An Overview of the Bacterial Carbonic Anhydrases

Abstract

Bacteria encode carbonic anhydrases (CAs, EC 4.2.1.1) belonging to three different genetic families, the α-, β-, and γ-classes. By equilibrating CO₂ and bicarbonate, these metalloenzymes interfere with pH regulation and other crucial physiological processes of these organisms. The detailed investigations of many such enzymes from pathogenic and non-pathogenic bacteria afford the opportunity to design both novel therapeutic agents, as well as biomimetic processes, for example, for CO₂ capture. Investigation of bacterial CA inhibitors and activators may be relevant for finding antibiotics with a new mechanism of action.

Keywords: CO2 capture; antibiotic; bacterial carbonic anhydrases; engineered bacteria; inhibitors.

Figure 1

Multi-alignment of the amino acid sequences of two human α-CAs (hCAI and hCAII) and of five bacterial α-CAs (SspCA, SazCA, NgoCA, VchCA, and HypyCA) was performed with the ClustalW program, version 2.1. The hCA I numbering system was used. Black bold indicates the amino acid residues of the catalytic triad; blue bold represents the “gate-keeper” residues; and red bold shows the “proton shuttle residue”. Box indicates the signal peptide. The asterisk (*) indicates identity at a position; the symbol (:) designates conserved substitutions, while (.) indicates semi-conserved substitutions. Multi-alignment was performed with the program Clustal W, version 2.1. Legend: hCAI, α-CA isoform I from Homo sapiens; hCAII, α-CA isoform II from Homo sapiens; SspCA, α-CA from Sulfurihydrogenibium yellowstonense; SazCA, α-CA from Sulfurihydrogenibium azorense; NgonCA, α-CA from Neisseria gonorrhea; VchCA, α-CA from Vibrio cholerae; HpyCA, α-CA from Helicobacter pylori.

Figure 2

Alignment of the amino acid sequences of bacterial 𝛽-CAs from different species. Zinc ligands are indicated in black bold; amino acids involved in the enzyme catalytic cycle are indicated in blue bold. Box indicates the signal peptide. The asterisk (*) indicates identity at a position; the symbol (:) designates conserved substitutions, while (.) indicates semi-conserved substitutions. Multi-alignment was performed with the program Clustal W, version 2.1. Pisum sativum numbering system was used. Legend: EcoCA, 𝛽-CA from Escherichia coli; VchCA, 𝛽-CA from Vibrio cholerae; bSuCA, 𝛽-CA from Brucella suis; HpyCA, 𝛽-CA from Helicobacter pylori; PgiCA, 𝛽-CA from Porphyromonas gingivalis.

Figure 3

Amino acid sequence alignment of the 𝛾-CAs from different bacterial sources, such as Vibrio cholerae, Sulfurihydrogenibium yellowstonense, Porphyromonas gingivalis, and Methanosarcina thermophila. The metal ion ligands (His81, His117, and His122) are indicated in black bold; the catalytically relevant residues of CAM, such as Asn73, Gln75, and Asn202, which participate in a network of hydrogen bonds with the catalytic water molecule, are indicated in red bold; the acidic loop residues containing the proton shuttle residues (Glu89) are colored in blue bold, but are missing in PgiCA. The CAM numbering system was used. Box indicates the signal peptide. Legend: VchCA (𝛾-CA from Vibrio cholerae), SspCA (𝛾-CA from Sulfurihydrogenibium yellowstonense), PgiCA (𝛾-CA from Porphyromonas gingivalis), and CAM (𝛾-CA from Methanosarcina thermophila). The asterisk (*) indicates identity at all aligned positions; the symbol (:) relates to conserved substitutions, while (.) means that semi-conserved substitutions are observed. The multi-alignment was performed with the program Clustal W.

Figure 4

Ribbon representation of the overall fold of α-CA (SspCA) from Sulfurihydrogenibium yellowstonense. (A): SspCA active monomer with the inhibitor acetazolamide (AAZ) showed; (B): SspCA active dimer.

Figure 5

Ribbon representation of the catalytically inactive monomer (A) and active tetramer (B) of 𝛽-CA (VchCA) from Vibrio cholerae.

Figure 6

Structural representation of the catalytically inactive monomer (A) and active trimer (B) of the CAM (γ-CA) enzyme from Methanosarcina thermophila.

Figure 7

Kinetic parameters for the CO₂ hydration reaction catalyzed by the human cytosolic isozymes hCA I and II (α-class CAs) and bacterial α-, β-, and γ-CAs, such as SazCA (α-CAs from Sulfurihydrogenibium azorense), SspCA (α-CAs from Sulfurihydrogenibium yellowstonense), HpyCA (α- and β-CAs from Helicobacter pylori), VchCA (α-, β-, and γ-CAs from Vibrio cholerae), PgiCA (β- and γ-CAs from Porphyromonas gingivalis), and CAM (γ-CA from Methanosarcina thermophila). All the measurements were done at 20 °C, pH 7.5 (α-class enzymes), and pH 8.3 (β- and γ-CAs) by a stopped flow CO₂ hydratase assay method.

Figure 8

Sulfonamides, sulfamates, and some of their derivatives investigated as bacterial CA inhibitors.

Figure 8

Sulfonamides, sulfamates, and some of their derivatives investigated as bacterial CA inhibitors.

Figure 9

Amino acid and amine CA activators 25–43 investigated for their interaction with bacterial CAs.

Figure 10

(A): Activation of the bacterial BpsCA (γ-CA) with L-Tyr; (B): Activation of the bacterial SspCA (α-CA) L-Phe. All measurements were carried out at 25 °C and pH 7.5, for the CO₂ hydration reaction.