Mutational analysis of 6-aminohexanoate-dimer hydrolase: relationship between nylon oligomer hydrolytic and esterolytic activities.
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.
Ohki
Taku
T
Department of Materials Science and Chemistry, Graduate School of Engineering, University of Hyogo, 2167 Shosha, Himeji, Hyogo 671-2280, Japan.
Wakitani
Yoshiaki
Y
Takeo
Masahiro
M
Yasuhira
Kengo
K
Shibata
Naoki
N
Higuchi
Yoshiki
Y
Negoro
Seiji
S
D000581:Amidohydrolases / Q000235:genetics* / Q000378:metabolism*; D001173:Arthrobacter / Q000201:enzymology*; D002384:Catalysis; D004252:DNA Mutational Analysis; D006868:Hydrolysis; D007700:Kinetics; D008049:Lipase / Q000378:metabolism*; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009757:Nylons / Q000737:chemistry*; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000378:metabolism; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
D009757:Nylons; D011994:Recombinant Proteins; D008049:Lipase; D000581:Amidohydrolases; C026250:6-aminohexanoate-dimer hydrolase