Dual Activity BLEG-1 from Bacillus lehensis G1 Revealed Structural Resemblance to B3 Metallo-β-Lactamase and Glyoxalase II: An Insight into Its Enzyme Promiscuity and Evolutionary Divergence
Abstract
:1. Introduction
2. Results
2.1. Phylogenetic Analysis and Sequence Alignment of BLEG-1
2.2. Steady-State Kinetics Analysis of BLEG-1
2.3. Overall X-ray Crystal Structure of BLEG-1
2.4. Structural Comparison of BLEG-1 with L1 B3 MBL Global Structure and Metal-Center
2.5. Structural Comparison of BLEG-1 with YcbL and GloB GLXIIs Global Structure and Metal-Center
2.6. Active Site Configuration of BLEG-1, L1 B3 MBL, YcbL, and GloB GLXIIs
2.7. In Silico Substrate Binding Analyses of BLEG-1, L1 MBL, YcbL GLXII, and GloB GLXII
2.7.1. Docking of BLEG-1 and L1 MBL with Ampicillin
2.7.2. Docking of BLEG-1, YcbL, and GloB GLXII with S-D-Lactoylglutathione (SLG)
3. Discussion
4. Materials and Methods
4.1. Bacterial Strains and Plasmids
4.2. Phylogenetic Analysis of BLEG-1
4.3. Sequence Alignment of BLEG-1
4.4. Overexpression and Purification of BLEG-1
4.5. Steady-State Kinetic Analysis of BLEG-1
4.6. Protein Crystallization, Diffraction, and Data Collection
4.7. Structure Determination, Refinement, and Validation
4.8. Docking Analyses of BLEG-1 and Selected MBL and GLXII
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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With Ampicillin | With SLG | |
---|---|---|
Vmax (mM s−1) | (4.20 ± 0.16) × 10−5 | (3.55 ± 0.26) × 10−5 |
KM (mM) | (7.27 ± 0.51) × 10−2 | (5.50 ± 0.12) × 10−2 |
kcat (s−1) | (4.20 ± 0.16) × 10−2 | (3.55 ± 0.26) × 10−2 |
kcat/KM (mM−1 s−1) | (5.78 ± 0.19) × 10−1 | (6.47 ± 0.77) × 10−1 |
Data Collection Statistics | |
---|---|
Data collection site | Malaysia Genome Institute |
Detector | CCD MSC PAD200sk |
Wavelength (Å) | 1.5418 |
Data resolution (Å) | 50.00–1.44 (1.46–1.44) |
Space group | P41212 |
Unit cell dimensions (a, b, c) (Å) | 55.36, 55.36, 143.09 |
Total number of reflections | 205685 |
Number of unique reflections | 40528 |
Multiplicity | 5.1 (3.1) |
Completeness (%) | 98.0 (97.9) |
Rmerge | 0.045 (0.254) |
<I/σ(I)> | 28.5 (4.0) |
Refinement Statistics | |
Resolution range (Å) | 30.26–1.44 (1.49–1.44) |
No: residues/water/ion/ligand | 209/245/11/2 |
R-factor (Rwork/Rfree) (%) | 18.44/20.85 |
Rms bond length deviation (Å) | 0.005 |
Rms bond angle deviation (°) | 0.804 |
Ramachandran angles (favored/allowed/disallowed) (%) | 98.55/1.45/0 |
Metal ion | Distance (Å) | First Shell Ligand | Second Shell Ligand | |
---|---|---|---|---|
Involved in Metal Coordination | Involved in Second Shell Effect | |||
Zn1 | 2.07 | His-54 NE2 | His-54 ND1 | Thr-53 OG1 |
Zn1 | 2.12 | His-56 ND1 | His-56 NE2 | Wat’ O, Arg-163 O |
Zn1 | 2.06 | His-131 NE2 | His-131 ND1 | Arg-159 O |
Zn1 | 2.62 | Asp-150 OD2 | ||
Zn1 | 1.94 | Wat1 O | ||
Zn2 | 2.34 | Asp-58 OD2 | ||
Zn2 | 2.06 | His-59 NE2 | His-59 ND1 | Asp-28 OD2 |
Zn2 | 2.05 | Asp-150 OD2 | ||
Zn2 | 2.03 | His-191 NE2 | His-191 O | Ser-189 OG |
Zn2 | 2.00 | Wat1 O | ||
Zn2 | 3.52 | Wat2 O |
BLEG-1 | YcbL | GloB |
---|---|---|
His-131 (Loop10)
| His-132 (Loop8)
| His-110 (Loop7)
|
Phe-153 (Loop12)
| Phe-154 (β10)
| Phe-130 (β11)
|
Ile-157 (Loop12)
| Val-158 (β11)
| Cys-134 (β12)
|
Gly-158 (Loop12)
| Gly-159 (Loop11)
| Gly-135 (Loop9)
|
Arg-159 (Loop12)
| Arg-160 (Loop11)
| Arg-136 (Loop9)
|
His-191 (Loop14)
| His-192 (Loop12)
| His-165 (Loop11)
|
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Au, S.X.; Dzulkifly, N.S.; Muhd Noor, N.D.; Matsumura, H.; Raja Abdul Rahman, R.N.Z.; Normi, Y.M. Dual Activity BLEG-1 from Bacillus lehensis G1 Revealed Structural Resemblance to B3 Metallo-β-Lactamase and Glyoxalase II: An Insight into Its Enzyme Promiscuity and Evolutionary Divergence. Int. J. Mol. Sci. 2021, 22, 9377. https://doi.org/10.3390/ijms22179377
Au SX, Dzulkifly NS, Muhd Noor ND, Matsumura H, Raja Abdul Rahman RNZ, Normi YM. Dual Activity BLEG-1 from Bacillus lehensis G1 Revealed Structural Resemblance to B3 Metallo-β-Lactamase and Glyoxalase II: An Insight into Its Enzyme Promiscuity and Evolutionary Divergence. International Journal of Molecular Sciences. 2021; 22(17):9377. https://doi.org/10.3390/ijms22179377
Chicago/Turabian StyleAu, Shaw Xian, Nur Syazana Dzulkifly, Noor Dina Muhd Noor, Hiroyoshi Matsumura, Raja Noor Zaliha Raja Abdul Rahman, and Yahaya M. Normi. 2021. "Dual Activity BLEG-1 from Bacillus lehensis G1 Revealed Structural Resemblance to B3 Metallo-β-Lactamase and Glyoxalase II: An Insight into Its Enzyme Promiscuity and Evolutionary Divergence" International Journal of Molecular Sciences 22, no. 17: 9377. https://doi.org/10.3390/ijms22179377