oxygenase domain
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2021 ◽  
Author(s):  
Molly J. McBride ◽  
Mrutyunjay A. Nair ◽  
Debangsu Sil ◽  
Jeffrey W. Slater ◽  
Monica Neugebauer ◽  
...  

ABSTRACTThe enzyme BesC from the β-ethynyl-L-serine biosynthetic pathway in Streptomyces cattleya fragments 4-chloro-L-lysine (produced from L-Lysine by BesD) to ammonia, formaldehyde, and 4-chloro-L-allylglycine and can analogously fragment L-Lys itself. BesC belongs to the emerging family of O2-activating non-heme-diiron enzymes with the "heme-oxygenase-like" protein fold (HDOs). Here we show that binding of L-Lys or an analog triggers capture of O2 by the protein’s diiron(II) cofactor to form a blue µ-peroxodiiron(III) intermediate analogous to those previously characterized in two other HDOs, the olefin-installing fatty acid decarboxylase, UndA, and the guanidino-N-oxygenase domain of SznF. The ∼ 5- and ∼ 30-fold faster decay of the intermediate in reactions with 4-thia-L-Lys and (4RS)-chloro-DL-lysine than in the reaction with L-Lys itself, and the primary deuterium kinetic isotope effects (D-KIEs) on decay of the intermediate and production of L-allylglycine in the reaction with 4,4,5,5-[2H]-L-Lys, imply that the peroxide intermediate or a successor complex with which it reversibly interconverts initiates the oxidative fragmentation by abstracting hydrogen from C4. Surprisingly, the sluggish substrate L-Lys can dissociate after triggering the intermediate to form, thereby allowing one of the better substrates to bind and react. Observed linkage between Fe(II) and substrate binding suggests that the triggering event involves a previously documented (in SznF) ordering of the dynamic HDO architecture that contributes one of the iron sites, a hypothesis consistent with the observation that the diiron(III) product cluster produced upon decay of the intermediate spontaneously degrades, as it has been shown to do in all other HDOs studied to date.


2019 ◽  
Vol 2019 (4) ◽  
Author(s):  
Timothy R. Billiar ◽  
Giuseppe Cirino ◽  
David Fulton ◽  
Roberto Motterlini ◽  
Andreas Papapetropoulos ◽  
...  

Nitric oxide synthases (NOS, E.C. 1.14.13.39) are a family of oxidoreductases that synthesize nitric oxide (NO.) via the NADPH and oxygen-dependent consumption of L-arginine with the resultant by-product, L-citrulline. There are 3 NOS isoforms and they are related by their capacity to produce NO, highly conserved organization of functional domains and significant homology at the amino acid level. NOS isoforms are functionally distinguished by the cell type where they are expressed, intracellular targeting and transcriptional and post-translation mechanisms regulating enzyme activity. The nomenclature suggested by NC-IUPHAR of NOS I, II and III [11] has not gained wide acceptance, and the 3 isoforms are more commonly referred to as neuronal NOS (nNOS), inducible NOS (iNOS) and endothelial NOS (eNOS) which reflect the location of expression (nNOS and eNOS) and inducible expression (iNOS). All are dimeric enzymes that shuttle electrons from NADPH, which binds to a C-terminal reductase domain, through the flavins FAD and FMN to the oxygenase domain of the other monomer to enable the BH4-dependent reduction of heme bound oxygen for insertion into the substrate, L-arginine. Electron flow from reductase to oxygenase domain is controlled by calmodulin binding to canonical calmodulin binding motif located between these domains. eNOS and nNOS isoforms are activated at concentrations of calcium greater than 100 nM, while iNOS shows higher affinity for Ca2+/calmodulin with great avidity and is essentially calcium-independent and constitutively active. Efficient stimulus-dependent coupling of nNOS and eNOS is achieved via subcellular targeting through respective N-terminal PDZ and fatty acid acylation domains whereas iNOS is largely cytosolic and function is independent of intracellular location. nNOS is primarily expressed in the brain and neuronal tissue, iNOS in immune cells such as macrophages and eNOS in the endothelial layer of the vasculature although exceptions in other cells have been documented. L-NAME and related modified arginine analogues are inhibitors of all three isoforms, with IC50 values in the micromolar range.


2018 ◽  
Author(s):  
John C. Salerno ◽  
Benjamin L. Hopper ◽  
Dipak. K. Ghosh ◽  
Israel M. Scott ◽  
Jonathan L. McMurry

AbstractEndothelial and neuronal nitric oxide synthases (eNOS, nNOS) are important signal generators in a number of processes including angiogenesis and neurotransmission. The homologous inducible isoform (iNOS) occupies a multitude of conformational states in a catalytic cycle, including subnanosecond input and output states and a distribution of ‘open’ conformations with average lifetimes of ~4.3 ns. In this study, fluorescence lifetime spectroscopy was used to probe conformational states of purified eNOS and nNOS in the presence of chaotropes, calmodulin, NADP+ and NADPH. Two-domain FMN/oxygenase constructs of nNOS were also examined with respect to calmodulin effects. Optical biosensing was used to analyze calmodulin binding in the presence of NADP+ and NADPH. Calmodulin binding induced a shift of the population away from the input and to the open and output states of NOS. NADP+ shifted the population towards the input state. The oxygenase domain, lacking the input state, provided a measure of calmodulin-induced open-output transitions. A mechanism for regulation by calmodulin and an elucidation of the catalytic mechanism are suggested by a ‘conformational lockdown’ model. Calmodulin speeds transitions between input and open and between open and output states, effectively reducing the conformational manifold, speeding catalysis. Conformational control of catalysis involves reorientation of the FMN binding domain, of which fluorescence lifetime is an indicator. The approach described herein is a new tool for biophysical and structural analysis of NOS enzymes, regulatory events and other homologous reductase-containing enzymes.A note to the readerThis manuscript has over the past several years been submitted to and rejected by several journals, usually on the basis of reviewer opinion that it was not an important enough result to merit inclusion in the journal. Owing to the passing of the first author and the loss of his expertise in fluorescence lifetime spectroscopy, it has become too onerous a task to continually revise the manuscript to suit the whims of reviewers who nevertheless still reject the work. We are thus simply releasing the final form of the manuscript to BioRxiv in the hopes that it finds a readership who will find, as we do, that the results are of value to the field.


ChemBioChem ◽  
2013 ◽  
Vol 14 (14) ◽  
pp. 1852-1857 ◽  
Author(s):  
Jérôme Santolini ◽  
Amandine Maréchal ◽  
Alain Boussac ◽  
Pierre Dorlet

2013 ◽  
Vol 450 (3) ◽  
pp. 607-617 ◽  
Author(s):  
Mohammad Mahfuzul Haque ◽  
Mekki Bayachou ◽  
Mohammed A. Fadlalla ◽  
Deborah Durra ◽  
Dennis J. Stuehr

The NOS (nitric oxide synthase; EC 1.14.13.39) enzymes contain a C-terminal flavoprotein domain [NOSred (reductase domain of NOS)] that binds FAD and FMN, and an N-terminal oxygenase domain that binds haem. Evidence suggests that the FMN-binding domain undergoes large conformational motions to shuttle electrons between the NADPH/FAD-binding domain [FNR (ferredoxin NADP-reductase)] and the oxygenase domain. Previously we have shown that three residues on the FMN domain (Glu762, Glu816 and Glu819) that make charge-pairing interactions with the FNR help to slow electron flux through nNOSred (neuronal NOSred). In the present study, we show that charge neutralization or reversal at each of these residues alters the setpoint [Keq(A)] of the NOSred conformational equilibrium to favour the open (FMN-deshielded) conformational state. Moreover, computer simulations of the kinetic traces of cytochrome c reduction by the mutants suggest that they have higher conformational transition rates (1.5–4-fold) and rates of interflavin electron transfer (1.5–2-fold) relative to wild-type nNOSred. We conclude that the three charge-pairing residues on the FMN domain govern electron flux through nNOSred by stabilizing its closed (FMN-shielded) conformational state and by retarding the rate of conformational switching between its open and closed conformations.


2009 ◽  
Vol 131 (20) ◽  
pp. 6940-6941 ◽  
Author(s):  
Joseph Sempombe ◽  
Bradley O. Elmore ◽  
Xi Sun ◽  
Andrea Dupont ◽  
Dipak K. Ghosh ◽  
...  

2009 ◽  
Vol 14 (3) ◽  
pp. 263-272 ◽  
Author(s):  
Philip R. Mallinder ◽  
Alan V. Wallace ◽  
Gary Allenby

Inducible nitric oxide synthase (iNOS) is active as a homodimer. A cell-based assay suitable for high-throughput screening (HTS) was generated to identify inhibitors of iNOS dimerization using the InteraX™ enzyme complementation technology of Applied Biosystems. The cells contain 2 chimeric proteins of complementing deletion mutants of β-galactosidase, each fused to the oxygenase domain of human iNOS. The assay was characterized using known iNOS dimerization inhibitors, which gave a decrease in β-galactosidase activity. Surprisingly, the assay was also able to identify compounds that have the same profile as known inhibitors of fully formed dimeric iNOS by causing an increase in β-galactosidase activity. The iNOS InteraX™ assay was used to screen ~800,000 compounds in a 384-well format. After hit confirmation, 3359 compounds were taken forward for full IC50 determination in InteraX™ and cytotoxicity assays. Of these compounds 40.5% were confirmed as greater than 10-fold more active in InteraX™ compared to a cytotoxicity assay and were classified as potential iNOS dimerization inhibitors as they did not inhibit β-galactosidase alone. In the same primary screen, 901 compounds gave a significant increase in β-galactosidase activity. Many of these were known inhibitors of iNOS. After IC50 determination in InteraX™ and cytotoxicity assays, 182 novel compounds remained as potential arginine-competitive inhibitors of dimeric iNOS. ( Journal of Biomolecular Screening 2009:263-272)


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