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J. Biol. Chem., Vol. 280, Issue 47, 39644-39652, November 25, 2005

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X-ray Crystallographic Analysis of 6-Aminohexanoate-Dimer Hydrolase

MOLECULAR BASIS FOR THE BIRTH OF A NYLON OLIGOMER-DEGRADING ENZYME^*

Seiji Negoro1, Taku Ohki, Naoki Shibata¶, Nobuhiro Mizuno, Yoshiaki Wakitani, Junya Tsurukame, Keiji Matsumoto, Ichitaro Kawamoto, Masahiro Takeo, and Yoshiki Higuchi¶

From the Department of Materials Science and Chemistry, Graduate School of Engineering, University of Hyogo, Hyogo 671-2201, the Department of Life Science, Graduate School of Life Science, University of Hyogo, Hyogo 678-1297, and ^¶RIKEN Harima Institute/SPring-8, Hyogo 679-5248, Japan

6-Aminohexanoate-dimer hydrolase (EII), responsible for the degradation of nylon-6 industry by-products, and its analogous enzyme (EII') that has only 0.5% 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 Ser¹¹² as a common active site both for nylon oligomer-hydrolytic and esterolytic activities. However, it requires at least two additional amino acid residues (Asp¹⁸¹ and Asn²⁶⁶) 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.

Received for publication, June 1, 2005 , and in revised form, September 7, 2005.

The atomic coordinates and structure factors (code 1WYB) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

^* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed: Dept. of Materials Science and Chemistry, Graduate School of Engineering, University of Hyogo, 2167 Shosha, Himeji, Hyogo 671-2201, Japan. Tel./Fax: 81-792-67-4891; E-mail: negoro{at}eng.u-hyogo.ac.jp.

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