A new procedure for the cloning, expression and purification of the β-carbonic anhydrase from the pathogenic yeastMalassezia globosa, an anti-dandruff drug target

Abstract

Malassezia yeasts are almost exclusively the single eukaryotic members of the fungal flora of the skin. Malassezia globosa and Malassezia restricta are found on the skin of practically all humans. Malassezia globosa is highly implicated in the pathogenesis of dandruff and its genome encodes for only one carbonic anhydrases (CAs, EC 4.2.1.1) belonging to the b-class (MgCA). It has been indeed demonstrated that in many pathogenic microorganisms, CAs are essential for their life cycle and their inhibition can lead to growth impairment and defects. In the previous work, the recombinant MgCA was investigated for its inhibition profile with sulfonamides, which in models of dandruff infection were able to protect animals from the fungal infection, allowing us to propose this enzyme as a new antidandruff target. MgCA was cloned as GST-fusion protein, but the yield was rather low and the protein was often found in inclusion bodies. Here, we propose an alternative procedure consisting in cloning the recombinant MgCA as His-Tag fusion protein. This procedure resulted in a good method to express and purify the active recombinant MgCA, and the protein recovery was better with respect to that used for preparing MG-CA (b-CA cloned as GST-fusion protein).

Sulfonamide Inhibition Profile of the β-Carbonic Anhydrase from Malassezia restricta, An Opportunistic Pathogen Triggering Scalp Conditions

“…All of these traditional drugs, including shampoos, creams, and lotions, have been used for years showing effectiveness, which is however limited to the duration of the treatment, and relapse occurs 3 to 6 weeks after the treatment is stopped [2]. We recently proposed exploring certain members of the ancient superfamily of ubiquitous metalloenzymes, known as carbonic anhydrases (CAs, EC 4.2.1.1) from Malassezia restricta, as a new antidandruff target [8,9]. CAs catalyze the simple but physiologically crucial reaction of CO 2 hydration to bicarbonate and protons (CO 2 + H 2 O HCO 3 − + H + ) [10] and they are the fastest among the known enzymes (k cat = 10 4 -10 6 s −1 ) [11].…”

Sulfonamide inhibition profile of the γ-carbonic anhydrase identified in the genome of the pathogenic bacterium Burkholderia pseudomallei the etiological agent responsible of melioidosis

“…[57][58][59][60] (iii) The inhibitory action against BpsCAc was very low for the following compounds: 8, 11-13, 16 and 24, which had K I s in the range of 10,800-24,500 nM. Again, they belong to heterogeneous classes of sulfonamides, with aromatic monosulfonamides (8,16,24), disulfonamides (11,12), and a heterocylic sulfonamide derivative, deacetylated acetazolamide 13 (Table 1). (iv) A number of the investigated sulfonamides did not inhibit BpsCAc up to 50 lM, which is the maximum concentration of inhibitor in the assay system.…”

“…The following structure-activity relationship (SAR) may be concluded by considering the inhibition data in Table 2: (i) BpsCAc is less sensitive to sulfonamide CAIs compared to other a-(e.g., hCA II) or c-CAs (e.g., VchCAc) investigated earlier, 8 for which many nanomolar (and low nanomolar) inhibitors were found ( Comparison of the kinetic parameters for the CO 2 hydration reaction catalyzed by BpsCAc, the human cytosolic isozymes hCA I and II (a-class CAs) and the c-CAs from Vibrio cholerae (VchCAc) and P. gingivalis (PgiCAc). Acetazolamide inhibition data are also shown.…”

“…[5][6][7][8][9] This superfamily includes seven distinct classes known as the a-, b-, c-, d-, f-, g-and ɵ-CAs. [10][11][12][13][14][15][16][17][18] These enzymes are involved in many physiologic processes, such as photosynthesis, respiration, CO 2 transport, as well as metabolism of xenobiotics (e.g., cyanate in Escherichia coli).…”

Cloning, expression and purification of the complete domain of theη-carbonic anhydrase fromPlasmodium falciparum

“…HCO À 3 , generated from CO 2 through the action of a CA, is the substrate of the carbamoyl phosphate synthetase II (PfCPSII), the first enzyme involved in the Plasmodia pyrimidine pathway 54 . Finally, we should mention that six different, genetically distinct CA families are known to date, the a-, b-, g-, d-, z-and Z-CAs 17,55,56 . Thus, the last important aspect concerning the P. falciparum CA, classified as belonging to the Z-CA class 17,40,45,53,[57][58][59][60] , is the observation that the metal ion coordination pattern is unique among all the genetic families encoding for such enzymes.…”