Biochemical and structural characterisation of a protozoan beta-carbonic anhydrase from Trichomonas vaginalis

Figures & data

Figure 1. Illustration of the pBVboostFG expression vector. The designed parts of the insert: 1. attL1, 2. Shine-Dalgarno, 3. Kozak, 4. Met-Ser-Tyr-Tyr, 5. 6 × His, 6. Asp-Tyr-Asp-Ile-Pro-Thr-Thr, 7. Lys-Val, 8. CA gene of interest, 9. 2 × stop codon, 10. attL2.

Table 1. Data collection and refinement statistics

Cell parameters
Space group	P212121
Cell dimensions (Å)	a = 47.3
	b = 77.3
	c = 90.7
Number of independent molecules	2
Data collection statistics
Wavelength (Å)	1.54178
Resolution limits (Å)	41.9–2.48
Total reflections	167801
Unique reflections	12403
Redundancy	13.5
Completeness (%)	99.9 (97.9)
R-merge^a	0.152 (0.572)
Rmeas^b	0.158 (0.646)
Rpim^c	0.042 (0.291)
<I>/<σ(I)>	15.9 (2.3)
Refinement statistics
Resolution limits (Å)	41.9–2.48
Rwork^d (%)	19.8
Rfree^d (%)	25.7
r.m.s.d. from ideal geometry:
Bond lengths (Å)	0.004
Bond angles (°)	1.0
Number of protein atoms	2790
Number of water molecules	64
Average B factor (Å^2)
All atoms	24.14
Protein atoms	25.28
Waters	17.80
PDB accession code	6Y04

^a R-merge = Σ_hklΣ_i|I_i(hkl) – <I(hkl)>|/Σ_hklΣ_iI_i(hkl), where I_i(hkl) is the intensity of an observation and < I(hkl)> is the mean value for its unique reflection; summations are over all reflections.

^b Rmeas = Σ_hkl{N(hkl)/[N(hkl)-1]}^1/2xΣ_i|I_i(hkl) – <I(hkl)>|/Σ_hklΣ_iI_i(hkl).

^c Rpim= Σ_hkl{1/[N(hkl)-1]}^1/2xΣ_i|I_i(hkl) – <I(hkl)>|/Σ_hklΣ_iI_i(hkl).

^d Rwork = Σ_hklǁFo(hkl)| − |Fc(hkl)ǁ/Σ_hkl|Fo(hkl)| calculated for the working set of reflections. Rfree is calculated as for Rwork, but from data of the test set that was not used for refinement (Test Set Size (%) = 8.0). Values in parentheses are referred to the highest resolution shell (2.52–2.48 Å).

Figure 2. SDS-PAGE of purified TvaCA1 with a 6xHis-tag (lane 1) and after removal of the tag (lane 2). All the polypeptide bands shown on the gel were identified as TvaCA1 protein by MS/MS. The standard molecular weight (M_w) marker is shown on the far left.

Figure 3. Light scattering data for the assessment of the oligomeric state and size of TvaCA1. The left Y-axis shows the UV absorption intensity at 280 nm and right-angle light scattering intensity (RALS). The right Y-axis shows the M_w calculated using static LS intensity.

Table 2. Kinetic data of TvaCA1. For comparison, kinetic parameters of hCA I, hCA II, and other representative β-CA enzymes are shown.

Enzyme	kcat (s^–1)	kcat/KM (M^–1 s^–1)	Ki (AAZ) (nM)
TvaCA1	4.9 × 10^5	8.0 × 10^7	391
hCA I^40	2.0 × 10^5	5.0 × 10^7	250
hCA II^40	1.4 × 10^6	1.5 × 10^8	12
SenCA1^41	1.0 × 10^6	8.3 × 10^6	59
SenCA2^41	7.9 × 10^5	5.2 × 10^7	84
LpnCA1^42	3.4 × 10^5	4.7 × 10^7	76
LpnCA2^42	8.3 × 10^5	8.5 × 10^7	72

AAZ: acetazolamide; SenCA: Salmonella enterica β-CA; LpnCA: Legionella pneumophila β-CA.

Figure 4. (A) Ribbon representation of the TvaCA1 monomer. (B) Enlarged view of the active site, showing Zn²⁺ coordination. (C) σA-weighted |2Fo-Fc| electron density map (contoured at 1.0 σ) relative to zinc ion coordination site.

Figure 5. Dimeric structure of TvaCA1, with one monomer coloured in magenta and the other in green. The catalytic zinc ions are depicted as yellow spheres.

Table 3. β-CAs whose crystal structure has been determined

Protein name	Source	Subclass	Assembly	PDB code
PsCA	Pisum sativum	Type I	Octamer	1EKJ^43
CoCA	Coccomyxa sp.	Type I	Tetramer	3UCO^47
ScCA	Saccharomyces cerevisiae	Type I	Dimer	3EYX^46
CAS1	Sordaria macrospora	Type I	Tetramer	4O1J^55
MtCab	Methanobacterium thermoautotrophicum	Type I	Dimer	1G5C^32
Rv1284	Mycobacterium tuberculosis	Type I	Dimer	1YLK^44
HnCA	Halothiobacillus neapolitanus	Type I	Dimer	2FGY^45
CcaA	Synechocystis sp. PCC 6803	Type I	Hexamer	5SWC^54
CafC	Aspergillus fumigatus	Type I	Dimer	6JQC^56
CaNce103p	Candida albicans	Type I	Tetramer	6GWU^57
PpCA	Porphyridium purpureum	Type II	Dimer	1DDZ^48
Can2	Cryptococcus neoformans	Type II	Dimer	2W3Q^51
CAS2	Sordaria macrospora	Type II	Tetramer	4O1K^55
EcCA	Escherichia coli	Type II	Tetramer	1I6P^49
HiCA	Haemophilus influenzae	Type II	Tetramer	2A8D^50
Rv3588c	Mycobacterium tuberculosis	Type II	Dimer	1YM3^44
VchCA	Vibrio cholerae	Type II	Tetramer	5CXK^52
psCA3	Pseudomonas aeruginosa	Type II	Dimer	4RXY^53
TvaCA1	Trichomonas vaginalis	Type I	Dimer	6Y04

Figure 6. Surface representation of (A) hCA II, chosen as a representative hCA isoform, and (B) TvaCA1. Residues delimiting the rim of the active site cavity are coloured in red. The metal ions are shown as yellow spheres. It is evident that in hCA II, the active site rim is larger (approximately 15 Å × 14 Å) and more accessible than that in TvaCA1 (8 Å × 6.5 Å).

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