Para -hydroxybenzoate hydroxylase inserts oxygen into substrates by means of the labile intermediate, flavin C(4a)-hydroperoxide. This reaction requires transient isolation and substrate from bulk solvent. Previous crystal structures have revealed position para during oxygenation but not how it enters active site. In this study, enzyme with ring displaced relative to protein were determined, was established that these or similar conformations also occur in solution. Movement appears be...

Heteroaromatic sulfones react with cysteine via nucleophilic aromatic substitution, providing a mechanistically selective and irreversible scaffold for conjugation. Here we evaluate library of heteroaromatic sulfides different oxidation states, heteroatom substitutions, series electron-donating electron-withdrawing substituents. Select substitutions profoundly influence reactivity stability compared to conventional conjugation reagents, increasing the reaction rate by >3 orders magnitude....

The ubiquitous flavin-dependent monooxygenases commonly catalyze oxygenation reactions by means of a transient C4a–peroxyflavin. A recent study, however, suggested an unprecedented flavin-oxygenating species, proposed as the flavin-N5-oxide (FlN5[O]), key to oxidative Favorskii-type rearrangement in biosynthesis bacterial polyketide antibiotic enterocin. This stable superoxidized flavin is covalently tethered enzyme EncM and converted into FADH2 (Flred) during substrate turnover. Subsequent...

The role of protein residues in activating the substrate reaction catalyzed by flavoprotein p-hydroxybenzoate hydroxylase was studied. X-ray crystallography (Schreuder, H. A., Prick, P.A.J., Wieringa, R.K., Vriend, G., Wilson, K.S., Hol, W.G. J., and Drenth, J. (1989) Mol. Biol. 208, 679-696) indicates that Tyr-201 Tyr-385 form a hydrogen bond network with 4-OH p-hydroxybenzoate. Therefore, site directed mutants were constructed, converting each these tyrosines into phenylalanines. Spectral...

In this contribution, the entangled two-photon absorption (ETPA) process on naturally occurring flavoproteins was studied. Low temperature responsive protein (LOT6P) and b-type dihydroorotate dehydrogenase (DHOD B), which possess flavin mononucleotide (FMN) adenine dinucleotide (FAD) chromophores embedded in environment, were investigated. The ETPA cross-section measured, we found that it increases when going from an aqueous solution of free chromophore to protein. This enhancement is...

Glutathione reductase is an important housekeeping enzyme for redox homeostasis both in human cells and the causative agent of tropical malaria, Plasmodium falciparum. inhibitors were shown to have anticancer antimalarial activity per se contribute reversal drug resistance. The development menadione chemistry has led selection 6-[2'-(3'-methyl)-1',4'-naphthoquinolyl]hexanoic acid, called M(5), as a potent reversible uncompetitive inhibitor P. falciparum glutathione reductases. Here we...

Flavin-binding LOV domains are broadly conserved in plants, fungi, archaea, and bacteria. These approximately 100-residue photosensory modules generally encoded within larger, multidomain proteins that control a range of blue light-dependent physiologies. The bacterium Caulobacter crescentus encodes soluble LOV-histidine kinase, LovK, regulates the adhesive properties cell. Full-length LovK is dimeric as series systematically truncated constructs containing only N-terminal sensory domain....

Many flavoenzymes—oxidases and monooxygenases—react faster with oxygen than free flavins do. There are many ideas on how enzymes cause this. Recent work has focused the importance of a positive charge near N5 reduced flavin. Fructosamine oxidase lysine its We measured rate constant 1.6 × 105 M–1 s–1 for reaction oxygen. The Lys276Met mutant reacted 291 s–1, suggesting an important role this in activation. dihydroorotate dehydrogenases from E. coli L. lactis also have They react O2 constants...

Significance Vitamins are often precursors for the biosynthesis of organic enzyme cofactors, as exemplified by ubiquitous vitamin B2-derived flavins. Enzymes employ flavins, e.g., to oxygenate substrates with help covalent flavin–oxygen adducts that serve oxygenating species. However, details preceding reaction O 2 reduced flavin cofactor gives rise these species remain scarce. We have now shown how a flavoenzyme interacts and controls formation an key oxidative catalysis. This knowledge...

The flavin of p-hydroxybenzoate hydroxylase (PHBH) adopts two conformations [Gatti, D. L., Palfey, B. A., Lah, M.-S., Entsch, B., Massey, V., Ballou, P., and Ludwig, M. L. (1994) Science 266, 110−114; Schreuder, H. Mattevi, Obmolova, G., Kalk, K. H., Hol, W. G. J., van der Bolt, F. J. T., Berkel, Biochemistry 33, 10161−10170]. Kinetic studies detected the movement from buried conformation to exposed caused by binding NADPH prior its reaction with flavin. pH dependence rate constant for...

Biocatalytic reactions embody many features of ideal chemical transformations, including the potential for impeccable selectivity, high catalytic efficiency, mild reaction conditions, and use environmentally benign reagents. These advantages have created a demand biocatalysts that expand portfolio complexity-generating available to synthetic chemists. However, trade-off often exists between substrate scope biocatalyst its selectivity limits application enzymes in synthesis. We recently...

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTCatalytic function of the conserved hydroxyl group in protein tyrosine phosphatase signature motifZhong-Yin Zhang, Bruce A. Palfey, Li Wu, and Yu ZhaoCite this: Biochemistry 1995, 34, 50, 16389–16396Publication Date (Print):December 19, 1995Publication History Published online1 May 2002Published inissue 19 December 1995https://pubs.acs.org/doi/10.1021/bi00050a020https://doi.org/10.1021/bi00050a020research-articleACS PublicationsRequest reuse...

Dihydrouridine synthases (DUSs) are flavin-dependent enzymes that catalyze site-specific reduction of uracils in tRNAs. The mechanism DUS 2 from Saccharomyces cerevisiae was studied. Previously published turnover rates for this were very low. Our studies show the catalytic cycle consists reductive and oxidative half-reactions. enzyme is reduced by NADPH rapidly but has a slow half-reaction using vitro transcribed tRNA substrates. Using tRNA(Leu) purified knockout strain yeast we obtained...

Dihydroorotate dehydrogenases (DHODs) oxidize dihydroorotate (DHO) to orotate using the FMN prosthetic group abstract a hydride equivalent from C6 and protein residue (Ser for Class 2 DHODs) deprotonate C5. The fundamental question of whether scission two DHO C-H bonds is concerted or stepwise was addressed enzymes, those Escherichia coli Homo sapiens, by determining kinetic isotope effects on flavin reduction in anaerobic stopped-flow experiments. Isotope were determined E. enzyme at pH...

NAD(P)H:quinone acceptor oxidoreductases are flavoenzymes expressed in the cytoplasm of many tissues and afford protection against cytotoxic effects electrophilic quinones by catalyzing a strict two‐electron reduction. Such enzymes have been reported from several mammalian sources, e.g. human, mouse rat, plant species. Here, we report identification Lot6p (YLR011wp), first soluble quinone reductase unicellular model organism Saccharomyces cerevisiae . Localization studies using an antibody...

YhdA, a thermostable NADPH:FMN oxidoreductase from Bacillus subtilis , reduces quinones via ping‐pong bi‐bi mechanism with pronounced preference for NADPH. The enzyme occurs as stable tetramer in solution. two extended dimer surfaces are packed against each other by 90° rotation of one respect to the other. This assembly is stabilized formation four salt bridges between K109 and D137 neighbouring protomers. To investigate importance ion pair contacts, K109L D137L single replacement variants,...

By using a Raman microscope, we show that it is possible to probe the conformational states in protein crystals and crystal fragments under growth conditions (in hanging drops). The flavin cofactor enzyme para -hydroxybenzoate hydroxylase can assume two conformations: buried matrix (“in”) or essentially solvent-exposed (“out”). difference spectroscopy, previously have identified characteristic marker bands for out conformers solution phase. Now be used these crystals, permitting comparison...

The native flavin, FMN, has been removed from the l -lactate oxidase of Aerococcus viridans , and apoprotein reconstituted with 12 FMN derivatives various substituents at flavin 6- 8-positions. Impressive linear relationships are exhibited between sum Hammett σ para ortho parameters redox potentials free flavins, enzyme-bound flavins. Rapid reaction kinetics studies enzymes substrates -mandelate show an increase in reduction rate constant increasing potential, except that, lactate, a...

Subunit activity and cooperativity of a homodimeric flavoenzyme, dihydroorotate dehydrogenase A (DHODA) from Lactococcus lactis , were characterized by employing single-molecule spectroscopy to follow the turnover kinetics individual DHODA molecules, eliminating ensemble averaging. Because enzyme-bound FMN is fluorescent in its oxidized state but not when reduced, single molecule exhibits stepwise fluorescence changes during turnover, providing signal determine reaction study cooperativity....