Benzoylformate dehydrogenase from Pseudomonas putida

Enzyme Description

Species

Extremophile

EC Number

4.1.1.7 ↗

Sequence

Length: 528 amino acids

MASVHGTTYELLRRQGIDTVFGNPGSNELPFLKDFPEDFRYILALQEACVVGIADGYAQASRKPAFINLHSAAGTGNAMGALSNAWNSHSPLIVTAGQQTRAMIGVEALLTNVDAANLPRPLVKWSYEPASAAEVPHAMSRAIHMASMAPQGPVYLSVPYDDWDKDADPQSHHLFDRHVSSSVRLNDQDLDILVKALNSASNPAIVLGPDVDAANANADCVMLAERLKAPVWVAPSAPRCPFPTRHPCFRGLMPAGIAAISQLLEGHDVVLVIGAPVFRYHQYDPGQYLKPGTRLISVTCDPLEAARAPMGDAIVADIGAMASALANLVEESSRQLPTAAPEPAKVDQDAGRLHPETVFDTLNDMAPENAIYLNESTSTTAQMWQRLNMRNPGSYYFCAAGGLGFALPAAIGVQLAEPERQVIAVIGDGSANYSISALWTAAQYNIPTIFVIMNNGTYGALRWFAGVLEAENVPGLDVPGIDFRALAKGYGVQALKADNLEQLKGSLQEALSAKGPVLIEVSTVSPVK

B. Lingen et al. (2002) — 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 · doi:10.1093/protein/15.7.585 ↗ · Activity - Classical

22 measurements ▼

Database ID

UniProt: P20906 ↗

Sequence Annotation

Inferred - from protein name

Protein Source

Recombinant, host bacterium Escherichia coli SG13009

Experimental Data (22 measurements)

22 measurements — page 2 of 2

Mutation	Property	Assay	Solvent	Solvent Volume	Aqueous Reference	WT Reference	Measured Value	Units	Extraction method	Data type	Solution	pH	Temperature	Substrate(s)	Product(s)	Cofactor(s)	Shaking	Comments
L476Q/S525G	Activity - Classical	Activity (as a percentage of the WT) measured by HPLC in the presence of organic solvent	Ethanol	1.5 Molar	245	100	373	%	Direct	Continuous	50 mM potassium phosphate buffer, 2.5 mM MgSO4	7	30°C	500 mM Acetaldehyde, 40 mM Benzaldehyde	1-Hydroxy-1-phenyl-2-propanone	0.5 mM ThDP	—	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	Dimethyl Sulfoxide (DMSO)	20 % v/v	395	100	506	%	Direct	Continuous	50 mM potassium phosphate buffer, 2.5 mM MgSO4	7	30°C	500 mM Acetaldehyde, 40 mM Benzaldehyde	1-Hydroxy-1-phenyl-2-propanone	0.5 mM ThDP	—	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

Dimethyl Sulfoxide (DMSO)

20 % v/v

382

100

423

Direct

Continuous

50 mM potassium phosphate buffer, 2.5 mM MgSO4

30°C

500 mM Acetaldehyde, 40 mM Benzaldehyde

1-Hydroxy-1-phenyl-2-propanone

0.5 mM ThDP

—

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 % v/v

237

100

427

Direct

Continuous

50 mM potassium phosphate buffer, 2.5 mM MgSO4

30°C

500 mM Acetaldehyde, 40 mM Benzaldehyde

1-Hydroxy-1-phenyl-2-propanone

0.5 mM ThDP

—

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 Molar

237

100

469

Direct

Continuous

50 mM potassium phosphate buffer, 2.5 mM MgSO4

30°C

500 mM Acetaldehyde, 40 mM Benzaldehyde

1-Hydroxy-1-phenyl-2-propanone

0.5 mM ThDP

—

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 % v/v

203

100

319

Direct

Continuous

50 mM potassium phosphate buffer, 2.5 mM MgSO4

30°C

500 mM Acetaldehyde, 40 mM Benzaldehyde

1-Hydroxy-1-phenyl-2-propanone

0.5 mM ThDP

—

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 Molar

203

100

362

Direct

Continuous

50 mM potassium phosphate buffer, 2.5 mM MgSO4

30°C

500 mM Acetaldehyde, 40 mM Benzaldehyde

1-Hydroxy-1-phenyl-2-propanone

0.5 mM ThDP

—

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 % v/v

244

100

411

Direct

Continuous

50 mM potassium phosphate buffer, 2.5 mM MgSO4

30°C

500 mM Acetaldehyde, 40 mM Benzaldehyde

1-Hydroxy-1-phenyl-2-propanone

0.5 mM ThDP

—

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 Molar

244

100

558

Direct

Continuous

50 mM potassium phosphate buffer, 2.5 mM MgSO4

30°C

500 mM Acetaldehyde, 40 mM Benzaldehyde

1-Hydroxy-1-phenyl-2-propanone

0.5 mM ThDP

—

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 % v/v

292

100

500

Direct

Continuous

50 mM potassium phosphate buffer, 2.5 mM MgSO4

30°C

500 mM Acetaldehyde, 40 mM Benzaldehyde

1-Hydroxy-1-phenyl-2-propanone

0.5 mM ThDP

—

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 Molar

292

100

592

Direct

Continuous

50 mM potassium phosphate buffer, 2.5 mM MgSO4

30°C

500 mM Acetaldehyde, 40 mM Benzaldehyde

1-Hydroxy-1-phenyl-2-propanone

0.5 mM ThDP

—

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 % v/v

328

100

398

Direct

Continuous

50 mM potassium phosphate buffer, 2.5 mM MgSO4

30°C

500 mM Acetaldehyde, 40 mM Benzaldehyde

1-Hydroxy-1-phenyl-2-propanone

0.5 mM ThDP

—

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 Molar

328

100

500

Direct

Continuous

50 mM potassium phosphate buffer, 2.5 mM MgSO4

30°C

500 mM Acetaldehyde, 40 mM Benzaldehyde

1-Hydroxy-1-phenyl-2-propanone

0.5 mM ThDP

—

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 Molar

395

100

727

Direct

Continuous

50 mM potassium phosphate buffer, 2.5 mM MgSO4

30°C

500 mM Acetaldehyde, 40 mM Benzaldehyde

1-Hydroxy-1-phenyl-2-propanone

0.5 mM ThDP

—

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 Molar

382

100

562

Direct

Continuous

50 mM potassium phosphate buffer, 2.5 mM MgSO4

30°C

500 mM Acetaldehyde, 40 mM Benzaldehyde

1-Hydroxy-1-phenyl-2-propanone

0.5 mM ThDP

—

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 % v/v

270

100

431

Direct

Continuous

50 mM potassium phosphate buffer, 2.5 mM MgSO4

30°C

500 mM Acetaldehyde, 40 mM Benzaldehyde

1-Hydroxy-1-phenyl-2-propanone

0.5 mM ThDP

—

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 Molar

270

100

515

Direct

Continuous

50 mM potassium phosphate buffer, 2.5 mM MgSO4

30°C

500 mM Acetaldehyde, 40 mM Benzaldehyde

1-Hydroxy-1-phenyl-2-propanone

0.5 mM ThDP

—

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 % v/v

290

100

350

Direct

Continuous

50 mM potassium phosphate buffer, 2.5 mM MgSO4

30°C

500 mM Acetaldehyde, 40 mM Benzaldehyde

1-Hydroxy-1-phenyl-2-propanone

0.5 mM ThDP

—

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 Molar

290

100

420

Direct

Continuous

50 mM potassium phosphate buffer, 2.5 mM MgSO4

30°C

500 mM Acetaldehyde, 40 mM Benzaldehyde

1-Hydroxy-1-phenyl-2-propanone

0.5 mM ThDP

—

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 % v/v

100

175

Direct

Continuous

50 mM potassium phosphate buffer, 2.5 mM MgSO4

30°C

500 mM Acetaldehyde, 40 mM Benzaldehyde

1-Hydroxy-1-phenyl-2-propanone

0.5 mM ThDP

—

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 Molar

100

142

Direct

Continuous

50 mM potassium phosphate buffer, 2.5 mM MgSO4

30°C

500 mM Acetaldehyde, 40 mM Benzaldehyde

1-Hydroxy-1-phenyl-2-propanone

0.5 mM ThDP

—

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 % v/v

245

100

325

Direct

Continuous

50 mM potassium phosphate buffer, 2.5 mM MgSO4

30°C

500 mM Acetaldehyde, 40 mM Benzaldehyde

1-Hydroxy-1-phenyl-2-propanone

0.5 mM ThDP

—

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

Ethanol

1.5 Molar

245

100

373

Direct

Continuous

50 mM potassium phosphate buffer, 2.5 mM MgSO4

30°C

500 mM Acetaldehyde, 40 mM Benzaldehyde

1-Hydroxy-1-phenyl-2-propanone

0.5 mM ThDP

—

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

Dimethyl Sulfoxide (DMSO)

20 % v/v

395

100

506

Direct

Continuous

50 mM potassium phosphate buffer, 2.5 mM MgSO4

30°C

500 mM Acetaldehyde, 40 mM Benzaldehyde

1-Hydroxy-1-phenyl-2-propanone

0.5 mM ThDP

—

Classical aqueous control (in %) | Control compared to WT activity

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Mutations in this dataset (11)

S181T/L476P L476A L476C L476G L476H L476K L476M L476Q L476Q/S525G L476S L476T

Visualization : Activity — Classical

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

Structure

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Benzoylformate dehydrogenase from Pseudomonas putida

Enzyme Description

Sequence

Experimental Data (22 measurements)

Visualization : Activity — Classical

Mutation Effect

Structure

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