Cysteine dioxygenase (CDO) is a non-heme iron enzyme that catalyzes the O2-dependent oxidation of l-cysteine to produce cysteinesulfinic acid. Bacterial CDOs have been subdivided as either “Arg-type” or “Gln-type” on the basis of the identity of conserved active site residues. To date, “Gln-type” enzymes remain largely uncharacterized. It was recently noted that the “Gln-type” enzymes are more homologous with another thiol dioxygenase [3-mercaptopropionate dioxygenase (MDO)] identified in Variovorax paradoxus, suggesting that enzymes of the “Gln-type” subclass are in fact MDOs. In this work, a putative “Gln-type” thiol dioxygenase from Azotobacter vinelandii (Av) was purified to homogeneity and characterized. Steady-state assays were performed using three substrates [3-mercaptopropionic acid (3mpa), l-cysteine (cys), and cysteamine (ca)]. Despite comparable maximal velocities, the “Gln-type” Av enzyme exhibited a specificity for 3mpa (kcat/KM = 72000 M−1 s−1) nearly 2 orders of magnitude greater than those for cys (110 M−1 s−1) and ca (11 M−1 s−1). Supporting X-band electron paramagnetic resonance (EPR) studies were performed using nitric oxide (NO) as a surrogate for O2 binding to confirm obligate-ordered addition of substrate prior to NO. Stoichimetric addition of NO to solutions of 3mpa-bound enzyme quantitatively yields an iron-nitrosyl species (Av ES-NO) with EPR features consistent with a mononuclear false(S=true32false) {FeNO}7 site. Conversely, two distinct substrate-bound conformations were observed in Av ES-NO samples prepared with cys and ca, suggesting heterogeneous binding within the enzymatic active site. Analytical EPR simulations are provided to establish the relative binding affinity for each substrate (3map > cys > ca). Both kinetic and spectroscopic results presented here are consistent with 3mpa being the preferred substrate for this enzyme.

The vast amount of research over the past decades has significantly added to our knowledge of phantom limb pain. Multiple factors including site of amputation or presence of preamputation pain have been found to have a positive correlation with the development of phantom limb pain. The paradigms of proposed mechanisms have shifted over the past years from the psychogenic theory to peripheral and central neural changes involving cortical reorganization. More recently, the role of mirror neurons in the brain has been proposed in the generation of phantom pain. A wide variety of treatment approaches have been employed, but mechanism-based specific treatment guidelines are yet to evolve. Phantom limb pain is considered a neuropathic pain, and most treatment recommendations are based on recommendations for neuropathic pain syndromes. Mirror therapy, a relatively recently proposed therapy for phantom limb pain, has mixed results in randomized controlled trials. Most successful treatment outcomes include multidisciplinary measures. This paper attempts to review and summarize recent research relative to the proposed mechanisms of and treatments for phantom limb pain.

Post-transcriptional modifications of tRNA are made to structurally diversify tRNA. These modifications alter noncovalent interactions within the ribosomal machinery, resulting in phenotypic changes related to cell metabolism, growth, and virulence. MiaE is a carboxylate bridged, nonheme diiron monooxygenase, which catalyzes the O2-dependent hydroxylation of a hypermodified-tRNA nucleoside at position 37 (2-methylthio-N(6)-isopentenyl-adenosine(37)-tRNA) [designated ms(2)i(6)A37]. In this work, recombinant MiaE was cloned from Salmonella typhimurium , purified to homogeneity, and characterized by UV-visible and dual-mode X-band EPR spectroscopy for comparison to other nonheme diiron enzymes. Additionally, three nucleoside substrate-surrogates (i(6)A, Cl(2)i(6)A, and ms(2)i(6)A) and their corresponding hydroxylated products (io(6)A, Cl(2)io(6)A, and ms(2)io(6)A) were synthesized to investigate the chemo- and stereospecificity of this enzyme. In the absence of the native electron transport chain, the peroxide-shunt was utilized to monitor the rate of substrate hydroxylation. Remarkably, regardless of the substrate (i(6)A, Cl(2)i(6)A, and ms(2)i(6)A) used in peroxide-shunt assays, hydroxylation of the terminal isopentenyl-C4-position was observed with >97% E-stereoselectivity. No other nonspecific hydroxylation products were observed in enzymatic assays. Steady-state kinetic experiments also demonstrate that the initial rate of MiaE hydroxylation is highly influenced by the substituent at the C2-position of the nucleoside base (v0/[E] for ms(2)i(6)A > i(6)A > Cl(2)i(6)A). Indeed, the >3-fold rate enhancement exhibited by MiaE for the hydroxylation of the free ms(2)i(6)A nucleoside relative to i(6)A is consistent with previous whole cell assays reporting the ms(2)io(6)A and io(6)A product distribution within native tRNA-substrates. This observation suggests that the nucleoside C2-substituent is a key point of interaction regulating MiaE substrate specificity.

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The pterin-dependent aromatic amino acid hydroxylases are non-heme iron enzymes that catalyze the hydroxylation of the aromatic side chain of their respective substrates using an FeO intermediate. While the eukaryotic enzymes are homotetramers with complex regulatory properties, bacterial phenylalanine hydroxylases are monomers that lack regulatory domains. As a result, the bacterial enzymes are more tractable for mechanistic studies. Using single turnover methods, the complete kinetic mechanism and intrinsic rate constants for Chromobacterium violaceum phenylalanine hydroxylase have been determined with both tetrahydrobiopterin and 6-methyltetrahyropterin as substrates. In addition the kinetics of formation of the enzyme-pterin complex have been determined with the unreactive 5-deaza, 6-methyltetrahydropterin. For all three pterins, binding of phenylalanine and pterin occurs in random order with binding of the pterin first the preferred pathway. The reaction of the ternary enzyme-phenylalanine-tetrahydropterin complex can be described by a mechanism involving reversible oxygen binding, formation of an early intermediate preceding formation of the FeO, and rate-limiting product release.