Molecular and biochemical characterization of carbonic anhydrases of Paracoccidioides

Abstract

Carbonic anhydrases (CA) belong to the family of zinc metalloenzymes that catalyze the reversible hydration of carbon dioxide to bicarbonate. In the present work, we characterized the cDNAs of four Paracoccidioides CAs (CA1, CA2, CA3, and CA4). In the presence of CO2, there was not a significant increase in fungal ca1, ca2 and ca4 gene expression. The ca1 transcript was induced during the mycelium-to-yeast transition, while ca2 and ca4 gene expression was much higher in yeast cells, when compared to mycelium and mycelium-to-yeast transition. The ca1 transcript was induced in yeast cells recovered directly from liver and spleen of infected mice, while transcripts for ca2 and ca4 were down-regulated. Recombinant CA1 (rCA1) and CA4 (rCA4), with 33 kDa and 32 kDa respectively, were obtained from bacteria. The enzymes rCA1 (β-class) and rCA4 (α-class) were characterized regarding pH, temperature, ions and amino acids addition influence. Both enzymes were stable at pHs 7.5-8.5 and temperatures of 30-35 °C. The enzymes were dramatically inhibited by Hg+2 and activated by Zn+2, while only rCA4 was stimulated by Fe2+. Among the amino acids tested (all in L configuration), arginine, lysine, tryptophan and histidine enhanced residual activity of rCA1 and rCA4.

Immunoproteomic Approach of Extracellular Antigens From Paracoccidioides Species Reveals Exclusive B-Cell Epitopes

“…Additionally, in Paracoccidioides sp., four coding genes for carbonic anhydrase have already been described, leading to the synthesis of CA1, CA2, and CA3 proteins for Class β and CA4 protein for Class α. In addition, this enzyme was positively regulated in Paracoccidioides yeast when compared to the mycelium phase of the fungus (Tomazett et al, 2016). In Stenocarpella macrospora, CA has already been described in the mitochondrial, cytoplasmic, and extracellular compartments (Elleuche and Poggeler, 2010).…”

Detection of a multi‐disease biomarker in saliva with graphene field effect transistors

“…Considering the molecular weight of CA1 (33 kDa) (Tomazett et al, 2016), the measured concentration range is equivalent to 330 fM <ρ <3 nM. The achieved lower detection limit of 10 pg/ml is well below the reported critical value of 2.6 ng/ml indicative of non-small cell lung cancer .…”

“…The expected increase in voltage shift was observed with increasing concentration of CA1 ranging between 10 pg/ml <ρ < 100 ng/ml. Considering the molecular weight of CA1 (33 kDa) (Tomazett et al, 2016), the measured concentration range is equivalent to 330 fM <ρ <3 nM. The achieved lower detection limit of 10 pg/ml is well below the reported critical value of 2.6 ng/ml indicative of non‐small cell lung cancer (Wang, et al, 2016).…”

“…Thus, the charges associated with different biomarkers and their distance from the graphene surface could significantly affect the sensitivity of such sensors. Given the size of the aptamers (~2.5 nm see Figure 2a) and the CA1 (~4.25 nm) (Erickson, 2009; Tomazett et al, 2016), it is crucial to carefully consider the Debye screening length of the employed buffer. Specifically, if the Debye length is too small it is likely to dramatically reduce the induced electrostatic charges in the graphene upon binding of CA1 with the aptamers, resulting in the lower sensitivity of G‐FET biosensor.…”

Evaluation of the antidermatophytic activity of potassium salts of N-acylhydrazinecarbodithioates and their aminotriazole-thione derivatives

“…Based on docking simulations, fungal carbonic anhydrase (CA) was proposed as one of the putative enzymatic targets for N-acylhydrazinecarbodithioates derivatives 31 . CAs are found in Eukarya, Bacteria, as well as Archea and are essential for carbon dioxide (CO 2 ) sensing and metabolism 32 . As zinc-containing metalloenzymes, they are responsible for the hydration of CO 2 to bicarbonate (HCO 3 − ) and proton (H + ) 33 .…”