Taurine/alpha-ketoglutarate dioxygenase (TauD), a non-heme mononuclear Fe(II) oxygenase, liberates sulfite from taurine in a reaction that requires the oxidative decarboxylation of alpha-ketoglutarate (alphaKG). The lilac-colored alphaKG-Fe(II)TauD complex (lambda(max) = 530 nm; epsilon(530) = 140 M(-)(1) x cm(-)(1)) reacts with O(2) in the absence of added taurine to generate a transient yellow species (lambda(max) = 408 nm, minimum of 1,600 M(-)(1) x cm(-)(1)), with apparent first-order rate constants for formation and decay of approximately 0.25 s(-)(1) and approximately 0.5 min(-)(1), that transforms to yield a greenish brown chromophore (lambda(max) = 550 nm, 700 M(-)(1) x cm(-)(1)). The latter feature exhibits resonance Raman vibrations consistent with an Fe(III) catecholate species presumed to arise from enzymatic self-hydroxylation of a tyrosine residue. Significantly, (18)O labeling studies reveal that the added oxygen atom derives from solvent rather than from O(2). The transient yellow species, identified as a tyrosyl radical on the basis of EPR studies, is formed after alphaKG decomposition. Substitution of two active site tyrosine residues (Tyr73 and Tyr256) by site-directed mutagenesis identified Tyr73 as the likely site of formation of both the tyrosyl radical and the catechol-associated chromophore. The involvement of the tyrosyl radical in catalysis is excluded on the basis of the observed activity of the enzyme variants. We suggest that the Fe(IV) oxo species generally proposed (but not yet observed) as an intermediate for this family of enzymes reacts with Tyr73 when substrate is absent to generate Fe(III) hydroxide (capable of exchanging with solvent) and the tyrosyl radical, with the latter species participating in a multistep TauD self-hydroxylation reaction.

Isolation of DNA from blood and buccal swabs in adequate quantities is an integral part of forensic research and analysis. The present study was performed to determine the quality and the quantity of DNA extracted from four commonly available samples and to estimate the time duration of the ensuing PCR amplification. Here, we demonstrate that hair and urine samples can also become an alternate source for reliably obtaining a small quantity of PCR-ready DNA. We developed a rapid, cost-effective, and noninvasive method of sample collection and simple DNA extraction from buccal swabs, urine, and hair using the phenol-chloroform method. Buccal samples were subjected to DNA extraction, immediately or after refrigeration (4 -6°C) for 3 days. The purity and the concentration of the extracted DNA were determined spectrophotometerically, and the adequacy of DNA extracts for the PCR-based assay was assessed by amplifying a 1030-bp region of the mitochondrial D-loop. Although DNA from all the samples was suitable for PCR, the blood and hair samples provided a good quality DNA for restriction analysis of the PCR product compared with the buccal swab and urine samples. In the present study, hair samples proved to be a good source of genomic DNA for PCR-based methods. Hence, DNA of hair samples can also be used for the genomic disorder analysis in addition to the forensic analysis as a result of the ease of sample collection in a noninvasive manner, lower sample volume requirements, and good storage capability.

Recent reports indicate that manganese (Mn), applied as a foliar fertilizer in tank mixtures with glyphosate, has the potential to antagonize glyphosate efficacy and reduce weed control. It was hypothesized that Mn2+ complexed with glyphosate in a similar manner to Ca2+, forming salts that were not readily absorbed and, thereby, reducing glyphosate efficacy. This study was conducted to confirm the interaction of Mn2+ and glyphosate and to measure the effect of Mn on glyphosate absorption and translocation in velvetleaf. In aqueous solutions, Mn2+ binds with solvent molecules and with chelating agents to form hexacoordinate complexes. The distribution of paramagnetic species, both the free manganous ion ([Mn{H2O}6]2+) and the Mn2+–glyphosate complex, in Mn–glyphosate solutions at various pH values were analyzed using electron paramagnetic resonance (EPR) spectroscopy. Glyphosate interaction with Mn appeared to increase as the pH was increased from spray solution levels (2.8 to 4.5) to levels common in the plant symplast (7.5). Growth chamber bioassays were conducted to measure absorption and translocation of 14C-labeled glyphosate in solution with four Mn fertilizers: Mn-ethylaminoacetate (Mn-EAA), Mn-ethylenediaminetetraacetate (Mn-EDTA), Mn-lignin sulfonate (Mn-LS), and Mn-sulfate (MnSO4). Mn-EDTA did not interfere with glyphosate efficacy, absorption, or translocation. However, both MnSO4 and Mn-LS reduced glyphosate efficacy, absorption, and translocation. Mn-EAA severely antagonized glyphosate efficacy, and although glyphosate in tank mixtures with Mn-EAA was absorbed rapidly, little was translocated from the treated leaf. The Mn-EAA fertilizer contained approximately 0.5% iron (Fe) not reported on the fertilizer label. Iron is presumed to be partially responsible for the very limited translocation of glyphosate from the treated leaf in Mn-EAA tank mixtures. Adding ammonium sulfate increased the efficacy, absorption, and translocation of glyphosate for each Mn fertilizer tank mixture.

The structural relationship between substrate taurine and the non-heme Fe(II) center of taurine/alpha-ketoglutarate (alphaKG) dioxygenase (TauD) was measured using electron spin echo envelope modulation (ESEEM) spectroscopy. Studies were conducted on TauD samples treated with NO, cosubstrate alphaKG, and either protonated or specifically deuterated taurine. Stimulated echo ESEEM data were divided to eliminate interference from 1H and 14N modulations and accentuate modulations from 2H. For taurine that was deuterated at the C1 position (adjacent to the sulfonate group), 2H ESEEM spectra show features that arise from dipole-dipole and deuterium nuclear quadrupole interactions from a single deuteron. Parallel measurements taken for taurine deuterated at both C1 and C2 show an additional ESEEM feature at the deuterium Larmor frequency. Analysis of these data at field positions ranging from g = 4 to g = 2 have allowed us to define the orientation of substrate taurine with respect to the magnetic axes of the Fe(II)-NO, S = 3/2, paramagnetic center. These results are discussed in terms of previous X-ray crystallographic studies and the proposed catalytic mechanism for this family of enzymes.