2′-Deoxyribosyltransferase from Bacillus psychrosaccharolyticus: A Mesophilic-Like Biocatalyst for the Synthesis of Modified Nucleosides from a Psychrotolerant Bacterium
Abstract
:1. Introduction
2. Results
2.1. Biochemical Characterization of Recombinant BpNDT
2.2. Substrate Specificity
2.3. Structural Analysis of BpNDT
2.4. Active Site of BpNDT
2.5. Enzymatic Production of Nucleoside Analogues
3. Discussion
4. Materials and Methods
4.1. Chemicals
4.2. Cloning, Sequencing and Tagging of BpNDT
4.3. Production and Purification of Recombinant His-Tag BpNDT
4.4. Site Directed Mutagenesis of His-tag BpNDT
4.5. N-Deoxyribosyltransferase Assay
4.6. Thermal Inactivation Studies
4.7. Effect of Ionic Strength and Cations on Enzyme Activity
4.8. Analytical Ultracentrifugation Analysis
4.9. Enzyme Crystallization and Data Collection
4.10. Structure Solution and Refinement
4.11. Spectroscopic Studies
4.12. Differential Scanning Calorimetry Studies
4.13. Kinetic Studies
4.14. Accession Number
5. Conclusions
Acknowledgments
Author Contributions
Conflicts of Interest
References
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Additive | Relative Activity (%) at 1 mM | Relative Activity (%) at 5 mM |
---|---|---|
None | 100 | 100 |
K2SO4 | 105.8 | 96.1 |
KCl | 105.1 | 104.8 |
LiCl | 102.0 | 100 |
Na2SO4 | 97.1 | 95.7 |
NaCl | 96.6 | 98.2 |
RbCl | 95.9 | 101.8 |
BaCl2 | 98.1 | 91.8 |
CaCl2 | 84.0 | 74.7 |
CoCl2 | 33.0 | 21.8 |
CuSO4 | 63.7 | 22.3 |
MgSO4 | 86.6 | 95.7 |
MgCl2 | 88.5 | 90.1 |
MnCl2 | 32.6 | 8.1 |
ZnSO4 | 91.5 | 57.2 |
(NH4)2SO4 | 103.8 | 98.8 |
2-mercaptoethanol | 98.9 | 97.5 |
Al2(SO4)3 | 90.7 | 18.2 |
FeCl3 | 95.6 | 114.8 |
EDTA | 101.3 | 110.8 |
Temperature (°C) | kd (h−1) | t1/2 (h) |
---|---|---|
60 | 0.032 | 21.8 |
70 | 0.118 | 5.9 |
Donor | Specific Activity (IU/mg Protein) with Acceptor | ||||
---|---|---|---|---|---|
Ade | Ura | Cyt | Thy | Hyp | |
dAdo | -- | 24.4 | 36.8 | 26.1 | 43.5 |
dUrd | 40.0 | -- | 45.4 | 24.9 | 41.7 |
dCyd | 61.2 | 60.0 | -- | 47.8 | 84.6 |
dThd | 51.0 | 26.1 | 31.3 | -- | 38.3 |
dIno | 20.7 | 15.4 | 18.0 | 40.6 | -- |
dGuo | 38.8 | 16.2 | 36.3 | 8.1 | 22.4 |
BpNDT | |
---|---|
PDB code | 6EVS |
Data collection | |
Synchrotron source | ESRF |
Beamline | ID29 |
Wavelength (Å) | 0.9792 |
Space group | R3 |
Unit-cell parameters | a = b = 107.55, c = 61.24 α = β = 90°, γ = 120° |
Resolution range (Å) | 37.07–1.90 |
No. of measured reflections a | 108,568 (14,833) |
No. of unique reflections | 20,796 (3009) |
Mean (I/σI) | 14.8 (2.9) |
Completeness (%) | 100 (100) |
Multiplicity | 5.2 (4.9) |
Rmeas (%); Rpim (%) | 5.7 (59.8); 2.5 (26.7) |
CC1/2 | 0.988 (0.2845) |
B-factor (Wilson plot, Å2) | 32.7 |
Molecules/non-H atoms | |
Protein | 2/2257 |
Water | 104/104 |
Refinement statistics | |
Rwork(%)/Rfree(%) | 17.7/21.6 |
Average B-factors (Å2) | |
protein | 47.3 |
water | 47.2 |
Rms deviation bond length (Å) | 0.009 |
Rms deviation angles (°) | 0.927 |
Ramachandran | |
Favoured (%) | 95.6 |
Disallowed (%) | 1.48 |
Mutation | None | Tyr5Phe | Tyr5His | Gln40Glu | Gln40Lys | Asp59Asn | Asp59His | Asp79Asn |
---|---|---|---|---|---|---|---|---|
Relative activity (%) | 100 | 1.17 | 1.38 | 0 | 5.91 | 5.8 | 0 | 0 |
Mutation | Asp79His | Glu85Asp | Glu85Gln | Glu85His | Asn107Asp | Asn107His | Lys142Tyr | ΔK142 |
Relative activity (%) | 0 | 0 | 0 | 0 | 0 | 2.12 | 97.72 | 21.5 |
Acceptor | Specific Activity, IU/mg Protein (Conversion, %) with Donor: | |
---|---|---|
dUrd | dCyd | |
5-Azacytosine (5-Acyt) | 4.2 (42) | ND |
Benzimidazole (B) | 7 (75) | 6.2 (68) |
5-Ethyluracil (5-Eura) | 3.6 (40) | 3.5 (40) |
2-Fluoroadenine (2-FAde) | 7.5 (86) | 6.2 (72) |
5-(trifluoromethyl)pyrimidine-2,4(1H,3H)-dione (TFThy) | 4.2 (42) | 3.5 (35.5) |
2,6-Diaminopurine (DAP) | 8.9 (89) | 7.4 (82) |
6-Mercaptopurine (6-M) | 5.5 (55) | 4 (40) |
5-Chlorouracil (5-ClUra) | 3.8 (38) | 3.5 (35) |
5-Fluorocytosine (5-FCyt) | ND | 5 (50) |
5-Fluoro-2-methoxy-4(1H)pyrimidinone (FMP) | 1.15 (18) | 0.4 (5) |
5-Fluorouracil (5-FUra) | 4.0 (40) | 4.0 (40) |
5-Bromouracil (5-BrUra) | 0.5 (3.6) | 1.0 (3.5) |
5-Iodouracil (5-IUra) | 4.1 (41) | 3.9 (39) |
7-Deaza-6-hydroxypurine (DHP) | 1.6 (15) | 1.6 (16) |
5-Hydroxymethyluracil (5-HMUra) | ND | 4 (42) |
5-Methylcytosine (5-MCyt) | 0.9 | 0.7 |
Theophylline (Teo) | 4.5 (45) | 4.2 (42) |
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Fresco-Taboada, A.; Fernández-Lucas, J.; Acebal, C.; Arroyo, M.; Ramón, F.; De la Mata, I.; Mancheño, J.M. 2′-Deoxyribosyltransferase from Bacillus psychrosaccharolyticus: A Mesophilic-Like Biocatalyst for the Synthesis of Modified Nucleosides from a Psychrotolerant Bacterium. Catalysts 2018, 8, 8. https://doi.org/10.3390/catal8010008
Fresco-Taboada A, Fernández-Lucas J, Acebal C, Arroyo M, Ramón F, De la Mata I, Mancheño JM. 2′-Deoxyribosyltransferase from Bacillus psychrosaccharolyticus: A Mesophilic-Like Biocatalyst for the Synthesis of Modified Nucleosides from a Psychrotolerant Bacterium. Catalysts. 2018; 8(1):8. https://doi.org/10.3390/catal8010008
Chicago/Turabian StyleFresco-Taboada, Alba, Jesús Fernández-Lucas, Carmen Acebal, Miguel Arroyo, Fernando Ramón, Isabel De la Mata, and José Miguel Mancheño. 2018. "2′-Deoxyribosyltransferase from Bacillus psychrosaccharolyticus: A Mesophilic-Like Biocatalyst for the Synthesis of Modified Nucleosides from a Psychrotolerant Bacterium" Catalysts 8, no. 1: 8. https://doi.org/10.3390/catal8010008