Activation studies of the α- and β-carbonic anhydrases from the pathogenic bacterium Vibrio cholerae with amines and amino acids

Andrea Angeli,Sonia Del Prete,Sameh M Osman,Fatmah A S Alasmary,Zeid Alothman,William A Donald,Clemente Capasso,Claudiu T Supuran

doi:10.1080/14756366.2017.1412316

Abstract

The α- and β-class carbonic anhydrases (CAs, EC 4.2.1.1) from the pathogenic bacterium Vibrio cholerae, VchCAα, and VchCAβ, were investigated for their activation with natural and non-natural amino acids and amines. The most effective VchCAα activators were L-tyrosine, histamine, serotonin, and 4-aminoethyl-morpholine, which had KAs in the range of 8.21–12.0 µM. The most effective VchCAβ activators were D-tyrosine, dopamine, serotonin, 2-pyridyl-methylamine, 2-aminoethylpyridine, and 2-aminoethylpiperazine, which had KAs in the submicromolar – low micromolar range (0.18–1.37 µM). The two bacterial enzymes had very different activation profiles with these compounds, between each other, and in comparison to the human isoforms hCA I and II. Some amines were selective activators of VchCAβ, including 2-pyridylmethylamine (KA of 180 nm for VchCAβ, and more than 20 µM for VchCAα and hCA I/II). The activation of CAs from bacteria, such as VchCAα/β has not been considered previously for possible biomedical applications. It would be of interest to study in more detail the extent that CA activators are implicated in the virulence and colonisation of the host by such pathogenic bacteria, which for Vibrio cholerae, is highly dependent on the bicarbonate concentration and pH in the surrounding tissue.

Highlights

Carbonic anhydrases (CAs, EC 4.2.1.1) are a superfamily of ubiquitous metalloenzymes with the catalytically active form represented by a metal hydroxide derivative acting as a potent nucleophile on CO2 or other electrophiles (e.g. COS, CS2, esters, etc.)[1–15]
The metal ion from the CA active site is crucial for catalysis, and is coordinated by three His residues in the a, c, d, and probably the h-classes; by one His, and two Cys residues in b- and f-CAs or by two His and one Gln residues in the g-class, with the fourth ligand being a water molecule/hydroxide ion acting as nucleophile in the catalytic cycle of the enzyme[1–15]
The activators 1–19 were included in this study, as they were employed for investigations as CA activators (CAAs) against many classes of CAs, including the bacterial ones from Burkholderia pseudomallei, BpsCAb/c36c,45

Summary

Introduction

Carbonic anhydrases (CAs, EC 4.2.1.1) are a superfamily of ubiquitous metalloenzymes with the catalytically active form represented by a metal hydroxide derivative acting as a potent nucleophile on CO2 (the physiological substrate) or other electrophiles (e.g. COS, CS2, esters, etc.)[1–15]. CAs catalyse only one simple but physiologically highly relevant reaction, which is the reversible hydration of carbon dioxide to bicarbonate and protons[5,6,13,15] These enzymes are grouped in seven genetically distinct families, named a-, b-, c-, d-, f-, g- and Õ-CAs, and they share a low sequence similarity and protein three dimensional structure, all of them possess a high efficiency as catalysts for the transformation of the metabolically crucial gas CO2 into soluble products, HCO3À and Hþ ions[5,6,8,13,15–17]. The rate determining step in the CA catalytic cycle is the formation of the metal hydroxide species of the enzyme from the acidic one in which a water molecule is coordinated as the fourth ligand to the metal centre[3–6,9] This process is usually assisted by amino acid residues placed in the middle or at the rim of the active site, which can shuttle protons between the metal centre and the reaction medium by means of moieties possessing a pKa in the region of 6–8 pH units, such as imidazoles (from His residues), carboxylates (from Asp or Glu residues), etc.[3–6,9].

Methods

Results

Conclusion