Mutational analysis of 6-aminohexanoate-dimer hydrolase: Relationship between nylon oligomer hydrolytic and esterolytic activities

• Published in: FEBS letters 2006-09, Vol.580 (21), p.5054-5058
• Main Authors: Ohki, Taku, Wakitani, Yoshiaki, Takeo, Masahiro, Yasuhira, Kengo, Shibata, Naoki, Higuchi, Yoshiki, Negoro, Seiji
• Format: Article
• Language: English
• Subjects: 2,3-dimercaptopropan-1-ol tributyroate; 6-aminohexanoate; 6-Aminohexanoate-dimer hydrolase; 6-aminohexanoate-linear dimer; a protein with 88% homology to EII encoded on plasmid pOAD2; Ahx; Ald; Amidohydrolases - genetics; Amidohydrolases - metabolism; Amp; ampicillin; an EII/EII′ hybrid protein; Arthrobacter; Arthrobacter - enzymology; BALB; Catalysis; DNA Mutational Analysis; EII; Esterase; gene for Hyb-24; glyceryltributyrate; H-EII; H-Hyb-24; His-tagged EII; His-tagged Hyb-24; Hyb-24; Hydrolysis; Kinetics; Lipase - metabolism; Models, Molecular; Mutagenesis, Site-Directed; nylB24; Nylon oligomer; Nylons - chemistry; PCR; phenylmethylsulfonyl fluoride; Plasmid pOAD2; PMSF; polymerase chain reaction; Protein Structure, Secondary; Random mutagenesis; Recombinant Proteins - metabolism; Structure-Activity Relationship; Substrate Specificity; Tributyrin; β-lactamase
• ISSN: 0014-5793; 1873-3468
• DOI: 10.1016/j.febslet.2006.08.031

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.