2922-83-0Relevant articles and documents
Structures of bacterial kynurenine formamidase reveal a crowded binuclear zinc catalytic site primed to generate a potent nucleophile
Diaz-Saez, Laura,Srikannathasan, Velupillai,Zoltner, Martin,Hunter, William N.
, p. 581 - 589 (2014)
Tryptophan is an important precursor for chemical entities that ultimately support the biosynthesis of key metabolites. The second stage of tryptophan catabolism is catalysed by kynurenine formamidase, an enzyme that is different between eukaryotes and prokaryotes. In the present study, we characterize the catalytic properties and present the crystal structures of three bacterial kynurenine formamidases. The structures reveal a new amidase protein fold, a highly organized and distinctive binuclear Zn2+ catalytic centre in a confined, hydrophobic and relatively rigid active site. The structure of a complex with 2-aminoacetophenone delineates aspects of molecular recognition extending to the observation that the substrate itself may be conformationally restricted to assist binding in the confined space of the active site and for subsequent processing. The cations occupy a crowded environment, and, unlike most Zn2+ -dependent enzymes, there is little scope to increase co-ordination number during catalysis.We propose that the presence of a bridging water/hydroxide ligand in conjunction with the placement of an active site histidine supports a distinctive amidation mechanism.
In vitro modulation of cytochrome p450 reductase supported indoleamine 2,3-dioxygenase activity by allosteric effectors cytochrome b5 and methylene blue
Pearson, Josh T.,Siu, Sophia,Meininger, David P.,Wienkers, Larry C.,Rock, Dan A.
, p. 2647 - 2656 (2010)
Indoleamine 2,3-dioxygenase (IDO) is a heme-containing dioxygenase involved in the degradation of several indoleamine derivatives and has been indicated as an immunosuppressive. IDO is an attractive target for therapeutic intervention in diseases which are known to capitalize on immune suppression, including cancer, HIV, and inflammatory diseases. Conventionally, IDO activity is measured through chemical reduction by the addition of ascorbate and methylene blue. Identification of potential coenzymes involved in the reduction of IDO in vivo should improve in vitro reconstitution systems used to identify potential IDO inhibitors. In this study we show that NADPH-cytochrome P450 reductase (CPR) is capable of supporting IDO activity in vitro and that oxidation of L-Trp follows substrate inhibition kinetics (kcat = 0.89 ± 0.04 s -1 , Km = 0.72 ±0.15 μM, and Ki = 9.4 ± 2.0 μM). Addition of cytochrome b5 to CPR-supported L-Trp incubations results in modulation from substrate inhibition to sigmoidal kinetics (kcat = 1.7 ± 0.3 s-1, Km = 1.5 ± 0.9 μM, and Ki = 1.9 ± 0.3). CPR-supported D-Trp oxidations (±cytochrome b5) exhibit Michaelis-Menten kinetics. Addition of methylene blue (minus ascorbate) to CPR-supported reactions resulted in inhibition of DTrp turnover and modulation of L-Trp kinetics from allosteric to Michaelis-Menten with a concurrent decrease in substrate affinity for IDO. Our data indicate that CPR is capable of supporting IDO activity in vitro and oxidation of tryptophan by IDO displays substrate stereochemistry dependent atypical kinetics which can be modulated by the addition of cytochrome b5.
Evaluation of the Edman degradation product of vancomycin bonded to core-shell particles as a new HPLC chiral stationary phase
Hellinghausen, Garrett,Lopez, Diego A.,Lee, Jauh T.,Wang, Yadi,Weatherly, Choyce A.,Portillo, Abiud E.,Berthod, Alain,Armstrong, Daniel W.
, p. 1067 - 1078 (2018/08/01)
A modified macrocyclic glycopeptide-based chiral stationary phase (CSP), prepared via Edman degradation of vancomycin, was evaluated as a chiral selector for the first time. Its applicability was compared with other macrocyclic glycopeptide-based CSPs: TeicoShell and VancoShell. In addition, another modified macrocyclic glycopeptide-based CSP, NicoShell, was further examined. Initial evaluation was focused on the complementary behavior with these glycopeptides. A screening procedure was used based on previous work for the enantiomeric separation of 50 chiral compounds including amino acids, pesticides, stimulants, and a variety of pharmaceuticals. Fast and efficient chiral separations resulted by using superficially porous (core-shell) particle supports. Overall, the vancomycin Edman degradation product (EDP) resembled TeicoShell with high enantioselectivity for acidic compounds in the polar ionic mode. The simultaneous enantiomeric separation of 5 racemic profens using liquid chromatography-mass spectrometry with EDP was performed in approximately 3?minutes. Other highlights include simultaneous liquid chromatography separations of rac-amphetamine and rac-methamphetamine with VancoShell, rac-pseudoephedrine and rac-ephedrine with NicoShell, and rac-dichlorprop and rac-haloxyfop with TeicoShell.
Synthesis of peptides containing 5-hydroxytryptophan, oxindolylalanine, N-formylkynurenine and kynurenine
Todorovski, Toni,Fedorova, Maria,Hennig, Lothar,Hoffmann, Ralf
scheme or table, p. 256 - 262 (2012/01/13)
ROS, continuously produced in cells, can reversibly or irreversibly oxidize proteins, lipids, and DNA. At the protein level, cysteine, methionine, tryptophan, and tyrosine residues are particularly prone to oxidation. Here, we describe the solid phase synthesis of peptides containing four different oxidation products of tryptophan residues that can be formed by oxidation in proteins in vitro and in vivo: 5-HTP, Oia, Kyn, and NFK. First, we synthesized Oia and NFK by selective oxidation of tryptophan and then protected the ${bf alpha}$-amino group of both amino acids, and the commercially available 5-HTP, with Fmoc-succinimide. High yields of Fmoc-Kyn were obtained by acid hydrolysis of Fmoc-NFK. All four Fmoc derivatives were successfully incorporated, at high yields, into three different peptide sequences from skeletal muscle actin, creatin kinase (M-type), and ${bf beta}$-enolase. The correct structure of all modified peptides was confirmed by tandem mass spectrometry. Interestingly, isobaric peptides containing 5-HTP and Oia were always well separated in an acetonitrile gradient with TFA as the ion-pair reagent on a C18-phase. Such synthetic peptides should prove useful in future studies to distinguish isobaric oxidation products of tryptophan.
