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

Abstract

Nylon hydrolase (NylC) encoded by Arthrobacter plasmid pOAD2 (NylC p2 ) was expressed in Escherichia coli JM109 and purified by ammonium sulfate fractionation, anion-exchange column chromatography and gel-filtration chromatography. NylC p2 was crystallized by the sitting-drop vapour-diffusion method with ammonium sulfate as a precipitant in 0.1 M HEPES buffer pH 7.5 containing 0.2 M NaCl and 25% glycerol. Diffraction data were collected from the native crystal to a resolution of 1.60 Å . The obtained crystal was spindle shaped and belonged to the C-centred orthorhombic space group C222 1 , with unit-cell parameters a = 70.84, b = 144.90, c = 129.05 Å . A rotation and translation search gave one clear solution containing two molecules per asymmetric unit.

Structural basis of the correct subunit assembly, aggregation, and intracellular degradation of nylon hydrolase

“…In contrast, the M AV of wild-type NylC p2 was 85,602 based on sedimentation equilibrium analysis. This value was in good agreement with the molecular weight (93,000) estimated by gel-filtration chromatography 11 . These results suggest that the trimeric structure of αβ-heterodimers (3.7 S ), as well as the monomeric (2.1 S ) and dimeric (3.2 S ) structures, was formed in the case of NylC p2 .…”

“…Such a large number of monomers in one unit cell is not suitable for structural analysis at high resolution. Therefore, we screened and established a new crystallization condition, which generated crystals with only two molecules (corresponding to the A/B dimer) related by a non-crystallographic two-fold axis in an asymmetric unit (space group C 222 1 ) 11 . We performed X-ray-crystallographic studies of wild-type NylC p2 and the seven typical mutants at 1.05–2.00 Å resolution (Table S1 ).…”

“…The culture of E . coli clones and subsequent enzyme purification were performed as previously reported 9 , 11 . In the NylC activity assays, the enzyme solution (0.1 ml) was mixed with an Ahx-cyclic oligomer solution (0.9 ml) [4 mg ml −1 of Ahx-cyclic oligomer in 20 mM phosphate buffer (pH 7.3) containing 10% glycerol (buffer A)] and incubated at 30 °C (standard assay conditions).…”

“…In the NylC activity assays, the enzyme solution (0.1 ml) was mixed with an Ahx-cyclic oligomer solution (0.9 ml) [4 mg ml −1 of Ahx-cyclic oligomer in 20 mM phosphate buffer (pH 7.3) containing 10% glycerol (buffer A)] and incubated at 30 °C (standard assay conditions). An increase in the concentration of the amino group was found using trinitrobenzene sulfonic acid (TNBS) 9 , 11 . Kinetic studies were performed under standard assay conditions, with the exception of the different Ahx-cyclic oligomer concentrations used.…”

Enzymatic hydrolysis of nylons: quantification of the reaction rate of nylon hydrolase for thin-layered nylons

“…The cell lysates were obtained by ultrasonication, and the enzyme was purified using ammonium sulfate fractionation, anion exchange chromatography, and gel filtration chromatography (see "Supplementary Text") (Yasuhira et al 2011;Nagai et al 2013). The enzyme obtained at the final purification step gave a single band on native PAGE (supplementary Fig.…”