A meta-cleavage pathway for the aerobic degradation of aromatic hydrocarbons is catalyzed by extradiol dioxygenases via a two-step mechanism: catechol substrate binding and dioxygen incorporation. The binding of substrate triggers the release of water, thereby opening a coordination site for molecular oxygen. The crystal structures of AkbC, a type I extradiol dioxygenase, and the enzyme substrate (3-methylcatechol) complex revealed the substrate binding process of extradiol dioxygenase. AkbC is composed of an N-domain and an active C-domain, which contains iron coordinated by a 2-His-1-carboxylate facial triad motif. The C-domain includes a -hairpin structure and a C-terminal tail. In substrate-bound AkbC, 3-methylcatechol interacts with the iron via a single hydroxyl group, which represents an intermediate stage in the substrate binding process. Structure-based mutagenesis revealed that the C-terminal tail and -hairpin form part of the substrate binding pocket that is responsible for substrate specificity by blocking substrate entry. Once a substrate enters the active site, these structural elements also play a role in the correct positioning of the substrate. Based on the results presented here, a putative substrate binding mechanism is proposed.A general comparison of the major pathways for the aerobic degradation of aromatic hydrocarbons in bacteria has revealed that the initial conversion steps are carried out by different enzymes, but that the compounds are transformed into a limited number of central intermediates, including (substituted) catechols (1). These dihydroxylated intermediates are channeled into one of two possible pathways: an ortho-cleavage pathway or a meta-cleavage pathway (2). The former pathway is catalyzed by intradiol dioxygenases, which utilize a non-heme ferric iron to cleave the aromatic ring ortho to the hydroxyl substituents, while the latter pathway is catalyzed by extradiol dioxygenases, which utilize a non-heme ferrous iron or other divalent metal ion to cleave the aromatic ring meta to the hydroxyl substituents. Additionally, it is generally understood that extradiol dioxygenase is more versatile than its intradiol counterpart (3).Two decade-long research on the reaction mechanism of extradiol dioxygenases has revealed that the catalytic process occurs via a two-step mechanism: binding of catechol substrates and incorporation of the dioxygen atoms of molecular oxygen into the substrate (3-7). As depicted in Fig. 1, the iron atom in the active site is coordinated by the so-called 2-His-1-carboxylate facial triad motif (two histidines and one glutamate (or aspartate)), which occupies only one face of the coordination sphere. The opposite face is occupied by a displaceable water molecule and the hydroxyl oxygens of the catechol substrate. The binding of the substrate triggers the release of the water, thereby opening a coordination site for molecular oxygen, which is then apparently activated by an electron transferred from the substrate through the iron. Several conserved amino...
Overview:Effective sample extraction from endoscope channels is crucial for monitoring manual cleaning adequacy as well as for ensuring optimal sensitivity for culture after disinfection. The objective of this study was to compare the efficacy of Turbulent Fluid Flow (TFF) to Flush (F) or Flush-Brush-Flush (FBF) methods.Materials & Methods: Pseudomonas aeruginosa and Enterococcus faecalis in artificial test soil-2015 (ATS2015) were used as bacterial markers while protein and carbohydrate were the organic markers for biofilm formed inside 3.2-mm and 1.37-mm polytetrafluoroethylene (PTFE) channels. TFF was generated using compressed air and sterile water to provide friction for sample extraction. Extraction for biofilm coated PTFE channels as well as for colonoscope channels perfused with ATS2015 containing 10 8 CFU/mL P. aeruginosa, E. faecalis and Candida albicans was determined using TFF compared to FBF and F.
Results:The extraction ratio for P. aeruginosa and E. faecalis from biofilm extracted by TFF compared to the positive control was significantly better than F for 1.37-mm channels (≥ 0.94 for both bacteria by TFF versus 0.69 to 0.72 by F for P. aeruginosa and E. faecalis, respectively) but not significantly different between TFF and FBF for 3.2-mm channels. F was also ineffective for extraction of protein and carbohydrate from 1.37-mm channels. Extraction efficacy by TFF from inoculated colonoscope channels was >98% for all test markers.
Conclusions:The novel TFF method for extraction of samples from colonoscope channels is a more effective method than the existing FBF and F methods.
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