DNA topoisomerases I from Pseudomonas aeruginosa and vaccinia virus and their use as drug targets

Pseudomonas aeruginosa encodes a putative topoisomerase with sequence similarity to the type IB topoisomerase enzyme from vaccinia virus. Residues in the active site are conserved, notably Tyr292 which would be predicted to form the transient covalent bond to DNA. The gene encoding the P. aeruginosa topoisomerase I (PAT) was cloned and expressed in E. coli. The enzyme relaxes supercoiled DNA, while a mutant of P. aeruginosa containing a Tyr292 to Phe substitution at the active site was found to be catalytically inert. This is consistent with the role of Tyr in forming the covalent intermediate. Like vaccinia topoisomerase (VT), PAT relaxes DNA in the absence of ATP. Unlike VT, PAT does not relax supercoiled DNA without MgCl2 (or MnCl2) present. In addition, high concentration of NaCl is not able to substitute for MgCl2 as seen for VT. A truncated derivative of the topoisomerase lacking residues 1-98 relaxes DNA in the absence of the N-terminal domain, with both full length and truncated enzyme exhibiting equivalent requirements for divalent cations. Data shows that P. aeruginosa encodes a functional topoisomerase with significant similarity to the type IB enzyme encoded by poxviruses. Fluoroquinolones are antibacterial agents in clinical use with activity against DNA gyrase and DNA topoisomerase IV. P. aeruginosa is an opportunistic pathogen causing life-threatening diseases. Sparfloxacin, enrofloxacin and norfloxacin fluoroquinolones, are able to inhibit the PAT at high concentrations but other drugs belonging to this family are unable to do so. VT is ¡Ö32 KDa and one-third the size of the human topoisomerase ¡Ö 100 KDa. It shares sequence and biochemical similarities with the human topoisomerase. The VT binds duplex DNA with stringent specificity for transesterification at 5`-(C/T) CCTT site, where the 3` phosphate of the incised strand is linked to the Tyr274 of the enzyme to form a covalent cleavage complex. The fluoroquinolone enrofloxacin inhibits relaxation of supercoiled DNA by VT in a Mg2+-dependent fashion. Further results indicate that the mechanism by which enrofloxacin inhibits VT is by preventing formation of the covalent complex which suggest that fluoroquinolones may be structurally optimized to target type IB topoisomerases

A functional type I topoisomerase from Pseudomonas aeruginosa

<p>Abstract</p> <p>Background</p> <p><it>Pseudomonas aeruginosa </it>encodes a putative topoisomerase with sequence similarity to the eukaryotic type IB topoisomerase from Vaccinia virus. Residues in the active site are conserved, notably Tyr292 which would be predicted to form the transient covalent bond to DNA.</p> <p>Results</p> <p>The gene encoding the <it>P. aeruginosa </it>topoisomerase I was cloned and expressed in <it>E. coli</it>. The enzyme relaxes supercoiled DNA, while a mutant containing a Tyr292 to Phe substitution at the active site was found to be catalytically inert. This is consistent with the role of Tyr in forming the covalent intermediate. Like Vaccinia topoisomerase, the <it>P. aeruginosa topoisomerase </it>relaxes DNA in the absence of ATP, but unlike Vaccinia topoisomerase, <it>P. aeruginosa </it>topoisomerase does not relax supercoiled DNA without MgCl₂present. In addition, high concentration of NaCl is not able to substitute for MgCl₂as seen for Vaccinia topoisomerase. A truncated derivative of the topoisomerase lacking residues 1–98 relaxes DNA, with both full length and truncated enzyme exhibiting equivalent requirements for divalent cations and the ability to relax DNA to completion, suggesting a shared domain organization. DNA-binding assays suggest an only modest preference for the CCCTT pentameric sequence required for transesterification by Vaccinia topoisomerase IB.</p> <p>Conclusion</p> <p><it>P. aeruginosa </it>encodes a functional topoisomerase with significant similarity to the type IB enzyme encoded by poxviruses. In contrast to the Vaccinia-encoded homolog, the <it>P. aeruginosa</it>-encoded enzyme requires divalent cations for catalytic activity, relaxes DNA to completion, and does not exhibit a strong preference for the pentameric sequence stringently required by the Vaccinia-encoded homolog. A comparison with the structure of poxviral topoisomerase in complex with DNA suggests that bacterial homologs of the eukaryotic type IB topoisomerase may exhibit a relaxed sequence preference due to the lack of conservation of certain residues involved in sequence-specific DNA contacts, and that interaction with an only modestly preferred sequence may result in suboptimal positioning of catalytic residues.</p

Postural Hypotension and Cognitive Function in Older Adults

Background: Cognitive decline in the elderly is associated with chronic cerebral hypoperfusion. While many forms of exercise can slow or reverse cognitive decline, compliance in unsupervised exercise programs is poor. Objective: We address whether passive exercise, that is, muscle stimulation, is capable of reversing postural hypotension in an older adult population sufficiently to significantly improve cognitive function as measured by executive function tests. Subjects and Methods: In this study, 50- to 80-year-old women underwent cognitive testing, long-duration cardiac hemodynamic recordings during quiet sitting, and 60 min of soleus muscle stimulation with continued hemodynamic recording. Results: Two thirds of our subjects were hypotensive (diastolic blood pressure [DBP] < 70 mmHg) after 30 min of quiet sitting. Cognitive performance was significantly better in individuals with higher DBPs (0.79 s per 1-mmHg increase in DBP). Soleus muscle stimulation resulted in an average increase in DBP of 6.1 mmHg, which could translate into a 30% or greater improvement in cognitive performance. Conclusions: Incongruent Stroop testing provides high statistical power for distinguishing differential cognitive responses to resting DBP levels. These results set the stage to investigate whether regular use of calf muscle pump stimulation could effectively reverse age-related cognitive impairment

Ancestral Variations of the PCDHG Gene Cluster Predispose to Dyslexia in a Multiplex Family

Dyslexia is a heritable neurodevelopmental disorder characterized by difficulties in reading and writing. In this study, we describe the identification of a set of 17 polymorphisms located across 1.9 Mb region on chromosome 5q31.3, encompassing genes of the PCDHG cluster, TAF7, PCDH1 and ARHGAP26, dominantly inherited with dyslexia in a multi-incident family. Strikingly, the non-risk form of seven variations of the PCDHG cluster, are preponderant in the human lineage, while risk alleles are ancestral and conserved across Neanderthals to non-human primates. Four of these seven ancestral variations (c.460A > C [p.Ile154Leu], c.541G > A [p.Ala181Thr], c.2036G > C [p.Arg679Pro] and c.2059A > G [p.Lys687Glu]) result in amino acid alterations. p.Ile154Leu and p.Ala181Thr are present at EC2: EC3 interacting interface of γA3-PCDH and γA4-PCDH respectively might affect trans-homophilic interaction and hence neuronal connectivity. p.Arg679Pro and p.Lys687Glu are present within the linker region connecting trans-membrane to extracellular domain. Sequence analysis indicated the importance of p.Ile154, p.Arg679 and p.Lys687 in maintaining class specificity. Thus the observed association of PCDHG genes encoding neural adhesion proteins reinforces the hypothesis of aberrant neuronal connectivity in the pathophysiology of dyslexia. Additionally, the striking conservation of the identified variants indicates a role of PCDHG in the evolution of highly specialized cognitive skills critical to reading