Molecular and biochemical characterization of carbonic anhydrases of Paracoccidioides.

Mariana Vieira Tomazett,Clayton Luiz Borges,Elisa Flávia Cardoso Bailão,Alexandre Melo Bailão,Fabiana Fonseca Zanoelo,Célia Maria De Almeida Soares

doi:10.1590/1678-4685-gmb-2015-0213

Mariana Vieira Tomazett, Clayton Luiz Borges + Show 4 more

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https://doi.org/10.1590/1678-4685-gmb-2015-0213

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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.

Highlights

Fungi belonging to the genus Paracoccidioides are human pathogens that cause paracoccidioidomycosis (PCM), the most prevalent systemic mycosis in Latin America
The proteins were purified and enzymatic parameters were determined in vitro. These results provide original information of the relevance of Carbonic anhydrases (CA) in the physiology of Paracoccidioides
Comparisons of the predicted protein sequences allowed their classification based on conserved regions of carbonic anhydrase, as follows: CAs signature; Zn binding amino acids and highly conserved amino acid residues in CAs, possibly important for enzyme activity (Kerry and Ferry, 2000)

Summary

Introduction

Fungi belonging to the genus Paracoccidioides are human pathogens that cause paracoccidioidomycosis (PCM), the most prevalent systemic mycosis in Latin America. The species of this genus grow as yeast cells at 36 °C or in vertebrate tissues, and as mycelia at 23 °C or in the soil (Franco, 1987; Restrepo et al, 2001). Carbonic anhydrases (CAs) are metalloenzymes, that catalyze the reversible hydration of CO2 to generate a proton and HCO3-(Supuran, 2008a). CAs are widely found in all life kingdoms (Eukarya, Bacteria and Archaea) playing important roles in the global

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