Biochemical characterization of the δ-carbonic anhydrase from the marine diatomThalassiosira weissflogii, TweCA

Abstract

Diatom genome sequences clearly reveal the presence of different systems for HCO 3 À uptake. Carbon-concentrating mechanisms (CCM) based on HCO 3 À transport and a plastid-localized carbonic anhydrase (CA, EC 4.2.1.1) appear to be more probable than the others because CAs have been identified in the genome of many diatoms. CAs are key enzymes involved in the acquisition of inorganic carbon for photosynthesis in phytoplankton, as they catalyze efficiently the interconversion between carbon dioxide and bicarbonate. Five genetically distinct classes of CAs exist, a-, b-, g-, d-and z and all of them are metalloenzymes. Recently we investigated for the first time the catalytic activity and inhibition of the d-class CA from the marine diatom Thalassiosira weissflogii, named TweCA. This enzyme is an efficient catalyst for the CO 2 hydration and its inhibition profile with sulfonamide/sulfamate and anions have also been investigated. Here, we report the detailed biochemical characterization and chemico-physical properties of the d-CA of T. weissflogii. The d-CA encoding gene was cloned and expressed in Artic Express cells and the recombinant protein purified to homogeneity. Interesting to note that TweCA has no intrinsic esterase activity with 4-nitrophenyl acetate (pNpA) as substrate although the phylogenetic analysis showed that d-CAs are closer to the a-CAs than to the other classes of such enzymes.

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

“…Six different, genetically distinct CA families are known to date, the a-, b-, g-, d-, z-and Z-CAs [8][9][10][11] . The a-, b-, d-and Z-CAs use Zn(II) ions at the active site, the g-CAs are probably Fe(II) enzymes (but they are active also with bound Zn(II) or Co(II) ions), whereas the z-class uses Cd(II) or Zn(II) to perform the physiologic reaction catalysis 5,[11][12][13][14][15][16][17][18][19][20][21][22][23][24][25][26][27][28] . Many representatives of all these enzyme classes have been crystallized and characterized in detail, except d-and Z-CAs.…”

“…Many representatives of all these enzyme classes have been crystallized and characterized in detail, except d-and Z-CAs. The three-dimensional analysis of CAs present a substantial variability between the different classes: the overall shape of the molecules, the protein folding patterns as well as the oligomeric organization of the six genetic CA families are very much different 5,[11][12][13][14][15][16][17][18][19][20][21][22][23][24][25][26][27][28] . 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.…”

“…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] .…”

Sulfonamide inhibition studies of the γ-carbonic anhydrase from the Antarctic bacterium Pseudoalteromonas haloplanktis

“…4,5,[30][31][32][33][34][35][36][37][38] These enzymes are involved in a multitude of physiologic processes in organisms all over the phylogenetic tree, with six genetically distinct CA classes known to date: the a-, b-, c-, d-, f-and g-CAs. 26,[39][40][41][42][43][44][45][46][47][48][49][50] Their biochemical features are known in detail for at least four classes, together with their distribution and role in various organisms. 32,33,41,[48][49][50][51][52][53][54][55][56][57][58][59][60][61][62] Inhibition and activation studies of many such enzymes from vertebrates, protozoa, fungi and bacteria have shown that they are drug targets for obtaining pharmacological agents of the diuretic, antiglaucoma, antiobesity, antiepileptic, anticancer or anti-infective type.…”

“…26,[39][40][41][42][43][44][45][46][47][48][49][50] Their biochemical features are known in detail for at least four classes, together with their distribution and role in various organisms. 32,33,41,[48][49][50][51][52][53][54][55][56][57][58][59][60][61][62] Inhibition and activation studies of many such enzymes from vertebrates, protozoa, fungi and bacteria have shown that they are drug targets for obtaining pharmacological agents of the diuretic, antiglaucoma, antiobesity, antiepileptic, anticancer or anti-infective type. [55][56][57][58]60 Many such enzymes also possess biotechnologic applications for biomimetic CO 2 capture processes.…”