Functional and structural analysis of carbonic anhydrases from the filamentous ascomycete Sordaria macrospora

Abstract

Carbonic anhydrases (CAs) are metalloenzymes catalyzing the rapid and reversible hydration of carbon dioxide to bicarbonate (hydrogen carbonate) and protons. CAs have been identified in archaea, bacteria and eukaryotes and can be classified into five groups (α, β, γ, δ, ζ) that are unrelated in sequence and structure. The filamentous ascomycete Sordaria macrospora encodes four CAs, three of the β-class (cas1, cas2 and cas3) and one of the α-class (cas4). The CAS4 protein exhibits a functional N-terminal signal peptide for translocation into the endoplasmic reticulum and is posttranslationally glycosylated and targeted to the supernatant. The knockout strain Δcas4 had a significantly reduced rate of ascospore germination but showed no significant involvement into sexual development and vegetative growth. Vegetative growth rate of the quadruple mutant lacking all cas genes was drastically reduced compared to the wild type and the mutant invaded the agar under normal air conditions. Likewise, the fruiting bodies that were formed only after elongated incubation time were embedded in the agar and completely devoid of mature ascospores. The phenotypic defects could only be partially restored by elevated CO₂-levels and the fruiting bodies that were formed after prolonged incubation were immature without ascospores. In an in-vivo assay, CAS1 and CAS2 could substitute for the S. cerevisiae β-CA Nce103p. Both proteins could be easily produced in E. coli and purified to high purity and exhibited noticeable in-vitro CO₂ hydration activity. In addition, CAS1 and CAS2 were only weakly inhibited by the widely used sulfonamide drug acetazolamide. The best anionic inhibitors for both enzymes were sulfamide, sulfamate, phenylboronic acid and phenylarsonic acid. In contrast, the activity of both CAs was only weakly inhibited by nitrite and nitrate anions and some other anions. To further investigate the structural properties of CAS1 and CAS2 their crystal structures were determined to a resolution of 2.7 Å and 1.8 Å, respectively. The oligomeric state of both proteins in the crystals as well as in solution is tetrameric. With exception of the active site composition, no further major differences could be observed. In contrast to other organisms, the filamentous ascomycete S. macrospora appears to be able to use traces of HCO₃⁻ for growth without CA genes in a standard CO₂ atmosphere. With no functional CAs, S. macrospora switched from preferred growth at the air/medium interface to submerged growth. This work provides for the first time crystal structures of two β-CA enzymes from a filamentous ascomycete that form tetrameric assemblies, unlike other fungal β-CAs.