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Literature summary for 1.14.13.92 extracted from

  • Seo, E.; Kim, M.; Park, S.; Park, S.; Oh, D.; Bornscheuer, U.; Park, J.
    Enzyme access tunnel engineering in Baeyer-Villiger monooxygenases to improve oxidative stability and biocatalyst performance (2022), Adv. Synth. Catal., 364, 555-564 .
No PubMed abstract available

Application

Application Comment Organism
synthesis the H2O2-resistant enzyme variants are robust biocatalysts for synthetic applications Thermobifida fusca

Protein Variants

Protein Variants Comment Organism
C65D site-directed mutagenesis Thermobifida fusca
C65D/M446I site-directed mutagenesis Thermobifida fusca
C65D/M446I/Y495I site-directed mutagenesis, the M446I and Y495I mutations do not have significant influence on the NADPH oxidation activities. The triple mutant, which shows the greatest stability to H2O2, exhibits the highest catalytic activity (kcat) for NADPH oxidation. Thus, the oxidative stability is not markedly related to the NADPH oxidation and H2O2 generation rates Thermobifida fusca
C65D/M446I/Y517I site-directed mutagenesis, the M446I mutation does not have significant influence on the NADPH oxidation activities. The residual activity of this triple mutant variant remains unchanged during incubation with externally added H2O2. The variant completely loses the catalytic activity after 1 hour when H2O2 is generated in situ from NADPH oxidation. This fast deactivation of the C65D/M446I/Y517I variant leads to oxidation of only 10% of NADPH added. The Y517I mutation in C65D/M446I variant appears to result in blocking of the H2O2 exit and entrance path. The H2O2 generated in the active site might remain there, oxidizing amino acid residues in vicinity of the active site. The low catalytic activity of the C65D/M446I/Y517I variant for NADPH oxidation suggests that the Y517I mutation results in not only blocking of the H2O2 migration path but also modification of the active site structure Thermobifida fusca
additional information a rational approach is used to improve the robustness of enzymes, in particular, Baeyer-Villiger monooxygenases (BVMOs) against H2O2. The enzyme access tunnels, which may serve as exit paths for H2O2 from the active site to the bulk, are predicted by using the CAVER and/or protein energy landscape exploration (PELE) software for mutant PAMO_C65D from Thermobifida fusca. The amino acid residues, which are susceptible to oxidation by H2O2 (e.g. methionine and tyrosine) and located in vicinity of the predicted H2O2 migration paths, are substituted with less reactive or inert amino acids (e.g. leucine and isoleucine), leading to design of H2O2-resistant enzyme variants Thermobifida fusca

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
additional information
-
additional information steady-state kinetics of wild-type and mutant enzymes Thermobifida fusca

Natural Substrates/ Products (Substrates)

Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
phenylacetone + NADPH + H+ + O2 Thermobifida fusca
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benzyl acetate + NADP+ + H2O
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?

Organism

Organism UniProt Comment Textmining
Thermobifida fusca Q47PU3
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-

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
additional information the enzyme produces H2O2 in a side uncoupling reaction. The enzyme access tunnels, which may serve as exit paths for H2O2 from the active site to the bulk, are predicted by using the CAVER and/or protein energy landscape exploration (PELE) software for the phenylacetone monooxygenase variant (PAMO_C65D) from Thermobifida fusca. The simplified mechanism of flavin-dependent monooxygenases (e.g. BVMOs) consists of NAD(P)H-dependent reduction of the flavin prosthetic group, followed by activation of molecular oxygen as a (hydro)peroxyflavin, and substrate oxygenation. The catalytic cycle is closed after elimination of water and reformation of the oxidized flavin. Alternatively, the (hydro)peroxyflavin can eliminate H2O2 spontaneously (uncoupling reaction) into the oxidized flavin Thermobifida fusca ?
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phenylacetone + NADPH + H+ + O2
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Thermobifida fusca benzyl acetate + NADP+ + H2O
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?

Synonyms

Synonyms Comment Organism
PAMO
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Thermobifida fusca

Temperature Optimum [┬░C]

Temperature Optimum [┬░C] Temperature Optimum Maximum [┬░C] Comment Organism
30
-
assay at Thermobifida fusca

pH Optimum

pH Optimum Minimum pH Optimum Maximum Comment Organism
8
-
assay at Thermobifida fusca

Cofactor

Cofactor Comment Organism Structure
FAD
-
Thermobifida fusca
NADPH
-
Thermobifida fusca

General Information

General Information Comment Organism
additional information construction of an enzyme structure model exhibiting most of the conserved motifs of the BVMOs, including the FAD binding domain, the NADP(H) binding domain, the flexible linkers, and the signature motif, overview Thermobifida fusca