ABCD1 p.Ala61Val
| Predicted by SNAP2: | C: N (78%), D: N (93%), E: N (93%), F: N (87%), G: N (97%), H: N (87%), I: N (93%), K: N (97%), L: N (93%), M: N (93%), N: N (97%), P: N (93%), Q: N (97%), R: N (97%), S: N (97%), T: N (97%), V: N (93%), W: N (57%), Y: N (66%), |
| Predicted by PROVEAN: | C: N, D: N, E: N, F: N, G: N, H: N, I: N, K: N, L: N, M: N, N: N, P: N, Q: N, R: N, S: N, T: N, V: N, W: N, Y: N, |
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[hide] Two alternative modes for optimizing nylon-6 bypro... Protein Sci. 2009 Aug;18(8):1662-73. Ohki T, Shibata N, Higuchi Y, Kawashima Y, Takeo M, Kato D, Negoro S
Two alternative modes for optimizing nylon-6 byproduct hydrolytic activity from a carboxylesterase with a beta-lactamase fold: X-ray crystallographic analysis of directly evolved 6-aminohexanoate-dimer hydrolase.
Protein Sci. 2009 Aug;18(8):1662-73., [PMID:19521995]
Abstract [show]
Promiscuous 6-aminohexanoate-linear dimer (Ald)-hydrolytic activity originally obtained in a carboxylesterase with a beta-lactamase fold was enhanced about 80-fold by directed evolution using error-prone PCR and DNA shuffling. Kinetic studies of the mutant enzyme (Hyb-S4M94) demonstrated that the enzyme had acquired an increased affinity (K(m) = 15 mM) and turnover (k(cat) = 3.1 s(-1)) for Ald, and that a catalytic center suitable for nylon-6 byproduct hydrolysis had been generated. Construction of various mutant enzymes revealed that the enhanced activity in the newly evolved enzyme is due to the substitutions R187S/F264C/D370Y. Crystal structures of Hyb-S4M94 with bound substrate suggested that catalytic function for Ald was improved by hydrogen-bonding/hydrophobic interactions between the Ald--COOH and Tyr370, a hydrogen-bonding network from Ser187 to Ald--NH(3) (+), and interaction between Ald--NH(3) (+) and Gln27-O(epsilon) derived from another subunit in the homo-dimeric structure. In wild-type Ald-hydrolase (NylB), Ald-hydrolytic activity is thought to be optimized by the substitutions G181D/H266N, which improve an electrostatic interaction with Ald--NH(3) (+) (Kawashima et al., FEBS J 2009; 276:2547-2556). We propose here that there exist at least two alternative modes for optimizing the Ald-hydrolytic activity of a carboxylesterase with a beta-lactamase fold.
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No. Sentence Comment
47 Plasmid pS1M77 was found to encode the enzyme (Hyb-24 A61V/A253T/F264C/D370Y) (designated as Hyb-S1M77) with the highest Ald-hydrolytic activity among the 350 clones in the first-cycle pS1M mutant library [Fig. 2(b)].17 The plasmids pS1M164, pS1M197, pS1M258, and pS1M259 were found to produce enzymes having the second to fifth highest activity.
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ABCD1 p.Ala61Val 19521995:47:54
status: NEW54 Nucleotide sequencing of the 1.4 kb EcoRI/HindIII-fragment encoding the nylB/nylB0 region in pS4M94 revealed that the mutant enzyme (Hyb-S4M94) had six amino acid alterations (A61V, A124V, R187S, F264C, G291R, and G338A) in addition to the D370Y mutation.
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ABCD1 p.Ala61Val 19521995:54:176
status: NEW76 X-ray crystallographic analysis To analyze the structural basis for the increased activities, we determined the substrate-bound and unbound crystal structures of Hyb-24Y and Hyb-S4M94 by X-ray crystallographic analysis at resolutions of 1.501.58 A˚ (Supporting Information Table S3) and compared the structures with that of Hyb-24DN, which had been solved previously.9 In the three-dimensional structure model of Hyb-S4M94, three of the substitutions (R187S/F264C/D370Y) are located in the catalytic cleft, whereas three others (A61V/G291R/G338A) are located on the surface, and the remaining one (A124V) is located on the inside of the protein molecule [Fig. 4(a)].
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ABCD1 p.Ala61Val 19521995:76:535
status: NEW109 Among the seven alterations differing between Hyb-24 and Hyb-S4M94, three (R187S/F264C/D370Y) were able to effectively increase the Ald-hydrolytic activity and are shown in blue, whereas the remaining four (A61V, A124V, G291R, and G338A) are shown in purple.
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ABCD1 p.Ala61Val 19521995:109:207
status: NEW[hide] Mutational analysis of 6-aminohexanoate-dimer hydr... FEBS Lett. 2006 Sep 18;580(21):5054-8. Epub 2006 Aug 28. Ohki T, Wakitani Y, Takeo M, Yasuhira K, Shibata N, Higuchi Y, Negoro S
Mutational analysis of 6-aminohexanoate-dimer hydrolase: relationship between nylon oligomer hydrolytic and esterolytic activities.
FEBS Lett. 2006 Sep 18;580(21):5054-8. Epub 2006 Aug 28., [PMID:16949580]
Abstract [show]
Carboxylesterase (EII') from Arthrobacter sp. KI72 has 88% homology to 6-aminohexanoate-dimer hydrolase (EII) and possesses ca. 0.5% of the level of 6-aminohexanoate-linear dimer (Ald)-hydrolytic activity of EII. To study relationship between Ald-hydrolytic and esterolytic activities, random mutations were introduced into the gene for Hyb-24 (an EII/EII' hybrid with the majority of the sequence deriving for EII' and possessing an EII'-like level of Ald-hydrolytic activity). Either a G181D or a D370Y substitution in Hyb-24 increased the Ald-hydrolytic activity ca. 10-fold, and a G181D/D370Y double substitution increased activity ca. 100-fold. On the basis of kinetic studies and the three-dimensional structure of the enzyme, we suggest that binding of Ald is improved by these mutations. D370Y increased esterolytic activity for glycerylbutyrate ca. 30-50-fold, whereas G181D decreased the activity to 30% of the parental enzyme.
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No. Sentence Comment
86 HPLC analysis revealed that the specific activity for Ald-hydrolysis was increased approximately ten-fold in two clones, pR1M9 (containing a single G181D substitution) and pS1M77 (containing A61V, A253T, F264C and D370Y substitutions) (Fig. 3).
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ABCD1 p.Ala61Val 16949580:86:191
status: NEW84 HPLC analysis revealed that the specific activity for Ald-hydrolysis was increased approximately ten-fold in two clones, pR1M9 (containing a single G181D substitution) and pS1M77 (containing A61V, A253T, F264C and D370Y substitutions) (Fig. 3).
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ABCD1 p.Ala61Val 16949580:84:191
status: NEW