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

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 ␣/␤ fold, typically found in the amidase signature superfamily; this fold was especially similar to the fold of glutamyl-tRNA Gln 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. Nylon-6 is produced by ring cleavage polymerization of ⑀-caprolactam and consists of more than 100 units of 6-aminohexanoate. During the polymerization reaction, however, some molecules fail to polymerize and remain oligomers, whereas others undergo head-to-tail condensation to form cyclic oligomers (1, 2). These byproducts (designated nylon oligomers) contribute to an increase in industrial waste material into the environment. Therefore, biodegradation of the xenobiotic compounds is important in terms of environmental points of view. In addition, the biological system for degradation provides us with a suitable system to study how microorganisms have evolved specific enzymes responsible for this degradation (1, 2).Previous biochemical studies in a nylon oligomer-degrading bacterium Arthrobacter sp. strain KI72 have revealed that three enzymes, 6-aminohexanoate cyclic dimer hydrolase (NylA), 4 6-aminohexanoate dimer hydrolase (NylB), and endo-type 6-aminohexanoate oligomer hydrolase (NylC), are responsible for the degradation of nylon oligomers (1, 2). NylA specifically hydrolyzes one of the two equivalent amide bonds in Acd, generating a 6-aminohexanoate linear dimer (Ald) (see Fig. 1A). However, NylA is barely active on Ald (substrate for NylB), 6-aminohexanoate cyclic oligomers (degree of polymerization Ͼ 3, substrates for NylC), and more than 60 kinds of various amide compounds, including peptides and ␤-lactams (1-3). NylA is a homodimeric enzyme, and Acd hydrolytic activity is inhibited by diisopropylfluorophosphate, a Ser-enzyme-specific inhibitor, suggesting that Ser is involved in the catalytic function (3). NylA has also been found in Pseudomonas sp. NK87 and six alkalophilic strains, including Kocuria sp. KY2 (4 -6). All of the NylA proteins identified so far have been * This work was supported in part by a grant-in-aid for scientific research from the Japan Society for Promotion of Science and by grants from the Global Centers of Excellence Program, the National Project on Protein Structural and Functional Analyses, the Core Research for Evolutional Science and Technolo...

Structure-Based Engineering of Amidase from Pantoea sp. for Efficient 2-Chloronicotinic Acid Biosynthesis

“…bonds between the residues and substrate. The oxyanion hole of amidases plays a crucial role in catalysis by stabilizing the tetrahedral intermediate (TI) or the transition state (TS) for formation and hydrolysis of the acyl-enzyme intermediate (17)(18)(19)25). It was found in our study that mutation of Gly175 to Ala175 resulted in a 3.2-fold increase in the activity of Pa-Ami, while mutation of Gly175 to any other amino acids or mutation of other amino acid residues located near the oxyanion hole abolished or led to extremely low activities toward 2-chloronicotinamide (data not shown).…”

“…To further elucidate the mechanism of the improved catalytic activity of the double mutant toward 2-chloronicotinamide, the substrate access tunnels of the wild type and the G175A/A305T mutant ( Fig. 3; Table 5) were also built and analyzed using the software program PyMOL with plugin CAVER3.0 (23)(24)(25). The tunnel analysis indicated that the bottleneck radius and length of the catalytic tunnel in the G175A/A305T mutant were 1.44 Å and 11.64 Å, respectively, compared with 1.13 Å and 11.99 Å in the wild type.…”

The Biochemistry and Molecular Biology of Xenobiotic Polymer Degradation by Microorganisms

“…Negoro's group also explained the catalytic mechanisms and evolution of enzymes from the ancestor enzymes NylB and NylA, based on X-ray analysis of crystallized enzymes. 89,90) "-Caprolactam is a starting material for nylon-6 manufacturing, and is assimilated by a P. aeruginosa strain. 91) A thermophilic bacterium, Geobacillus thermocatenulatus, has been suggested as a possible degrader of nylon-12 and 66 but not nylon-6, 92) but its enzymes have yet to be characterized.…”

Crystallization and X-ray diffraction analysis of nylon hydrolase (NylC) fromArthrobactersp. KI72

“…We have studied the degradation of polymeric nylon-6 and the byproducts of the manufacture of nylon-6, namely 6-aminohexanoate oligomers (nylon oligomers), as a model system (Okada et al, 1983;Negoro, 2000). We found that three enzymes, 6-aminohexanoatecyclic dimer hydrolase (NylA; Yasuhira et al, 2010), 6-aminohexanoate-dimer hydrolase (NylB; Negoro et al, 2005;Ohki et al, 2006Ohki et al, , 2009Kawashima et al, 2009) and nylon hydrolase (NylC), are responsible for the degradation of nylon-6 and its related compounds.…”