Steady-state and pre-steady state kinetic analysis of ornithine 4,5-aminomutase.

Ornithine 4,5-aminomutase (4,5-OAM) is a pyridoxal 5'-phosphate and adenosylcobalamin-dependent enzyme that catalyzes a 1,2-rearrangement of the terminal amine of d-ornithine to form (2R, 4S)-diaminopentanoate. The gene encoding ornithine 4,5-aminomutase is clustered with other genes that function in the oxidative l-ornithine metabolic pathway present in a number of anaerobic bacteria. This chapter discusses the methodology for measuring 4,5-OAM activity using NAD+-dependent diaminopentanoate dehydrogenase, which functions downstream of 4,5-OAM in the l-ornithine metabolic pathway. The use of ornithine racemace, which functions upstream of 4,5-OAM, for the synthesis of d,l-ornithine-3,3,4,4,5,5-d6 is also presented. Finally, this chapter describes the anaerobic stopped-flow spectrophotometric analysis of 4,5-OAM. Information is provided on the integration of a stopped-flow system in the anaerobically-maintained glove, the preparation of anaerobic solutions, and the experimental approach.

Off-Label Therapeutic Potential of Crisaborole.

Crisaborole, a topical phosphodiesterase-4 inhibitor, was recently approved in 2016 for the treatment of mild to moderate atopic dermatitis in adults and children greater than 2 years of age. Since that time, several case reports and a small randomized controlled trial have been published regarding the off-label use of crisaborole for the treatment of other inflammatory dermatologic disorders. This paper reviews the current, albeit limited, evidence for off-label use of crisaborole for psoriasis, seborrheic dermatitis, vitiligo, and inflammatory linear verrucous epidermal nevus. Additional potential therapeutic uses for crisaborole are also postulated, based on its mechanism of action. Future studies are required to elucidate the full therapeutic potential of crisaborole; however, it is a welcome addition to the current nonsteroid topical treatments for inflammatory dermatologic disease.

Oral Ketone Supplementation Acutely Increases Markers of NLRP3 Inflammasome Activation in Human Monocytes.

SCOPE: Cell culture studies indicate that the ketone ß-hydroxybutyrate (ß-OHB) directly inhibits the NLRP3 inflammasome, a key regulator of inflammation. However, direct evidence demonstrating this effect in humans is lacking. METHODS AND RESULTS: To determine the effects of acutely raising blood ß-OHB in healthy humans, two separate randomized double-blind placebo-controlled experiments are conducted using similar methods but each employed different exogenous ketone supplements. Participants' blood ß-OHB is directly elevated by ketone salts (0.3 g ß-OHB per kg; Study 1, N = 10 males) or ketone monoester (0.482 g ß-OHB per kg; Study 2, N = 18, equal males/females). Markers of NLRP3 inflammasome activation include caspase-1, IL-1ß secretion, and IL1B and NLRP3 mRNA in LPS-stimulated whole blood collected at the baseline and 30 minutes following supplementation. Caspase-1 activation increases after ketone salt (Study 1: condition × time interaction, p = 0.012) and monoester supplementation (Study 2: condition × time interaction, p = 0.016) compared to placebo. IL-1ß secretion increases (main effect of condition, p = 0.024; Study 2) while IL1B and NLRP3 mRNA remain unchanged. CONCLUSION: Measures of NLRP3 activation increases when blood ß-OHB is elevated using ketone supplements, suggesting that increasing ß-OHB exogenously may have unintended effects that augment inflammatory activation.

Optimal electrostatic interactions between substrate and protein are essential for radical chemistry in ornithine 4,5-aminomutase.

Ornithine 4,5-aminomutase (OAM) from Clostridium sticklandii is an adenosylcobalamin (AdoCbl) and pyridoxal 5'-phosphate (PLP)-dependent enzyme that catalyzes a 1,2-amino shift, interconverting d-ornithine and 2S, 4R-diaminopentanoate. The reaction occurs via a radical-based mechanism whereby a PLP-bound substrate radical undergoes intramolecular isomerization via an azacyclopropylcarbinyl radical intermediate. Herein, we investigated the catalytic role of active site residues that form non-covalent interactions with PLP and/or substrate, d-ornithine. Kinetic analyses revealed that residues that form salt bridges to the α-carboxylate (R297) or the α-amine (E81) of d-ornithine are most critical for OAM activity as conservative substitutions of these residues results in a 300-600-fold reduction in catalytic turnover and a more pronounced 1000- to 14,000-fold decrease in catalytic efficiency. In contrast, mutating residues that solely interact with the PLP cofactor led to more modest decreases (10-60-fold) in kcat and kcat/Km. All but one variant (S162A) elicited an increase in the kinetic isotope effect on kcat and kcat/Km with d,l-ornithine-3,3,4,4,5,5-d6 as the substrate, which indicates that hydrogen atom abstraction is more rate determining. Electron paramagnetic resonance spectra of the variants reveal that while the substitutions decrease the extent of CoC bond homolysis, they do not affect the structural integrity of the active site. Our experimental results, discussed in context with published computational work, suggests that the protonation state of the PLP cofactor has less of a role in radical-mediated chemistry compared to electrostatic interactions between the substrate and protein.

Does Viperin Function as a Radical S-Adenosyl-l-methionine-dependent Enzyme in Regulating Farnesylpyrophosphate Synthase Expression and Activity?

Viperin is an endoplasmic reticulum-associated antiviral responsive protein that is highly up-regulated in eukaryotic cells upon viral infection through both interferon-dependent and independent pathways. Viperin is predicted to be a radical S-adenosyl-l-methionine (SAM) enzyme, but it is unknown whether viperin actually exploits radical SAM chemistry to exert its antiviral activity. We have investigated the interaction of viperin with its most firmly established cellular target, farnesyl pyrophosphate synthase (FPPS). Numerous enveloped viruses utilize cholesterol-rich lipid rafts to bud from the host cell membrane, and it is thought that by inhibiting FPPS activity (and therefore cholesterol synthesis), viperin retards viral budding from infected cells. We demonstrate that, consistent with this hypothesis, overexpression of viperin in human embryonic kidney cells reduces the intracellular rate of accumulation of FPPS but does not inhibit or inactivate FPPS. The endoplasmic reticulum-localizing, N-terminal amphipathic helix of viperin is specifically required for viperin to reduce cellular FPPS levels. However, although viperin reductively cleaves SAM to form 5'-deoxyadenosine in a slow, uncoupled reaction characteristic of radical SAM enzymes, this cleavage reaction is independent of FPPS. Furthermore, mutation of key cysteinyl residues ligating the catalytic [Fe4S4] cluster in the radical SAM domain, surprisingly, does not abolish the inhibitory activity of viperin against FPPS; indeed, some mutations potentiate viperin activity. These observations imply that viperin does not act as a radical SAM enzyme in regulating FPPS.

Isotope effects for deuterium transfer and mutagenesis of Tyr187 provide insight into controlled radical chemistry in adenosylcobalamin-dependent ornithine 4,5-aminomutase.

Adenosylcobalamin-dependent ornithine 4,5-aminomutase (OAM) from Clostridium sticklandii utilizes pyridoxal 5'-phosphate (PLP) to interconvert d-ornithine to 2,4-diaminopentanoate via a multistep mechanism that involves two hydrogen transfer steps. Herein, we uncover features of the OAM catalytic mechanism that differentiate it from its homologue, the more catalytically promiscuous lysine 5,6-aminomutase. Kinetic isotope effects (KIEs) with dl-ornithine-3,3,4,4,5,5-d6 revealed a diminished (D)kcat/Km of 2.5 ± 0.4 relative to a (D)kcat of 7.6 ± 0.5, suggesting slow release of the substrate from the active site. In contrast, a KIE was not observed on the rate constant associated with Co-C bond homolysis as this step is likely "gated" by the formation of the external aldimine. The role of tyrosine 187, which lies planar to the PLP pyridine ring, was also investigated via site-directed mutagenesis. The 25- and 1260-fold reduced kcat values for Y187F and Y187A, respectively, are attributed to a slower rate of external aldimine formation and a diminution of adenosylcobalamin Co-C bond homolysis. Notably, electron paramagnetic resonance studies of Y187F suggest that the integrity of the active site is maintained as cob(II)alamin and the PLP organic radical (even at lower concentrations) remain tightly exchange-coupled. Modeling of d-lysine and l-ß-lysine into the 5,6-LAM active site reveals interactions between the substrate and protein are weaker than those in OAM and fewer in number. The combined data suggest that the level of protein-substrate interactions in aminomutases not only influences substrate specificity, but also controls radical chemistry.

Kinetic analysis of cytochrome P450 reductase from Artemisia annua reveals accelerated rates of NADH-dependent flavin reduction.

Cytochrome P450 reductase from Artemisia annua (aaCPR) is a diflavin enzyme that has been employed for the microbial synthesis of artemisinic acid (a semi-synthetic precursor of the anti-malarial drug, artemisinin) based on its ability to transfer electrons to the cytochrome P450 monooxygenase, CYP71AV1. We have isolated recombinant aaCPR (with the N-terminal transmembrane motif removed) from Escherichia coli and compared its kinetic and thermodynamic properties with other CPR orthologues, most notably human CPR. The FAD and FMN redox potentials and the macroscopic kinetic constants associated with cytochrome c(3+) reduction for aaCPR are comparable to that of other CPR orthologues, with the exception that the apparent binding affinity for the oxidized coenzyme is ~ 30-fold weaker compared to human CPR. CPR from A. annua shows a 3.5-fold increase in uncoupled NADPH oxidation compared to human CPR and a strong preference (85 100-fold) for NADPH over NADH. Strikingly, reduction of the enzyme by the first and second equivalent of NADPH is much faster in aaCPR, with rates of > 500 and 17 s(-1) at 6 °C. We also optically detect a charge-transfer species that rapidly forms in < 3 ms and then persists during the reductive half reaction. Additional stopped-flow kinetic studies with NADH and (R)-[4-(2) H]NADPH suggest that the accelerated rate of flavin reduction is attributed to the relatively weak binding affinity of aaCPR for NADP(+) .

Mutagenesis of a conserved glutamate reveals the contribution of electrostatic energy to adenosylcobalamin co-C bond homolysis in ornithine 4,5-aminomutase and methylmalonyl-CoA mutase.

Binding of substrate to ornithine 4,5-aminomutase (OAM) and methylmalonyl-CoA mutase (MCM) leads to the formation of an electrostatic interaction between a conserved glutamate side chain and the adenosyl ribose of the adenosylcobalamin (AdoCbl) cofactor. The contribution of this residue (Glu338 in OAM from Clostridium sticklandii and Glu392 in human MCM) to AdoCbl Co-C bond labilization and catalysis was evaluated by substituting the residue with a glutamine, aspartate, or alanine. The OAM variants, E338Q, E338D, and E338A, showed 90-, 380-, and 670-fold reductions in catalytic turnover and 20-, 60-, and 220-fold reductions in k(cat)/K(m), respectively. Likewise, the MCM variants, E392Q, E392D, and E392A, showed 16-, 330-, and 12-fold reductions in k(cat), respectively. Binding of substrate to OAM is unaffected by the single-amino acid mutation as stopped-flow absorbance spectroscopy showed that the rates of external aldimine formation in the OAM variants were similar to that of the native enzyme. The decrease in the level of catalysis is instead linked to impaired Co-C bond rupture, as UV-visible spectroscopy did not show detectable AdoCbl homolysis upon binding of the physiological substrate, d-ornithine. AdoCbl homolysis was also not detected in the MCM mutants, as it was for the native enzyme. We conclude from these results that a gradual weakening of the electrostatic energy between the protein and the ribose leads to a progressive increase in the activation energy barrier for Co-C bond homolysis, thereby pointing to a key role for the conserved polar glutamate residue in controlling the initial generation of radical species.

Role of histidine 225 in adenosylcobalamin-dependent ornithine 4,5-aminomutase.

Pyridoxal 5'-phosphate (PLP), in the active site of ornithine 4,5-aminomutase (OAM), forms a Schiff base with N(δ) of the d-ornithine side chain and facilitates interconversion of the amino acid to (2R, 4S) 2,4-diaminopentanoic acid via a radical-based mechanism. The crystal structure of OAM reveals that His225 is within hydrogen bond distance to the PLP phenolic oxygen, and may influence the pK(a) of the Schiff base during radical rearrangement. To evaluate the role of His225 in radical stabilization and catalysis, the residue was substituted with a glutamine and alanine. The H225Q and H225A variants have a 3- and 10-fold reduction in catalytic turnover, respectively, and a decrease in catalytic efficiency (7-fold for both mutants). Diminished catalytic performance is not linked to an increase in radical-based side reactions leading to enzyme inactivation. pH-dependence studies show that k(cat) increases with the ionization of a functional group, but it is not attributed to His225. Binding of 2,4-diaminobutyric acid to native OAM leads to formation of an overstabilized 2,4-diaminobutyryl-PLP derived radical. In the H225A and the H225Q mutants, the radical forms and then decays, as evidenced by accumulation of cob(III)alamin. From these data, we propose that His225 enhances radical stability by acting as a hydrogen bond acceptor to the phenolic oxygen, which favors the deprotonated state of the imino nitrogen and leads to greater resonance stabilization of the 2,4-diaminobutyryl-PLP radical intermediate. The potential role of His225 in lowering the activation energy barrier to mediate PLP-dependent radical rearrangement is discussed.