X-ray crystallographic analysis of the 6-aminohexanoate cyclic dimer hydrolase: Catalytic mechanism and evolution of an enzyme responsible for nylon-6 byproduct degradation

Article abstract of DOI:10.1074/jbc.M109.041285

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-tRNA^Gln amido-transferase 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 A 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.

Full text of DOI:10.1074/jbc.M109.041285

Enzyme Catalysis and Regulation:
X-ray Crystallographic Analysis of the
6-Aminohexanoate Cyclic Dimer
Hydrolase: CATALYTIC MECHANISM
AND EVOLUTION OF AN ENZYME
RESPONSIBLE FOR NYLON-6
BYPRODUCT DEGRADATION
Kengo Yasuhira, Naoki Shibata, Go
Mongami, Yuki Uedo, Yu Atsumi, Yasuyuki
Kawashima, Atsushi Hibino, Yusuke Tanaka,
Young-Ho Lee, Dai-ichiro Kato, Masahiro
Takeo, Yoshiki Higuchi and Seiji Negoro
J. Biol. Chem. 2010, 285:1239-1248.
doi: 10.1074/jbc.M109.041285 originally published online November 3, 2009
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THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 285, NO. 2, pp. 1239–1248, January 8, 2010
© 2010 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.
X-ray Crystallographic Analysis of the 6-Aminohexanoate
Cyclic Dimer Hydrolase
CATALYTIC MECHANISM AND EVOLUTION OF AN ENZYME RESPONSIBLE FOR NYLON-6
□
S
BYPRODUCT DEGRADATION*
Received for publication, July 13, 2009, and in revised form, October 14, 2009 Published, JBC Papers in Press, November 3, 2009, DOI 10.1074/jbc.M109.041285
Kengo Yasuhira‡1, Naoki Shibata§¶1, Go Mongami‡, Yuki Uedo‡, Yu Atsumi‡, Yasuyuki Kawashima‡, Atsushi Hibino‡,
Yusuke Tanaka‡, Young-Ho Leeʈ, Dai-ichiro Kato‡, Masahiro Takeo‡, Yoshiki Higuchi§¶2, and Seiji Negoro‡3
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, the ¶RIKEN Harima
Institute, SPring-8 Center, Hyogo 679-5148, and the ʈInstitute for Protein Research, Osaka University, Osaka 565-0871, Japan
We performed x-ray crystallographic analyses of the contrast, substitutions at position 316 specifically affected Acd
6-aminohexanoate cyclic dimer (Acd) hydrolase (NylA) from hydrolytic activity, suggesting that Cys316 is responsible for Acd
Arthrobacter sp., an enzyme responsible for the degradation of binding. On the basis of the structure and functional analysis, we
the nylon-6 industry byproduct. The fold adopted by the 472- discussed the catalytic mechanisms and evolution of NylA in com-
amino acid polypeptide generated a compact mixed ␣/␤ fold, parison with other Ser-reactive hydrolases.
typically found in the amidase signature superfamily; this fold
was especially similar to the fold of glutamyl-tRNAGln amido-
transferase subunit A (z score, 49.4) and malonamidase E2 (z
score, 44.8). Irrespective of the high degree of structural sim-
ilarity 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-defi-
Nylon-6 is produced by ring cleavage polymerization of ⑀-ca-
prolactam and consists of more than 100 units of 6-aminohex-
anoate. During the polymerization reaction, however, some
molecules fail to polymerize and remain oligomers, whereas
others undergo head-to-tail condensation to form cyclic oli-
gomers (1, 2). These byproducts (designated nylon oligomers)
contribute to an increase in industrial waste material into the
environment. Therefore, biodegradation of the xenobiotic com-
pounds 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 spe-
cific enzymes responsible for this degradation (1, 2).
˚
cient S174A mutant of NylA and Acd, performed at 1.8 A
resolution, suggested the following enzyme/substrate inter-
actions: a Ser174-cis-Ser150-Lys72 triad constitutes the cata-
lytic center; the backbone N in Ala171 and Ala172 are involved
␥
in oxyanion stabilization; Cys316-S forms a hydrogen bond
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, gen-
erating 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 polymeriza-
tion Ͼ 3, substrates for NylC), and more than 60 kinds of vari-
ous amide compounds, including peptides and ␤-lactams
(1–3). NylA is a homodimeric enzyme, and Acd hydrolytic
activity is inhibited by diisopropylfluorophosphate, a Ser-en-
zyme-specific inhibitor, suggesting that Ser is involved in the
catalytic function (3). NylA has also been found in Pseudomo-
nas sp. NK87 and six alkalophilic strains, including Kocuria sp.
KY2 (4–6). All of the NylA proteins identified so far have been
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
* Thisworkwassupportedinpartbyagrant-in-aidforscientificresearchfrom
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
Technology Program, “Development of the Foundation for Nano-Interface
Technology” from JST, and the Japan Aerospace Exploration Agency
project.
□S
The on-line version of this article (available at http://www.jbc.org) contains
supplemental Table S1 and Figs. S1–S4.
The atomic coordinates and structure factors (codes 3A2P and 3A2Q) have been
deposited in the Protein Data Bank, Research Collaboratory for Structural
Bioinformatics, Rutgers University, NewBrunswick, NJ(http://www.rcsb.org/).
1 These authors contributed equally to this work.
2 To whom correspondence may be addressed: Dept. of Life Science, Gradu-
ate School of Life Science, University of Hyogo, 3-2-1 Koto, Kamigori-cho,
Ako-gun, Hyogo 678-1297, Japan. Tel. and Fax: 81-791-58-0179; E-mail:
hig@sci.u-hyogo.ac.jp.
4 The abbreviations used are: NylA, 6-aminohexanoate cyclic dimer hydro-
lase; Acd, 6-aminohexanoate cyclic dimer; Ald, 6-aminohexanoate linear
dimer; NylA-A174, NylA containing the S174A substitution; AS, amidase sig-
nature; GatA, glutamyl-tRNAGln amidotransferase subunit A; MAE2, malo-
namidase E2; PAM, peptide amidase.
3 To whom correspondence may be addressed: Dept. of Materials Science
and Chemistry, Graduate School of Engineering, University of Hyogo, 2167
Shosha, Himeji, Hyogo 671-2280, Japan. Tel. and Fax: 81-792-67-4891;
E-mail: negoro@eng.u-hyogo.ac.jp.
JANUARY 8, 2010•VOLUME 285•NUMBER 2
JOURNAL OF BIOLOGICAL CHEMISTRY 1239

Three-dimensional Structure of Nylon Oligomer Hydrolase
found to be immunologically identical and possess similar sub- ously (22). Buffer A (20 mM phosphate buffer containing 10%
unit molecular masses of 52 kDa (5, 6). NylA from Pseudomo- glycerol, pH7.0) was used throughout for purification. Cell
nas is 99% identical in amino acid sequence to the homolog extracts obtained by ultrasonication were used as crude
from Arthrobacter, and both nylA genes are encoded on a plas- enzyme. NylA and its mutant enzymes were fractionated by
mid (4, 7).
(NH4)2SO4 (25–55% saturated). The enzyme sample dissolved
NylA is classified as an amidase signature (AS) superfamily in buffer A was desalted using a PD-10 desalting column (GE
protein, characterized by the presence of a conserved stretch Healthcare), and the protein-containing fractions were loaded
of ϳ130 amino acid residues (AS sequence) (8–21). AS onto a Hi-Trap Q-Sepharose (GE Healthcare) column equili-
superfamily enzymes are widely distributed among microor- brated with buffer A. After the column was washed with buffer
ganisms, animals, and plants; they are involved in various A, the enzyme was eluted by increasing the NaCl concentration
biological functions, including transamidation of misacy- from 0 to 0.25 M (22). Fractions containing NylA or its mutants
lated Glu-tRNAGln (8, 9), formation of the plant hormone were identified as highly expressed 52-kDa bands on SDS-
indoleacetic acid (10, 11), and hydrolysis of malonamide (12, PAGE, which were absent in cell extracts of E. coli JM109 har-
13), fatty acid amides (14, 15), and peptide amides (16, 17). In boring pUC18. At the final stage, the enzymes were judged to be
addition, amidases from Rhodococcus rhodochrous and Sul- homogeneous by analyzing the SDS-PAGE, native PAGE, and
folobus solfataricus possess nitrilase activity, catalyzing the light-scattering diffraction patterns.
cleavage of the carbon-nitrogen triple bond in a nitrile (18,
Assay
19). Recently, NylA from Arthrobacter was also shown to exhibit
98% homology to ␻-laurolactam hydrolase from Rhodococcus
To measure Acd hydrolase activity, enzyme reactions were
(dbj͉BAG70960.1͉), Cupriavidus (dbj͉BAH09869.1͉), and Sphin- performed at 30 °C using 10 mM Acd (in buffer A) (standard
gomonas (dbj͉BAH09872.1͉), which are potentially used for pro- assay condition). Amino groups liberated by the reaction were
duction of 12-aminolauryl acid (material for nyon-12 production) quantified by colorimetry using trinitrobenzenesulfonic acid
(20). Moreover, NylA is closely related to an aryl acylamidase from (3). Enzyme reactions were similarly performed using 20 mM
Nocardia farcinica, which cleaves the amide bond in a polyamide malonamide (malonamidase activity) or 20 mM indoleacet-
model compound (adipic acid bishexylamide) and the ester bond amide (indoleacetamide hydrolase activity), and release of
in bis(benzoyloxyethyl)terephthalate and increases the hydrophi- ammonia formed by the hydrolytic reactions was quantified by
licity of polyamide 6 (21). Thus, from fundamental, industrial, and colorimetry using trinitrobenzenesulfonic acid (3).
environmental points of view, it would be interesting to know the
For kinetic study, Acd hydrolytic activity was assayed under
structuralfactorsthatdeterminetheuniquesubstratespecificityof standard assay conditions, except that various concentrations
NylA recognizing a cyclic amide compound having 6-aminohex- of Acd were used. Kinetic parameters (kcat and Km values) were
anoate as a monomeric unit.
evaluated by directly fitting the Michaelis-Menten equation to
Recently, we have reported the crystallization conditions of the data using GraphPad prism, version 5.01 (GraphPad, San
NylA from strain KI72 (22). In this paper, we performed x-ray Diego, CA). The kcat values were expressed as turnover num-
crystallographic analysis of NylA, identified the amino acid res- bers/subunit (Mr of the subunit: 53,000).
idues responsible for catalytic function based on the three-di-
For semi-quantitative detection of Acd hydrolase and glutamin-
mensional structures, estimated the catalytic mechanisms, and ase activity, the enzyme reactions were performed using purified
discussed the evolution of nylon oligomer-degrading enzymes. NylA (0.1–1 mg/ml) and 10 mM Acd or 20 mM Gln. After the
reaction proceeded at 30 °C for 2–48 h, 10-␮l aliquots were sam-
EXPERIMENTAL PROCEDURES
pled, and the reactions were stopped by incubating the aliquots in
Plasmids and Site-directed Mutagenesis
boiling water for 3 min. The reaction mixtures (1 ␮l) were spotted
onto a silica gel plate. The samples were developed by solvent mix-
ture (1-propanol:water:ethyl acetate:ammonia ϭ 24:12:4:1.3), and
degradation products were detected by spraying with 0.2% ninhy-
drin solution (in butanol saturated with water) (6).
ThehybridplasmidpUEFA, containingawild-type nylAgenein
the vector pUC18, was used as the parental plasmid DNA (22). To
obtain mutant enzyme versions of NylA, site-directed mutagene-
sis was carried out by the modification of restriction site method
(23) (for K72A, S150A, S174A, C316G, C316D, C316E, and
C316S substitutions) or PrimeSTAR mutagenesis kit (Takara
Bio Inc., Otsu, Japan) (for N125A, C316A, C316K, and C316N
substitutions) using the primers shown in supplemental Table
S1 and plasmid pUEFA as a template DNA. After sequencing the
entire nylA region, we confirmed that a single desired substitution
was integrated in the wild-type nylA sequence, and recombinant
plasmid expressing the mutant nylA genes were constructed using
the pUC18 vector, as described previously (22).
To measure carboxylesterase activity, enzyme reactions were
performed at 30 °C using 0.2 mM each of p-nitrophenylacetate
(C2-ester), p-nitrophenylbutyrate (C4-ester), and p-nitrophenyl-
octanoate(C8-ester)dissolvedin50mM phosphatebuffercontain-
ing 0.43% Tween 20 (pH 7.0). The release of p-nitrophenol
Ϫ1
(⑀400 nm ϭ 6,710 M cmϪ1) was assayed by absorbance at 400
nm. As a control, we confirmed that spontaneous hydrolysis of
p-nitrophenylesters in buffer A is not observed during the 30 min
of the reaction. Carboxylesterase activities were assayed in tripli-
cate, and the average values and standard errors were calculated.
Enzyme Purification
Crystallographic Analysis
Because cultivation at temperatures 30 °C or higher results in
insoluble NylA, Escherichia coli JM109 cells expressing NylA
Crystallization and Data Collection of Diffraction—Hexago-
and mutant enzymes were grown at 25 °C, as described previ- nal prismatic crystals (0.3 ϫ 0.3 ϫ 0.5 mm) were obtained by the
1240 JOURNAL OF BIOLOGICAL CHEMISTRY
VOLUME 285•NUMBER 2•JANUARY 8, 2010

Three-dimensional Structure of Nylon Oligomer Hydrolase
sitting drop vapor diffusion method as reported previously (22). probity (29) Ramachandran analysis showed 467 residues
The droplets were prepared by mixing 2–3 ␮l of purified NylA (97.1%) in favored regions, 481 residues (99.8%) in allowed
solution (10 mg mlϪ1 protein in 20 mM phosphate buffer con-
taining 10% glycerol, pH 7.3) and the same volume of reservoir
solution containing 1.0 M sodium citrate as a precipitant in 0.1 M
imidazole buffer (pH 8.0) and 12.5% glycerol and equilibrated
against 100 ␮l of reservoir solution at 10 °C. The crystal be-
longed to space group P62, with unit cell parameters a ϭ b ϭ
130.75, c ϭ 58.23 Å (see Table 1). For native crystals, the crystals
were soaked for 24 h in cryoprotectant solution (1.0 M sodium
citrate, 0.1 M imidazole, pH 8.0, 25% glycerol). Heavy atom
derivatives were prepared by soaking the crystals for 24 h in
cryoprotectant solution containing 1 mM HgCl2. For analysis
of enzymeꢀsubstrate complexes, the activity-deficient S174A
mutant of NylA (NylA-A174) was used to prevent hydrolysis of
the substrate during crystallographic analysis. the NylA-
A174ꢀAcd complex was prepared by soaking the crystals in the
cryoprotectant solution containing 20 mM Acd for 3 h. Cryo-
cooling was performed by blowing cold nitrogen steam onto the
crystals at 100 K. The diffraction data sets of the NylA crystal
and NylA-A174ꢀAcd complex were collected at the SPring-8
(Hyogo, Japan) Beamlines BL38B1 (equipped with a Rigaku
Jupiter CCD detector system) and BL41XU (equipped with an
ADSC Quantum 315 detector system), respectively. For HgCl2
derivatives, diffraction data were recorded on Beamline BL-5A
at the Photon Factory (Tsukuba, Japan) using an ADSC Quan-
tum 315 detector system. The following parameters were cho-
sen for data collection: wavelength, 1.0000 Å; crystal to detector
distance, 180 mm for native crystals, 300 mm for mercury
derivatives, and 200 mm for NylA-A174ꢀAcd complexes; oscil-
lation range/image, 0.5° for native crystals and mercury deriva-
tives or 1.0° for NylA-A174ꢀAcd complexes. Indexing, integra-
tion, and scaling of reflections were performed using the
HKL2000 program package (24). The diffraction data were col-
lected from native NylA crystal, NylA-mercury (II) dichloride
derivative, and NylA-A174ꢀAcd complex crystal to resolutions
of 1.90, 2.06, and 1.80 Å, respectively (see Table 1).
regions, and 1 residue (Thr75) (0.2%) in outliers for NylA and
465 residues (96.9%) in favored regions, 479 residues (99.8%) in
allowed regions, and 1 residue (Ser416) (0.2%) in outliers for the
NylA-A174ꢀAcd complex. The electron density map of Thr75
and Ser416, located in outliers, was clear enough to assign the
structures without ambiguity. The results of the crystal struc-
ture analysis are summarized in Table 1. Figures of three-di-
mensional models of proteins were generated with the program
MolFeat (version 3.6; FiatLux Co., Tokyo, Japan).
RESULTS AND DISCUSSION
Overall Structure of NylA and Structural Comparison with
Proteins in Protein Data Bank
The structure of NylA from Arthrobacter sp. KI72 was solved
with single anonymous dispersion phasing using a mercury
derivative crystal of the enzyme at 2.2 Å resolution, and models
of the native enzymes were constructed at 1.9 Å resolution
(Table 1). NylA consists of a single polypeptide chain of 472
amino acid residues (3). The overall structure of the molecule is
a compact mixed ␣/␤ fold containing a central irregular ␤-sheet
composed of 11 ␤-strands surrounded by 20 ␣-helices. Each
␤-strand is sequentially rotated in the same direction with
respect to an adjacent ␤-strand, such that the ␤-strand at one
end of the molecule is rotated ϳ180o relative to the ␤-strand at
the other end of the molecule (see Fig. 2A). A homology search
based on the NylA structure was carried out using the DALI
program. Among the ϳ55,000 proteins registered in the Pro-
tein Data Bank, five proteins classified as “AS superfamily,” glu-
tamyl-tRNAGln amidotransferase subunit A (GatA) (z score,
49.4) (Protein Data Bank code 2df4) (9), probable amidase (z
score, 45.0) (Protein Data Bank code 2dc0), malonamidase E2
(MAE2) (z score, 44.8) (Protein Data Bank code 1ocm) (12),
peptide amidase (PAM) (z score, 44.0) (Protein Data Bank code
1m21) (16), and fatty acid amide hydrolase (z score, 38.2) (Pro-
tein Data Bank code 1mt5) (14), and their mutants were
extracted as proteins with high z scores. The z scores of other
proteins in Protein Data Bank were found to be lower than 4.5.
Bacterial glutamyl-tRNAGln amidotransferase is a heterotri-
mer (GatCAB). GatA receives top scores in a Protein Data Bank
homology search; the protein possesses glutaminase activity for
generating an ammonia donor for recruitment of the amido-
transferase reaction of misacylated Glu-tRNAGln to a corrected
Gln-tRNAGln. MAE2 and PAM catalyze the hydrolysis of mal-
onamide and peptide amide, respectively. Fatty acid amide
hydrolase is a transmembrane protein that catalyzes the hydro-
lysis of fatty-acid amide. Functionally, GatA, MAE2, and PAM
catalyze the hydrolysis of terminal amide bonds, releasing
ammonia, whereas NylA specifically hydrolyzes internal amide
linkages of Acd (Fig. 1). Although “probable amidase” (Protein
Data Bank code 2dc0) was extracted as the second highest z
score, its function has not been identified. Accordingly, struc-
tural and functional comparison mainly focuses on GatA,
MAE2, and PAM, which have z scores higher than 40.
Phase Determination, Model Building, and Crystallographic
Refinement—The NylA structure was determined by the single
wavelength anomalous diffraction method using the HgCl2
derivative data. The mercury substructure for the derivative
crystal was solved at 2.1 Å resolution by the program BnP (25)
using the anomalous signal of the mercury atoms. Initial phase
parameters were determined using the program SHARP (26).
The electron density map was automatically traced with ARP/
wARP. The models for the untraced regions, except for amino
acids at position 1–4, 19–26, 403–410, and 486–493, were
constructed by manual model building using XFIT (27). Rigid
body refinement was performed using the coordinates of the
initial model to fit the unit cell of the NylA crystal, followed by
positional and B-factor refinement with the program CNS (28).
For the NylA-A174ꢀAcd complex, rigid body refinement was
performed using the coordinates of the refined NylA structure
to fit the unit cell of the NylA-A174ꢀAcd complex, followed by
positional and B-factor refinement with the program CNS.
With several cycles of a manual model rebuilding by XFIT,
R-factor and Rfree were 18.1 and 19.5% (for NylA) and 18.3 and
The structurally superimposable regions comprise 451 resi-
19.5% (for the NylA-A174ꢀAcd complex), respectively. The Mol- dues (GatA), 405 residues (MAE2), or 439 residues (PAM), and
JANUARY 8, 2010•VOLUME 285•NUMBER 2
JOURNAL OF BIOLOGICAL CHEMISTRY 1241

Three-dimensional Structure of Nylon Oligomer Hydrolase
TABLE 1
Data collection statistics
The values in parentheses are for the outer resolution shell.
Data collection
NylA (native)
NylA-A174ꢀAcd complex
Mercury (II) chloride derivative
Space group
P62
P62
P62
Unit cell a ϭ b (Å)
c (Å)
130.75
130.66
131.39
58.23
58.63
58.07
Wavelength (Å)
Resolution (Å)
Total reflections
Unique reflections
Completeness (%)
1.0000
1.0000
1.0000
30-1.90 (1.97-1.90)
485,579
30-1.80 (1.86-1.80)
567,837
30-2.06 (2.13-2.06)
363,039
44,300 (4,021)
98.6 (90.4)
3.8 (30.4)
24.5 (5.2)
52,953 (5,212)
99.7 (99.1)
5.4 (48.9)
22.3 (5.0)
34,955 (2,980)
97.2 (82.3)
5.8 (33.4)
16.5 (6.6)
R
merge (%)
ϽIϾ/Ͻ␴(I)Ͼ
Phasing
Figure of merit, centric/acentric
Phasing power
RCurris
0.106/0.380
1.67
0.649
Refinement
Resolution range (Å)
28.0–1.90 (1.97–1.90)
50–1.80 (1.86–1.80)
R
R
work (%)a
free (%)b
18.1 (26.2)
21.4 (28.4)
3,602
0
18.7 (27.9)
22.2 (33.0)
3,592
16
No. of protein atoms
No. of ligand atoms
No. of water molecules
Root mean square bond distances (Å)
Root mean square bond angles (°)
Average B-factor
445
521
0.005
1.2
0.005
1.2
Protein
38.6
50.3
35.0
45.7
47.9
Ligand
Solvent
͉Rwork ϭ ͚ ͉͉Fobs ͉ Ϫ k͉Fcalc͉͉(͚ ͉Fobs͉)Ϫ1; k, scaling factor.
a
hkl
hkl
hkl
b Rwork ϭ ͚ ͉͉F ͉ Ϫ k͉Fcalc͉͉(͚ ͉Fobs͉)Ϫ1, where all of the reflections belong to a test set of randomly selected data.
hkl obs
sponding region in the other AS superfamily enzymes (supple-
mental Figs. S1 and S2). As discussed below, we suggest that
dynamic motion of this flexible loop is important for the cata-
lytic function of NylA.
Functional Comparison with Typical AS Superfamily Enzymes
To examine the activity of NylA on substrates recognized by
typical AS superfamily enzymes, we examined glutaminase,
malonamidase, and indoleacetamide hydrolase activity in NylA
(Fig. 1, B–D). TLC analyses demonstrated that the product
(Ald) was detected from Acd within 2 h of the reaction using
purified NylA (0.1 mg mlϪ1). In contrast, no detectable amount
of product (Glu) was obtained from the substrate (Gln), even
after continuing the reaction for 48 h using 1 mg mlϪ1 of NylA
(data not shown). This suggests that the glutaminase activity
caused by NylA was less than 0.5% of the level of Acd hydrolytic
activity. Moreover, the hydrolytic activity of NylA for mal-
onamide and indoleacetamide is ϳ0.1% of the level of its activ-
ity for Acd. These results demonstrate that NylA specifically
recognizes Acd as a preferred substrate irrespective of the
structural conservations with other AS superfamily enzymes. In
contrast, NylA had hydrolytic activity for p-nitrophenylacetate
(C2-ester), p-nitrophenylbutyrate (C4-ester), and p-nitrophe-
nyloctanoate (C8-ester), which are almost equivalent to the
level of activity for Acd (Fig. 1E and Table 2). The structural
basis for substrate recognition, on the basis of the enzyme/sub-
strate interaction, is discussed below.
FIGURE 1. Hydrolysis of various amide and ester compounds. A, Acd. B,
Gln. C, malonamide. D, indoleacetamide. E, p-nitrophenylacetate (C2-ester).
Enzyme/Substrate Interaction
Catalytic Residues—In AS superfamily enzymes, the Ser-cis-
the root mean square deviations of the superimposed C atoms Ser-Lys triad has been proposed to make up the catalytic resi-
were calculated to be 2.2–2.4 Å. The loop region flanke␣d by ␤9 dues (8–21). The NylA triad (Ser174-cis-Ser150-Lys72) spatially
and H17 in NylA was 10–14 residues longer than the corre- shares a similar position with that of MAE2 (Ser155-cis-Ser131
-
1242 JOURNAL OF BIOLOGICAL CHEMISTRY
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Three-dimensional Structure of Nylon Oligomer Hydrolase
TABLE 2
Effect of amino acid substitution in NylA on Acd hydrolytic and esterolytic activity
The effects of amino acid substitutions at positions 72, 150, 174, 125, and 316 were tested using purified NylA and its mutant enzymes. Enzyme activities for 10 mM Acd,
0.2 mM p-nitrophenylacetate (C2-ester), 0.2 mM p-nitrophenylbutyrate (C4-ester), and 0.2 mM p-nitrophenyloctanoate (C8-ester) were assayed. The specific activity (␮mol
minϪ1(units) mgϪ1) was estimated for each enzyme. The numbers in parentheses indicate the activity relative to the specific activity of wild-type NylA. Acd hydrolytic
activity was assayed in duplicate, and the average value was calculated. The deviations were found to be less than 2%.
Esterase activity
Acd hydrolytic activity
C2-ester
C4-ester
C8-ester
Ϫ1
Ϫ1
units mg
units mg
Wild type
K72A
4.17 (100)
Ͻ0.002
1.91 Ϯ 0.02 (100)
Ͻ0.001
1.78 Ϯ 0.06 (100)
Ͻ0.001
1.48 Ϯ 0.08 (100)
Ͻ0.001
S150A
S174A
N125A
C316A
C316S
C316G
C316D
C316E
C316N
C316K
Ͻ0.001
Ͻ0.001
Ͻ0.001
Ͻ0.001
Ͻ0.001
Ͻ0.001
Ͻ0.001
Ͻ0.001
0.57 (13.7)
0.73 (17.5)
4.46 (107)
7.70 (185)
0.063 (1.51)
0.040 (0.96)
0.18 (4.32)
0.15 (3.60)
1.37 Ϯ 0.01 (71.7)
1.73 Ϯ 0.01 (90.6)
3.10 Ϯ 0.14 (162)
2.38 Ϯ 0.06 (125)
0.82 Ϯ 0.03 (42.9)
1.13 Ϯ 0.02 (59.2)
0.83 Ϯ 0.01 (43.5)
0.84 Ϯ 0.05 (44.0)
1.11 Ϯ 0.02 (62.3)
1.92 Ϯ 0.03 (108)
2.53 Ϯ 0.14 (142)
2.29 Ϯ 0.08 (129)
1.48 Ϯ 0.08 (83.1)
1.50 Ϯ 0.09 (84.3)
1.12 Ϯ 0.06 (62.9)
1.11 Ϯ 0.01 (62.4)
1.05 Ϯ 0.02 (70.9)
1.49 Ϯ 0.04 (101)
3.33 Ϯ 0.23 (225)
1.83 Ϯ 0.07 (124)
1.01 Ϯ 0.01 (68.2)
0.96 Ϯ 0.06 (64.9)
0.75 Ϯ 0.05 (50.7)
0.82 Ϯ 0.02 (55.4)
mM Acd). Single S174A, S150A, or
K72A mutations resulted in drastic
decreases in Acd hydrolytic and
esterolytic activities to undetectable
levels (Ͻ0.001 unit mgϪ1) (Table 2),
indicating that the Ser174-cis-
Ser150-Lys72 triad is responsible for
NylA function.
To analyze the enzyme/substrate
interactions, we performed x-ray
crystallographic analysis of NylA-
A174 (S174A mutant of NylA)ꢀAcd
complex at 1.80 Å resolution (Table
1). Amino acid residues and Acd
substrate in the catalytic cleft gave a
clear electron density distribution
for which structural models could
be determined (Fig. 3). Superimpo-
sition of the Acd-bound structure
with the unbound structure re-
vealed that the root mean square
␣
deviation at C is smaller than 0.1 Å
for the overall structures. Interest-
ingly, Acd is entirely trapped inside
the protein molecules upon Acd
binding (Figs. 2A, 3B, and 4). Acces-
sibility of the substrate and water
molecules to the catalytic center is
discussed below.
In most Ser-reactive hydrolases,
the reaction steps of catalysis are
divided into acylation and deacyla-
tion steps (30, 31). During acylation,
the catalytic reactions are initiated
FIGURE 2. Stereoviews of NylA structures. A, ribbon diagram of the overall structure of NylA. ␣-Helices and
␤-strands are colored in green and orange, respectively, with the number shown in Fig. S1. Substrate Acd and
catalytic residues (Lys72, cis-Ser150, and Ser174) are shown by a “ball and stick diagram” colored in red and blue,
respectively. B, superimposition of NylA structure (green) with glutamyl-tRNAGln amidotransferase subunit A
(Protein Data Bank code 2df4; blue), malonamidase E2 (Protein Data Bank code 1ocm; purple), and peptide
amidase (Protein Data Bank code 1m21; olive). Superimposition was carried out for the Ser-cis-Ser-Lys catalytic
triad and its linked region on the transformation matrix generated by secondary structure matching (45).
␥Ϫ
by nucleophilic attack of Ser-O to
Lys62), GatA (Ser178-cis-Ser154-Lys79), and PAM (Ser226-cis-
the electrophilic carbonyl carbons
Ser202-Lys123) (Fig. 2B). To examine whether the NylA triad is
responsible for catalytic function, we individually replaced
Ser174, cis-Ser150, and Lys72 with Ala by site-directed mutagen-
esis, purified the mutant enzymes to homogeneity, and assayed
of amide bonds in the substrate. Therefore, questions are raised
as to what residue functions as a nucleophile. X-ray crystallo-
graphic analysis of NylA and the NylA-A174ꢀAcd complex sug-
␥
gested that the environment around Ser174-O is not suffi-
␥
the enzymatic activities under “standard assay conditions” (10 ciently polar to promote deprotonation of Ser174-O H. In
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Three-dimensional Structure of Nylon Oligomer Hydrolase
3C). This spatial location should
␥
enable Ser174-O to form hydrogen
bonds simultaneously with cis-
␥
Ser150-O and cis-Ser150-N. Because
of the dual hydrogen bonding, it is
␥
likely that Ser174-O favors a depro-
tonated form even at physiological
pH, resulting in the increase in
nucleophilicity of Ser174 (Fig. 5, A
and B).
It should be noted that the CD
spectrum of the Ala150 mutant was
found to be similar to that of wild-
type NylA (supplemental Fig. S3A).
This result indicates that substitu-
tion of Ser150, which has an unusual
cis-conformer, with Ala does not
cause misfolded proteins. More-
over, the Ala150 mutant has no
detectable activity (Ͻ0.001 unit
mgϪ1) under any assay conditions
using different Acd concentrations
(0.625, 1.25, 2.5, 5.0, and 10.0 mM;
almost maximum solubility in
water) and an enzyme sample that
has been concentrated 20-fold (2.07
mg mlϪ1). On the basis of these
findings, we estimated that turnover
(kcat) of the Ala150 mutant is Ͻ0.001
s
Ϫ1. Similar roles of cis-Ser for
increasing the nucleophilicity of
catalytic Ser have been proposed for
PAM and MAE2 (12, 16).
Substrate-binding Residues—
Structural analysis of NylA and the
NylA-A174ꢀAcd complex revealed
that six hydrogen bonds are possible
between NylA and Acd (Fig. 3B).
Among the four hydrogen bonds
connecting to cleaved amide link-
ages in Acd, two bonds, from Acd-
C1-carbonyl O to Ala171-N (back-
bone) and to Ala172-N (backbone),
are estimated to be responsible for
oxyanion stabilization (see “Pro-
posed Catalytic Mechanisms”),
whereas the other two bonds, from
Acd-N14 to Gly124-O (backbone)
FIGURE 3. Stereoviews of catalytic cleft of NylA and NylA-A174ꢀAcd complex. A and B, 2Fo Ϫ Fc electron
density maps of NylA (green) (A) and the NylA-A174ꢀAcd complex (orange) (B) contoured at 1.0 ␴. Side chain
atoms of catalytic/binding residues (Lys72, Gly124, Asn125, cis-Ser150, Ala171, Ala172, Ser174 (Ala174), and Cys316),
water molecules, and substrate Acd are shown. C, structure around catalytic residues of NylA (green) was
superimposed with that around corresponding residues of the NylA-A174ꢀAcd complex (orange). Carbon, nitro-
gen, and oxygen atoms in substrate Acd are shown in yellow, blue, and red, respectively. Possible hydrogen
bonds are indicated as red dotted lines with the distances in angstroms.
␥
(3.0 Å) and to cis-Ser150-O (2.8 Å),
are estimated to be involved in
addition, because Ser174-O H is ϳ4.8 Å away from Lys72-N , it Acd binding (Fig. 3C). In addition, at the uncleaved amide
␥
␨
is unlikely that Lys72 directly participates in eliminating the linkage in Acd, two hydrogen bonds, from Acd-N7 to
⑀
proton from Ser174-O H (Fig. 3). However, Ser150 exhibited an Cys316-S (3.2 Å) and from Acd-C8-carbonyl O to Asn125-N
␥
␥
unusual cis-conformation. We estimate that low steric hin- (2.7 Å), were identified (Fig. 3B). In the multiple three-di-
drance of Ser150 for the backbone structures, because of the mensional structural alignments, Cys316 in NylA is altered to
presence of consecutive Gly148/Gly149 residues, should allow Ala313 (GatA), Gln277 (MAE2), or Leu363 (PAM), suggesting
the unusual cis-conformation of Ser150. Ser174-O is spatially that the interaction at position 316 is unique for NylA. Sim-
␥
2.8 Å from either cis-Ser150-O or cis-Ser150-N (backbone) (Fig. ilarly, Asn125 in NylA is altered to Met129 (GatA) and Ser112
␥
1244 JOURNAL OF BIOLOGICAL CHEMISTRY
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Three-dimensional Structure of Nylon Oligomer Hydrolase
protein structures were not affected
much by substitutions at the sub-
strate-binding sites, because the
CD spectra of the three mutants
(N125A, C316S, and C316D) were
very similar to the spectrum of wild-
type enzyme (supplemental Fig.
S3B). Substitution to acidic (Glu
and Asp), basic (Lys), or other polar
(Asn) residues decreased the activ-
ity to 1–4% of the wild-type level
(Table 3). Although the C316S sub-
stitution had no significant effect on
activity under standard assay condi-
tions, a kinetic study showed that
the Km for Acd had decreased from
3.5 mM (wild-type NylA) to 1.8 mM
(Ser316 mutant) without affecting
the kcat. It is likely that possible
hydrogen bonding between the
␥
Acd-N7 and Ser316-O more stably
positions the substrate than hydro-
␥
gen bonding with Cys316-S . Unex-
pectedly, kcat was enhanced in the
Gly316 mutant without affecting the
Km, even though the C316G muta-
tion should have resulted in the loss
of hydrogen bonding at position
316. In contrast, the C316D muta-
tion significantly decreased activity.
Because of the limited solubility of
Acd in water, it is possible to per-
FIGURE 4. Surface structures of the entrance of the catalytic cleft of NylA-A174ꢀAcd complex. A, stereoview
form kinetic studies only at concen-
trations lower than 10 mM Acd.
Although the standard error for the
calculated Km value is quite large
because of extrapolation to higher
concentrations, we found that the
of the entrance of the catalytic cleft. The flexible loop region (positions 400–414) is shown in yellow, whereas
rest of the region is shown in gray. B, stereoview of cross-section, including catalytic/binding residues and
substrate Acd. The main chain folding (ribbon diagram) and side chain (ball and stick diagram) of catalytic/
binding residues (Lys72, Gly124, Asn125, cis-Ser150, Ala171, Ala172, Ser174 (Ala174), and Cys316) are shown. The
surface is shown as a gray layer. Carbon, nitrogen, and oxygen atoms in substrate Acd are shown in yellow, blue,
and red, respectively.
(MAE2), although Asn125 is conserved as Asn172 in PAM calculated Km value of the Asp316 mutant was ϳ20 mM, which
(supplemental Fig. S1).
exceeds the saturated Acd concentration, and the kcat value was
To examine the effect of amino acid substitutions at posi- ϳ3% the level of wild-type NylA (Table 3). Thus, the binding
tions 125 and 316 on catalytic function, we constructed the affinity and turnover of catalysis are estimated to be drastically
N125A and C316A mutants by site-directed mutagenesis. decreased by C316D substitutions.
Assays under standard conditions showed that hydrolytic activ-
However, it should be demonstrated that amino acid substi-
ity was ϳ14% (Ala125 mutant) and 18% (Ala316 mutant) of the tutions at position 316 had less effect on carboxylesterase activ-
wild-type NylA level (Table 2). A kinetic study showed that ity than Acd hydrolytic activity. Namely, the Acd hydrolytic
the N125A mutation increased the Km ϳ4-fold, suggesting that activity varied 200-fold (0.96–185% the activity of wild-type
the decrease in substrate-binding is due to the loss of hydrogen NylA) among the mutants, whereas the activity for C2-, C4-,
bonding between the Acd-C8-carbonyl O and the Asn125-N . and C8-esters was only within the range of 43–225% (Table 2).
⑀
Similarly, the C316A substitution should result in the loss of Substrate recognition between amides and carboxylesters is
hydrogen bonding between Acd-N7 and Cys316-S . However, discussed below with other Ser-reactive hydrolases.
␥
the C316A substitution decreased the Km to approximately
Proposed Catalytic Mechanisms
one-quarter the level of wild-type NylA (Table 3). Moreover,
these two mutations also decreased the kcat values to 23%
From the spatial location of catalytic/binding residues and
(Ala125 enzyme) and 14% (Ala316 enzyme) of the wild-type NylA functional analysis by site-directed mutagenesis, we estimated
level.
The effects of amino acid substitutions at position 316 were
the catalytic mechanisms of NylA (Fig. 5).
Nucleophilic Attack of Ser174 and Oxyanion Stabilization—
more extensively examined. It should be noted that the overall Nucleophilic Ser174-O activated by cis-Ser150 should attack
␥Ϫ
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Three-dimensional Structure of Nylon Oligomer Hydrolase
Formation of Acyl-enzyme—To
break down the tetrahedral inter-
mediate to the acyl-enzyme, the
tetrahedral intermediate should
accept a proton from a general acid.
␥
Because cis-Ser150-O is close to
Acd-N14 (nitrogen of cleaved amide
linkage in Acd) (distance, 2.8 Å)
(Fig. 3C), it should be possible that
␥
cis-Ser150-O H functions as the
general acid, providing a proton to
the departing amino group (Fig.
5C). The proton transfer should be
facilitated by the amino group of
ϩ
Lys72-NH3 , which can stabilize the
␥Ϫ
deprotonated Ser150-O
(2.7 Å
ϩ
from Lys72-NH3 ) (Figs. 3C and
5C). Thus, cis-Ser150 and Lys72
should be involved in facilitating
proton transfer and maintaining the
optimum electrostatic environment
to promote the acylation of Ser174
.
Deacylation—Conversion of the
acyl-enzyme to free enzyme (deacy-
lation) generally requires two steps,
i.e. formation of a tetrahedral in-
termediate through nucleophilic
attack of a water molecule (Fig. 5D)
and regeneration of free enzyme
(Fig. 5E). In NylA, water molecules
responsible for deacylation have not
been found around the catalytic
center in the Acd-bound structure
(Fig. 3B). However, as discussed
below, we estimate that the enzyme
accepts water molecules for deacy-
lation from solvent water through
FIGURE 5. Proposed catalytic mechanism of NylA. The steps in the catalytic reaction are shown. A, free
enzyme. B, enzyme ϩ substrate. C, tetrahedral intermediate. D, acyl-enzyme. E, tetrahedral intermediate.
the dynamic motion of a flexible loop region (Phe400–Phe414
)
(Fig. 4 and supplemental Fig. S4). The incorporated water mol-
ecule should function as a nucleophile to form a tetrahedral
intermediate, and the resulting tetrahedral intermediate should
TABLE 3
Kinetic parameters of NylA mutant enzymes
Acd hydrolytic activity was assayed using the purified enzymes under standard assay
conditions, except that various concentrations of Acd were used.
Kinetic study
Mutation
ϩ
ϩ
be stabilized by Ala171-N and Ala172-N (Fig. 5E), in a similar
fashion as the acylation step. Finally, the intermediate should be
hydrolyzed to Ald plus free enzyme, which reinitiates the suc-
cessive catalytic cycle.
Km
mM
kcat
s
kcat/Km
s
Ϫ1
Ϫ1
Ϫ1 mM
Wild type
N125A
C316A
C316S
3.51 Ϯ 0.16
13.2 Ϯ 0.75
0.85 Ϯ 0.04
1.80 Ϯ 0.20
3.66 Ϯ 0.27
19 Ϯ 10
4.97 Ϯ 0.10
1.12 Ϯ 0.04
0.71 Ϯ 0.01
4.83 Ϯ 0.17
9.15 Ϯ 0.28
0.14 Ϯ 0.05
1.42
0.085
0.83
2.68
Accessibility of Substrate to Catalytic Center
C316G
C316D
2.50
0.007
The catalytic (Ser174, cis-Ser150, Lys72) and binding (Asn125
and Cys316) residues in NylA are located at ␤2 (Lys72), H7 (cis-
the electrophilic carbonyl carbon of the substrate (Fig. 5B), with Ser150), H8 (Ser174), H6 (Asn125), and H14 (Cys316) (supple-
the subsequent formation of a tetrahedral intermediate con- mental Fig. S1). Through these interactions, NylA should hold
taining an unstable oxyanion (Fig. 5C). Therefore, stabilization Acd inside the catalytic cleft, surrounded by stable secondary
of the oxyanion is important for catalytic function. The car- structures (Fig. 4B). Therefore, questions are raised regarding
bonyl oxygen in Acd is spatially 2.9 and 3.0 Å from the main how the Acd substrate can penetrate close to the catalytic res-
chain nitrogens of Ala171 and Ala172, respectively (Fig. 3C). idues located near the center of the globular protein molecule.
Therefore, we estimate that the oxyanion formed during Acd As stated above, the loop region (Thr388–Phe414), flanked by ␤9
hydrolytic reaction is stabilized by the positively charged nitro- and H17 in NylA, is 10–14 residues longer than the corre-
gens at Ala171 and Ala172 (Fig. 5C).
sponding regions in MAE2, PAM, and GatA (supplemental Fig.
1246 JOURNAL OF BIOLOGICAL CHEMISTRY
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Three-dimensional Structure of Nylon Oligomer Hydrolase
S1). It is likely that the longer loop makes it more flexible. Actu- Comparison with Other Ser-reactive Hydrolases and
Evolutionary Implications
ally, crystallographic analysis of NylA revealed that the atomic
B-factors of amino acid residues are significantly large (80–95
Å2), especially at Phe400–Arg410, compared with helices,
␤-strands, and the other loop regions (supplemental Fig. S4).
Because the atomic B-factors correlate with the degree of ther-
mal motion and spatial disorder in the crystal structures, the
structure of the loop should significantly deviate during the
Typical Ser-proteases (trypsin, etc.) and lipase/esterases pos-
sess a classical Ser-His-Asp(Glu) triad, in which His abstracts
the proton of Ser-OH and Asp (Glu) promotes the role of His by
stabilizing the positively charged His-imidazole (30, 31).
Another catalytic triad, Ser-Tyr-Lys, has been identified in
NylB (Ald-hydrolase) (37–41), NylBЈ carboxylesterase (37–
41), EstB carboxylesterase (42), DD-peptidase (43), and class C
␤-lactamase (44). NylB and a NylB/NylBЈ hybrid protein
(Hyb24DN; having a NylB-level of Ald hydrolytic activity)
effectively recognize substrates having 6-aminohexanoate
(nylon unit) as N-terminal residues in the substrate but possess
activities for various carboxylesters (37–41).
enzymatic reaction. Interestingly, the flexible loop (Phe410
–
Phe414) region forms a catalytic cleft with a long helix (H14) and
short helix (H15), including catalytic/binding residues (Fig. 4).
We suggest that the dynamic motion of the loop located
between ␤-strand 9 (␤9) and Helix 17 (H17) allows Acd to
incorporate into the cleft, including the catalytic center.
Assuming that the substrate is incorporated in this manner, the
catalytic triads (Ser174-cis-Ser150-Lys72) are located at the bot-
tom of the catalytic cleft, whereas the binding residues (Asn125
and Cys316) are localized closer to the entrance of the cleft.
X-ray crystallographic analysis of Hyb-24DN (a NylB/NylBЈ
hybrid) and its Ald-bound structure demonstrates that Ald
binding induced the transition from the open (substrate-un-
bound) form to the closed (substrate-bound) form through the
dynamic motion of the loop region and the flip-flop of Tyr170
,
whereas the enzyme performs the catalytic function as a relaxed
open form during the entire catalytic cycle as a carboxylesterase
(38). This model is consistent with our finding that Ald hydro-
lytic activity is significantly affected by amino acid substitutions
at positions 170, 181, 187, 264, 266, and 370, responsible for Ald
binding, whereas amino acid substitutions at these positions
barely affect the esterase activity (37–41). A molecular dynamic
simulation for NylB/NylBЈ has suggested that the substrate-
unbound open form is maintained at an energy minimum and
requires activation energy to transition to the substrate-bound
closed form (38). In addition, water molecules incorporated
into the catalytic cleft are excluded in the closed form but
regained from the solvent water at deacylation with a back tran-
sition to the open form. We have speculated that the release of
6-aminohexanoate (corresponding to the C-terminal half of
Ald) triggers the penetration of water molecules to the catalytic
center (38). On the basis of these results, we have proposed that
NylB has evolved from a pre-existing esterase with a ␤-lacta-
mase fold (37–41).
Water Molecules
In most hydrolases, the catalytic sites are open to solvent, so
the enzyme can accept water molecules for deacylation from
the massive solvent environment. In class A ␤-lactamase,
hydrolytic water, activated by Glu166, is considered to be
responsible for either the acylation or deacylation step (32–34).
In NylA, 521 and 445 water molecules were identified for Acd-
bound and unbound structures, respectively. Larger numbers
of water molecules in substrate-bound structures are thought
to be due to the higher resolutions. In unbound structures,
seven water molecules (Wat34, Wat181, Wat230, Wat231,
Wat232, Wat233, and Wat260) were identified in the cleft
around the catalytic residues (Fig. 3A). However, in the
enzymeꢀsubstrate complex, these water molecules are replaced
with Acd, and no water molecules generating hydrogen bonds
with substrate or catalytic residues can be identified (Fig. 3B).
Wat354 in the NylA-A174ꢀAcd complex (Wat361 in the
⑀
unbound enzyme) is 3.5 Å from Asn125-N . However, Wat354 is
close to uncleaved amide linkages in Acd and therefore is not
considered to be responsible for deacylation. These results sug-
gest that the enzyme accepts water molecules responsible for
deacylation during the dynamic motion of the flexible loop
regions.
Present x-ray crystallographic analysis revealed that NylA is
classified as a member of the AS superfamily and utilizes the
distinct catalytic triad Ser-cis-Ser-Lys. Although NylA pos-
sesses broad specificity for carboxylesters, we estimate that the
unique substrate specificity of NylA recognizing the nylon-6
byproduct should be achieved by incorporating amino acid res-
idues responsible for Acd-binding while utilizing the common
Ser-cis-Ser-Lys catalytic triad conserved in the AS superfamily.
Thus, the new enzyme acting on the nylon-6 byproduct seems
to diverge from ancestral proteins to create families composed
of highly specialized enzymes.
Even after completing the stage of acylation, no reaction
product should be released from NylA utilizing cyclic amide
compounds as substrate (Fig. 5D), whereas first reaction prod-
uct is released from ordinary protease/esterase utilizing the lin-
ear amide/ester compounds. Therefore, the bulky N-(6-amino-
hexanoyl)-6-aminohexanoyl group in the acyl-NylA enzyme
transiently alters the position of the flexible loop, which allows
the penetration of water molecules to the catalytic center.
Alternatively, the loop region flexibly fluctuates regardless of
the presence or absence of substrate and freely accepts water
molecules from the solvent environment. In either case,
dynamic motion of the flexible loop associated with the uptake
of substrate/water molecules and release of product should be
essential for the catalytic reaction of NylA.
In malonamidase E2 (MAE2, an AS family enzyme), the cat-
alytic activity is drastically decreased by R158E and R158Q
mutations, and the kcat value of the R158K mutant is similar to
that of wild type with a Km value increased by 100-fold (13). In
Ϫ
Hyb-24DN, Asp181-COO is responsible for electrostatic sta-
ϩ
bilization of Ald-NH3 , and D181K and D181H substitutions
decreased the activity by Ͼ10,000-fold (37, 38). In contrast to
the drastic effect by electrostatic stabilization, D370Y substitu-
JANUARY 8, 2010•VOLUME 285•NUMBER 2
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Three-dimensional Structure of Nylon Oligomer Hydrolase
B. F. (2002) Science 298, 1793–1796
tion in NylBЈ-type carboxylesterase (Hyb-24) increased the Ald
hydrolytic activity by 8.2-fold, concomitant with formation of a
hydrogen bond with substrate Ald. Thus, we roughly estimate
that the energetic contribution of a hydrogen bond contributes
a ϳ10-fold difference in binding affinity (39–41). However, the
“improvements” in NylA activity caused by substitution at posi-
tion 316 are extremely modest. We suggest that that the effects
of amino acid substitution at the Acd-binding sites in NylA are
summarized as follows: (i) Even if the hydrogen bond at Acd-N7
is eliminated, close contact with the substrate and inner surface
of the protein molecule still stably holds the substrate inside the
globular protein. (ii) Because of the inflexible compact struc-
ture of Acd trapped in the catalytic cleft, amino acid substitu-
tions interacting at the uncleaved site (Acd-N7) affect the suit-
able positioning of the cleaved site (Acd-C1/Acd-N14) against
the catalytic residues, resulting in the decrease in kcat. (iii) Sub-
stitution of Cys316 to a bulky/polar residue (Glu, Asp, Asn, and
Lys), located at the entrance of the catalytic cleft, has a negative
effect on the incorporation of the substrate into the catalytic
cleft, resulting in a decrease in enzyme activity. In particular,
replacement to acidic residues (Glu and Asp) significantly
decreases the activity, probably because of an electrostatic
effect. Finally, it should be noted that plasmid-encoded NylA
from Arthrobacter and Pseudomonas and ␻-laurolactam
hydrolase from Rhodococcus, Cupriavidus, and Sphingomonas
exhibit 98–99% overall homology, although these strains have
been isolated independently and classified as different genera.
These results may imply that the nylA-related genes have been
recently distributed among microorganisms in the course of
evolution, probably by plasmid-mediated gene transfer (4–7,
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