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ABSTRACT: We performed x-ray crystallographic analyses of the 6-aminohexanoate cyclic dimer (Acd) hydrolase (NylA) from Arthrobacter sp., an enzyme responsible for the degradation of the nylon-6 industry byproduct. The fold adopted by the 472-amino acid
polypeptide generated a compact mixed α/β fold, typically found in the amidase signature superfamily; this fold was especially
similar to the fold of glutamyl-tRNAGln amidotransferase subunit A (z score, 49.4) and malonamidase E2 (z score, 44.8). Irrespective of the high degree of structural similarity to the typical amidase signature superfamily enzymes,
the specific activity of NylA for glutamine, malonamide, and indoleacetamide was found to be lower than 0.5% of that for Acd.
However, NylA possessed carboxylesterase activity nearly equivalent to the Acd hydrolytic activity. Structural analysis of
the inactive complex between the activity-deficient S174A mutant of NylA and Acd, performed at 1.8 Å resolution, suggested
the following enzyme/substrate interactions: a Ser174-cis-Ser150-Lys72 triad constitutes the catalytic center; the backbone N in Ala171 and Ala172 are involved in oxyanion stabilization; Cys316-Sγ forms a hydrogen bond with nitrogen (Acd-N7) at the uncleaved amide bond in two equivalent amide bonds of Acd. A single S174A, S150A, or K72A substitution in NylA by
site-directed mutagenesis decreased the Acd hydrolytic and esterolytic activities to undetectable levels, indicating that
Ser174-cis-Ser150-Lys72 is essential for catalysis. In contrast, substitutions at position 316 specifically affected Acd hydrolytic activity, suggesting
that Cys316 is responsible for Acd binding. On the basis of the structure and functional analysis, we discussed the catalytic mechanisms
and evolution of NylA in comparison with other Ser-reactive hydrolases.
Journal of Biological Chemistry 11/2009; 285(2):1239-48. DOI:10.1074/jbc.M109.041285 · 4.57 Impact Factor