Anion inhibition profiles of the γ-carbonic anhydrase from the pathogenic bacterium Burkholderia pseudomallei responsible of melioidosis and highly drug resistant to common antibiotics

Article abstract of DOI:10.1016/j.bmc.2016.11.021

Burkholderia pseudomallei is a Gram-negative saprophytic bacterium responsible of melioidosis, an endemic disease of tropical and sub-tropical regions of the world. A recombinant γ-CA (BpsγCA) identified in the genome of this bacterium was cloned and puri

Full text of DOI:10.1016/j.bmc.2016.11.021

Accepted Manuscript
Anion inhibition profiles of the γ-carbonic anhydrase from the pathogenic bac-
terium Burkholderia pseudomallei responsible of melioidosis and highly drug
resistant to common antibiotics
Sonia Del Prete, Daniela Vullo, Pietro di Fonzo, Sameh M. Osman, Zeid
AlOthman, Claudiu T. Supuran, Clemente Capasso
PII:
S0968-0896(16)30963-4
DOI:
http://dx.doi.org/10.1016/j.bmc.2016.11.021
BMC 13388
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
13 October 2016
11 November 2016
12 November 2016
Please cite this article as: Del Prete, S., Vullo, D., di Fonzo, P., Osman, S.M., AlOthman, Z., Supuran, C.T., Capasso,
C., Anion inhibition profiles of the γ-carbonic anhydrase from the pathogenic bacterium Burkholderia
pseudomallei responsible of melioidosis and highly drug resistant to common antibiotics, Bioorganic & Medicinal
Chemistry (2016), doi: http://dx.doi.org/10.1016/j.bmc.2016.11.021
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers
we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and
review of the resulting proof before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Anion inhibition profiles of the γ-carbonic anhydrase from the pathogenic bacterium
Burkholderia pseudomallei responsible of melioidosis and highly drug resistant to common
antibiotics
Sonia Del Pretea,b, Daniela Vullob, Pietro di Fonzoa, Sameh M. Osman,c Zeid AlOthman, c
Claudiu T. Supuranc,d* and Clemente Capassoa*
aIstituto di Bioscienze e Biorisorse, CNR, Via Pietro Castellino 111, Napoli, Italy.
bUniversità degliStudi di Firenze, Dipartimento Di Chimica, Laboratorio di Chimica Bioinorganica,
Polo Scientifico, Via della Lastruccia 3, 50019 Sesto Fiorentino, Florence, Italy.
cDepartment of Chemistry, College of Science, King Saud University, P.O. Box 2455 Riyadh
11451, Saudi Arabia.
dUniversità degli Studi di Firenze, Dipartimento Neurofarba, Sezione di ScienzeFarmaceutiche e
Nutraceutiche, Via U. Schiff 6, 50019 Sesto Fiorentino, Florence, Italy.
Abstract. Burkholderia pseudomallei is a Gram-negative saprophytic bacterium responsible of
melioidosis, an endemic disease of tropical and sub-tropical regions of the world. A recombinant γ-
CA (BpsγCA) identified in the genome of this bacterium was cloned and purified. Its catalytic
activity and anion inhibition profiles were investigated. The enzyme was an efficient catalyst for the
CO2 hydration showing a kcat of 5.3x105 s-1 and kcat/Km of 2.5 x107 M-1 x s-1. The best BpsγCA
inhibitors were sulfamide, sulfamic acid, phenylboronic acid and phenylarsonic acid, which showed
KI in the range of 49 – 83 µM (these inhibitors showed millimolar inhibition constant against hCA
II), followed by diethyldithiocarbamate, selenate, tellurate, perrhenate, selenocyanate,
trithiocarbonate, tetraborato, pyrophosphate, stannate, carbonate, bicarbonate, azide, cyanide,
thiocyanate and cyanate with KIs in the range of 0.55–9.1 mM. In our laboratories, work is in
progress to resolve the X-ray crystal structures of BpsγCA, which may allow the development of
small molecule inhibitors with desired properties for targeting and inhibiting specifically the
bacterial over the human CAs, considering the fact that B. pseudomallei is involved in a serious
bacterial disease.
Keywords: carbonic anhydrase; metalloenzymes; γ-class; inhibitor; anion; Burkholderia
pseudomallei
_______
*Corresponding authors: Tel/Fax: +39-0816132559. E-mail: clemente.capasso@ibbr.cnr.it
(Clemente Capasso); Tel/Fax: +39-055-4573729, E-mail: claudiu.supuran@unifi.it (Claudiu T.
Supuran).

1. Introduction
Many Gram-negative and Gram-positive bacteria no longer respond to many classes of
1-2
antibiotic clinically used.
Antibiotic resistance, in fact, to all pharmacological classes represents
3
a worldwide problem. Therefore, the existence of antibiotic resistance prompts us to identify new
therapeutic targets to fight bacterial diseases. Exploring alternative pathways involved in the
inhibition of the virulence factors or enzymes/proteins fundamental for the life cycle of the
pathogenic microorganisms it has been found a class of metalloenzymes belonging to the carbonic
anhydrases superfamily (CAs, EC 4.2.1.1). These enzymes catalyze a simple but physiologically
relevant process: the hydration of carbon dioxide to bicarbonate and protons. 4-8 CAs are grouped in
seven genetically distinct families, named α-, β-, γ-, δ-, ζ-, - and ɵ-CAs, with a similar structure,
7-11, 4, 6, 12-16
an hydratase activity but a lower sequence similarity.
The - and ɵ-CAs were
discovered recently analyzing the amino acid sequences of CAs from Plasmodia and the Cys-Gly-
His-rich protein family of the pyrenoid-penetrating thylakoid of Phaeodactylum tricornutum
(marine diatom), respectively. 11, 14, 17-19 CAs are known to contain divalent metal ions (usually Zn)
at their active site but, the metal coordination patter is almost variegating. In fact, it is formed by
three His residues in the α-, γ-, δ- and ɵ-classes; one His, and two Cys residues in the β- and ζ-CAs;
two His and one Gln residues in the -CA. 7-9, 11, 20-24 Bacteria encode for enzymes belonging to the
4-6, 9, 22-23, 25-29
α-, β-, and γ-CA classes.
By catalyzing the simple but highly important hydration of
carbon dioxide to bicarbonate and protons, bacterial CAs are probably involved in critical steps of
the bacterial life cycle, which are important for survival, invasion, and pathogenicity. 4-8, 12, 30-31
The genus Burkholderia includes more than twenty species. The three main human
32
pathogens are Burkholderia mallei, Burkholderia pseudomallei and Burkholderia cepacia.
Among them, Burkholderia pseudomallei is a Gram-negative saprophytic bacterium responsible of
the melioidosis, which is an endemic disease of tropical and sub-tropical regions of the world.
Melioidosis is a disease that affects both humans and animals. B. pseudomallei is a common soil
33
inhabitants, but it thrives also in water.
Humans and animals acquire melioidosis infection
through skin inoculation or inhalation or ingestion of contaminated water and soil. In humans, the
clinical manifestation of melioidosis is represented by bacteremia with septic shock, pneumonia,
pericarditis, genitourinary infections, inflammation of parotid glands, central nervous system
33
infection, septic arthritis, abscess involving spleen, liver or adrenals.
Moreover, Burkholderia
mallei and Burkholderia pseudomallei due to their high virulence by inhalation are considered
32
potential bioterrorism agents.
Burkholderia pseudomallei is inherently resistance to penicillin,
ampicillin, first-generation and second-generation cephalosporins, macrolides, quinolones and most
34
aminoglycosides.
The limiting therapeutic options addressed us to investigate the presence of

CAs in the genome of B. pseudomallei as promising targets for obtaining antibacterials devoid of
the resistance problems. Furthermore, it was reported that the optimal proliferation temperature of
B. pseudomallei is around 40°C in neutral or slightly acidic conditions (pH 6.8–7.0), which might
be controlled by CAs, enzymes known to participate in pH homeostasis in organisms all over the
phylogenetic tree. 32
B. pseudomallei genome encodes for at least three β-CAs and one γ-CA, but not for a α-
CAs. Here, we describe the recombinant B. pseudomallei γ-CA, designated as BpsγCA, which is an
active CA for the physiologic hydration of CO2 to form bicarbonate and protons. We also
investigated the inhibition profiles of BpsγCA and compared it with those obtained for human CAs
(α-CAs) in the presence of inorganic anions and other small molecules known to interfere with the
activity of metalloenzymes. The organic anions are less efficient inhibitors when compared to the
sulfonamides, but their study is important for understanding the inhibition/catalytic mechanisms
BpsγCA. It will be possible to design new types of inhibitors, which may have clinical applications.
2. Results and Discussion
2.1. Purification, sequence analysis and phylogeny.
IPTG induction of Escherichia coli BL21 (DE3) cells transformed with the plasmid pET-
100/BpsγCA resulted in the production of the recombinant BpsγCA as a fusion protein with a
HisTag tail. It was isolated and purified to homogeneity from E. coli (DE3) cell extract. Most of the
CA activity was recovered in the soluble fraction of cell extract after sonication and centrifugation.
Using the affinity column (His-select HF Nickel affinity gel), BpsγCA was purified to apparent
homogeneity, as indicated by a single protein band after SDS-PAGE (Figure 1). A subunit with an
apparent molecular weight of about 23 kDa was estimated by SDS-PAGE, while the recombinant
BpsγCA fused to the HisTag tail had a theoretical molecular mass of 22kDa as calculated on the
basis of the amino acid composition deduced from the nucleotide sequence. It is know that γ-CAs
catalyze the hydration of carbon dioxide to bicarbonate and protons when the γ-CAs monomers
assemble into a trimer to form a triangular motif with a real molecular weight of 66 kDa. The
apparent molecular weight of 23 kDa on the SDS-PAGE is due to the SDS concentration, which
determines the separation of the trimeric state of the protein allowing the migration of the enzyme
as a monomer (see Figure 1).

Figure 1. SDS-PAGE of the recombinant BpsγCA overexpressed in E. coli. Lane STD: molecular
markers; lane 1: purified BpsγCA from His-tag affinity column.
The full nucleotide sequence encoding for BpsγCA without the HisTag showed an open-reading
frame of 174 residues polypeptide chain (see Figure 2).
Figure 2. Nucleotide and amino acid sequence of the BpsγCA. Legend: lower case are the
nucleotides; upper case are the amino acid residues; * indicate the stop codon
In Figure 3 we aligned the amino acid sequence of BpsγCA and compared it with those of
Porphyromonas gingivalis (PgiCAγ), Nostoc commune (NcoCA, Antarctic γ-CA), CAM and
CAMH, which belong to the γ-CA class and were isolated from the archaeon Metanosarcina
thermophila. Data of Figure 3 show that the three His residues involved in the Zn(II) coordination

(His81, 117 and 122, CAM numbering system) are conserved in BpsγCA, similar to all other γ-
class CAs. In the γ-CAs, or at least for CAM, the residue acting as a proton shuttle seems to be a
glutamic acid (Glu84, see Figure 3).
Figure 3. Multialignment of the amino acid sequences of γ-CAs from BpsγCA, B. pseudomallei;
PgiCAγ, P. gingivalis; CAMH, M. thermophila; CAM, M. thermophila; NcoCA, Nostoc commune.
Multialignment was performed with the program Muscle. The zinc ligands His81, 117 and 122 are
indicated in bold and are conserved in all these enzymes. The residue acting as a proton shuttle
(Glu84) is colored in red. The CAMH numbering system was used. The asterisk (*) indicates
identity at all aligned positions; the symbol (:) relates to conserved substitutions, while (.) means
that semi-conserved substitutions are observed.
Using PhylML, a phylogenetic analysis was carried out to better understand the relationships of the
BpsγCA with other γ-CA identified in mesophilic, thermophilic and psychrophilic bacteria. As
shown in Figure 4, the dendrogram shows two distinct and well-separated clusters: one formed by
Cam and CAMH and the other made by all the other γ-CAs. BpsCAγ appears very similar to the γ-
CA identified in pathogen Brucella suis (bsCA). Intriguingly, with the exception of CAM and
CAMH, BpsCA and bsCA formed a cluster well separated from the group containing the Antarctic,
the mesophilic and thermophilic γ-CAs. From these results, it seems that, apparently, BpsCA
belongs to a subclass of γ-CAs different from those forming the cluster CAM and CAMH and also

from those developing the cluster, which include the remaining γ-CAs considered in this
phylogenetic analysis.
Figure 4. Phylogenetic analysis constructed using the amino acid sequences of the γ-CAs from different
species of bacteria, such as Burkholderia pseudomallei (BpsCA, mesophilic γ-CA); Brucella suisꢀ (bsCA,
mesophilic γ-CA); Porphyromonas gingivalis (PgiCA, mesophilic γ-CA); Methanosarcina thermophila
(CAMH, thermophilic γ-CA); Methanosarcina thermophila (CAM, thermophilic γ-CA); Nostoc commune
(NcoCA, Antarctic γ-CA), Sulfurihydrogenibium yellowstonense (SspCA, thermophilic γ-CA);
Sulfurihydrogenibium azorense (SazCA, thermophilic γ-CA); Escherichia coliꢀ (EcoCA); Vibrio cholerae
(VchCA, mesophilic γ-CA); Pseudoalteromonas haloplanktis (PhaCA, Antarctic γ-CA)
2.2. Kinetic properties of BpsγCA
We compared the catalytic efficiency of recombinant BpsγCA for the physiologic reaction, CO2
hydration to bicarbonate and protons, and compared the kinetic parameters of this new enzyme with
those of other CAs belonging to the - or γ-CA classes, identified and characterized in other
organisms such as H. sapiens (hCA I and II, the physiologically dominant isoforms) and Archaea or
7, 39-43
Bacteria among which Methanosarcina thermophila,35-38 Porphyromonas gingivalis,
Nostoc
23, 44-45
commune,
and Pseudoalteromonas haloplanktis 46-47 (Table 1). Data of Table 1 show that
BpsγCA possess a moderate but significant CO2 hydrase activity, comparable to that of the
widespread and physiologically relevant human isoform hCA I, or the bacterial γ-CAs from various
pathogenic or non-pathogenic bacteria (e.g., Porphyromonas gingivalis, an oral cavity pathogenic
bacterium). The kinetic parameters for the new enzyme were: kcat of 5.3x105 s-1 and kcat/Km of 2.5
x107 M-1x s-1(Table 1). Furthermore, this activity was inhibited (KI of 149 nM) by the clinically
used sulfonamide inhibitor acetazolamide (5-acetamido-1,3,4-thiadiazole-2-sulfonamide).

Table 1. Kinetic parameters for the CO2 hydration reaction catalysed by the human cytosolic
isozymes hCA I and II (-class CAs) at 20 °C and pH 7.5 in 10 mM HEPES buffer and 20 mM
Na2SO4, and the -CAs CAM (Methanosarcina thermophila), PgiCA (P. gingivalis), NcoCA
(Nostoc commune), PhaCAγ (P. haloplanktis) and BpsγCA (B. pseudomalleii) measured at 20 °C,
pH 8.3 in 20 mM TRIS buffer and 20 mM NaClO4.48 Acetazolamide inhibition data are also shown.
Enzyme
Activity level Class kcat
(s-1)
kcat/Km
(M-1x s-1)
KI (acetazolamide)
(nM)
hCA I
moderate
very high
low
α
α
γ
γ
γ
2.0x105
1.4x106
6.1 x104
4.1x105
9.5 x105
1.4x105
5.3x105
5.0x107
1.5x108
8.7 x105
5.4 x107
8.3 x107
1.9 x106
2.5 x107
250
12
hCA II
CAM
63
PgiCA
NcoCA
PhaCAγ
BpsγCA
moderate
high
324
75.8
403
149
medium-low γ
moderate
γ
2.3. Anion inhibition profiles of BpsγCA
Inorganic anions constitute an important class of CA inhibitors (CAIs), which were
extensively investigated for their interaction with enzymes belonging to all classes, including the
bacterial γ-CAs. 45, 47, 49-53 Such data may be relevant for understanding details of the environment in
which these organisms evolved and may thrive, but also for designing pharmacologically useful
inhibitors (although generally the inorganic anions are weak – medium potency CAIs).54 Here, we
report a comparison of the anion inhibition profiles of the recombinant BpsγCA identified in the
genome of B. pseudomallei with those obtained for the human α-CA isoform (hCA II) and for the
bacterial γ-CAs identified in pathogenic and non pathogenic bacteria (Table 2). In the study were
included a wide range of anionic inhibitors, including simple and complex inorganic anions as well
as sulfamide, sulfamate, phenylboronic acid and phenylarsonic acid (Table 2).
Table 2. Inhibition constants of anionic inhibitors against the -CA isoform hCA II, and the -CAs
CAM, PgiCA, NcoCA, PhaCAγ, and BpsγCA for the CO2 hydration reaction, at 20 °C.48

Inhibitor§
KI [mM]#
PgiCA
hCA II
CAM
NcoCA PhaCAγ BpsγCA
F-
Cl-
Br-
I-
CNO-
SCN-
CN-
>300
200
63
>200
>200
160
160
0.09
7.0
0.68
5.8
42
0.95
0.94
0.92
8.7
>100
8.7
>100
>100
8.7
>100
>100
>100
>100
2.7
9.3
26
8.9
>100
1.7
0.03
1.6
0.71
0.093
0.041
0.073
0.96
0.89
8.5
6.0
8.1
9.3
4.6
0.02
1.5
6.7
5.9
7.6
-
N3
30.5
4.5
>100
6.2
9.1
-
HCO3
85
4.2
2-
CO3
73
6.7
36.5
6.8
0.05
11.7
>200
nt
2.4
2.5
8.3
-
NO3
35
27.6
8.8
5.6
24.1
6.8
-
NO2
HS-
63
3.1
6.7
0.04
89
0.092
9.3
20.4
14.9
>100
5.4
8.4
3.6
-
HSO3
24.1
>100
6.7
>100
>100
4.9
2-
SO4
>200
0.83
112
0.92
48.50
0.57
0.95
0.75
0.69
0.084
0.086
0.0088
3.1
8.7
2-
SnO3
10.8
85.5
16.7
78.9
18.5
71.6
0.93
5.3
2-
SeO4
nt
8.5
6.1
3.1
2-
TeO4
nt
7.7
8.5
4.4
4-
P2O7
nt
9.1
>100
>100
8.0
5.1
4-
V2O7
nt
7.6
>100
7.7
2-
B4O7
nt
6.4
-
ReO4
nt
8.8
>100
8.7
9.0
-
RuO4
nt
7.6
17.9
>100
3.9
2-
S2O8
SeCN-
nt
8.7
9.5
9.1
nt
0.91
0.097
0.004
>200
> 200
89.5
nt
8.0
7.8
2-
CS3
nt
8.9
34.6
0.96
>100
>100
7.7
3.3
-
Et2NCS2
nt
0.80
>100
>100
9.5
0.55
> 100
> 100
>100
12.6
15.7
-
ClO4
>200
>200
0.46
nt
nt
-
BF4
nt
-
FSO3
nt
-
PF6
nt
7.4
>100
>100
CF3SO3-
nt
nt
nt
7.3

2-
NH(SO3)2
0.76
1.13
0.39
23.1
49.2
nt
nt
nt
nt
nt
7.7
6.9
6.5
2.5
H2NSO2NH2
H2NSO3H
0.0092
0.093
0.0098
0.090
0.081
0.090
0.070
0.084
0.082
0.091
0.088
0.087
0.077
0.083
0.074
0.049
Ph-B(OH)2
Ph-AsO3H2
§As sodium salt; nt = not tested.
# Errors were in the range of 3-5 % of the reported values, from three different assays.
The following was noted on the inhibition data of Table 2:
i)
The most efficient inhibitors of BpsγCA were sulfamide, sulfamic acid, phenylboronic acid
and phenylarsonic acid, with showed KI in the range of 49 – 83 µM. It should be observed
that these were also the best bacterial γ-CA inhibitors (except for CAM, which was not
investigated with these anions) when compared with the human enzyme.
ii)
Anions acting as weak inhibitors, with inhibition constants in the range of 2.5-9.1 mM, were
selenate, tellurate, perrhenate, selenocyanate, trithiocarbonate, tetraborato, pyrophosphate,
stannate, carbonate, bicarbonate, azide, cyanide, thiocyanate and cyanate. Some of these
anions (e.g., bicarbonate, catbonate and pyrophosphate) act as much stronger inhibitors of
BpsγCA compared to the widespread human isoform hCA II. On the other hand, only
diethyldithiocarbamate had a submillimolar inhibition constant against BpsγCA among the
investigated anions (except the four ones mentioned above), with a KI of 0.55 mM.
Weaker inhibitory activity against BpsγCA was observed for nitrate, perrhuthenate,
hexafluorophosphate and trifluoromethanesulfonate (KI of 12.6 – 24.1 mM).
iii)
iv)
Fluoride, chloride, bromide, iodide, perchlorate, tetrafluoroborate, bisulfite, sulfate,
divanadate, peroxydisulfate anions known for their low affinity for metal ions in
metalloenzymes (and sometimes also in solution) were not inhibitory against BpsγCA (KI >
-
100 mM), a behavior that they did not show against all bacterial γ-CAs. For example, SO4
had a KI of 8.7 mM for the recombinant γ-CA (PgiCA) identified in pathogen P. gingivalis.
Bromide and iodide act as week inhibitors with KI in the range of 0.92-9.3 mM for NcoCA
(Antarctic γ-CA) and PgiCA.
v)
The overall anion inhibition profile of BpsγCA and of the other γ-CA is rather different
from that obtained from the human isoform hCA II belonging to the α-CA class. This aspect
represents a good chance of a rational drug design for the production of selective inhibitors
over the human isoforms.

3. Conclusion
We cloned and purified the recombinant γ-CA (BpsγCA) identified in the genome of the Gram-
negative bacteria B. pseudomallei. Its catalytic activity and anion inhibition profiles were
investigated. The enzyme was an efficient catalyst for CO2 hydration showing a kcat of 5.3x105 s-1
and kcat/Km of 2.5 x107 M-1 x s-1. The best BpsγCA inhibitors were sulfamide, sulfamic acid,
phenylboronic acid and phenylarsonic acid, which showed KI in the range of 49 – 83 µM, followed
by diethyldithiocarbamate, selenate, tellurate, perrhenate, selenocyanate, trithiocarbonate,
tetraborato, pyrophosphate, stannate, carbonate, bicarbonate, azide, cyanide, thiocyanate and
cyanate with a KIs in the range of 0.55–9.1 mM. In our laboratories, work is in progress to resolve
the X-ray crystal structures of BpsγCA, which may allow the development small molecule
inhibitors with desired properties for targeting and inhibiting specifically the bacterial over the
human CAs, considering the fact that B. pseudomallei is involved in a serious bacterial disease.
4. Experimental
4.1. Gene Identification
The identification of the gene encoding for B. pseudomallei γ-CA (BpsγCA) was performed at the
link http://www.ncbi.nlm.nih.gov/ genome/selecting the genome of ‘‘B. pseudomallei’’. The γ-CA
gene of B. pseudomallei (accession number: WP_038762492.1) was identified running the
‘‘BLAST’’ program and using the nucleotide sequences of bacterial γ-CAs as query sequence.
4.2. Cloning, expression and purification of the recombinant BpsγCA
The GeneArt Company (Invitrogen), specialized in gene synthesis, designed the synthetic BpsγCA
gene (BpsγCA-DNA) encoding for the BpsγCA (β-CA of 358 amino acid residues) containing four
base-pair sequences (CACC) necessary fordirectional cloning at the 5' end of the PfCAdom gene.
The recovered PfCAdom gene and the linearized expression vector (pET-100/D-TOPO) were
ligated by T4 DNA ligase to form the expression vector pET-100/BpsγCA. Arctic Express DE3
competent cells (Agilent) were transformed with pET-100/BpsγCA, grown at 20 °C and induced
with 1 mM IPTG. After 30 min was added ZnSO4 (0.5 mM) to the culture medium and cells were
growth for additional 6 h. Subsequently, cells were harvested and resuspended in the following
buffer: 50 mM Tris/HCl, pH 8.0, 0.5 mM PMSF, and 1 mM benzamidine. Cells were then disrupted
by sonication at 4 °C. After centrifugation at 12,000×g for 45 min, the supernatant was incubated
with His Select HF nickel affinity gel resin (Sigma) equilibrated in lysis buffer for 30 min.
Following centrifugation at 2,000xg, the resin was washed in wash buffer (50 mM Tris/HCl, pH

8.0, 500 mM KCl, 20 mM imidazole). The protein was eluted with the wash buffer containing 200
mM imidazole. Collected fractions were dialyzed against 50 mM Tris/HCl, pH 8.0. At this stage of
purification the protein was at least 85% pure and the obtained recovery was of 0.1 mg of the
recombinant protein.
4.3. SDS-PAGE
Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) was performed as
described previously, using 12% gels. 55
4.4. Amino acid and phylogenetic analysis
Multialignment of amino acid sequences was performed using the program MUSCLE,56 a new
computer program for creating multiple alignments of protein sequence. A phylogenetic tree was
constructed using the program PhyML 3.0 searching for the tree with the highest probability. 57
4.5. BpsγCA kinetic and inhibition assay
An Applied Photophysics stopped-flow instrument has been used for assaying the CA catalyzed
48
CO hydration activity . Phenol red (at a concentration of 0.2 mM) has been used as indicator,
2
working at the absorbance maximum of 557 nm, with 20 mM TRIS (pH 8.3) as buffer, and 20 mM
NaClO (for maintaining constant the ionic strength), following the initial rates of the CA-catalyzed
4
CO hydration reaction for a period of 10-100 s. The CO concentrations ranged from 1.7 to 17
2
2
mM for the determination of the kinetic parameters (by Lineweaver-Burk plots) and inhibition
constants. For each inhibitor at least six traces of the initial 5-10% of the reaction have been used
for determining the initial velocity. The uncatalyzed rates were determined in the same manner and
subtracted from the total observed rates. Stock solutions of inhibitor (10 -100 mM) were prepared in
distilled-deionized water and dilutions up to 0.01 mM were done thereafter with the assay buffer.
Inhibitor and enzyme solutions were preincubated together for 15 min at room temperature prior to
assay, in order to allow for the formation of the E-I complex or for the eventual active site mediated
hydrolysis of the inhibitor. The inhibition constants were obtained by non-linear least-squares
methods using PRISM 3 and the Cheng-Prusoff equation, as reported earlier, and represent the
mean from at least three different determinations. All CA isoforms were recombinant ones obtained
in-house. All salts/small molecules were of the highest purity available, from Sigma-Aldrich
(Milan, Italy).
Acknowledgments

This work was supported in part by an FP7 European Union Project (Gums & Joints, Grant
agreement Number HEALTH-F2-2010-261460) and by a Distinguished Scientist Fellowship
Program (DSFP) from King Saud University, Saudi Arabia.
References and note
1. Miller, W.R.; Bayer, A.S.; Arias, C.A. Cold Spring Harb Perspect Med 2016, doi:
10.1101/cshperspect.a026997
2. Blaser, M.J. Science 2016, 352, 544.
3. Arenz, S.; Wilson, D.N. Mol Cell 2016, 61, 3.
4. Capasso, C.; Supuran, C.T. Expert Opin Ther Targets 2015, 19, 1689.
5. Capasso, C.; Supuran, C.T. Curr Med Chem 2015, 22, 2130.
6. Capasso, C.; Supuran, C.T. J Enzyme Inhib Med Chem 2015, 30, 325.
7. Capasso, C.; Supuran, C.T. Curr Top Med Chem 2016, 16, 2359.
8. Ozensoy Guler, O.; Capasso, C.; Supuran, C.T. J Enzyme Inhib Med Chem 2016, 31, 689.
9. Supuran, C.T.; Capasso, C. J Enzyme Inhib Med Chem 2016, 31, 1254.
10. Del Prete, S.; Vullo, D.; De Luca, V.; Carginale, V.; Ferraroni, M.; Osman, S.M.; AlOthman, Z.;
Supuran, C.T.; Capasso, C. Bioorg Med Chem 2016, 24, 1115.
11. Del Prete, S.; De Luca, V.; De Simone, G.; Supuran, C.T.; Capasso, C. J Enzyme Inhib Med
Chem 2016, 1.
12. Supuran, C.T.; Capasso, C. Expert Opin Ther Targets 2015, 19, 551.
13. Del Prete, S.; De Luca, V.; Supuran, C.T.; Capasso, C. J Enzyme Inhib Med Chem 2015, 30,
920.
14. De Simone, G.; Di Fiore, A.; Capasso, C.; Supuran, C.T. Bioorg Med Chem Lett 2015, 25, 1385.
15. De Luca, V.; Del Prete, S.; Supuran, C.T.; Capasso, C. J Enzyme Inhib Med Chem 2015, 30,
277.
16. Alafeefy, A.M.; Abdel-Aziz, H.A.; Vullo, D.; Al-Tamimi, A.M.; Awaad, A.S.; Mohamed, M.A.;
Capasso, C.; Supuran, C.T. J Enzyme Inhib Med Chem 2015, 30, 52.
17. Kikutani, S.; Nakajima, K.; Nagasato, C.; Tsuji, Y.; Miyatake, A.; Matsuda, Y. Proc Natl Acad
Sci U S A 2016, 113, 9828.
18. Del Prete, S.; Vullo, D.; De Luca, V.; Carginale, V.; Osman, S.M.; AlOthman, Z.; Supuran, C.T.;
Capasso, C. Bioorg Med Chem Lett 2016, 26, 4184.
19. Del Prete, S.; Vullo, D.; Fisher, G.M.; Andrews, K.T.; Poulsen, S.A.; Capasso, C.; Supuran, C.T.
Bioorg Med Chem Lett 2014, 24, 4389.
20. Vullo, D.; Del Prete, S.; Capasso, C.; Supuran, C.T. Bioorg Med Chem Lett 2016, 26, 1381.
21. Del Prete, S.; De Luca, V.; Vullo, D.; Osman, S.M.; AlOthman, Z.; Carginale, V.; Supuran, C.T.;
Capasso, C. J Enzyme Inhib Med Chem 2016, 31, 1156.
22. Del Prete, S.; De Luca, V.; Capasso, C.; Supuran, C.T.; Carginale, V. Bioorg Med Chem 2016,
24, 220.
23. De Luca, V.; Del Prete, S.; Vullo, D.; Carginale, V.; Di Fonzo, P.; Osman, S.M.; AlOthman, Z.;
Supuran, C.T.; Capasso, C. J Enzyme Inhib Med Chem 2016, 31, 810.
24. Annunziato, G.; Angeli, A.; D'Alba, F.; Bruno, A.; Pieroni, M.; Vullo, D.; De Luca, V.; Capasso,
C.; Supuran, C.T.; Costantino, G. ChemMedChem 2016, 11, 1904.
25. Prete, S.D.; Vullo, D.; Osman, S.M.; Scozzafava, A.; AlOthman, Z.; Capasso, C.; Supuran, C.T.
Bioorg Med Chem 2014, 22, 4537.
26. Nishimori, I.; Vullo, D.; Minakuchi, T.; Scozzafava, A.; Osman, S.M.; AlOthman, Z.; Capasso,
C.; Supuran, C.T. Bioorg Med Chem Lett 2014, 24, 1127.
27. Nishimori, I.; Vullo, D.; Minakuchi, T.; Scozzafava, A.; Capasso, C.; Supuran, C.T. Bioorg Med
Chem 2014, 22, 2939.

28. Migliardini, F.; De Luca, V.; Carginale, V.; Rossi, M.; Corbo, P.; Supuran, C.T.; Capasso, C. J
Enzyme Inhib Med Chem 2014, 29, 146.
29. Alafeefy, A.M.; Abdel-Aziz, H.A.; Vullo, D.; Al-Tamimi, A.M.; Al-Jaber, N.A.; Capasso, C.;
Supuran, C.T. Bioorg Med Chem 2014, 22, 141.
30. Capasso, C.; Supuran, C.T. J Enzyme Inhib Med Chem 2014, 29, 379.
31. Capasso, C.; Supuran, C.T. Expert Opin Ther Pat 2013, 23, 693.
32. Gilad, J.; Schwartz, D.; Amsalem, Y. Int J Biomed Sci 2007, 3, 144.
33. Stephens, D.P.; Thomas, J.H.; Ward, L.M.; Currie, B.J. Crit Care Med 2016, 44, 1500.
34. Jabbar, Z.; Currie, B.J. Nephrology (Carlton) 2013, 18, 169.
35. Zimmerman, S.; Domsic, J.F.; Tu, C.; Robbins, A.H.; McKenna, R.; Silverman, D.N.; Ferry, J.G.
Arch Biochem Biophys 2013, 529, 11.
36. Innocenti, A.; Zimmerman, S.; Ferry, J.G.; Scozzafava, A.; Supuran, C.T. Bioorg Med Chem
Lett 2004, 14, 3327.
37. Tripp, B.C.; Ferry, J.G. Biochemistry 2000, 39, 9232.
38. Iverson, T.M.; Alber, B.E.; Kisker, C.; Ferry, J.G.; Rees, D.C. Biochemistry 2000, 39, 9222.
39. Alafeefy, A.M.; Ceruso, M.; Al-Tamimi, A.M.; Del Prete, S.; Supuran, C.T.; Capasso, C. J
Enzyme Inhib Med Chem 2015, 30, 592.
40. Vullo, D.; Del Prete, S.; Osman, S.M.; De Luca, V.; Scozzafava, A.; Alothman, Z.; Supuran, C.T.;
Capasso, C. Bioorg Med Chem Lett 2014, 24, 240.
41. Del Prete, S.; De Luca, V.; Vullo, D.; Scozzafava, A.; Carginale, V.; Supuran, C.T.; Capasso, C. J
Enzyme Inhib Med Chem 2014, 29, 532.
42. Ceruso, M.; Del Prete, S.; AlOthman, Z.; Osman, S.M.; Scozzafava, A.; Capasso, C.; Supuran,
C.T. Bioorg Med Chem Lett 2014, 24, 4006.
43. Del Prete, S.; Vullo, D.; De Luca, V.; Carginale, V.; Scozzafava, A.; Supuran, C.T.; Capasso, C.
Bioorg Med Chem Lett 2013, 23, 4067.
44. Vullo, D.; De Luca, V.; Del Prete, S.; Carginale, V.; Scozzafava, A.; Capasso, C.; Supuran, C.T.
Bioorg Med Chem 2015, 23, 1728.
45. De Luca, V.; Del Prete, S.; Carginale, V.; Vullo, D.; Supuran, C.T.; Capasso, C. Bioorg Med
Chem Lett 2015, 25, 4970.
46. Vullo, D.; De Luca, V.; Del Prete, S.; Carginale, V.; Scozzafava, A.; Capasso, C.; Supuran, C.T.
Bioorg Med Chem Lett 2015, 25, 3550.
47. De Luca, V.; Vullo, D.; Del Prete, S.; Carginale, V.; Scozzafava, A.; Osman, S.M.; AlOthman, Z.;
Supuran, C.T.; Capasso, C. Bioorg Med Chem 2015, 23, 4405.
48. Khalifah, R.G. J Biol Chem 1971, 246, 2561.
49. Vullo, D.; Del Prete, S.; De Luca, V.; Carginale, V.; Ferraroni, M.; Dedeoglu, N.; Osman, S.M.;
AlOthman, Z.; Capasso, C.; Supuran, C.T. Bioorg Med Chem Lett 2016, 26, 1406.
50. Del Prete, S.; Vullo, D.; De Luca, V.; Carginale, V.; di Fonzo, P.; Osman, S.M.; AlOthman, Z.;
Supuran, C.T.; Capasso, C. Bioorg Med Chem 2016, 24, 3413.
51. De Luca, V.; Vullo, D.; Del Prete, S.; Carginale, V.; Osman, S.M.; AlOthman, Z.; Supuran, C.T.;
Capasso, C. Bioorg Med Chem 2016, 24, 835.
52. Vullo, D.; Del Prete, S.; Osman, S.M.; Scozzafava, A.; Alothman, Z.; Supuran, C.T.; Capasso, C.
Bioorg Med Chem Lett 2014, 24, 4402.
53. Del Prete, S.; Vullo, D.; Scozzafava, A.; Capasso, C.; Supuran, C.T. Bioorg Med Chem 2014, 22,
531.
54. Alterio, V.; Di Fiore, A.; D'Ambrosio, K.; Supuran, C.T.; De Simone, G. Chem Rev 2012, 112,
4421.
55. Laemmli, U.K. Nature 1970, 227, 680.
56. Edgar, R.C. Nucleic Acids Res 2004, 32, 1792.
57. Guindon, S.; Lethiec, F.; Duroux, P.; Gascuel, O. Nucleic Acids Res 2005, 33, W557.

Anion inhibition profiles of the γ-carbonic anhydrase from the pathogenic bacterium
Burkholderia pseudomallei responsible of melioidosis and highly drug resistant to common
antibiotics
Sonia Del Pretea,b, Daniela Vullob, Pietro di Fonzoa, Sameh M. Osman,c Zeid AlOthman, c
Claudiu T. Supuranc,d* and Clemente Capassoa*
bacterial CA
(BpsγCA)
KI= 84µM
KI= 87µM
KI= 81µM
KI= 23mM
KI= 91µM
human CA
(hCA II)
KI= 1.13mM
KI= 0.39mM
KI= 49.2mM