Toluene/o-xylene monooxygenase hydroxylase (ToMOH), a diiron-containing enzyme, can activate dioxygen to oxidize aromatic substrates. To elucidate the role of a strictly conserved T201 residue during dioxygen activation of the enzyme, T201S, T201G, T201C, and T201V variants of ToMOH were prepared by site-directed mutagenesis. X-ray crystal structures of all the variants were obtained. Steady-state activity, regiospecificity, and single-turnover yields were also determined for the T201 mutants. Dioxygen activation by the reduced T201 variants was explored by stopped-flow UV-vis and Mössbauer spectroscopy. These studies demonstrate that the dioxygen activation mechanism is preserved in all T201 variants; however, both the formation and decay kinetics of a peroxodiiron(III) intermediate, T201 peroxo, were greatly altered, revealing that T201 is critically involved in dioxygen activation. A comparison of the kinetics of O 2 activation in the T201S, T201C, and T201G variants under various reaction conditions revealed that T201 plays a major role in proton transfer, which is required to generate the peroxodiiron(III) intermediate. A mechanism is postulated for dioxygen activation and possible structures of oxygenated intermediates are discussed.lippard@mit.edu. SUPPORTING INFORMATION Detailed experimental methods, a table of X-ray data collection, phase determination, and refinement statistics for the structures, a table of product distribution in toluene and o-xylene oxidation, a figure of active sites, a figure of Michaelis-Menten kinetics, time-dependent Mössbauer and UV-vis spectral changes upon the reaction with dioxygen, and the kinetics of T201 peroxo under various conditions in T201 variants. This material is available free of charge via the Internet at http://pubs.acs.org. NIH Public Access NIH-PA Author ManuscriptA role for a strictly conserved threonine residue at the active sites of bacterial multicomponent monooxygenase (BMM) hydroxylases has been discussed for more than a decade. [1][2][3][4][5] In particular, it has been conjectured that the threonine might stabilize a hydrogen-bonding network leading from the protein surface to a metal-bound, active dioxygen moiety, facilitating proton transfer required to generate and stabilize oxygenated intermediates. Such speculation for the function of the threonine residue is based in part by analogy to studies of cytochrome P450 monooxygenase (P450), where a similarly positioned threonine in the distal pocket of the heme site facilitates proton transfer to the distal oxygen atom to form an iron(III) hydroperoxo species, which then converts to an oxoiron(IV) porphyrin π-cation radical. [6][7][8][9] Replacing threonine with alanine uncouples this process, liberating hydrogen peroxide. Unlike P450, however, few studies of diiron monooxygenases have been conducted in sufficient detail to determine whether the threonine residue does play a role during O 2 activation and, if so, whether it operates in a manner similar to that of P450. The present communicatio...

No abstract

No abstract

A Fourier Transform (FT) based pattern-matching algorithm was adapted for use in medical image registration. This algorithm obtained the FT of two images, determined the normalized cross-power spectrum of the transformed images, and then applied an inverse FT. The result was a delta function with a maximum value at the location corresponding to the distance between the two images; a similar method was used to recover rotations. This algorithm was first tested using a simple two-dimensional image, with induced shifts of ±20 pixels and ±10 degrees. All translations were recovered with no error and all rotations were recovered within 0.18 degrees. Subsequently, this algorithm was tested on eight clinical kV images drawn from four different body sites. Twenty-five random shifts and rotations were applied to each image. The average mean error of the registration solution was −0.002 ± 0.077 mm in the x direction, 0.002 ± 0.075 mm in the y direction, and −0.012 ± 0.099 degrees. These initial results suggest that a FT algorithm has a high degree of accuracy when registering clinical kV images.

mean time between RP and IPP was 72 months, with the mean ages of RP and IPP being 59 and 66 years respectively. Increased time between RP and IPP were correlated with lower shim scores. Additionally, each month that passed caused a decrease of 1.5% in SHIM scores (p< 0.05), as well as a greater likely hood of developing vasculogenic ED (p[ 0.036). Increased age at IPP was correlated with lower shim scores (p[ 0.049), while RALP was found to be a predictor of higher patient satisfaction (p[ 0.02).CONCLUSIONS: Our expedited algorithm proposes that patients who fail PDE-5 inhibitors following RP be evaluated by PDUS 3 months post-operatively. Those patients found to have venoocclusive etiology should have early surgical intervention limiting patient suffering and known penile atrophy regardless of rehabilitation protocol. If etiology is arterial insufficiency or unknown, then neuropraxia should be considered and patients can be informed to wait for definitive therapy. Early intervention can be fully endorsed for veno-occlusive patients once a prospective study is designed to determine whether they worsen or improve and addresses nerve-sparing status.