Three-dimensional structure of nylon hydrolase and mechanism of nylon-6 hydrolysis

Abstract

We performed x-ray crystallographic analyses of the 6-aminohexanoate oligomer hydrolase (NylC) from Agromyces sp. at 2.0 Å-resolution. This enzyme is a member of the N-terminal nucleophile hydrolase superfamily that is responsible for the degradation of the nylon-6 industry byproduct. We observed four identical heterodimers (27 kDa + 9 kDa), which resulted from the autoprocessing of the precursor protein (36 kDa) and which constitute the doughnut-shaped quaternary structure. The catalytic residue of NylC was identified as the N-terminal Thr-267 of the 9-kDa subunit. Furthermore, each heterodimer is folded into a single domain, generating a stacked αββα core structure. Amino acid mutations at subunit interfaces of the tetramer were observed to drastically alter the thermostability of the protein. In particular, four mutations (D122G/H130Y/D36A/E263Q) of wild-type NylC from Arthrobacter sp. (plasmid pOAD2-encoding enzyme), with a heat denaturation temperature of T(m) = 52 °C, enhanced the protein thermostability by 36 °C (T(m) = 88 °C), whereas a single mutation (G111S or L137A) decreased the stability by ∼10 °C. We examined the enzymatic hydrolysis of nylon-6 by the thermostable NylC mutant. Argon cluster secondary ion mass spectrometry analyses of the reaction products revealed that the major peak of nylon-6 (m/z 10,000-25,000) shifted to a smaller range, producing a new peak corresponding to m/z 1500-3000 after the enzyme treatment at 60 °C. In addition, smaller fragments in the soluble fraction were successively hydrolyzed to dimers and monomers. Based on these data, we propose that NylC should be designated as nylon hydrolase (or nylonase). Three potential uses of NylC for industrial and environmental applications are also discussed.

FIGURE 1.

Stereo view of quaternary structure of NylC_A. A, the quaternary structure is shown, with different colors highlighting the individual molecules A (green), B (blue), C (red), and D (yellow). The catalytic residue Thr-267 (the N terminus of the β-subunit) is shown as a space-filling model (red). B, an enlarged view of molecule A and its interfaces with the adjacent molecules B and D. Six residues selected for mutagenesis (Asp-36 (Ala-36 in NylC_K), Ser-111, Gly-122, Tyr-130, Ala-137, and Met-225) are shown as space-filling models. Because of the poor electron density distribution of the C-terminal region in the α-subunit, including Glu-263 (Gln-263 in NylC_K), the adjacent Pro-260 is shown as a space-filling model (magenta). C, surface structure of NylC_A. The α-subunit and β-subunit in a single heterodimer (molecule A) are highlighted in dark green and light green, respectively. The other three heterodimers are shown in different shades of gray.

FIGURE 2.

Stereo view of subunit structure and catalytic center of NylC_A. A, the overall structure of the heterodimer (molecule A) is shown as a ribbon diagram. Ten helices (H1–H10) and eighteen β-strands (S1–S18) are colored in green and orange, respectively. H1–H6 and H8–H10 are α-helices. H7 is a 3₁₀ helix. B, the structure around the catalytic residue Thr-267 of NylC_A (green) is superimposed onto the structure of DmpA (PDB ID code, 1B65; magenta) and BapA (PDB ID code, 3N33; orange). Possible hydrogen bonds in NylC_A are indicated as dotted lines with the distances listed in angstroms.

FIGURE 3.

Thermostability of NylC mutants. A, thermal transition curves of the various NylC mutant enzymes. CD measurements were performed at 220 nm from 25 to 95 °C (1 °C min⁻¹). The results are expressed as the mean residue molar ellipticity [θ]. Protein concentrations of 0.1 mg ml⁻¹ were used. deg, degree. B, the cumulative effects of amino acid mutations on the melting temperatures of denaturation (T_m) are shown.

FIGURE 4.

Possible interactions at the subunit interfaces. A–D, the possible interactions at the subunit interfaces around Gly-122 (A), Asp-36/Tyr-130 (B), Ser-111/Ala-137 (C), and Met-225 (D) are shown. Molecule A, molecule B, and molecule D in the quaternary structure are colored in green, blue, and yellow, respectively. Possible hydrogen bonds and contacts between two atoms are indicated as dotted lines with the distances listed in angstroms.

FIGURE 5.

Nylon degradation tests using argon cluster SIMS and TLC. Nylon-6 powder (10 mg) was pretreated in triplicate in buffer A (180 μl) at 120 °C for 20 min. The NylC_p2-G¹²²Y¹³⁰A³⁶Q²⁶³ mutant (1 mg ml⁻¹, 20 μl) was then added and incubated at 60 °C for 2 h. The experiment was performed in triplicate. A, the reaction products (both the soluble and the insoluble fractions) were spotted onto a silicon plate (1 cm²). The m/z range of 5,000–35,000 was analyzed. Red line, pretreated nylon-6 at 120 °C in buffer A; blue line, enzyme-treated nylon-6; black line, enzyme alone. B, the solid fraction was washed with distilled water, lyophilized, and dissolved in trifluoroethanol (0.2 ml), and a fraction (0.02 ml) was spotted onto a silicon plate (1 cm²). The m/z range of 1,500–15,000 was analyzed. C, the reaction products (both the soluble and the insoluble fractions) were spotted onto a silicon plate (1 cm²). The m/z range of 0–1,500 was analyzed. The positions of Ahx monomer to heptamer (marked by 1–7; m/z = 113, 226, 339, 452, 565, 678, 791) and those of the potassium-bound forms (marked by 3K–7K) are shown. D, the soluble fractions (1 μl) were spotted onto a thin layer plate and developed, and the degradation products were detected by the ninhydrin reaction (5). Slot 1, 6-aminohexanoate; slot 2, 6-aminohexanoate-dimer; slot 3, pretreated nylon-6 at 120 °C in buffer A; and slot 4, enzyme-treated nylon-6.

FIGURE 6.

Mode of nylon-6 degradation. The polymer chains are stabilized by hydrogen bonds with adjacent chains aligned in the reverse orientation (α-Crystal) (1). Some chains constitute amorphous regions. The circles indicate the cyclic oligomers attached to the polymers. Long arrows indicate the direction of the polymer chains. Short arrows indicate cleavage by the enzyme. The 6-aminohexanoate-oligomers were converted to 6-aminohexanoate by the subsequent NylB reaction.