Sulfonamide inhibition studies of the γ-carbonic anhydrase from the Antarctic cyanobacterium Nostoc commune

Abstract

A carbonic anhydrase (CA, EC 4.2.1.1) belonging to the γ-class has been cloned, purified and characterized from the Antarctic cyanobacterium Nostoc commune. The enzyme showed a good catalytic activity for the physiologic reaction (hydration of carbon dioxide to bicarbonate and a proton) with the following kinetic parameters, k_cat of 9.5 × 10⁵ s⁻¹ and k_cat/K_M of 8.3 × 10⁷ M⁻¹ s⁻¹, being the γ-CA with the highest catalytic activity described so far. A range of aromatic/heterocyclic sulfonamides and one sulfamate were investigated as inhibitors of the new enzyme, denominated here NcoCA. The best NcoCA inhibitors were some sulfonylated sulfanilamide derivatives possessing elongated molecules, aminobenzolamide, acetazolamide, benzolamide, dorzolamide, brinzolamide and topiramate, which showed inhibition constants in the range of 40.3–92.3 nM. As 1,5-bisphosphate carboxylase/oxygenase (RubisCO) and γ-CAs are closely associated in carboxysomes of cyanobacteria for enhancing the affinity of RubisCO for CO₂ and the efficiency of photosynthesis, investigation of this new enzyme and its affinity for modulators of its activity may bring new insights in these crucial processes.

Introduction

Carbonic anhydrases (CAs, EC 4.2.1.1) are biological catalysts for the interconversion of CO₂ and water to bicarbonate (HCO₃⁻) and protons (H⁺).1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 CAs are multifunctional enzymes which play a central role in different physiological and biochemical processes, such as respiratory gas exchange; acid–base homeostasis; electrolytes secretion; biosynthetic reactions (e.g., ureagenesis, gluconeogenesis, synthesis of fatty acids); ionic transport; muscular contraction (in vertebrates) and photosynthesis (in cyanobacteria, plants and algae).19, 20, 21 Recently many studies revealed that CAs are also widely distributed in prokaryotes where their role may be much more important than previously thought. Six different, genetically distinct CA families are known to date, the α-, β-, γ-, δ-, ζ- and η-CAs.22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 Except for δ- and η-CAs, the three-dimensional structure of all the other CA classes has been resolved by X-ray diffraction techniques.26, 28, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 α-CAs are normally monomers and rarely dimers; β-CAs are dimers, tetramers or octamers; γ-CAs are trimers, whereas the δ- and ζ-CAs are less well understood at this moment. For example, the most investigated ζ-CA (from the marine diatom Thalassiosira weissflogii) has three slightly different active sites on the same polypeptide chain.⁵²

Bacteria encode for enzymes belonging to the α-, β-, and γ-CA classes.53, 54 All these enzymes contain a zinc ion (Zn²⁺) in their active site, coordinated by three histidine residues and a water molecule/hydroxide ion (in the α- and γ-CAs) or by two Cys and one His residues (in the β class), with the fourth ligand being a water molecule/hydroxide ion acting as nucleophile in the catalyzed reactions.48, 53, 55, 56, 57, 58

Few data are available in the literature on CAs from Antarctic organisms59, 60, 61 and most such data deal with CAs isolated from mammals, prokaryotes or other mesophilic sources, these organisms living at physiological temperatures of around 37 °C.15, 22, 24, 25, 27, 29, 30, 31, 33, 34, 35, 36, 37, 38, 39, 62, 63, 64, 65, 66, 67 Recently, our group studied CAs from extremophiles, microorganisms living at temperatures ranging from 70 °C to 110 °C, demonstrating that these enzymes are thermostable, thermoactive and stable to the common enzyme denaturants.41, 68, 69, 70, 71, 72, 73

Antarctica can be considered as a huge natural laboratory and many scientists share the interest of elucidating evolutionary mechanisms developed by Antarctic organisms to adapt to such an extreme habitat.74, 75, 76, 77, 78, 79

In the present paper, we focused our attention on the γ-CA identified in the genome of the diazotrophic cyanobacterium Nostoc commune, isolated from freshwater of Antarctic lakes. Nostoc includes several species, which thrive in nutrient-poor freshwaters and harsh semi-terrestrial environments characterized by extended drought and/or freezing cycles, high levels of radiation and prolonged dormancy periods.⁸⁰ As its name indicates, Nostoc commune is the most widespread such species, being present in snow, sea ice, glaciers, permafrost, and ice clouds both in the Arctic and the Antarctic.⁸⁰ The analysis of genomic DNA from the aforementioned cyanobacterium revealed that it encodes for CAs belonging to the α-, β- and γ-classes.

Carbon fixation in cyanobacteria is possible due to the presence of cytosolic organelles called carboxysomes, which contain two enzymes: 1,5-bisphosphate carboxylase/oxygenase (RubisCO) and CA.⁸¹ RubisCO is a rather inefficient enzyme, as it has a low affinity for CO₂, possesses a low catalytic rate, and O₂ can also act as an alternative substrate, impairing thus the binding of CO₂. On the other hand, CAs are generally highly efficient enzymes for the CO₂ hydration reaction/bicarbonate dehydration to CO₂, possessing turnover numbers among the highest known for any enzyme.82, 83, 84, 85, 86, 87 These two proteins, RubisCO and CA, act in concert for the carbon dioxide fixation through a process known as carbon concentrating mechanism (CCM).⁸⁸ Inorganic carbon (as CO₂) is actively pumped into the cytosol, where it is converted by the CA hydrase activity and accumulates as HCO₃⁻, which thereafter passively diffuses into the carboxysomes, where it is re-converted to CO₂ by the CA.⁸⁸ Using the aforementioned mechanism, CO₂ is provided to RubisCO at high concentration (up to 50 mM), compensating the inefficiency of RubisCO and its low affinity for this substrate.⁸⁸

Few cyanobacterial CAs were investigated up until now. Recently, our groups reported the catalytic activity and inhibition of the β-class CA (CahB1), from the relict cyanobacterium, Coleofasciculus chthonoplastes (previously denominated Microcoleus chthonoplastes).⁸¹ The enzyme showed good activity as a catalyst for the CO₂ hydration, with a k_cat of 2.4 × 10⁵ s⁻¹ and a k_cat/K_M of 6.3 × 10⁷ M⁻¹ s⁻¹. A range of inorganic anions and small molecules were investigated as inhibitors of CahB1. The best inhibitors were N,N-diethyldithiocarbamate, sulfamate, sulfamide, phenylboronic acid and phenylarsonic acid, with K_Is in the range of 8–75 μM, whereas acetazolamide inhibited the enzyme with a K_I of 76 nM.⁸¹

Here we report the kinetic characterization of the recombinant γ-CA identified in the Antarctic cyanobacterium Nostoc commune and its inhibition profile with sulfonamides and their bioisosteres, such as sulfamates. The new Antarctic γ-CA was named NcoCA. These studies are relevant at the level of the molecular structure because Antarctic CAs probably preserve their biochemical features even though the enzyme works in an ‘extreme’ environment characterized by very low temperatures. The enzymes isolated from Antarctic organisms may thus represent a useful tool for studying the relations among structure, stability and function of proteins in organisms adapted to constantly live at low temperatures.