ABCMdb

PMID: 19521995

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., [PubMed]

Sentences

No. Mutations Sentence Comment

47 ABCD1 p.Ala61Val 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. Login to comment 54 ABCD1 p.Ala61Val 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. Login to comment 76 ABCD1 p.Ala61Val 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)]. Login to comment 109 ABCD1 p.Ala61Val 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. Login to comment