Crystal structures of two tetrameric β‐carbonic anhydrases from the filamentous ascomycete Sordaria macrospora

Abstract

Carbonic anhydrases (CAs) are metalloenzymes catalyzing the 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 fungal β‐class has only recently attracted attention. In the present study, we investigated the structure and function of the plant‐like β‐CA proteins CAS1 and CAS2 from the filamentous ascomycete Sordaria macrospora. We demonstrated that both proteins can substitute for the Saccharomyces cerevisiae β‐CA Nce103 and exhibit an in vitro CO2 hydration activity (kcat/Km of CAS1: 1.30 × 106 m−1·s−1; CAS2: 1.21 × 106 m−1·s−1). To further investigate the structural properties of CAS1 and CAS2, we determined their crystal structures to a resolution of 2.7 Å and 1.8 Å, respectively. The oligomeric state of both proteins is tetrameric. With the exception of the active site composition, no further major differences have been found. In both enzymes, the Zn2+ ‐ion is tetrahedrally coordinated; in CAS1 by Cys45, His101 and Cys104 and a water molecule and in CAS2 by the side chains of four residues (Cys56, His112, Cys115 and Asp58). Both CAs are only weakly inhibited by anions, making them good candidates for industrial applications. Structured digital abstract CAS1 and CAS2 bind by x-ray crystallography ( View interaction) Database Structural data have been deposited in the Protein Data Bank database under accession numbers http://www.rcsb.org/pdb/search/structidSearch.do?structureId=4O1J for CAS1 and http://www.rcsb.org/pdb/search/structidSearch.do?structureId=4O1K for CAS2.

An overview of the alpha-, beta- and gamma-carbonic anhydrases fromBacteria: can bacterial carbonic anhydrases shed new light on evolution of bacteria?

“…The 3D fold of the five CA classes is very different: a-CAs are normally monomers and rarely dimmers; b-CAs are dimers, tetramers or octamers; g-CAs are trimers, whereas the d-and z-CAs are less well understood (the only z-CA crystallized so far has three slightly different active sites on the same polypeptide chain whereas no X-ray crystal structures of d-CAs are available so far) [19][20][21] . Many representatives of all these enzyme classes have been crystallized and characterized in detail, except the d-CAs [23][24][25][26][27][28][29] . Bacteria encode for enzymes belonging to the a-, b-, and g-CA classes [30][31][32][33][34][35][36][37][38][39][40][41][42][43] .…”

Protonography, a technique applicable for the analysis ofη-carbonic anhydrase activity

“…For example, a-CAs are normally monomers and rarely dimmers; b-CAs are dimers, tetramers or octamers; g-CAs are trimers, whereas the d-and z-CAs are less well understood. The only z-CA crystallized so far has three slightly different active sites on the same polypeptide chain, whereas X-ray crystal structures of d-and Z-CAs are not available so far [12][13][14][29][30][31][32][33][34][35] . Many classes of CA inhibitors (CAIs) are known to date: the metal complexing anions and the unsubstituted sulfonamides which bind to the Zn(II) ion of the enzyme either by substituting the non-protein zinc ligand or add to the metal coordination sphere, generating trigonal-bipyramidal species are the classical, are the most frequently investigated ones 15,[36][37][38][39][40][41][42][43][44][45][46][47][48][49][50][51][52] .…”

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

“…Only the z-CA crystallized has three slightly different active sites on the same polypeptide chain 26 , whereas no X-ray crystal structures of d-and Z-CAs are available up to now. CAs contain Zn 2+ in their active site, coordinated by three histidine residues and a water molecule/ hydroxide ion (in the a-and g-CAs) or two Cys and one His residues (in the b class), with the fourth ligand being a water molecule/hydroxide ion 22,23,27 . In the Z-CAs, the coordination pattern of the Zn(II) ion is totally different from the other classes, with one Gln and two His acting as ligands 21 .…”