Crystal Structure of a Tetrameric Type II β-Carbonic Anhydrase from the Pathogenic Bacterium Burkholderia pseudomallei

Abstract

Carbonic anhydrase (CA) is a zinc enzyme that catalyzes the reversible conversion of carbon dioxide to bicarbonate and proton. Currently, CA inhibitors are widely used as antiglaucoma, anticancer, and anti-obesity drugs and for the treatment of neurological disorders. Recently, the potential use of CA inhibitors to fight infections caused by protozoa, fungi, and bacteria has emerged as a new research line. In this article, the X-ray crystal structure of β-CA from Burkholderia pseudomallei was reported. The X-ray crystal structure of this new enzyme was solved at 2.7 Å resolution, revealing a tetrameric type II β-CA with a “closed” active site in which the zinc is tetrahedrally coordinated to Cys46, Asp48, His102, and Cys105. B. pseudomallei is known to encode at least two CAs, a β-CA, and a γ-CA. These proteins, playing a pivotal role in its life cycle and pathogenicity, offer a novel therapeutic opportunity to obtain antibiotics with a different mechanism of action. Furthermore, the new structure can provide a clear view of the β-CA mechanism of action and the possibility to find selective inhibitors for this class of CAs.

Structure–Activity Relationship Studies of Acetazolamide-Based Carbonic Anhydrase Inhibitors with Activity against Neisseria gonorrhoeae

“…[23][24][25] These roles have made CAs prime drug targets as FDA-approved carbonic anhydrase inhibitors are used to treat various disorders including glaucoma, 26,27 as a diuretic for kidney health, 28 treatment for congestive heart failure, 29 and recently certain isoforms have gained momentum as promising cancer targets. [30][31][32] Aside from human targets, CAs have been identified in a handful of parasites and pathogenic bacteria, 33,34 including Vibrio cholera, 35 Burkholderia spp 36,37 and N. gonorrhoeae. In bacteria, altering bicarbonate homeostasis was revealed to perturb the proton motive force and reduce bacterial fitness.…”

“…Aside from human targets, CAs have been identified in a handful of parasites and pathogenic bacteria, , including Vibrio cholera , Burkholderia spp , , and N. gonorrhoeae . In bacteria, altering bicarbonate homeostasis was revealed to perturb the proton motive force and reduce bacterial fitness .…”

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

“…Within a general research project aimed at developing new anti-melioidosis drugs, we have undertaken a detailed characterization of these two enzymes. In particular, both enzymes were produced in Escherichia coli and kinetically characterized, showing to be moderately active in catalyzing the CO 2 hydration reaction (k cat = 1.6·10 5 s −1 , k cat /K m = 3.4·10 7 M −1 s −1 for BpsβCA and k cat = 5.3·10 5 s −1 , k cat /K m = 2.5·10 7 M −1 s −1 for BpsγCA) [38] , [39] , [40] ; furthermore their inhibition profile with different classes of molecules was deeply investigated [39] , [40] , [41] , [42] , [43] , [44] , [45] , [46] , [47] , [48] . However, while the crystallographic structure of BpsβCA was determined [38] , to date any structural information on BpsγCA is missing.…”

“…In particular, both enzymes were produced in Escherichia coli and kinetically characterized, showing to be moderately active in catalyzing the CO 2 hydration reaction (k cat = 1.6·10 5 s −1 , k cat /K m = 3.4·10 7 M −1 s −1 for BpsβCA and k cat = 5.3·10 5 s −1 , k cat /K m = 2.5·10 7 M −1 s −1 for BpsγCA) [38] , [39] , [40] ; furthermore their inhibition profile with different classes of molecules was deeply investigated [39] , [40] , [41] , [42] , [43] , [44] , [45] , [46] , [47] , [48] . However, while the crystallographic structure of BpsβCA was determined [38] , to date any structural information on BpsγCA is missing. Here we completed the characterization of this enzyme investigating on its catalytic activity at different pH values, reporting its crystallographic structure and dissecting the role of residues involved in the catalytic mechanism by means of theoretical pKa calculations.…”

Inhibition of the β-carbonic anhydrase from the protozoan pathogen Trichomonas vaginalis with sulphonamides

“…For this reason, β-CAs may represent an interesting target for finding anti-infectives with a novel mechanism of action 16 . Accordingly, Flaherty’s group 17 recently reported that sulphonamide CA inhibitors (CAIs), structurally related to acetazolamide (AAZ), have potent anti-bacterial effects against pathogenic drug resistant bacteria (which normally contain α-, β-, γ- and/or ι-CAs) 18 , 19 . They demonstrated this effect in vancomycin-resistant enterococci, validating thus in vivo the bacterial CAs as drug targets 16–17 .…”