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Literature summary for 4.1.1.7 extracted from
- Using site-saturation mutagenesis to explore mechanism and substrate specificity in thiamin diphosphate-dependent enzymes (2013), FEBS J., 280, 6395-6411.
Protein Variants
| Protein Variants | Comment | Organism |
|---|---|---|
| A460I | site-directed mutagenesis, the mutant enzymes is an excellent 2-ketohexanoate decarboxylase | Pseudomonas putida |
| A460Y | site-directed mutagenesis, the mutant shows altered substrate specificity compared to the wild-type enzyme | Pseudomonas putida |
| H281A | site-directed mutagenesis, the mutant shows about 600fold reduced catalytic efficiency compared to the wild-type enzyme | Pseudomonas putida |
| H281F | site-saturation mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme | Pseudomonas putida |
| H281Y | site-saturation mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme | Pseudomonas putida |
| H70A | site-directed mutagenesis, the mutant shows about 4000fold reduced catalytic efficiency compared to the wild-type enzyme | Pseudomonas putida |
| H70F | site-saturation mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme | Pseudomonas putida |
| H70L | site-saturation mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme | Pseudomonas putida |
| L403E | kcat value of the L403E variant is only 18fold lower than that of wild-type BFDC | Pseudomonas putida |
| L403F | L403F variant shows about 10fold increased Km value for the substrate compared to the wild-type enzyme, the cofactor is displaced with the thiazolium ring away | Pseudomonas putida |
| L403X | half the L403X colonies screened have at least 10% of wild-type activity | Pseudomonas putida |
| L476P | the mutant variant shows improved carboligase activity in organic solvents compared to the wild-type enzyme | Pseudomonas putida |
| L476Q | the mutant variant shows improved carboligase activity in organic solvents compared to the wild-type enzyme | Pseudomonas putida |
| additional information | site-saturation mutagenesis is used to evolve stereoselective enzymes as catalysts for synthetic organic chemistry | Pseudomonas putida |
| S26A | site-directed mutagenesis, the mutant shows about 600fold reduced catalytic efficiency compared to the wild-type enzyme, the S26A variant showed a 30fold increase in Km for benzoylformate and a 100fold increase in Ki value for R-mandelate | Pseudomonas putida |
| S26L | site-saturation mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme | Pseudomonas putida |
| S26M | site-saturation mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme | Pseudomonas putida |
| T377L | site-directed mutagenesis, the mutant shows altered substrate specificity compared to the wild-type enzyme | Pseudomonas putida |
| T377L/A460Y | site-directed mutagenesis, the mutant shows altered substrate specificity and kinetics compared to the wild-type enzyme | Pseudomonas putida |
Inhibitors
| Inhibitors | Comment | Organism | Structure |
|---|---|---|---|
| R-mandelate | a substrate-analogue inhibitor, the carboxylate of the inhibitor is in an apparent hydrogen bond with Ser26, whereas one of the histidine residues, His70, is positioned to act as a proton donor in the formation of the first tetrahedral intermediate, mandelyl-ThDP | Pseudomonas putida |  |
KM Value [mM]
| KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
|---|---|---|---|---|---|
| 0.27 | - |
phenylglyoxylate | pH and temperature not specified in the publication, wild-type enzyme | Pseudomonas putida | |
| 0.38 | - |
phenylglyoxylate | pH and temperature not specified in the publication, mutant H70L | Pseudomonas putida | |
| 0.49 | - |
phenylglyoxylate | pH and temperature not specified in the publication, mutant H281Y | Pseudomonas putida | |
| 0.54 | - |
phenylglyoxylate | pH and temperature not specified in the publication, mutant H281F | Pseudomonas putida | |
| 0.8 | - |
phenylglyoxylate | pH and temperature not specified in the publication, mutant S26M | Pseudomonas putida | |
| 0.85 | - |
phenylglyoxylate | pH and temperature not specified in the publication, mutant H70F | Pseudomonas putida | |
| 1.2 | - |
phenylglyoxylate | pH and temperature not specified in the publication, mutant H281A | Pseudomonas putida | |
| 1.2 | - |
phenylglyoxylate | pH and temperature not specified in the publication, mutant S26L | Pseudomonas putida | |
| 1.5 | - |
phenylglyoxylate | pH and temperature not specified in the publication, mutant H70A | Pseudomonas putida | |
| 7.8 | - |
phenylglyoxylate | pH and temperature not specified in the publication, mutant S26A | Pseudomonas putida | |
Natural Substrates/ Products (Substrates)
| Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
|---|---|---|---|---|---|---|
| phenylglyoxylate | Pseudomonas putida | - |
benzaldehyde + CO2 | - |
ir | |
Organism
| Organism | UniProt | Comment | Textmining |
|---|---|---|---|
| Pseudomonas putida | - |
- |
- |
Substrates and Products (Substrate)
| Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
|---|---|---|---|---|---|---|
| additional information | the enzyme has the ability to carry out stereospecific carbon-carbon bond formation, with products including various 2-hydroxy ketones and benzoin derivatives, the enzyme also catalyzes the conversion of benzaldehyde and acetaldehyde into R-benzoin and, predominantly, S-2-hydroxypropiophenone, via stereospecific carboligation | Pseudomonas putida | ? | - |
? | |
| phenylglyoxylate | - |
Pseudomonas putida | benzaldehyde + CO2 | - |
ir | |
Subunits
| Subunits | Comment | Organism |
|---|---|---|
| dimer | - |
Pseudomonas putida |
Synonyms
| Synonyms | Comment | Organism |
|---|---|---|
| BFDC | - |
Pseudomonas putida |
Turnover Number [1/s]
| Turnover Number Minimum [1/s] | Turnover Number Maximum [1/s] | Substrate | Comment | Organism | Structure |
|---|---|---|---|---|---|
| 0.46 | - |
phenylglyoxylate | pH and temperature not specified in the publication, mutant H70A | Pseudomonas putida | |
| 2.1 | - |
phenylglyoxylate | pH and temperature not specified in the publication, mutant H281A | Pseudomonas putida | |
| 4.5 | - |
phenylglyoxylate | pH and temperature not specified in the publication, mutant H70F | Pseudomonas putida | |
| 6.9 | - |
phenylglyoxylate | pH and temperature not specified in the publication, mutant H281Y | Pseudomonas putida | |
| 14 | - |
phenylglyoxylate | pH and temperature not specified in the publication, mutant H70L | Pseudomonas putida | |
| 15 | - |
phenylglyoxylate | pH and temperature not specified in the publication, mutant S26A | Pseudomonas putida | |
| 26 | - |
phenylglyoxylate | pH and temperature not specified in the publication, mutant S26M | Pseudomonas putida | |
| 65 | - |
phenylglyoxylate | pH and temperature not specified in the publication, mutant H281F | Pseudomonas putida | |
| 132 | - |
phenylglyoxylate | pH and temperature not specified in the publication, mutant S26L | Pseudomonas putida | |
| 320 | - |
phenylglyoxylate | pH and temperature not specified in the publication, wild-type enzyme | Pseudomonas putida | |
Cofactor
| Cofactor | Comment | Organism | Structure |
|---|---|---|---|
| thiamine diphosphate | dependent on, structural binding environment, overview. The V-conformation is assisted by the presence of a fulcrum residue, Leu403, located directly below the cofactor, the V-conformation results in a N4'-C2 distance of 3.1 A | Pseudomonas putida | |
General Information
| General Information | Comment | Organism |
|---|---|---|
| metabolism | penultimate enzyme in the mandelate pathway, that catalyzes the non-oxidative decarboxylation of benzoylformate to yield carbon dioxide and benzaldehyde | Pseudomonas putida |
| additional information | the first step in catalysis by all ThDP-dependent enzymes is the deprotonation of the C2 carbon by the 4'-imino group to form the ylide. Both active sites contain two histidine residues in the catalytic dimer, three ionizable residues in the active site of the enzyme are Ser26, His70 and His281. His281 is the donor for the protonation of the enamine intermediate, Ser26 forms a hydrogen bond with the carboxylate of the substrate | Pseudomonas putida |
| physiological function | the enzyme enables the organism to utilize R-mandelate as their sole carbon source | Pseudomonas putida |
kcat/KM [mM/s]
| kcat/KM Value [1/mMs-1] | kcat/KM Value Maximum [1/mMs-1] | Substrate | Comment | Organism | Structure |
|---|---|---|---|---|---|
| 0.3 | - |
phenylglyoxylate | pH and temperature not specified in the publication, mutant H70A | Pseudomonas putida | |
| 1.7 | - |
phenylglyoxylate | pH and temperature not specified in the publication, mutant H281A | Pseudomonas putida | |
| 1.9 | - |
phenylglyoxylate | pH and temperature not specified in the publication, mutant S26A | Pseudomonas putida | |
| 5.3 | - |
phenylglyoxylate | pH and temperature not specified in the publication, mutant H70F | Pseudomonas putida | |
| 14 | - |
phenylglyoxylate | pH and temperature not specified in the publication, mutant H281Y | Pseudomonas putida | |
| 32 | - |
phenylglyoxylate | pH and temperature not specified in the publication, mutant S26M | Pseudomonas putida | |
| 36 | - |
phenylglyoxylate | pH and temperature not specified in the publication, mutant H70L | Pseudomonas putida | |
| 110 | - |
phenylglyoxylate | pH and temperature not specified in the publication, mutant S26L | Pseudomonas putida | |
| 120 | - |
phenylglyoxylate | pH and temperature not specified in the publication, mutant H281F | Pseudomonas putida | |
| 1180 | - |
phenylglyoxylate | pH and temperature not specified in the publication, wild-type enzyme | Pseudomonas putida | |
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