MPV004 Molecular investigations on the intracellular life cycle of Burkholderia pseudomallei

Abstract

We report the cloning, purification and characterization of BpsβCA, a β-class carbonic anhydrase (CA, EC 4.2.1.1) from the pathogenic bacterium Burkholderia pseudomallei, the etiological agent of melioidosis, and compare its activity and inhibition with those of the γ-CA from the same organism, BpsγCA, recently investigated by our groups. BpsβCA showed a significant catalytic activity for the physiologic, CO2 hydration reaction, with the following kinetic parameters, kcat of 1.6 × 105 s−1 and kcat/Km of 3.4 × 107 M−1 × s−1. The inhibition of BpsβCA with a group of anions and small molecules was also investigated. The best inhibitors were sulfamide, sulfamic acid and phenylarsonic acid, which showed KIs in the range of 83–92 µM, whereas phenylboronic acid, fluoride, cyanide, azide, bisulfite, tetraborate, perrhenate, perruthenate, peroxydisulfate, perchlorate, tetrafluoroborate, fluorosulfonate and hexafluorophosphate showed KIs > 100 mM. Other inhibitors of this new enzyme were bicarbonate, trithiocarbonate, some complex inorganic anions and N,N-diethyldithiocarbamate, which had inhibition constants of 0.32–8.6 mM. As little is known of the life cycle and virulence of this bacterium, this type of study may bring information of interest for the development of novel strategies to fight bacterial infection and drug resistance to commonly used antibiotics.