Biochemical and Bioinformatic Studies of Mutations of Residues at the Monomer–Monomer Interface of Human Ornithine Aminotransferase Leading to Gyrate Atrophy of Choroid and Retina
Abstract
:1. Introduction
2. Results and Discussion
2.1. In Silico Analyses of hOAT Interfaces and of the Mutation Sites
2.2. Expression and Purification of the hOAT Interface Variants
2.3. Impact of the G52D, G121D, R154L, Y158S, T181M and P199Q Mutations on the Spectroscopic Features and Thermal Stability of hOAT
2.4. Impact of the Interface Variants on the Quaternary Structure of hOAT
2.5. Impact of the G52D, G121D, R154L, Y158S, T181M and P199Q Mutations on the Kinetic Features of hOAT
3. Materials and Methods
3.1. Materials
3.2. Computational Studies
3.3. Site Directed Mutagenesis
3.4. Expression and Purification of OAT Variants
3.5. Western-Blot
3.6. Enzyme Activity Assays
3.7. Spectroscopic Measurements and Thermal Stability
3.8. Analytical Size-Exclusion Chromatography (SEC)
3.9. HPLC Analysis of the Coenzymes
4. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
Abbreviations
References
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Enzymatic Species | ΔΔG (kcal/mol) | ΔTm (°C) | KDtet-dim (µM) |
---|---|---|---|
OAT wild-type | 0 | 0 | 0.13 ± 0.01 |
G51D | 1.95 | −0.5 | 0.24 ± 0.01 |
G121D | 3.83 | −1.3 | 0.30 ± 0.01 |
R154L | 8.68 | −12 | >>10 |
Y158S | 2.07 | −12 | >>10 |
T181M | 11.52 | −8.9 | >10 |
P199Q | 10.82 | −10.5 | >>10 |
Enzyme | Substrate | Cosubstrate | kcat (s−1) | KM(L-Orn) (mM) | KM(α-KG) (mM) | kcat/KM (mM−1s−1) |
---|---|---|---|---|---|---|
OAT WT [5] | L-Orn | α-KG | 34.9 ± 0.6 | 6.5 ± 0.4 | 5.4 ± 0.3 | |
α-KG | L-Orn | 35.7 ± 0.7 | 3.9 ± 0.5 | 9.1 ± 1.2 | ||
T181M | L-Orn | α-KG | 6.8 ± 0.3 | 91 ± 10 | 0.075 ± 0.009 | |
α-KG | L-Orn | 7.2 ± 0.4 | 14.7 ± 2.8 | 0.49 ± 0.10 | ||
P199Q | L-Orn | α-KG | 0.25 ± 0.01 | 18.8 ± 1.9 | 1.3 × 10−3 ± 2 × 10−4 | |
α-KG | L-Orn | 0.39 ± 0.03 | 87.6 ± 0.5 | 4.4 × 10−4 ± 3 × 10−5 | ||
Y158S | L-Orn | α-KG | 1.55 ± 0.13 | 3.4 ± 0.7 | 0.45 ± 0.10 | |
α-KG | L-Orn | 2.19 ± 0.14 | 31.0 ± 6.4 | 0.07 ± 0.02 |
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Floriani, F.; Borri Voltattorni, C.; Cellini, B.; Montioli, R. Biochemical and Bioinformatic Studies of Mutations of Residues at the Monomer–Monomer Interface of Human Ornithine Aminotransferase Leading to Gyrate Atrophy of Choroid and Retina. Int. J. Mol. Sci. 2023, 24, 3369. https://doi.org/10.3390/ijms24043369
Floriani F, Borri Voltattorni C, Cellini B, Montioli R. Biochemical and Bioinformatic Studies of Mutations of Residues at the Monomer–Monomer Interface of Human Ornithine Aminotransferase Leading to Gyrate Atrophy of Choroid and Retina. International Journal of Molecular Sciences. 2023; 24(4):3369. https://doi.org/10.3390/ijms24043369
Chicago/Turabian StyleFloriani, Fulvio, Carla Borri Voltattorni, Barbara Cellini, and Riccardo Montioli. 2023. "Biochemical and Bioinformatic Studies of Mutations of Residues at the Monomer–Monomer Interface of Human Ornithine Aminotransferase Leading to Gyrate Atrophy of Choroid and Retina" International Journal of Molecular Sciences 24, no. 4: 3369. https://doi.org/10.3390/ijms24043369