting flavins and flavin derivatives as cofactors for an exceptionally wide variety of reactions. Future research will reveal whether other, so far hidden, flavoenzyme-catalyzed chemistries exit.</p

Flavin-Tag:A Facile Method for Site-Specific Labeling of Proteins with a Flavin Fluorophore

Site-specific protein labeling methods are highly valuable tools for research and applications. We present a new protein labeling method that allows covalent attachment of a chromo-and fluorogenic flavin (FMN) to any targeted protein using a short flavinylation peptide-Tag. We show that this peptide can be as short as 7 residues and can be located at the N-Terminus, C-Terminus, or in internal regions of the target protein. Analogous to kinase-catalyzed phosphorylation, the flavin is covalently attached via a stable phosphothreonyl linkage. The site-specific covalent tethering of FMN is accomplished by using a bacterial flavin transferase. The covalent coupling of FMN was shown to work in Escherichia coli and Saccharomyces cerevisiae cells and could be performed in vitro, rendering the "Flavin-Tag"method a powerful tool for the selective decoration of proteins with a biocompatible redox-Active fluorescent chromophore

A Tailor-Made Deazaflavin-Mediated Recycling System for Artificial Nicotinamide Cofactor Biomimetics

[Image: see text] Nicotinamide adenine dinucleotide (NAD) and its 2′-phosphorylated form NADP are crucial cofactors for a large array of biocatalytically important redox enzymes. Their high cost and relatively poor stability, however, make them less attractive electron mediators for industrial processes. Nicotinamide cofactor biomimetics (NCBs) are easily synthesized, are inexpensive, and are also generally more stable than their natural counterparts. A bottleneck for the application of these artificial hydride carriers is the lack of efficient cofactor recycling methods. Therefore, we engineered the thermostable F(420):NADPH oxidoreductase from Thermobifida fusca (Tfu-FNO), by structure-inspired site-directed mutagenesis, to accommodate the unnatural N1 substituents of eight NCBs. The extraordinarily low redox potential of the natural cofactor F(420)H(2) was then exploited to reduce these NCBs. Wild-type enzyme had detectable activity toward all selected NCBs, with K(m) values in the millimolar range and k(cat) values ranging from 0.09 to 1.4 min(–1). Saturation mutagenesis at positions Gly-29 and Pro-89 resulted in mutants with up to 139 times higher catalytic efficiencies. Mutant G29W showed a k(cat) value of 4.2 s(–1) toward 1-benzyl-3-acetylpyridine (BAP(+)), which is similar to the k(cat) value for the natural substrate NADP(+). The best Tfu-FNO variants for a specific NCB were then used for the recycling of catalytic amounts of these nicotinamides in conversion experiments with the thermostable ene-reductase from Thermus scotoductus (TsOYE). We were able to fully convert 10 mM ketoisophorone with BAP(+) within 16 h, using F(420) or its artificial biomimetic FOP (FO-2′-phosphate) as an efficient electron mediator and glucose-6-phosphate as an electron donor. The generated toolbox of thermostable and NCB-dependent Tfu-FNO variants offers powerful cofactor regeneration biocatalysts for the reduction of several artificial nicotinamide biomimetics at both ambient and high temperatures. In fact, to our knowledge, this enzymatic method seems to be the best-performing NCB-recycling system for BNAH and BAPH thus far

Introducing an Artificial Deazaflavin Cofactor in Escherichia coli and Saccharomyces cerevisiae

[Image: see text] Deazaflavin-dependent whole-cell conversions in well-studied and industrially relevant microorganisms such as Escherichia coli and Saccharomyces cerevisiae have high potential for the biocatalytic production of valuable compounds. The artificial deazaflavin FOP (FO-5′-phosphate) can functionally substitute the natural deazaflavin F(420) and can be synthesized in fewer steps, offering a solution to the limited availability of the latter due to its complex (bio)synthesis. Herein we set out to produce FOP in vivo as a scalable FOP production method and as a means for FOP-mediated whole-cell conversions. Heterologous expression of the riboflavin kinase from Schizosaccharomyces pombe enabled in vivo phosphorylation of FO, which was supplied by either organic synthesis ex vivo, or by a coexpressed FO synthase in vivo, producing FOP in E. coli as well as in S. cerevisiae. Through combined approaches of enzyme engineering as well as optimization of expression systems and growth media, we further improved the in vivo FOP production in both organisms. The improved FOP production yield in E. coli is comparable to the F(420) yield of native F(420)-producing organisms such as Mycobacterium smegmatis, but the former can be achieved in a significantly shorter time frame. Our E. coli expression system has an estimated production rate of 0.078 μmol L(–1) h(–1) and results in an intracellular FOP concentration of about 40 μM, which is high enough to support catalysis. In fact, we demonstrate the successful FOP-mediated whole-cell conversion of ketoisophorone using E. coli cells. In S. cerevisiae, in vivo FOP production by SpRFK using supplied FO was improved through media optimization and enzyme engineering. Through structure-guided enzyme engineering, a SpRFK variant with 7-fold increased catalytic efficiency compared to the wild type was discovered. By using this variant in optimized media conditions, FOP production yield in S. cerevisiae was 20-fold increased compared to the very low initial yield of 0.24 ± 0.04 nmol per g dry biomass. The results show that bacterial and eukaryotic hosts can be engineered to produce the functional deazaflavin cofactor mimic FOP

Elevated serum level and altered glycosylation of _1-acid glycoprotein in hyperimmunoglobulinemia D and periodic fever syndrome: evidence for persistent inflammation

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Конференции

Background Polypharmacy poses threats to patients’ health. The Systematic Tool to Reduce Inappropriate Prescribing (STRIP) is a drug optimization process for conducting medication reviews in primary care. To effectively and efficiently incorporate this method into daily practice, the STRIP Assistant—a decision support system that aims to assist physicians with the pharmacotherapeutic analysis of patients’ medical records—has been developed. It generates context-specific advice based on clinical guidelines. Objective The aim of this study was to validate the STRIP Assistant’s usability as a tool for physicians to optimize medical records for polypharmacy patients. Methods In an online experiment, 42 physicians were asked to optimize medical records for two comparable polypharmacy patients, one in their usual manner and one using the STRIP Assistant. Changes in effectiveness were measured by comparing respondents’ optimized medicine prescriptions with medication prepared by an expert panel of two geriatrician-pharmacologists. Efficiency was operationalized by recording the time the respondents took to optimize the two cases. User satisfaction was measured with the System Usability Scale (SUS). Independent and paired t tests were used for analysis. Results Medication optimization significantly improved with the STRIP Assistant. Appropriate decisions increased from 58 % without the STRIP Assistant to 76 % with it (p < 0.0001). Inappropriate decisions decreased from 42 % without the STRIP Assistant to 24 % with it (p < 0.0001). Participants spent significantly more time optimizing medication with the STRIP Assistant (24 min) than without it (13 min; p < 0.0001). They assigned it a below-average SUS score of 63.25. Conclusion The STRIP Assistant improves the effectiveness of medication reviews for polypharmacy patients

Photophysics of the electronic states S0 and S1 for the coplanar molecular structures of the α,ω-diphenylpolyenes DPH and DPO

Spectroscopy of the monoclinic and orthorhombic crystalline forms of all-trans-diphenylhexatriene (DPH) and all-trans-diphenyloctatetraene (DPO) show absorption and emission bands that do not generate the widely known Stokes shift of the polyene compounds, discovered by Hausser et al. in 1953 and repeatedly studied over the last 60 years. It can be concluded from our study that the crystallization system, whether in a monoclinic or orthorhombic system, does not significantly influence the photophysics of DPH and DPO in the crystal phas