Benzoylformate dehydrogenase from Pseudomonas putida
Enzyme Description
Sequence
MASVHGTTYELLRRQGIDTVFGNPGSNELPFLKDFPEDFRYILALQEACVVGIADGYAQASRKPAFINLHSAAGTGNAMGALSNAWNSHSPLIVTAGQQTRAMIGVEALLTNVDAANLPRPLVKWSYEPASAAEVPHAMSRAIHMASMAPQGPVYLSVPYDDWDKDADPQSHHLFDRHVSSSVRLNDQDLDILVKALNSASNPAIVLGPDVDAANANADCVMLAERLKAPVWVAPSAPRCPFPTRHPCFRGLMPAGIAAISQLLEGHDVVLVIGAPVFRYHQYDPGQYLKPGTRLISVTCDPLEAARAPMGDAIVADIGAMASALANLVEESSRQLPTAAPEPAKVDQDAGRLHPETVFDTLNDMAPENAIYLNESTSTTAQMWQRLNMRNPGSYYFCAAGGLGFALPAAIGVQLAEPERQVIAVIGDGSANYSISALWTAAQYNIPTIFVIMNNGTYGALRWFAGVLEAENVPGLDVPGIDFRALAKGYGVQALKADNLEQLKGSLQEALSAKGPVLIEVSTVSPVK
B. Lingen et al. (2002)
β Improving the carboligase activity of benzoylformate decarboxylase from Pseudomonas putida by a combination of directed evolution and site-directed mutagenesis
Protein Engineering, Design and Selection
Β· doi:10.1093/protein/15.7.585 β
Β· Activity - Classical
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Database ID
UniProt: P20906 β
Sequence Annotation
Inferred - from protein name
Protein Source
Recombinant, host bacterium Escherichia coli SG13009
Experimental Data (22 measurements)
22 measurements β page 1 of 2
| Mutation | Property | Assay | Solvent | Solvent Volume | Aqueous Reference | WT Reference | Measured Value | Units | Solution | pH | Temperature | Substrate(s) | Product(s) | Cofactor(s) | Shaking | Comments |
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| L476M | Activity - Classical | Activity (as a percentage of the WT) measured by HPLC in the presence of organic solvent | Dimethyl Sulfoxide (DMSO) | 20% | 382 | 100 | 423 | % | 50 mM potassium phosphate buffer, 2.5 mM MgSO4 | 7 | 30Β°C | 500 mM Acetaldehyde, 40 mM Benzaldehyde | 1-Hydroxy-1-phenyl-2-propanone | 0.5 mM ThDP | β | Classical aqueous control (in %) | Control compared to WT activity |
| L476G | Activity - Classical | Activity (as a percentage of the WT) measured by HPLC in the presence of organic solvent | Dimethyl Sulfoxide (DMSO) | 20% | 237 | 100 | 427 | % | 50 mM potassium phosphate buffer, 2.5 mM MgSO4 | 7 | 30Β°C | 500 mM Acetaldehyde, 40 mM Benzaldehyde | 1-Hydroxy-1-phenyl-2-propanone | 0.5 mM ThDP | β | Classical aqueous control (in %) | Control compared to WT activity |
| L476G | Activity - Classical | Activity (as a percentage of the WT) measured by HPLC in the presence of organic solvent | Ethanol | 1.5 Mol | 237 | 100 | 469 | % | 50 mM potassium phosphate buffer, 2.5 mM MgSO4 | 7 | 30Β°C | 500 mM Acetaldehyde, 40 mM Benzaldehyde | 1-Hydroxy-1-phenyl-2-propanone | 0.5 mM ThDP | β | Classical aqueous control (in %) | Control compared to WT activity |
| L476H | Activity - Classical | Activity (as a percentage of the WT) measured by HPLC in the presence of organic solvent | Dimethyl Sulfoxide (DMSO) | 20% | 203 | 100 | 319 | % | 50 mM potassium phosphate buffer, 2.5 mM MgSO4 | 7 | 30Β°C | 500 mM Acetaldehyde, 40 mM Benzaldehyde | 1-Hydroxy-1-phenyl-2-propanone | 0.5 mM ThDP | β | Classical aqueous control (in %) | Control compared to WT activity |
| L476H | Activity - Classical | Activity (as a percentage of the WT) measured by HPLC in the presence of organic solvent | Ethanol | 1.5 Mol | 203 | 100 | 362 | % | 50 mM potassium phosphate buffer, 2.5 mM MgSO4 | 7 | 30Β°C | 500 mM Acetaldehyde, 40 mM Benzaldehyde | 1-Hydroxy-1-phenyl-2-propanone | 0.5 mM ThDP | β | Classical aqueous control (in %) | Control compared to WT activity |
| L476A | Activity - Classical | Activity (as a percentage of the WT) measured by HPLC in the presence of organic solvent | Dimethyl Sulfoxide (DMSO) | 20% | 244 | 100 | 411 | % | 50 mM potassium phosphate buffer, 2.5 mM MgSO4 | 7 | 30Β°C | 500 mM Acetaldehyde, 40 mM Benzaldehyde | 1-Hydroxy-1-phenyl-2-propanone | 0.5 mM ThDP | β | Classical aqueous control (in %) | Control compared to WT activity |
| L476A | Activity - Classical | Activity (as a percentage of the WT) measured by HPLC in the presence of organic solvent | Ethanol | 1.5 Mol | 244 | 100 | 558 | % | 50 mM potassium phosphate buffer, 2.5 mM MgSO4 | 7 | 30Β°C | 500 mM Acetaldehyde, 40 mM Benzaldehyde | 1-Hydroxy-1-phenyl-2-propanone | 0.5 mM ThDP | β | Classical aqueous control (in %) | Control compared to WT activity |
| L476K | Activity - Classical | Activity (as a percentage of the WT) measured by HPLC in the presence of organic solvent | Dimethyl Sulfoxide (DMSO) | 20% | 292 | 100 | 500 | % | 50 mM potassium phosphate buffer, 2.5 mM MgSO4 | 7 | 30Β°C | 500 mM Acetaldehyde, 40 mM Benzaldehyde | 1-Hydroxy-1-phenyl-2-propanone | 0.5 mM ThDP | β | Classical aqueous control (in %) | Control compared to WT activity |
| L476K | Activity - Classical | Activity (as a percentage of the WT) measured by HPLC in the presence of organic solvent | Ethanol | 1.5 Mol | 292 | 100 | 592 | % | 50 mM potassium phosphate buffer, 2.5 mM MgSO4 | 7 | 30Β°C | 500 mM Acetaldehyde, 40 mM Benzaldehyde | 1-Hydroxy-1-phenyl-2-propanone | 0.5 mM ThDP | β | Classical aqueous control (in %) | Control compared to WT activity |
| L476T | Activity - Classical | Activity (as a percentage of the WT) measured by HPLC in the presence of organic solvent | Dimethyl Sulfoxide (DMSO) | 20% | 328 | 100 | 398 | % | 50 mM potassium phosphate buffer, 2.5 mM MgSO4 | 7 | 30Β°C | 500 mM Acetaldehyde, 40 mM Benzaldehyde | 1-Hydroxy-1-phenyl-2-propanone | 0.5 mM ThDP | β | Classical aqueous control (in %) | Control compared to WT activity |
| L476T | Activity - Classical | Activity (as a percentage of the WT) measured by HPLC in the presence of organic solvent | Ethanol | 1.5 Mol | 328 | 100 | 500 | % | 50 mM potassium phosphate buffer, 2.5 mM MgSO4 | 7 | 30Β°C | 500 mM Acetaldehyde, 40 mM Benzaldehyde | 1-Hydroxy-1-phenyl-2-propanone | 0.5 mM ThDP | β | Classical aqueous control (in %) | Control compared to WT activity |
| L476Q | Activity - Classical | Activity (as a percentage of the WT) measured by HPLC in the presence of organic solvent | Ethanol | 1.5 Mol | 395 | 100 | 727 | % | 50 mM potassium phosphate buffer, 2.5 mM MgSO4 | 7 | 30Β°C | 500 mM Acetaldehyde, 40 mM Benzaldehyde | 1-Hydroxy-1-phenyl-2-propanone | 0.5 mM ThDP | β | Classical aqueous control (in %) | Control compared to WT activity |
| L476M | Activity - Classical | Activity (as a percentage of the WT) measured by HPLC in the presence of organic solvent | Ethanol | 1.5 Mol | 382 | 100 | 562 | % | 50 mM potassium phosphate buffer, 2.5 mM MgSO4 | 7 | 30Β°C | 500 mM Acetaldehyde, 40 mM Benzaldehyde | 1-Hydroxy-1-phenyl-2-propanone | 0.5 mM ThDP | β | Classical aqueous control (in %) | Control compared to WT activity |
| L476C | Activity - Classical | Activity (as a percentage of the WT) measured by HPLC in the presence of organic solvent | Dimethyl Sulfoxide (DMSO) | 20% | 270 | 100 | 431 | % | 50 mM potassium phosphate buffer, 2.5 mM MgSO4 | 7 | 30Β°C | 500 mM Acetaldehyde, 40 mM Benzaldehyde | 1-Hydroxy-1-phenyl-2-propanone | 0.5 mM ThDP | β | Classical aqueous control (in %) | Control compared to WT activity |
| L476C | Activity - Classical | Activity (as a percentage of the WT) measured by HPLC in the presence of organic solvent | Ethanol | 1.5 Mol | 270 | 100 | 515 | % | 50 mM potassium phosphate buffer, 2.5 mM MgSO4 | 7 | 30Β°C | 500 mM Acetaldehyde, 40 mM Benzaldehyde | 1-Hydroxy-1-phenyl-2-propanone | 0.5 mM ThDP | β | Classical aqueous control (in %) | Control compared to WT activity |
| L476S | Activity - Classical | Activity (as a percentage of the WT) measured by HPLC in the presence of organic solvent | Dimethyl Sulfoxide (DMSO) | 20% | 290 | 100 | 350 | % | 50 mM potassium phosphate buffer, 2.5 mM MgSO4 | 7 | 30Β°C | 500 mM Acetaldehyde, 40 mM Benzaldehyde | 1-Hydroxy-1-phenyl-2-propanone | 0.5 mM ThDP | β | Classical aqueous control (in %) | Control compared to WT activity |
| L476S | Activity - Classical | Activity (as a percentage of the WT) measured by HPLC in the presence of organic solvent | Ethanol | 1.5 Mol | 290 | 100 | 420 | % | 50 mM potassium phosphate buffer, 2.5 mM MgSO4 | 7 | 30Β°C | 500 mM Acetaldehyde, 40 mM Benzaldehyde | 1-Hydroxy-1-phenyl-2-propanone | 0.5 mM ThDP | β | Classical aqueous control (in %) | Control compared to WT activity |
| S181T/L476P | Activity - Classical | Activity (as a percentage of the WT) measured by HPLC in the presence of organic solvent | Dimethyl Sulfoxide (DMSO) | 20% | 66 | 100 | 175 | % | 50 mM potassium phosphate buffer, 2.5 mM MgSO4 | 7 | 30Β°C | 500 mM Acetaldehyde, 40 mM Benzaldehyde | 1-Hydroxy-1-phenyl-2-propanone | 0.5 mM ThDP | β | Classical aqueous control (in %) | Control compared to WT activity |
| S181T/L476P | Activity - Classical | Activity (as a percentage of the WT) measured by HPLC in the presence of organic solvent | Ethanol | 1.5 Mol | 66 | 100 | 142 | % | 50 mM potassium phosphate buffer, 2.5 mM MgSO4 | 7 | 30Β°C | 500 mM Acetaldehyde, 40 mM Benzaldehyde | 1-Hydroxy-1-phenyl-2-propanone | 0.5 mM ThDP | β | Classical aqueous control (in %) | Control compared to WT activity |
| L476Q/S525G | Activity - Classical | Activity (as a percentage of the WT) measured by HPLC in the presence of organic solvent | Dimethyl Sulfoxide (DMSO) | 20% | 245 | 100 | 325 | % | 50 mM potassium phosphate buffer, 2.5 mM MgSO4 | 7 | 30Β°C | 500 mM Acetaldehyde, 40 mM Benzaldehyde | 1-Hydroxy-1-phenyl-2-propanone | 0.5 mM ThDP | β | Classical aqueous control (in %) | Control compared to WT activity |
Mutations in this dataset (11)
S181T/L476P
L476A
L476C
L476G
L476H
L476K
L476M
L476Q
L476Q/S525G
L476S
L476T
Visualization : Activity β Classical
One bar per measurement. Colour = solvent, shade = solvent volume. β β β Reference value. Hover for details.
Mutation Effect
Mutation impact on enzyme stability and function in the presence of organic solvent: comparison of wild-type and mutant values in identical conditions.