Comparison of the anion inhibition profiles of the β- and γ-carbonic anhydrases from the pathogenic bacterium Burkholderia pseudomallei

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, k_cat of 1.6×10⁵s^-1 and k_cat/K_m of 3.4×10⁷M^-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 K_Is in the range of 83-92µM, whereas phenylboronic acid, fluoride, cyanide, azide, bisulfite, tetraborate, perrhenate, perruthenate, peroxydisulfate, perchlorate, tetrafluoroborate, fluorosulfonate and hexafluorophosphate showed K_Is>100mM. Other inhibitors of this new enzyme were bicarbonate, trithiocarbonate, some complex inorganic anions and N,N-diethyldithiocarbamate, which had inhibition constants of 0.32-8.6mM. 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.