Structural basis of the substrate recognition of hydrazidase isolated from Microbacterium sp. strain HM58-2, which catalyzes acylhydrazide compounds as its sole carbon source

Article abstract of DOI:10.1016/j.bbrc.2016.11.148

Hydrazidase was an enzyme that remained unidentified for a half century. However, recently, it was purified, and its encoding gene was cloned. Microbacterium sp. strain HM58-2 grows with acylhydrazides as its sole carbon source; it produces hydrazidase and degrades acylhydrazides to acetate and hydrazides. The bacterial hydrazidase belongs to the amidase signature enzyme family and contains a Ser-cisSer-Lys catalytic motif. The condensation of hydrazine and carbonic acid produces various hydrazides, some of which are raw materials for synthesizing pharmaceuticals and other useful chemicals. Although natural hydrazide compounds have been identified, the metabolic systems for hydrazides are not fully understood. Here, we report the crystal structure of hydrazidase from Microbacterium sp. strain HM58-2. The active site was revealed to consist of a Ser-cisSer-Lys catalytic triad, in which Ser179 forms a covalent bond with a carbonyl carbon of the substrate. 4-Hydroxybenzoic acid hydrazide bound to the S179A mutant, showing an oxyanion hole composed of the three backbone amide groups. Furthermore, H336 in the non-conserved region in the amidase family may define the substrate specificity, which was confirmed by mutation analysis. A wild-type apoenzyme structure revealed an unidentified molecule covalently bound to S179, representing a tetrahedral intermediate.

Full text of DOI:10.1016/j.bbrc.2016.11.148

Accepted Manuscript
Structural basis of the substrate recognition of hydrazidase isolated from
Microbacterium sp. strain HM58-2, which catalyzes acylhydrazide compounds as its
sole carbon source
Tomonori Akiyama, Misaki Ishii, Atsushi Takuwa, Ken-Ichi Oinuma, Yasuyuki Sasaki,
Naoki Takaya, Shunsuke Yajima
PII:
S0006-291X(16)32022-8
10.1016/j.bbrc.2016.11.148
YBBRC 36852
DOI:
Reference:
To appear in: Biochemical and Biophysical Research Communications
Received Date: 20 November 2016
Accepted Date: 27 November 2016
Please cite this article as: T. Akiyama, M. Ishii, A. Takuwa, K.-I. Oinuma, Y. Sasaki, N. Takaya, S.
Yajima, Structural basis of the substrate recognition of hydrazidase isolated from Microbacterium sp.
strain HM58-2, which catalyzes acylhydrazide compounds as its sole carbon source, Biochemical and
Biophysical Research Communications (2016), doi: 10.1016/j.bbrc.2016.11.148.
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ACCEPTED MANUSCRIPT
Structural basis of the substrate recognition of hydrazidase isolated from Microbacterium
sp. strain HM58-2, which catalyzes acylhydrazide compounds as its sole carbon source
Tomonori Akiyamaa, Misaki Ishiia, Atsushi Takuwab, Ken-Ichi Oinumab, Yasuyuki Sasakia,
Naoki Takayab, Shunsuke Yajimaa*
aDepartment of Bioscience, Tokyo University of Agriculture, Setagaya-ku, Tokyo 156-8502,
Japan
bFaculty of Life and Environmental Sciences, University of Tsukuba, Tennodai, Tsukuba
305-8572 Japan
*Corresponding author
E-mail address: yshun@nodai.ac.jp (S. Yajima).
Keywords: amidase signature family; Microbacterium; protein structure; substrate
specificity; X-ray crystallography
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Abstract
Hydrazidase was an enzyme that remained unidentified for a half century. However,
recently, it was purified, and its encoding gene was cloned. Microbacterium sp. strain
HM58-2 grows with acylhydrazides as its sole carbon source; it produces hydrazidase and
degrades acylhydrazides to acetate and hydrazides. The bacterial hydrazidase belongs to the
amidase signature enzyme family and contains a Ser-cisSer-Lys catalytic motif. The
condensation of hydrazine and carbonic acid produces various hydrazides, some of which
are raw materials for synthesizing pharmaceuticals and other useful chemicals. Although
natural hydrazide compounds have been identified, the metabolic systems for hydrazides
are not fully understood. Here, we report the crystal structure of hydrazidase from
Microbacterium sp. strain HM58-2. The active site was revealed to consist of a
Ser-cisSer-Lys catalytic triad, in which Ser179 forms a covalent bond with a carbonyl
carbon of the substrate. 4-Hydroxybenzoic acid hydrazide bound to the S179A mutant,
showing an oxyanion hole composed of the three backbone amide groups. Furthermore,
H336 in the non-conserved region in the amidase family may define the substrate
specificity, which was confirmed by mutation analysis. A wild-type apoenzyme structure
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revealed an unidentified molecule covalently bound to S179, representing a tetrahedral
intermediate.
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1. Introduction
Hydrazine and its derivatives are important compounds for various applications such as
pharmaceuticals, dyes, and herbicide synthesis. Acylated derivatives of hydrazine are
described as R1R2N-N(R3)C(=O)R4 and are called (acyl)hydrazides. An antituberculosis
agent, isonicotinic acid hydrazide, known as isoniazid (1) is a well-known hydrazide.
Isocarboxazid is used as an antidepressant (2), and other hydrazides are used as agricultural
herbicides, pesticides, insecticides, fungicides, and plant growth regulators (3). In industry,
hydrazides are applicable to cross-linking acrylic emulsions, curing epoxy resins, and
scavenging formaldehyde through their powerful reactivity with ketone and epoxy groups
(4).
Natural compounds possessing a hydrazide moiety have been also identified (5). Agaritine
from the commercial mushroom Agaricus bisporus and its relatives were the first natural
hydrazides to be discovered (6). A hydrazide degrading enzyme, with hydrazide
hydrolyzing activity, was discovered in Mycobacterium avium that could hydrolyze various
hydrazides, including isonicotinic acid hydrazide. It was named hydrazidase (7). However,
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the molecular identification of this enzyme had not been pursued.
Recently, we isolated Microbacterium sp. strain HM58-2 from soil; this strain grows with
an acylhydrazide such as 4-hydroxybenzoic acid 1-phenylethylidene hydrazide (HBPH) as
its sole carbon source, and we cloned the hydrazidase gene responsible for the reaction (8).
Hydrazidase hydrolyzes HBPH to 4-hydroxy benzoic acid and acetophenone hydrazone
(Fig. 1A). The amino acid sequence of the hydrazidase shares an amidase signature with a
Ser-Ser-Lys motif. The amidase signature enzyme family contains a signature region of
approximately 130 amino acids that includes a canonical catalytic triad and Gly/Ser-rich
motif (GGSS[GS]G) (9). The enzymes in this family exhibit a variety of functions, such as
peptide amidase (PAM), glutamyl-tRNA amidotransferase subunit
A
(GatA),
indole-acetamide hydrolase (IAAH), eukaryotic amidase group 1 (E1), eukaryotic amidase
group 2 (E2), and 6-aminohexanoate-cyclic-dimer hydrolase (AH) (10). The amino acid
sequence of hydrazidase shares homology with those of GatA and fatty acid amide
hydrolase (FAAH) (8), whose substrates are different from hydrazidase. To elucidate the
substrate recognition mechanism of hydrazidase, we determined the crystal structures of
hydrazidase for the wild-type and the mutant complexed with a hydrazide compound.
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These structures revealed critical residues for the catalytic reaction and substrate binding in
hydrazidase.
2. Materials and Methods
2.1. Protein expression and purification
The hydrazidase gene was cloned into pET28a(+), and the recombinant protein was
expressed in Escherichia coli BL21(DE3), as described previously (8). The E. coli strain
harboring hydrazidase was cultured in LB media with 25 µg/mL kanamycin at 37°C. The
protein was induced by isopropyl-thio-β-galactopyranoside, and then, the culture was
continued for 6 hr at 30°C. The supernatant from the cell lysate was loaded on a Ni-NTA
agarose column. The column was washed with buffer A [20 mM Tris-HCl, pH 7.5, 100 mM
NaCl, 20 mM imidazole, 10% glycerol], and then, the recombinant protein was eluted with
buffer B [20 mM Tris-HCl, pH 7.5, 100 mM NaCl, 500 mM imidazole, 10% glycerol]. The
eluted protein was dialyzed with buffer C [20 mM Tris-HCl, pH 7.5] followed by
purification with a MonoQ column. The collected fraction was further purified with a
HiLoad 16/60 column with buffer E [20 mM Tris-HCl, pH 7.5, 10% glycerol]. Hydrazidase
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was concentrated to 20 mg/mL with an Amicon Ultra 30K cartridge for crystallization.
2.2. Crystallization and structure determination
Crystals were grown at 4°C by the hanging-drop vapor-diffusion method in a drop
consisting of 1.0 µl of protein solution and 1.0 µl of a crystallization reservoir solution
containing 0.1 M imidazole, 0.17 M LiSO4, and 2 M (NH4)2SO4. Under these conditions,
two crystal forms were obtained, that for wild-type hydrazidase belongs to the space group
C2221 with a = 122.1 Å, b = 127.9 Å, c = 158.8 Å, and
α = β = γ = 90°, and that for the
S197A mutant belongs to the space group P21 with unit cell parameters a = 73.0 Å, b =
126.5 Å, c = 123.2 Å,
α = 90°, β = 105.7°, and γ = 90°. Prior to data collection, the crystal
was transferred instantly into a cryoprotectant solution [21%(w/v) xylitol in the reservoir
solution] and flash-cooled in a liquid-nitrogen gas stream at 95 K. Diffraction data were
collected on beamline AR BL-NW12A equipped with a Quantum 210 CCD detector or
BL-17A with a Quantum 270 detector (Area Detector Systems Corp., CA) at the Photon
Factory, High Energy Accelerator Research Organization, Tsukuba, Japan. The diffraction
data were processed using the HKL2000 package (11).
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The initial structure of wild-type hydrazidase was solved by molecular replacement using
MoRDa (12) followed by automated model building by Arp/wArp (13). The iterative model
refinement was carried out using Coot (14) and REFMAC5 (15). The initial structure of the
S179A mutant was solved by the molecular replacement method using MORLEP (16) in
the CCP4 suite (17) with the wild-type structure as a search model. Data collection and
refinement statistics are presented in Table 1.
To elucidate the structure of the hydrazidase-substrate complex, crystals were soaked
instantly with 3 mM 4-hydroxybenzoic acid hydrazide (HBH) (Sigma-Aldrich Co. LLC.
MO) in the cryoprotectant solution. All figures of protein structures were generated using
PyMOL (v.1.4.1; Schrödinger). The atomic coordinates and structure factors for the
wild-type and the S197A mutant have been deposited in the Protein Data Bank under
accession codes 5H6T and 5H6S, respectively.
2.3. Mutant preparation and enzyme assay
The plasmid pET-hydrazidase was mutagenized for single amino acid substitutions of
C129A, C129S, and H336A of hydrazidase using a PrimeSTAR Mutagenesis Basal Kit
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(TaKaRa Bio Inc.) according to the manufacturer’s instructions with each set of the
following primers: C129A: forward 5ʹ-GACCTGGCCATCCGATGGAACTCCGTG-3ʹ and
reverse
5ʹ-TCGGATGGCCAGGTCGGGGATTCCCGC-3ʹ,
C129S:
forward
reverse
5ʹ-GACCTGTCCATCCGATGGAACTCCGTG-3ʹ
5ʹ-TCGGATGGACAGGTCGGGGATTCCCGC-3ʹ,
CGGATGGCCATCGTGGACATGTTCGGC-3ʹ
and
and
H336A:
and
forward
5ʹ-
reverse
5ʹ-CACGATGGCCATCCGCTCGCTGGCGAT-3ʹ. Purified mutants were subjected to the
enzyme assay. Reaction mixtures contained 20 mM Tris-HCl, pH 7.5, 0.2 mM HBH, and
5%(v/v) dimethyl sulfoxide in a total volume of 200 µL. The enzyme was added, and the
reaction proceeded at 30°C for 60 min before being stopped by adding 200 µL of ice-cold
acetonitrile. The reaction product, 4-hydroxybenzoic acid, was measured using a
high-performance liquid chromatography (HPLC) system equipped with an ODS-4 column
(4.6 by 250 mm) by monitoring the absorption at 250 nm. The mobile-phase solvent system
contained 50 mM Tris-HCl (pH 7.5) and acetonitrile (6:4(v/v)).
3. Results and Discussion
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3.1. Overall structure of hydrazidase
Crystals of the wild-type and the S179A mutant of hydrazidase were obtained in different
space groups, which belong to C2221 and P21, respectively, in an identical reservoir [0.1 M
imidazole, pH 8.3, 0.17 M LiSO4, 2 M (NH4)2SO4]. These crystal structures were refined at
a 1.6 Å- and 1.8 Å-resolution, respectively. Both the wild-type and mutant hydrazidase
were composed of a homodimer structure (Fig. 1C) and one dimer and two dimers in an
asymmetric unit of C2221 and P21 crystals, respectively. In the C2221 structure, the electron
density for the His x 6 tag sequence at the N-terminus was clearly observed in one subunit
of the dimer, which contributed to a crystal contact. MoRDa software used for the
molecular replacement method selected the coordinates of the PDB code 3H0L for
glutamyl-tRNA amidotransferase subunit A (GatA) (18) as a search model. 3D structure
comparison using the Dali server (19) also revealed a top hit of GatA (PDB code: 2GI3)
(Z-score = 48.8, rmsd = 2.1 Å) followed by aryl acylamidase (PDB code: 4YJ6) (Z-score =
48.7, rmsd = 2.9 Å) (20) and a series of GatA proteins including 3H0L. Then, rat fatty acid
amino hydrolase (FAAH) (PDB code: 1MT5) (21) and Granulibacter bethesdensis
allophanate hydrolase (PDB code: 4GYS) (22) were also listed. Superposing the structures
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of the hydrazidase and the amidases listed by the Dali server revealed that most of their
overall structures fit well except for two regions around the entrances of the substrate
binding pocket (Fig. 2A, B, C). Amino acid sequence alignment of these amidases showed
that those two regions (N203-E215 and E311-D349) are located at the C-terminus of the
amidase signature and are not conserved (Fig. 2D). The amino-acid differences may cause
structural varieties and hence alter the substrate specificity of these enzymes. The average
b-factors of the whole hydrazidase chains A and B (wild-type) were 14.1 and 15.5,
respectively, whereas those of region G206-N211 were higher (32.9 and 30.8 for chains A
and B, respectively). These results agree with the flexible property of the entrance that
enables it to open/close upon binding the substrate.
3.2. Substrate binding
The amidase signature enzyme family contains three highly conserved residues
characterized by the Ser-cisSer-Lys catalytic triad. The amino acid sequence alignment (8)
as well as the crystal structure of the wild-type hydrazidase identified S179-cisS155-K80 as
the catalytic triad. To elucidate the substrate binding mode, we solved the crystal structure
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of the S179A mutant complexed with 4-hydroxybenzoic acid hydrazide (HBH) (Fig. 1B).
As shown in Figures 3A and 3B, HBH bound to the catalytic site with the carbonyl oxygen
formed three hydrogen bonds with the backbone amide groups of L176, G177, and S179A.
This coordination of the carbonyl oxygen binding represented the oxyanion hole formed by
these three amide groups, which is important to stabilize the tetrahedral transition states in
the reaction.
The 4-hydroxy oxygen of HBH formed a hydrogen bond with a water molecule, which
connected to H336 by another hydrogen bond. This binding was used to determine the
substrate specificity of the hydrazidase because H336 positions in a structurally
non-conserved region among the proteins described in the previous section (Fig. 2D). The
same binding mode using the water molecule and H336 was also observed in the wild-type
(see below). Another hydrogen bond was formed between a carbonyl oxygen of C129 and
the amide group of HBH (Fig. 3B). Hydrazidase hydrolyzes HBH more effectively than
4-hydroxybenzamide (HBA) (8). The only difference between these two compounds is the
additional amide group in HBH next to the amide nitrogen in HBA. Without this amide
group, the hydrogen bond is lost between C129 and HBA, which is expected to result in
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weak binding to HBA. The potential roles of the H336 and C129 residues in substrate
binding were investigated by analyzing the enzyme activities of the C129A, C129S, and
H336A mutants. The catalytic activity of the H336A mutant was markedly reduced to 1%
(Fig. 3E). Therefore, H336 is considered to be an important residue in determining
substrate specificity. The activities of C129A and C129S also decreased to 10% and 30%,
respectively, relative to that of the wild-type (Fig. 3E). These mutations may induce some
conformational change at the respective positions because HBH binds to C129 via a
backbone carbonyl oxygen.
When the structures of the wild-type and S179A mutant were superposed, the side chains of
the residues in the substrate binding site of both structures fit very well (Fig. 3D). The
distance between the OG atom of S179 in the wild-type and the carbonyl carbon of HBH in
the mutant structure was 1.7 Å (Fig. 3B). Therefore, in combination with the three
hydrogen bonds for the oxyanion hole, the bound position of HBH is considered to reflect
the catalytic site of hydrazidase.
HBPH, which is an effective substrate for hydrazidase, also has an imide group at the same
position as the amide of HBH. When we manually superposed HBPH with HBH using the
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4-hydroxybenzene ring, the acetophenone portion hydrophobically interacted mainly with
the residues L128 and Ile130 (Supplementary Fig. 1). This could lead to more a stable
binding of HBPH than in the case of HBH.
3.3. The catalytic site of the wild-type hydrazidase
At the catalytic site of the wild-type hydrazidase, a clear electron density was observed.
The shape of the density indicates that a cyclic compound bound covalently to the S179
residue. Attempts to fit various compounds originated from the protein solution, reservoir
solution, and cryoprotectant to the density did not identify any molecule. We, therefore,
attempted to fit a dummy molecule. Based on the shape of the electron density,
1,4-dimethyl cyclohexane was initially chosen to be fit. Then, based on the distances
between the atoms of the molecule and surrounding residues, some atoms were replaced
with oxygen atoms to make hydrogen bonds, resulting in 2,5-dihydroxy 1,3-dioxan. It is
also possible that some of them could be nitrogen atoms. This dummy molecule was fit to
the density (Fig. 4), and the resulting model indicated that the molecule forms hydrogen
bonds with the backbone amide group of G177, a carbonyl oxygen of C129, and the
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carbonyl oxygen and amide group of S155 and H336 by way of a water molecule (Fig. 3C).
The distance between the OG atom of S179 and the carbon atom of the dummy molecule
was 1.5 Å, which is consistent with the covalent binding of the molecule to S179 and
represents a tetrahedral conformation generated by nucleophilic attack of the side chain of
S179 to the substrate carbonyl carbon, as seen in the proteins in the amidase signature
enzyme family (9). The hydrazidase used in this study hydrolyses an artificial synthetic
substrate HBPH, and no natural substrate for the enzyme has been identified. This unknown
compound may be a candidate, but further study is needed.
Acknowledgments
This project was supported by JSPS KAKENHI Grant Number 15K07369 to S.Y. and
partially supported by the Platform Project for Supporting in Drug Discovery and Life
Science Research (Platform for Drug Discovery, Informatics, and Structural Life Science)
from the Japan Agency for Medical Research and Development (AMED). We thank the
beamline staff at Photon Factory for the technical support during data collection.
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Figure legends
Figure 1. Enzymatic reaction and overall structure of hydrazidase from Microbacterium sp.
strain HM58-2. A. Hydrazidase hydrolyzes HBPH to 4-hydroxy benzoic acid and
acetophenone hydrazone. B. The structure of 4-hydroxybenzoic acid hydrazide (HBH). C.
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The wild-type hydrazidase (C2221 form) is shown in a cartoon model. The portion colored
in pale pink corresponds to the N-terminal His x 6 – tag region. The spheres colored in
magenta indicate the catalytic residue S179.
Figure 2. Comparisons of the structures of hydrazidase and other amidases. A-C. The
structure of the wild-type hydrazidase is superposed by Chimera (23) with those of the
amidases indicated by PDB codes. The colored regions represent structurally
non-overlapped regions and correspond to regions 1 and 2 in the sequence alignment in
panel D. The green and cyan colors indicate hydrazidase and the indicated amidases,
respectively. The spheres colored in magenta and yellow represent HBPH and the residue
H336, respectively. D. Sequence alignment of hydrazidase and amidases. Sequence
alignment by ClustalW was performed for Hydr (hydrazidase, this study), 2GI3 (GatA),
4GYS (allophanate hydrolase), 4YJ6 (aryl acylamidase), and 1MT5 (FAAH). The black
triangles indicate the residues of the catalytic triad. The green and blue triangles indicate
the residues C129 and H336, respectively, which formed hydrogen bonds with the substrate.
The blue line indicates the conserved amidase signature region, and the orange lines
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indicate two regions, which constitute the entrance of the substrate binding pockets.
Figure 3. The catalytic site of hydrazidase. A. Fo-Fc omit map (1.5σ level) of HBH
calculated by CNS (24) bound in the catalytic site as in panel B. B. The binding mode of
HBH to the catalytic site of the S179A mutant is shown with a stick model. The black
dotted lines represent the distances between two atoms that are less than 2.8 Å. The
distance between OG of S156 and NE of K80 (a grey dotted line) is 3.3 Å. The cyan
colored S179 is from the wild-type when the S179A and the wild-type structures were
superposed. The pink dotted line indicates the distance of 1.7 Å. C, A dummy molecule
bound to the catalytic site of the wild-type. The black dotted lines represent the distances
between two atoms that are less than 3.0 Å, and the pink dotted line indicates a distance of
1.5 Å. D, Superposition of the wild-type and S197A structures. HBPH colored in yellow is
also superposed manually based on the position of HBH. E. Catalytic activities of mutant
hydrazidases. Catalytic activities of three mutants, C129A, C129S, and H336A, were
compared to that of the wild-type as 100%. The procedures for the assay and measurements
are described in the Materials and Methods section.
23

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Figure 4. An Fo-Fc omit map of an unknown compound and S179. The map was calculated
by omitting the residue S179 using CNS and contoured at 3σ. A dummy molecule colored
in cyan is placed in the electron density.
24

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Table 1
Data collection and refinement statistics
Data collection
Wild type
S179A+HBH
PF BL-17A
0.9800
Beamline
PF-AR BL-NW12A
Wavelength (Å)
Temperature (K)
Detector
1.0000
95
95
Quantum 210
C2221
Quantum 270
P21
Space group
Unit cell parameters (Å, °) a = 122.2, b = 128.1, c = a = 73.0, b = 126.5, c =
159.3,
α
=
β
=
γ
= 90
123.2,
= 90
4
α = 90, β = 105.7, γ
No. mol in ASU
Resolution range (Å)
No. of unique reflections
Redundancy
2
50 – 1.6 (1.63 – 1.6)
163385 (8126)
6.1 (5.9)
50 – 1.8 (1.82 – 1.8)
196526 (9687)
3.8 (3.7)
Completeness (%)
I/σI
99.9 (100)
97.9 (96.3)
47.2 (8.9)
29.9 (5.2)
*
Rmerge
0.054 (0.232)
0.065 (0.386)
Refinement
Resolution range (Å)
Completeness (%)
No. of reflections
Rwork/Rfree
34.0 – 1.6 (1.64 – 1.6)
99.7 (98.2)
40.6 – 1.8 (1.84 – 1.8)
97.6 (92.6)
155116 (11835)
186627 (13804)
0.165/0.178 (0.170/0.193)
0.172/0.192 (0.212/0.242)
No. of non-H atoms
Protein
7055
670
n.a.
13788
727
Water
Ligand (HBH)†
R.m.s. deviation
Bond (Å)
44
0.008
1.429
0.009
1.360
Angle (°)
Average B factors (Å2)

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Protein
14.3
23.5
n.a.
17.9
Water
26.1
25.1
Ligand (HBH)†
Ramachandran plot
Favoured (%)
Allowed (%)
dsiallowed (%)
97.2
2.7
97.1
2.9
0
0.1
*Rmerge = ΣhklΣi |Ii(hkl) - <I(hkl)>|/ΣhklΣiIi(hkl), where Ii(hkl) is the intensity of an observation
and <I(hkl)> is the mean value for the unique reflection; summations are over all reflections.
†n.a. denotes not applicable.

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