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

摘要: Carbonic anhydrases (CAs) are metalloenzymes catalyzing the rapid and reversible hydration of carbon dioxide to bicarbonate (hydrogen carbonate) protons. CAs have been identified in archaea, bacteria eukaryotes can be classified into five groups (α, β, γ, δ, ζ) that unrelated sequence structure. The filamentous ascomycete Sordaria macrospora encodes four CAs, three β-class (cas1, cas2 cas3) one α-class (cas4). CAS4 protein exhibits a functional N-terminal signal peptide for translocation endoplasmic reticulum is posttranslationally glycosylated targeted supernatant. knockout strain Δcas4 had significantly reduced rate ascospore germination but showed no significant involvement sexual development vegetative growth. Vegetative growth quadruple mutant lacking all cas genes was drastically compared wild type invaded agar under normal air conditions. Likewise, fruiting bodies were formed only after elongated incubation time embedded completely devoid mature ascospores. phenotypic defects could partially restored by elevated CO 2 -levels prolonged immature without In an in-vivo assay, CAS1 CAS2 substitute S. cerevisiae β-CA Nce103p. Both proteins easily produced E. coli purified high purity exhibited noticeable in-vitro activity. In addition, weakly inhibited widely used sulfonamide drug acetazolamide. best anionic inhibitors both enzymes sulfamide, sulfamate, phenylboronic acid phenylarsonic acid. contrast, activity nitrite nitrate anions some other anions. To further investigate structural properties their crystal structures determined resolution 2.7 A 1.8 A, respectively. oligomeric state crystals as well solution tetrameric. With exception active site composition, major differences observed. In contrast organisms, appears able use traces HCO 3 − CA standard atmosphere. switched from preferred at air/medium interface submerged This work provides first two form tetrameric assemblies, unlike fungal β-CAs.