1.14.14.9: 4-hydroxyphenylacetate 3-monooxygenase
This is an abbreviated version!
For detailed information about 4-hydroxyphenylacetate 3-monooxygenase, go to the full flat file.
Word Map on EC 1.14.14.9
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1.14.14.9
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flavin
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3,4-dihydroxyphenylacetate
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baumannii
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flavin-dependent
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tyrosol
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hydroxytyrosol
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fmnh
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two-protein
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piceatannol
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synthesis
- 1.14.14.9
- flavin
- 3,4-dihydroxyphenylacetate
- baumannii
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flavin-dependent
- tyrosol
- hydroxytyrosol
- fmnh
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two-protein
- piceatannol
- synthesis
Reaction
Synonyms
4 HPA 3-hydroxyylase, 4-HPA hydroxylase, 4-hydroxyphenylacetate 3-hydroxylase, 4-hydroxyphenylacetic acid 3-hydroxylase, 4HPA 3-monooxygenase, 4HPA3H, C2-hpah, EC 1.14.13.3, HPA 3-hydroxylase, HpaB, hpaBC, HpaC, HPAH, More, p-hydroxyphenylacetate 3-hydroxylase, p-hydroxyphenylacetate hydroxylase, p-hydroxyphenylacetic 3-hydroxylase, TPY_2462, two-component p-hydroxyphenylacetate hydroxylase
ECTree
1.14.14.9 4-hydroxyphenylacetate 3-monooxygenaseAdvanced search results
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results (Engineering
Engineering on EC 1.14.14.9 - 4-hydroxyphenylacetate 3-monooxygenase
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PROTEIN VARIANTS 
ORGANISM
UNIPROT
COMMENTARY 
LITERATURE
H120D
mutant can form C4a-hydroperoxy-FMN, a reactive intermediate necessary for hydroxylation, but cannot hydroxylate 4-hydroxyphenylacetate
H120E
mutant can form C4a-hydroperoxy-FMN, a reactive intermediate necessary for hydroxylation, but cannot hydroxylate 4-hydroxyphenylacetate
H120K
catalyzes hydroxylation with efficiency comparable to that of the wild-type enzyme, the hydroxylation rate constant for H120K is 5.7 per s and the product conversion ratio is 75%, compared to values of 16 s-1 and 90% for the wild-type enzyme
H120N
mutant can form C4a-hydroperoxy-FMN, a reactive intermediate necessary for hydroxylation, but cannot hydroxylate 4-hydroxyphenylacetate
H120Q
mutant can form C4a-hydroperoxy-FMN, a reactive intermediate necessary for hydroxylation, but cannot hydroxylate 4-hydroxyphenylacetate
H120Y
mutant can form C4a-hydroperoxy-FMN, a reactive intermediate necessary for hydroxylation, but cannot hydroxylate 4-hydroxyphenylacetate
H396A
mutation of oxygenase component, decrease in hydroxylation efficiency. pKa value is 7.1 compared to 9.8 for wild-type
H396N
mutation of oxygenase component, decrease in hydroxylation efficiency. pKa value is 9.3 compared to 9.8 for wild-type
H396V
mutation of oxygenase component, decrease in hydroxylation efficiency. pKa value is 7.3 compared to 9.8 for wild-type
R263A
mutation of oxygenase component, 72% hydroxylation efficiency of phydroxyphenylacetate, 7% hydroxylation of tyramine
R263D
mutation of oxygenase component, variant can catalyze hydroxylation of tyramine to form dopamine with the highest yield (57%) while maintaining 86% hydroxylation efficiency of phydroxyphenylacetate
R263E
mutation of oxygenase component, 73% hydroxylation efficiency of phydroxyphenylacetate, no hydroxylation of tyramine
S146A
S146C
product formation decreases from about 65% at pH 6.0 to 27% at pH 10.0
H155A
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drastically decreased hydroxylase activity with substrate 3-hydroxyphenylacetate
R113A
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drastically decreased hydroxylase activity with substrate 3-hydroxyphenylacetate
R379G
contrary to wild-type, mutant is not able to grow on 3-hydroxyphenylacetic acid. Residue 379 is located in the vicinity of the 4-hydroxyphenylacetic acid binding site, and plays an important role in mediating the entrance and stable binding of substrates to the active site
R379S
contrary to wild-type, mutant is not able to grow on 3-hydroxyphenylacetic acid
synthesis
construction of biosynthetic pathways for the production of tyrosol acetate and hydroxytyrosol acetate in Escherichia coli. Escherichia coli YeaE is the best aldehyde reductase for tyrosol accumulation. Tyrosol acetate production is achieved by overexpression of alcohol acetyltransferase ATF1 from Saccharomyces cerevisiae, and hydroxytyrosol acetate production by overexpression of 4-hydroxyphenylacetate 3-hydroxylase genes HpaBC
Y117A
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drastically decreased hydroxylase activity with substrate 3-hydroxyphenylacetate
R379G
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contrary to wild-type, mutant is not able to grow on 3-hydroxyphenylacetic acid. Residue 379 is located in the vicinity of the 4-hydroxyphenylacetic acid binding site, and plays an important role in mediating the entrance and stable binding of substrates to the active site
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R379S
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contrary to wild-type, mutant is not able to grow on 3-hydroxyphenylacetic acid
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synthesis
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construction of biosynthetic pathways for the production of tyrosol acetate and hydroxytyrosol acetate in Escherichia coli. Escherichia coli YeaE is the best aldehyde reductase for tyrosol accumulation. Tyrosol acetate production is achieved by overexpression of alcohol acetyltransferase ATF1 from Saccharomyces cerevisiae, and hydroxytyrosol acetate production by overexpression of 4-hydroxyphenylacetate 3-hydroxylase genes HpaBC
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S171A
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the mutant shows reduced activity compared to the wild type enzyme
S171A
Thermus thermophilus HB8 / ATCC 27634 / DSM 579
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the mutant shows reduced activity compared to the wild type enzyme
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additional information
S146A
product formation is pH-independent and constant at about 70% over a pH range of 6-10
S146A
mutation in oxygenase component C2, mutant is more effective than the wild-type in catalyzing the hydroxylation of 4-aminophenylacetate. Both variants first hydroxylate to give 3-hydroxy-4-aminophenylacetate, which is further hydroxylated to give 3,5-dihydroxy-4-aminophenylacetate
additional information
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3B-3 mutant deficient in the metabolism of 4-hydroxyphenylacetic acid are created by exposure to N-methyl-N'-nitro-N-nitrosoguanidine
additional information
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3B-3 mutant deficient in the metabolism of 4-hydroxyphenylacetic acid are created by exposure to N-methyl-N'-nitro-N-nitrosoguanidine
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additional information
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creation of 4-hydroxyphenylacetate-negative mutants by minimal salt medium with ethylmethanesulfonate
additional information
changing the amino acid residues of section I of the flexible loop (mutant XS2, amino acids 207-211, GlyPheGlySerAla) into larger ones does not change the original secondary structure and the flexibility of the loop. The mutants show decreased activity towards substrates 4-coumaric acid, umbelliferone, resveratrol and naringenin. Changing the amino acid residues of section III of the flexible loop (mutant XS3, amino acids 215-217, GlyGluAsn) increases the Km values of towards p-coumaric acid and umbelliferone by 1.7fold and 1.2fold, respectively. The kcat value towards umbelliferone decreases by almost 2fold. Mutant XS4 incorporating the mutations in both XS2 and XS3 still keeps the secondary structure of the loop unchanged unchanged. In mutant XS5 the secondary structure of the loop is changed by replacing residues in section II of the loop (GlnValMet) with GlySerGly. Mutant XS5 shows reduced catalytic activity towards p-coumaric acid, umbelliferone and resveratrol. Mutant XS6 employs a loop containing mainly Gly, Ser, and Asp residues and shows the almost same specificity constant values towards p-coumaric acid and umbelliferone as wild-type
additional information
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changing the amino acid residues of section I of the flexible loop (mutant XS2, amino acids 207-211, GlyPheGlySerAla) into larger ones does not change the original secondary structure and the flexibility of the loop. The mutants show decreased activity towards substrates 4-coumaric acid, umbelliferone, resveratrol and naringenin. Changing the amino acid residues of section III of the flexible loop (mutant XS3, amino acids 215-217, GlyGluAsn) increases the Km values of towards p-coumaric acid and umbelliferone by 1.7fold and 1.2fold, respectively. The kcat value towards umbelliferone decreases by almost 2fold. Mutant XS4 incorporating the mutations in both XS2 and XS3 still keeps the secondary structure of the loop unchanged unchanged. In mutant XS5 the secondary structure of the loop is changed by replacing residues in section II of the loop (GlnValMet) with GlySerGly. Mutant XS5 shows reduced catalytic activity towards p-coumaric acid, umbelliferone and resveratrol. Mutant XS6 employs a loop containing mainly Gly, Ser, and Asp residues and shows the almost same specificity constant values towards p-coumaric acid and umbelliferone as wild-type
additional information
Escherichia coli B / ATCC 11303
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creation of 4-hydroxyphenylacetate-negative mutants by minimal salt medium with ethylmethanesulfonate
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additional information
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changing the amino acid residues of section I of the flexible loop (mutant XS2, amino acids 207-211, GlyPheGlySerAla) into larger ones does not change the original secondary structure and the flexibility of the loop. The mutants show decreased activity towards substrates 4-coumaric acid, umbelliferone, resveratrol and naringenin. Changing the amino acid residues of section III of the flexible loop (mutant XS3, amino acids 215-217, GlyGluAsn) increases the Km values of towards p-coumaric acid and umbelliferone by 1.7fold and 1.2fold, respectively. The kcat value towards umbelliferone decreases by almost 2fold. Mutant XS4 incorporating the mutations in both XS2 and XS3 still keeps the secondary structure of the loop unchanged unchanged. In mutant XS5 the secondary structure of the loop is changed by replacing residues in section II of the loop (GlnValMet) with GlySerGly. Mutant XS5 shows reduced catalytic activity towards p-coumaric acid, umbelliferone and resveratrol. Mutant XS6 employs a loop containing mainly Gly, Ser, and Asp residues and shows the almost same specificity constant values towards p-coumaric acid and umbelliferone as wild-type
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additional information
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creation of 4-hydroxyphenylacetate-negative mutants by minimal salt medium with ethylmethanesulfonate
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additional information
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mutant AG813, defective in the 4-hydroxyphenylacetate hydroxylase
additional information
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several mutants are created, e.g. strains with enzyme defectives for analysis of the 4-hydroxyphenylacetate pathway
additional information
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hydroxylase deficient mutant P23X6 created by exposure to ethylmethane sulfonate
additional information
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several mutants are created, e.g. strains with enzyme defectives for analysis of the 4-hydroxyphenylacetate pathway
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