Engineered phosphite dehydrogenase (gene Rs-PtxD) from Ralstonia sp. 4506

Enzyme Description

Species
Ralstonia sp. 4506 β†—
Extremophile
No
EC Number
1.20.1.1 β†—

Sequence

Length: 335 amino acids
KPKVVLTHWVHPEIIELLSASADVIPNTTRETLPRSEVIARAKDADALMAFMPDSIDSAFLEECPKLRVIGAALKGYDNFDVNACTRHGVWLTIVPDLLTIPTAELTIGLLLGLTRHMLEGDRQIRSGHFQGWRPTLYGSGLTGKTLGIIGMGAVGRAIAQRLAGFEMNLLYCDPIPLNAEQEKAWHVQRVTLDELLEKCDYVVPMVPMAAETLHLIDATALAKMKTGSYLINACRGSVVDENAVIAALASGKLAGYAADVFEMEEWIRADRPQAIPKALLDNTAQTFFTPHLGSAVKEVRLEIERQAAMNIIQALAGEKPMGAINQPYPGVKAA
Gamal Nasser Abdel-Hady et al. (2021) β€” Engineering Cofactor Specificity of a Thermostable Phosphite Dehydrogenase for a Highly Efficient and Robust NADPH Regeneration System
Frontiers in Bioengineering and Biotechnology  Β· doi:10.3389/fbioe.2021.647176 β†—  Β· Stability - Incubation
60 measurements ▼
Database ID
β€”
Sequence Annotation
Explicit - Provided
Protein Source
Recombinant, host bacterium Escherichia coli BL21 pLysS

Experimental Data (60 measurements)

60 measurements β€” page 1 of 3
Mutation Property Assay Solvent Solvent Volume Aqueous ReferenceWT Reference Measured Value Units Solution pH Temperature Substrate(s) Product(s) Cofactor(s) Shaking Comments
C173H/D174A/P175R Stability - Incubation Activity after incubation (6 hours at 25Β°C), measured by absorbance spectrophotometry (NADH absorbance measurement, 340 nm) in aqueous phase Dimethylformamide (DMF) 10% 100 β€” 94 % Incubation: 50 mM MOPS buffer, without incubation cofactor, Assay: 100 mM MOPS buffer Incubation: 7.3, Assay: 7.3 Incubation: 25Β°C, Assay: 37Β°C 1 mM Phosphite Phosphate Assay: 0.5 mM NADP β€” Non-incubated control (in %), assay in aqueous phase | Uncertainty about whether assay really was in aqueous phase
C173H/D174A/P175R/I176K/P177A Stability - Incubation Activity after incubation (6 hours at 25Β°C), measured by absorbance spectrophotometry (NADH absorbance measurement, 340 nm) in aqueous phase Dimethylformamide (DMF) 20% 100 β€” 39 % Incubation: 50 mM MOPS buffer, with 0.5 mM NADP, Assay: 100 mM MOPS buffer Incubation: 7.3, Assay: 7.3 Incubation: 25Β°C, Assay: 37Β°C 1 mM Phosphite Phosphate Assay: 0.5 mM NADP β€” Non-incubated control (in %), assay in aqueous phase | Uncertainty about whether assay really was in aqueous phase
C173H/D174A/P175R/I176K/P177A Stability - Incubation Activity after incubation (6 hours at 25Β°C), measured by absorbance spectrophotometry (NADH absorbance measurement, 340 nm) in aqueous phase Dimethylformamide (DMF) 30% 100 β€” 8 % Incubation: 50 mM MOPS buffer, with 0.5 mM NADP, Assay: 100 mM MOPS buffer Incubation: 7.3, Assay: 7.3 Incubation: 25Β°C, Assay: 37Β°C 1 mM Phosphite Phosphate Assay: 0.5 mM NADP β€” Non-incubated control (in %), assay in aqueous phase | Uncertainty about whether assay really was in aqueous phase
C173H/D174A/P175R/I176K/P177A Stability - Incubation Activity after incubation (6 hours at 25Β°C), measured by absorbance spectrophotometry (NADH absorbance measurement, 340 nm) in aqueous phase Dimethylformamide (DMF) 40% 100 β€” 0 % Incubation: 50 mM MOPS buffer, with 0.5 mM NADP, Assay: 100 mM MOPS buffer Incubation: 7.3, Assay: 7.3 Incubation: 25Β°C, Assay: 37Β°C 1 mM Phosphite Phosphate Assay: 0.5 mM NADP β€” Non-incubated control (in %), assay in aqueous phase | Uncertainty about whether assay really was in aqueous phase
C173H/D174A/P175R/I176K/P177A Stability - Incubation Activity after incubation (6 hours at 25Β°C), measured by absorbance spectrophotometry (NADH absorbance measurement, 340 nm) in aqueous phase Dimethylformamide (DMF) 50% 100 β€” 0 % Incubation: 50 mM MOPS buffer, with 0.5 mM NADP, Assay: 100 mM MOPS buffer Incubation: 7.3, Assay: 7.3 Incubation: 25Β°C, Assay: 37Β°C 1 mM Phosphite Phosphate Assay: 0.5 mM NADP β€” Non-incubated control (in %), assay in aqueous phase | Uncertainty about whether assay really was in aqueous phase
C173H/D174A/P175R/I176R/P177A Stability - Incubation Activity after incubation (6 hours at 25Β°C), measured by absorbance spectrophotometry (NADH absorbance measurement, 340 nm) in aqueous phase Dimethylformamide (DMF) 10% 100 β€” 94 % Incubation: 50 mM MOPS buffer, with 0.5 mM NADP, Assay: 100 mM MOPS buffer Incubation: 7.3, Assay: 7.3 Incubation: 25Β°C, Assay: 37Β°C 1 mM Phosphite Phosphate Assay: 0.5 mM NADP β€” Non-incubated control (in %), assay in aqueous phase | Uncertainty about whether assay really was in aqueous phase
C173H/D174A/P175R/I176R/P177A Stability - Incubation Activity after incubation (6 hours at 25Β°C), measured by absorbance spectrophotometry (NADH absorbance measurement, 340 nm) in aqueous phase Dimethylformamide (DMF) 20% 100 β€” 29 % Incubation: 50 mM MOPS buffer, with 0.5 mM NADP, Assay: 100 mM MOPS buffer Incubation: 7.3, Assay: 7.3 Incubation: 25Β°C, Assay: 37Β°C 1 mM Phosphite Phosphate Assay: 0.5 mM NADP β€” Non-incubated control (in %), assay in aqueous phase | Uncertainty about whether assay really was in aqueous phase
C173H/D174A/P175R/I176R/P177A Stability - Incubation Activity after incubation (6 hours at 25Β°C), measured by absorbance spectrophotometry (NADH absorbance measurement, 340 nm) in aqueous phase Dimethylformamide (DMF) 30% 100 β€” 4 % Incubation: 50 mM MOPS buffer, with 0.5 mM NADP, Assay: 100 mM MOPS buffer Incubation: 7.3, Assay: 7.3 Incubation: 25Β°C, Assay: 37Β°C 1 mM Phosphite Phosphate Assay: 0.5 mM NADP β€” Non-incubated control (in %), assay in aqueous phase | Uncertainty about whether assay really was in aqueous phase
C173H/D174A/P175R/I176R/P177A Stability - Incubation Activity after incubation (6 hours at 25Β°C), measured by absorbance spectrophotometry (NADH absorbance measurement, 340 nm) in aqueous phase Dimethylformamide (DMF) 40% 100 β€” 1 % Incubation: 50 mM MOPS buffer, with 0.5 mM NADP, Assay: 100 mM MOPS buffer Incubation: 7.3, Assay: 7.3 Incubation: 25Β°C, Assay: 37Β°C 1 mM Phosphite Phosphate Assay: 0.5 mM NADP β€” Non-incubated control (in %), assay in aqueous phase | Uncertainty about whether assay really was in aqueous phase
C173H/D174A/P175R/I176R/P177A Stability - Incubation Activity after incubation (6 hours at 25Β°C), measured by absorbance spectrophotometry (NADH absorbance measurement, 340 nm) in aqueous phase Dimethylformamide (DMF) 50% 100 β€” 0 % Incubation: 50 mM MOPS buffer, with 0.5 mM NADP, Assay: 100 mM MOPS buffer Incubation: 7.3, Assay: 7.3 Incubation: 25Β°C, Assay: 37Β°C 1 mM Phosphite Phosphate Assay: 0.5 mM NADP β€” Non-incubated control (in %), assay in aqueous phase | Uncertainty about whether assay really was in aqueous phase
D174A/P175R Stability - Incubation Activity after incubation (6 hours at 25Β°C), measured by absorbance spectrophotometry (NADH absorbance measurement, 340 nm) in aqueous phase Dimethylformamide (DMF) 10% 100 β€” 99 % Incubation: 50 mM MOPS buffer, without incubation cofactor, Assay: 100 mM MOPS buffer Incubation: 7.3, Assay: 7.3 Incubation: 25Β°C, Assay: 37Β°C 1 mM Phosphite Phosphate Assay: 0.5 mM NADP β€” Non-incubated control (in %), assay in aqueous phase | Uncertainty about whether assay really was in aqueous phase
D174A/P175R Stability - Incubation Activity after incubation (6 hours at 25Β°C), measured by absorbance spectrophotometry (NADH absorbance measurement, 340 nm) in aqueous phase Dimethylformamide (DMF) 20% 100 β€” 94 % Incubation: 50 mM MOPS buffer, without incubation cofactor, Assay: 100 mM MOPS buffer Incubation: 7.3, Assay: 7.3 Incubation: 25Β°C, Assay: 37Β°C 1 mM Phosphite Phosphate Assay: 0.5 mM NADP β€” Non-incubated control (in %), assay in aqueous phase | Uncertainty about whether assay really was in aqueous phase
D174A/P175R Stability - Incubation Activity after incubation (6 hours at 25Β°C), measured by absorbance spectrophotometry (NADH absorbance measurement, 340 nm) in aqueous phase Dimethylformamide (DMF) 30% 100 β€” 0 % Incubation: 50 mM MOPS buffer, without incubation cofactor, Assay: 100 mM MOPS buffer Incubation: 7.3, Assay: 7.3 Incubation: 25Β°C, Assay: 37Β°C 1 mM Phosphite Phosphate Assay: 0.5 mM NADP β€” Non-incubated control (in %), assay in aqueous phase | Uncertainty about whether assay really was in aqueous phase
D174A/P175R Stability - Incubation Activity after incubation (6 hours at 25Β°C), measured by absorbance spectrophotometry (NADH absorbance measurement, 340 nm) in aqueous phase Dimethylformamide (DMF) 40% 100 β€” 0 % Incubation: 50 mM MOPS buffer, without incubation cofactor, Assay: 100 mM MOPS buffer Incubation: 7.3, Assay: 7.3 Incubation: 25Β°C, Assay: 37Β°C 1 mM Phosphite Phosphate Assay: 0.5 mM NADP β€” Non-incubated control (in %), assay in aqueous phase | Uncertainty about whether assay really was in aqueous phase
D174A/P175R Stability - Incubation Activity after incubation (6 hours at 25Β°C), measured by absorbance spectrophotometry (NADH absorbance measurement, 340 nm) in aqueous phase Dimethylformamide (DMF) 50% 100 β€” 0 % Incubation: 50 mM MOPS buffer, without incubation cofactor, Assay: 100 mM MOPS buffer Incubation: 7.3, Assay: 7.3 Incubation: 25Β°C, Assay: 37Β°C 1 mM Phosphite Phosphate Assay: 0.5 mM NADP β€” Non-incubated control (in %), assay in aqueous phase | Uncertainty about whether assay really was in aqueous phase
C173H/D174A/P175R/I176K/P177A Stability - Incubation Activity after incubation (6 hours at 25Β°C), measured by absorbance spectrophotometry (NADH absorbance measurement, 340 nm) in aqueous phase Dimethylformamide (DMF) 10% 100 β€” 106 % Incubation: 50 mM MOPS buffer, with 0.5 mM NADP, Assay: 100 mM MOPS buffer Incubation: 7.3, Assay: 7.3 Incubation: 25Β°C, Assay: 37Β°C 1 mM Phosphite Phosphate Assay: 0.5 mM NADP β€” Non-incubated control (in %), assay in aqueous phase | Uncertainty about whether assay really was in aqueous phase
C173H/D174A/P175R Stability - Incubation Activity after incubation (6 hours at 25Β°C), measured by absorbance spectrophotometry (NADH absorbance measurement, 340 nm) in aqueous phase Dimethylformamide (DMF) 20% 100 β€” 63 % Incubation: 50 mM MOPS buffer, without incubation cofactor, Assay: 100 mM MOPS buffer Incubation: 7.3, Assay: 7.3 Incubation: 25Β°C, Assay: 37Β°C 1 mM Phosphite Phosphate Assay: 0.5 mM NADP β€” Non-incubated control (in %), assay in aqueous phase | Uncertainty about whether assay really was in aqueous phase
C173H/D174A/P175R Stability - Incubation Activity after incubation (6 hours at 25Β°C), measured by absorbance spectrophotometry (NADH absorbance measurement, 340 nm) in aqueous phase Dimethylformamide (DMF) 30% 100 β€” 0 % Incubation: 50 mM MOPS buffer, without incubation cofactor, Assay: 100 mM MOPS buffer Incubation: 7.3, Assay: 7.3 Incubation: 25Β°C, Assay: 37Β°C 1 mM Phosphite Phosphate Assay: 0.5 mM NADP β€” Non-incubated control (in %), assay in aqueous phase | Uncertainty about whether assay really was in aqueous phase
C173H/D174A/P175R Stability - Incubation Activity after incubation (6 hours at 25Β°C), measured by absorbance spectrophotometry (NADH absorbance measurement, 340 nm) in aqueous phase Dimethylformamide (DMF) 40% 100 β€” 0 % Incubation: 50 mM MOPS buffer, without incubation cofactor, Assay: 100 mM MOPS buffer Incubation: 7.3, Assay: 7.3 Incubation: 25Β°C, Assay: 37Β°C 1 mM Phosphite Phosphate Assay: 0.5 mM NADP β€” Non-incubated control (in %), assay in aqueous phase | Uncertainty about whether assay really was in aqueous phase
C173H/D174A/P175R Stability - Incubation Activity after incubation (6 hours at 25Β°C), measured by absorbance spectrophotometry (NADH absorbance measurement, 340 nm) in aqueous phase Dimethylformamide (DMF) 50% 100 β€” 0 % Incubation: 50 mM MOPS buffer, without incubation cofactor, Assay: 100 mM MOPS buffer Incubation: 7.3, Assay: 7.3 Incubation: 25Β°C, Assay: 37Β°C 1 mM Phosphite Phosphate Assay: 0.5 mM NADP β€” Non-incubated control (in %), assay in aqueous phase | Uncertainty about whether assay really was in aqueous phase
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Mutations in this dataset (4)

C173H/D174A/P175R C173H/D174A/P175R/I176K/P177A C173H/D174A/P175R/I176R/P177A D174A/P175R

Visualization : Stability β€” Incubation

One bar per measurement. Colour = solvent, shade = solvent volume.

Structure

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