Crystal structures of Burkholderia cenocepacia dihydropteroate synthase in the apo-form and complexed with the product 7,8-dihydropteroate

<p>Abstract</p> <p>Background</p> <p>The enzyme dihydropteroate synthase (DHPS) participates in the <it>de novo </it>synthesis of folate cofactors by catalyzing the formation of 7,8-dihydropteroate from condensation of <it>p</it>-aminobenzoic acid with 6-hydroxymethyl-7,8-dihydropteroate pyrophosphate. DHPS is absent from humans, who acquire folates from diet, and has been validated as an antimicrobial therapeutic target by chemical and genetic means. The bacterium <it>Burkholderia cenocepacia </it>is an opportunistic pathogen and an infective agent of cystic fibrosis patients. The organism is highly resistant to antibiotics and there is a recognized need for the identification of new drugs against <it>Burkholderia </it>and related Gram-negative pathogens. Our characterization of the DHPS active site and interactions with the enzyme product are designed to underpin early stage drug discovery.</p> <p>Results</p> <p>An efficient recombinant protein expression system for DHPS from <it>B. cenocepacia </it>(<it>Bc</it>DHPS) was prepared, the dimeric enzyme purified in high yield and crystallized. The structure of the apo-enzyme and the complex with the product 7,8-dihydropteroate have been determined to 2.35 Å and 1.95 Å resolution respectively in distinct orthorhombic crystal forms. The latter represents the first crystal structure of the DHPS-pterin product complex, reveals key interactions involved in ligand binding, and reinforces data generated by other structural studies. Comparisons with orthologues identify plasticity near the substrate-binding pocket and in particular a range of loop conformations that contribute to the architecture of the DHPS active site. These structural data provide a foundation for hit discovery. An intriguing observation, an artifact of the analysis, that of a potential sulfenamide bond within the ligand complex structure is mentioned.</p> <p>Conclusion</p> <p>Structural similarities between <it>Bc</it>DHPS and orthologues from other Gram-negative species are evident as expected on the basis of a high level of sequence identity. The presence of 7,8-dihydropteroate in the binding site provides details about ligand recognition by the enzyme and the different states of the enzyme allow us to visualize distinct conformational states of loops adjacent to the active site. Improved drugs to combat infections by <it>Burkholderia sp. </it>and related Gram-negative bacteria are sought and our study now provides templates to assist that process and allow us to discuss new ways of inhibiting DHPS.</p

Principles and practice of sleep medicine

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Precision of circadian wake and activity onset timing in the mouse

In each circadian cycle, a mouse begins its major activity period with discrete wake onset and activity onset events. The precision with which these events are timed in constant darkness was analyzed using the approach outlined by Pittendrigh and Daan (1976). 1. Negative serial correlations of observed circadian period values (mean r1=−0.471 for wake data, −0.409 for activity data) imply that deviations in period tend to be compensated by opposite deviations in the following cycle. 2. As a result, precision of the circadian pacemaker must be better than that of observed rhythms. Standard deviation of the pacemaker periodσ(Τ) was estimated at 5.1 min. Some individual data series had estimates s(Τ)=0, implying a nearly perfect pacemaker. 3. Previous speculation was that wake onset would be under more direct pacemaker control than activity onset, and would therefore be timed more precisely (Pittendrigh and Daan 1976; Richardson et al. 1985). Contrary to this prediction, intervals between successive wake onsets exhibited significantly greater variance than intervals between successive activity onsets. Two possible interpretations of this finding were proposed