Biochemical, structural, and computational studies of a γ-carbonic anhydrase from the pathogenic bacterium Burkholderia pseudomallei

Anna Di Fiore; Viviana De Luca; Emma Langella; Alessio Nocentini; Martina Buonanno; Simona Maria Monti; Claudiu T Supuran; Clemente Capasso; Giuseppina De Simone

doi:10.1016/j.csbj.2022.07.033

Biochemical, structural, and computational studies of a γ-carbonic anhydrase from the pathogenic bacterium Burkholderia pseudomallei

Comput Struct Biotechnol J. 2022 Jul 27:20:4185-4194. doi: 10.1016/j.csbj.2022.07.033. eCollection 2022.

Authors

Anna Di Fiore¹, Viviana De Luca², Emma Langella¹, Alessio Nocentini³, Martina Buonanno¹, Simona Maria Monti¹, Claudiu T Supuran³, Clemente Capasso², Giuseppina De Simone¹

Affiliations

¹ Institute of Biostructures and Bioimaging-CNR, via Pietro Castellino 111, 80131 Napoli, Italy.
² Institute of Biosciences and Bioresources-CNR, via Pietro Castellino 111, 80131 Napoli, Italy.
³ NEUROFARBA Department, Pharmaceutical and Nutraceutical Section, University of Firenze, Via Ugo Schiff 6, 50019 Sesto Fiorentino, Italy.

Abstract

Melioidosis is a severe disease caused by the highly pathogenic gram-negative bacterium Burkholderia pseudomallei. Several studies have highlighted the broad resistance of this pathogen to many antibiotics and pointed out the pivotal importance of improving the pharmacological arsenal against it. Since γ-carbonic anhydrases (γ-CAs) have been recently introduced as potential and novel antibacterial drug targets, in this paper, we report a detailed characterization of BpsγCA, a γ-CA from B. pseudomallei by a multidisciplinary approach. In particular, the enzyme was recombinantly produced and biochemically characterized. Its catalytic activity at different pH values was measured, the crystal structure was determined and theoretical pKa calculations were carried out. Results provided a snapshot of the enzyme active site and dissected the role of residues involved in the catalytic mechanism and ligand recognition. These findings are an important starting point for developing new anti-melioidosis drugs targeting BpsγCA.

Keywords: BME, 2-betamercaptoethanol; BSA, Bovine Serum Albumin; BpsβCA, β-CA from B. pseudomallei; BpsγCA, γ-CA from B. pseudomallei; Burkholderia pseudomallei; CA, Carbonic Anhydrase; CA_D, γ-CA from Discovery Deep Brine Pool; CCD, Charge Coupled Device; CD, circular dichroism; Cag, γ-CA from G. kaustophilus; Cam, γ-CA from M. thermophila; Carbonic anhydrases; Crystal structure; Cyt C, horse Cytochrome C; Melioidosis; PDB, Protein Data Bank; PEG, Polyethylene glycol; PSR, proton shuttle residue; RicA, γ-CA from B. abortus; SEC, size exclusion chromatography; TeCcmM, γ-CA from T. elongatus; TtCA, γ-CA from T. thermophilus HB8; Yrda, γ-CA from E. coli; Zn-Cap, γ-CA from P. horikoshii; pKa calculations; r.s.m.d., root mean square deviation.