Biochar does not attenuate triclosan's impact on soil bacterial communities.

Triclosan, a broad-spectrum antimicrobial, has been widely used in pharmaceutical and personal care products. It undergoes limited degradation during wastewater treatment and is present in biosolids, most of which are land applied in the United States. This study assessed the impact of triclosan (0-100 mg kg-1) with and without biochar on soil bacterial communities. Very little 14C-triclosan was mineralized to 14CO2 (<7%) over the course of the study (42 days). While biochar (1%) significantly lowered mineralization of triclosan, analysis of 16S rRNA gene sequences revealed that biochar impacted very few OTUs and did not alter the overall structure of the community. Triclosan, on the other hand, significantly affected bacterial diversity and community structure (alpha diversity, ANOVA, p < 0.001; beta diversity, AMOVA, p < 0.01). Dirichlet multinomial mixtures (DMM) modeling and complete linkage clustering (CLC) revealed a dose-dependent impact of triclosan. Non-Parametric Metastats (NPM) analysis showed that 150 of 734 OTUs from seven main phyla were significantly impacted by triclosan (adjusted p < 0.05). Genera harboring opportunistic pathogens such as Flavobacterium were enriched in the presence of triclosan, as was Stenotrophomonas. The latter has previously been implicated in triclosan degradation via stable isotope probing. Surprisingly, Sphingomonads, which include well-characterized triclosan degraders were negatively impacted by even low doses of triclosan. Analyses of published genomes showed that triclosan resistance determinants were rare in Sphingomonads which may explain why they were negatively impacted by triclosan in our soil.

Active-site models for complexes of quinolinate synthase with substrates and intermediates.

Quinolinate synthase (QS) catalyzes the condensation of iminoaspartate and dihydroxyacetone phosphate to form quinolinate, the universal precursor for the de novo biosynthesis of nicotinamide adenine dinucleotide. QS has been difficult to characterize owing either to instability or lack of activity when it is overexpressed and purified. Here, the structure of QS from Pyrococcus furiosus has been determined at 2.8 Šresolution. The structure is a homodimer consisting of three domains per protomer. Each domain shows the same topology with a four-stranded parallel ß-sheet flanked by four α-helices, suggesting that the domains are the result of gene triplication. Biochemical studies of QS indicate that the enzyme requires a [4Fe-4S] cluster, which is lacking in this crystal structure, for full activity. The organization of domains in the protomer is distinctly different from that of a monomeric structure of QS from P. horikoshii [Sakuraba et al. (2005), J. Biol. Chem. 280, 26645-26648]. The domain arrangement in P. furiosus QS may be related to protection of cysteine side chains, which are required to chelate the [4Fe-4S] cluster, prior to cluster assembly.

Kinetics of hydrolysis and mutational analysis of N,N-diethyl-m-toluamide hydrolase from Pseudomonas putida DTB.

The initial step in the biodegradation pathway of N,N-diethyl-m-toluamide (DEET) in Pseudomonas putida strain DTB is catalyzed by DEET hydrolase (DthA), which hydrolyzes the amide bond to yield 3-methylbenzoic acid and diethylamine. In order to extend our understanding of DthA, the enzyme was purified and characterized. The enzyme is most active at pH 7.9, and is probably a tetramer in its native state. The kinetic parameters of the wild-type enzyme are K(m) = 10.2 ± 0.8 µm, k(cat) = 5.53 ± 0.09 s(-1) , and k(cat) /K(m) = (5.4 ± 0.4) × 10(5) m(-1) ·s(-1) . Mild substrate inhibition was observed with DEET concentrations over 500 µm. A homology model of DthA was used to guide mutational analysis of the active site, confirming that the catalytic triad is formed by Ser166, Ap292, and His320. The oxyanion hole is formed by the side chain OH of Tyr84 and the backbone amide of Trp167, with the Tyr84 OH being essential for enzyme activity. The DthA model also revealed a hydrophobic substrate-binding pocket comprosed of Trp167, Met170, and Trp214. W167A and M170A mutations decreased enzymatic activity and exacerbated substrate inhibition, whereas Trp214, which probably plays a role in substrate recognition, was essential for enzymatic activity. The pH rate profile of DthA was fitted to two ionizable groups (pK(a1) = 6.1 and pK(a2) = 9.9) that probably correspond to Nε of His320 and the OH of Tyr84, respectively. In addition to catalyzing the hydrolysis of DEET, DthA hydrolyzed a variety of esters and amides.

Glycal formation in crystals of uridine phosphorylase.

Uridine phosphorylase is a key enzyme in the pyrimidine salvage pathway. This enzyme catalyzes the reversible phosphorolysis of uridine to uracil and ribose 1-phosphate (or 2'-deoxyuridine to 2'-deoxyribose 1-phosphate). Here we report the structure of hexameric Escherichia coli uridine phosphorylase treated with 5-fluorouridine and sulfate and dimeric bovine uridine phosphorylase treated with 5-fluoro-2'-deoxyuridine or uridine, plus sulfate. In each case the electron density shows three separate species corresponding to the pyrimidine base, sulfate, and a ribosyl species, which can be modeled as a glycal. In the structures of the glycal complexes, the fluorouracil O2 atom is appropriately positioned to act as the base required for glycal formation via deprotonation at C2'. Crystals of bovine uridine phosphorylase treated with 2'-deoxyuridine and sulfate show intact nucleoside. NMR time course studies demonstrate that uridine phosphorylase can catalyze the hydrolysis of the fluorinated nucleosides in the absence of phosphate or sulfate, without the release of intermediates or enzyme inactivation. These results add a previously unencountered mechanistic motif to the body of information on glycal formation by enzymes catalyzing the cleavage of glycosyl bonds.