1.14.13.92 A435Y the mutant is active only with bicyclo[3.2.0]hept-2-en-6-one 726716 1.14.13.92 A435Y the mutant shows less than 3% of wild type activity 726716 1.14.13.92 A442G the mutant shows 75% of wild type activity 726716 1.14.13.92 A442P random/saturation mutagenesis, the mutant is active with cyclohexanone, in contrast to the wild-type enzyme, and catalyzes its conversion to epsilon-caprolactone at 81% conversion rate 744548 1.14.13.92 A442P/ L443I/S444Q random and site-directed mutagenesis, the mutant is active with cyclohexanone, in contrast to the wild-type enzyme, and catalyzes its conversion to epsilon-caprolactone at 43% conversion rate 744548 1.14.13.92 A442P/ L443V/S444Q random and site-directed mutagenesis, the mutant is active with cyclohexanone, in contrast to the wild-type enzyme, and catalyzes its conversion to epsilon-caprolactone at 45% conversion rate 744548 1.14.13.92 A442P/L443I random/saturation mutagenesis, the mutant is active with cyclohexanone, in contrast to the wild-type enzyme, and catalyzes its conversion to epsilon-caprolactone at 45% conversion rate 744548 1.14.13.92 A442P/L443L/S444Q random and site-directed mutagenesis, the mutant is active with cyclohexanone, in contrast to the wild-type enzyme, and catalyzes its conversion to epsilon-caprolactone at 41% conversion rate 744548 1.14.13.92 A442P/L443T/S444Q random and site-directed mutagenesis, the mutant is active with cyclohexanone, in contrast to the wild-type enzyme, and catalyzes its conversion to epsilon-caprolactone at 56% conversion rate 744548 1.14.13.92 A442P/L443V random/saturation mutagenesis, the mutant is active with cyclohexanone, in contrast to the wild-type enzyme, and catalyzes its conversion to epsilon-caprolactone at 90% conversion rate 744548 1.14.13.92 A442P/L443W random/saturation mutagenesis, the mutant is active with cyclohexanone, in contrast to the wild-type enzyme, and catalyzes its conversion to epsilon-caprolactone at 74% conversion rate 744548 1.14.13.92 A442P/L443W/ S444Q random and site-directed mutagenesis, the mutant is active with cyclohexanone, in contrast to the wild-type enzyme, and catalyzes its conversion to epsilon-caprolactone at 33% conversion rate 744548 1.14.13.92 C65D site-directed mutagenesis 763862 1.14.13.92 C65D site-directed mutagenesis, the engineered TfPAMO variant acts as an NADPH oxidase, it shows an extremely high rate of uncoupling compared with the wild-type enzyme. The mutant is not effective in stabilizing the C(4alpha)-peroxyflavin intermediate. For TfPAMO C65D, hydrogen peroxide and superoxide levels are highest at pH 9.0 763827 1.14.13.92 C65D/M446I site-directed mutagenesis 763862 1.14.13.92 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 763862 1.14.13.92 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 763862 1.14.13.92 C65V the mutant shows 94% of wild type activity 726716 1.14.13.92 C65V/I67T the mutant shows 47% of wild type activity 726716 1.14.13.92 C65V/I67T/Q152F/S441A/A442G the mutant shows 31% of wild type activity 726716 1.14.13.92 C65V/I67T/Q93W the mutant shows 54% of wild type activity 726716 1.14.13.92 H220A site-directed mutagenesis 710979 1.14.13.92 H220D site-directed mutagenesis 710979 1.14.13.92 H220E site-directed mutagenesis, H220E mutant performs worse than wild-type PAMO with both coenzymes NADPH and NADH 710979 1.14.13.92 H220F site-directed mutagenesis 710979 1.14.13.92 H220N site-directed mutagenesis, the mutant shows about 3fold improvement in the catalytic efficiency with NADH while the catalytic efficiency with NADPH is hardly affected 710979 1.14.13.92 H220Q site-directed mutagenesis, the mutant shows about 3fold improvement in the catalytic efficiency with NADH while the catalytic efficiency with NADPH is hardly affected 710979 1.14.13.92 H220Q/K336H site-directed mutagenesis 710979 1.14.13.92 H220Q/K336N site-directed mutagenesis 710979 1.14.13.92 H220T site-directed mutagenesis 710979 1.14.13.92 H220W site-directed mutagenesis 710979 1.14.13.92 I339S the mutant shows 81% of wild type activity 726716 1.14.13.92 I67A site-directed mutagenesis, the mutant shows no activity with cyclohexanone like the wild-type enzyme 764409 1.14.13.92 I67A/P440F site-directed mutagenesis, the mutant shows high activity with cyclohexanone, 95.1% conversion after 4 h 764409 1.14.13.92 I67C site-directed mutagenesis, the mutant shows no activity with cyclohexanone like the wild-type enzyme 764409 1.14.13.92 I67C/P440F site-directed mutagenesis, the mutant shows high activity with cyclohexanone, 92.4% conversion after 4 h 764409 1.14.13.92 I67C/P440F/A442F/L443D site-directed mutagenesis, the mutant shows moderate activity with cyclohexanone 764409 1.14.13.92 I67C/P440Y site-directed mutagenesis, the mutant shows moderate activity with cyclohexanone 764409 1.14.13.92 I67G site-directed mutagenesis, the mutant shows no activity with cyclohexanone like the wild-type enzyme 764409 1.14.13.92 I67G/P440F site-directed mutagenesis, the mutant shows high activity with cyclohexanone, 87.5% conversion after 4 h 764409 1.14.13.92 I67T the mutant shows 16% of wild type activity 726716 1.14.13.92 I67T/L338P the mutant shows less than 3% of wild type activity 726716 1.14.13.92 I67T/L338P/A435Y/A442G the mutant shows less than 3% of wild type activity 726716 1.14.13.92 I67T/L338P/A435Y/A442G/L443F/S444C the mutant shows less than 3% of wild type activity 726716 1.14.13.92 I67Y site-directed mutagenesis, the mutant shows no activity with cyclohexanone like the wild-type enzyme 764409 1.14.13.92 I67Y/P440F site-directed mutagenesis, the mutant shows high activity with cyclohexanone, 97.8% conversion after 4 h 764409 1.14.13.92 I67Y/P440Y site-directed mutagenesis, the mutant shows high activity with cyclohexanone 764409 1.14.13.92 K336H site-directed mutagenesis 710979 1.14.13.92 K336N site-directed mutagenesis 710979 1.14.13.92 L153G inactive 726716 1.14.13.92 L153G the mutant shows less than 3% of wild type activity 726716 1.14.13.92 L338P the mutant shows 59% of wild type activity 726716 1.14.13.92 L443F the mutant shows 65% of wild type activity 726716 1.14.13.92 L443V random/saturation mutagenesis, the mutant is active with cyclohexanone, in contrast to the wild-type enzyme, and catalyzes its conversion to epsilon-caprolactone at 53% conversion rate 744548 1.14.13.92 L443V/S444M random/saturation mutagenesis, the mutant is active with cyclohexanone, in contrast to the wild-type enzyme, and catalyzes its conversion to epsilon-caprolactone at 53% conversion rate 744548 1.14.13.92 L443V/S444Q random/saturation mutagenesis, the mutant is active with cyclohexanone, in contrast to the wild-type enzyme, and catalyzes its conversion to epsilon-caprolactone at 40-45% conversion rate 744548 1.14.13.92 L443V/S444Q random/saturation mutagenesis, the mutant is active with cyclohexanone, in contrast to the wild-type enzyme, and catalyzes its conversion to epsilon-caprolactone at 59% conversion rate 744548 1.14.13.92 L443V/S444T random/saturation mutagenesis, the mutant is active with cyclohexanone, in contrast to the wild-type enzyme, and catalyzes its conversion to epsilon-caprolactone at 57% conversion rate 744548 1.14.13.92 L447P the mutant shows 85% of wild type activity 726716 1.14.13.92 M446G enzyme has altered activity and substrate specificity 697754 1.14.13.92 M446G the mutant retains wild type thermostability and produces an altered substrate binding pocket, leading to substantial changes in substrate specificity and enantioselectivity towards sulfides and ketones 727379 1.14.13.92 M446G the mutant shows 49% of wild type activity and is able to convert 1-indanone to 1-isochromanone 726716 1.14.13.92 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 763862 1.14.13.92 additional information addition of the dimerization-docking and anchoring domain (RIDD-RIAD) system to the C-terminus of the NADPH-dependent Baeyer-Villiger monooxygenase phenylacetone monooxygenase (PAMO) and the NADPH-regenerating enzyme (phosphite dehydrogenase, PTDH, EC 1.20.1.1) allowing self-assembly based on specific protein-protein interactions between both peptides and allow tuning of the ratio of the targeted enzymes as the RIAD peptide binds to two RIDD peptides. Several RIDD/RIAD-tagged PAMO and PTDH variants are successfully overproduced in Escherichia coli and subsequently purified. Complementary tagged enzymes are mixed and analyzed for their oligomeric state, stability, and activity. Complexes are formed in the case of some specific combinations (PAMORIAD-PTDHRIDD and PAMORIAD/RIAD-PTDHRIDD). These enzyme complexes display similar catalytic activity when compared with the PTDH-PAMO fusion enzyme. The thermostability of PAMO in these complexes is retained while PTDH displays somewhat lower thermostability 764264 1.14.13.92 additional information construction of three mutants P1-P3 by elimination of a bulge loop region, involving residues Ser441, Ala442, and Leu443, leading to enhanced substrate enantioselectivity of Baeyer-Villiger reactions while maintaining high thermal stability, overview 671189 1.14.13.92 additional information directed evolution of phenylacetone monooxygenase as an active catalyst for the Baeyer-Villiger conversion of cyclohexanone to caprolactone using iterative saturation mutagenesis, mutant screening, overview. Molecular dynamics simulations and induced fit docking of wild-type and mutant enzymes with cyclohexanone. The mutants are used in the whole cell system of Escherichia coli cells 744548 1.14.13.92 additional information engineering of three highly stereoselective mutants of the thermally stable phenylacetone monooxygenase as practical catalysts for enantioselective Baeyer-Villiger oxidations of several ketones on a preparative scale under in vitro conditions, optimization of the method including a coupled cofactor-regeneration system, reaction mechanism, overview 671682 1.14.13.92 additional information expanding the substrate scope of a thermostable phenylacetone monooxygenase (PAMO) to cyclohexanone by using site-directed mutagenesis. Several mutants are found to be active with cyclohexanone for which wild-type PAMO shows no activity. There is possible additive or cooperative effect existing between I67 and P440. Based on the thermostable PAMO scaffold, a chimeric PAMO-CHMO enzyme mutant is created, which shows no activity on cyclohexanone 764409 1.14.13.92 additional information rational engineering of enzyme PAMO for wide applications in industrial biocatalysis, in particular, in the biotransformation of long-chain aliphatic oils into potential biodiesels 745991 1.14.13.92 additional information rational engineering of PAMO for wide applications in industrial biocatalysis, in particular, in the biotransformation of long-chain aliphatic oils into potential biodiesels 765535 1.14.13.92 P253F/G254A/R258M/L443F site-directed mutagenesis, the engineered mutant quadruple enzyme variant P253F/G254A/R258M/L443F exhibits significantly improved activity towards 2-octanone compared to wild-type. A remarkable movement of L289 is crucial for a reshaping of the active site of the quadruple variant so as to prevent the aliphatic substrate from moving away from the C4a-peroxyflavin, thus enabling it to keep a catalytically relevant pose during the oxygenation process, substrate specificity compared to wild-type 765535 1.14.13.92 P253F/G254A/R258M/L443F the mutant shows the same thermostability as the wild type enzyme while it displays an extended substrate spectrum 726793 1.14.13.92 P440F higher subtrate variability, temperature optimum at 50C with range from 45-56C 698487 1.14.13.92 P440F site-directed mutagenesis, the mutant shows high activity with cyclohexanone, 50.2% conversion after 4 h 764409 1.14.13.92 P440H higher subtrate variability, temperature optimum at 50C with range from 45-56C 698487 1.14.13.92 P440H site-directed mutagenesis, the mutant shows low activity with cyclohexanone 764409 1.14.13.92 P440I higher subtrate variability, temperature optimum at 50C with range from 45-56C 698487 1.14.13.92 P440I site-directed mutagenesis, the mutant shows low activity with cyclohexanone 764409 1.14.13.92 P440L higher subtrate variability, temperature optimum at 50C with range from 45-56C 698487 1.14.13.92 P440N higher subtrate variability, temperature optimum at 50C with range from 45-56C 698487 1.14.13.92 P440T higher subtrate variability, temperature optimum at 50C with range from 45-56C 698487 1.14.13.92 P440W site-directed mutagenesis, the mutant shows high acticity with cyclohexanone 764409 1.14.13.92 P440Y higher subtrate variability, temperature optimum at 50C with range from 45-58C 698487 1.14.13.92 P440Y site-directed mutagenesis, the mutant shows low activity with cyclohexanone 764409 1.14.13.92 Q152F the mutant shows 35% of wild type activity 726716 1.14.13.92 Q152F/A442G the mutant shows 37% of wild type activity 726716 1.14.13.92 Q152F/S441A/A442G the mutant shows 33% of wild type activity 726716 1.14.13.92 Q93N/P94D/P440F random/saturation mutagenesis, the mutant is active with cyclohexanone, in contrast to the wild-type enzyme, and catalyzes its conversion to epsilon-caprolactone at low rate 744548 1.14.13.92 Q93W the mutant shows 65% of wild type activity 726716 1.14.13.92 Q93W/A442G/S444C/M446G/L447P the mutant shows 38% of wild type activity 726716 1.14.13.92 Q93W/S441A/A442G/S444C/M446G/L447P the mutant shows 40% of wild type activity 726716 1.14.13.92 R217A site-directed mutagenesis 710979 1.14.13.92 R217L site-directed mutagenesis 710979 1.14.13.92 R258A site-directed mutagenesis, when the substrate 2-octanone binds to the R258A mutant, a significant change in the position of the hexyl tail of the substrate is observed, altered substrate specificity compared to wild-type 765535 1.14.13.92 R258M site-directed mutagenesis, the R258M mutation significantly affects pose of 2-octanone, since the hexyl tail moves towards M258, substrate specificity compared to wild-type 765535 1.14.13.92 R337A site-directed mutagenesis, the mutant is still able to form and stabilize the C4a-peroxyflavin intermediate, but loses the ability to convert phenylacetone or benzyle methylsulfide 685231 1.14.13.92 R337K site-directed mutagenesis, the mutant is still able to form and stabilize the C4a-peroxyflavin intermediate, but loses the ability to convert phenylacetone or benzyle methylsulfide 685231 1.14.13.92 S441A the mutant shows 73% of wild type activity 726716 1.14.13.92 S441A/A442G the mutant shows 56% of wild type activity 726716 1.14.13.92 S441A/A442G/S444C/M446G/L447P the mutant shows 41% of wild type activity 726716 1.14.13.92 S441D/A442E random/saturation mutagenesis, the mutant is active with cyclohexanone, in contrast to the wild-type enzyme, and catalyzes its conversion to epsilon-caprolactone at 73% conversion rate 744548 1.14.13.92 S441G/A442P/L443T/S444Q site-directed mutagenesis, the mutant is active with cyclohexanone, in contrast to the wild-type enzyme, and catalyzes its conversion to epsilon-caprolactone at about 90% conversion rate 744548 1.14.13.92 S441G/A442T random/saturation mutagenesis, the mutant is active with cyclohexanone, in contrast to the wild-type enzyme, and catalyzes its conversion to epsilon-caprolactone at 48% conversion rate 744548 1.14.13.92 S441H random/saturation mutagenesis, the mutant is active with cyclohexanone, in contrast to the wild-type enzyme, and catalyzes its conversion to epsilon-caprolactone at 34% conversion rate 744548 1.14.13.92 S441H/A442P random/saturation mutagenesis, the mutant is active with cyclohexanone, in contrast to the wild-type enzyme, and catalyzes its conversion to epsilon-caprolactone at 78% conversion rate 744548 1.14.13.92 S444C the mutant shows 66% of wild type activity 726716 1.14.13.92 T218A site-directed mutagenesis 710979 1.14.13.92 V54I the mutant shows 65% of wild type activity 726716 1.14.13.92 V54I/C65V/I67T/Q93W/I339S/S441A/A442G/S444C/M446G/L447P the mutant shows 5% of wild type activity 726716 1.14.13.92 V54I/C65V/I67T/Q93W/Q152F/I339S/S441A/A442G/S444C/M446G/L447P the mutant shows less than 3% of wild type activity 726716 1.14.13.92 V54I/C65V/I67T/Q93W/Q152F/L153G/I339S/S441A/A442G/S444C/M446G/L447P the mutant shows less than 3% of wild type activity 726716 1.14.13.92 W501A the mutant shows reduced activity compared to the wild type enzyme 726793