A carboxylesterase with a β‐lactamase fold from Arthrobacter possesses a low level of hydrolytic activity (0.023 μmol·min−1·mg−1) when acting on a 6‐aminohexanoate linear dimer byproduct of the nylon‐6 industry (Ald). G181D/H266N/D370Y triple mutations in the parental esterase increased the Ald‐hydrolytic activity 160‐fold. Kinetic studies showed that the triple mutant possesses higher affinity for the substrate Ald (Km = 2.0 mm) than the wild‐type Ald hydrolase from Arthrobacter (Km = 21 mm). In addition, the kcat/Km of the mutant (1.58 s−1·mm−1) was superior to that of the wild‐type enzyme (0.43 s−1·mm−1), demonstrating that the mutant efficiently converts the unnatural amide compounds even at low substrate concentrations, and potentially possesses an advantage for biotechnological applications. X‐ray crystallographic analyses of the G181D/H266N/D370Y enzyme and the inactive S112A‐mutant–Ald complex revealed that Ald binding induces rotation of Tyr370/His375, movement of the loop region (N167–V177), and flip‐flop of Tyr170, resulting in the transition from open to closed forms. From the comparison of the three‐dimensional structures of various mutant enzymes and site‐directed mutagenesis at positions 266 and 370, we now conclude that Asn266 makes suitable contacts with Ald and improves the electrostatic environment at the N‐terminal region of Ald cooperatively with Asp181, and that Tyr370 stabilizes Ald binding by hydrogen‐bonding/hydrophobic interactions at the C‐terminal region of Ald.

Carboxylesterase (EII 0 ) 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 0 hybrid with the majority of the sequence deriving for EII 0 and possessing an EII 0 -like level of Ald-hydrolytic activity). Either a G181D or a D370Y substitution in Hyb-24 increased the Ald-hydrolytic activity ca. 10-fol d, 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.

6-Aminohexanoate-dimer hydrolase (EII), responsible for the degradation of nylon-6 industry by-products, and its analogous enzyme (EII) that has only ϳ0.5% of the specific activity toward the 6-aminohexanoate-linear dimer, are encoded on plasmid pOAD2 of Arthrobacter sp. (formerly Flavobacterium sp.) KI72. Here, we report the three-dimensional structure of Hyb-24 (a hybrid between the EII and EII proteins; EII-level activity) by x-ray crystallography at 1.8 Å resolution and refined to an R-factor and R-free of 18.5 and 20.3%, respectively. The fold adopted by the 392-amino acid polypeptide generated a two-domain structure that is similar to the folds of the penicillin-recognizing family of serine-reactive hydrolases, especially to those of D-alanyl-D-alanine-carboxypeptidase from Streptomyces and carboxylesterase from Burkholderia. Enzyme assay using purified enzymes revealed that EII and Hyb-24 possess hydrolytic activity for carboxyl esters with short acyl chains but no detectable activity for D-alanyl-D-alanine. In addition, on the basis of the spatial location and role of amino acid residues constituting the active sites of the nylon oligomer hydrolase, carboxylesterase, D-alanyl-D-alanine-peptidase, and ␤-lactamases, we conclude that the nylon oligomer hydrolase utilizes nucleophilic Ser 112 as a common active site both for nylon oligomer-hydrolytic and esterolytic activities. However, it requires at least two additional amino acid residues (Asp 181 and Asn 266 ) specific for nylon oligomer-hydrolytic activity. Here, we propose that amino acid replacements in the catalytic cleft of a preexisting esterase with the ␤-lactamase fold resulted in the evolution of the nylon oligomer hydrolase.Microorganisms are believed to be highly adaptable toward environmental conditions. This can be elucidated from the observations that microorganisms capable of degrading unnatural synthetic compounds can be isolated relatively easily. Unnatural synthetic compounds include various chemicals such as endocrine disrupters and toxic compounds, which have unfavorable effects on living cells. A suitable system to enhance the biodegradability of these compounds is important from an environmental point of view. We have been studying the degradation of a by-product of nylon-6 manufacture (i.e. 6-aminohexanoate oligomers (namely nylon oligomers)) (Fig. 1), by Flavobacterium sp. KI72 as a model for studying the adaptation of microorganisms toward unnatural compounds (1, 2).2 Three enzymes, 6-aminohexanoate-cyclic dimer hydrolase (3), 6-aminohexanoate-dimer hydrolase (EII) 3 (4), and endotype 6-aminohexanoate-oligomer hydrolase (5), encoded on the plasmid pOAD2 (45,519 bp) (6) in strain KI72, were found to be responsible for the degradation of the nylon oligomers. It was also established that the EII-analogous protein (EIIЈ) is located on a different part of the pOAD2 (7,8). EIIЈ has 88% homology to EII (7) but has very low catalytic activity ( 1 ⁄ 200 of EII activity) toward the 6-aminohexanoate-linear dimer (Ald), suggesting that EII h...

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