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D. Meng et al. / Biochimica et Biophysica Acta xxx (2013) xxx–xxx
Table 1
t1:1
75
76
77
78
79
80
81
82
83
84
85
86
87
88
89
90
91
92
93
94
95
96
97
98
99
Agaricus bisporus (J.E. Lange) Imbach is a macro-fungus, which is a
Oligonucleotides used for cloning and expression of wild-type and mutant Ab-ACO t1:2
proteins.
“medium-evolved” species between plants and microorganisms, that
also produces ethylene during differentiation and development. A rela-
tionship between the sporocarp development and the appearance of
the peaks of ethylene production has been investigated [12,13]. Never-
theless, neither evidence for a similar regulatory role of ethylene as in
higher plants nor information on the route of ethylene biosynthesis
has been found in A. bisporus. A homologous gene encoding ACO was
recently isolated from A. bisporus during the genome project [14].
However, definite information on the nature of catalysing the produc-
tion of ethylene in A. bisporus by Ab-ACO, which could regulate growth
and development, is currently lacking.
By contrast, plant ACC oxidase has been scrutinised to various
degrees. ACO is a member of a superfamily of oxygenases and oxidases,
most members of which couple the reaction of the oxidative decar-
boxylation of α-ketoglutarate (α-KG) to substrate oxidation [15]. ACO
is unusual as the enzyme does not require α-KG as a cosubstrate but
uses the cosubstrate ascorbate and the activator CO2 instead [16,17].
ACO couples the two-electron oxidation of ACC and ascorbate to pro-
duce ethylene, HCN, CO2, and dehydroascorbate, using a single non-
heme Fe (II) ion and dioxygen [16,18]. The active site of ACO contains
the conserved Fe (II) binding residues, namely, a 2His–1Asp facial
triad and the putative co-substrate hydrogen-binding residues (RXS)
[19]. Thus, it is of interest and of significant importance to know if
ACO in A. bisporus (Ab-ACO) has similar biochemical and structural
t1:3
Oligonucleotide sequencea
Proteins
WT_for
WT_rev
H216D_f
5´-GGGAATTCCATATGACTATCATAACCCAGCCTCCCG-3´
5´-CGCGGATCCATTATAATGCCGAAGTAAAACACCG-3´
5´-CGGAGTCTGGTTGAAAGGAAATACTGACTT-3´
5´-TTCCTTTCAACCAGACTCCGCCGGATTTAT-3´
5´-GGTTGAAAGGAAATACTGAGTTTATGACGC-3´
5´-CTCAGTATTTCCTTTCAACCAGACTCCGCC-3´
H216D_v
H216D/D218E_f
H216D/D218E_v
H273Q_f
5´-ACAAGCCGACAATCCAAAGAGTGCGCC-3´
5´-TTGGATTGTCGGCTTGTAGTAGCCTCC-3´
5´-ACAAGCCGACAATCCAAAGAGTGCGCC-3´
H273Q_v
H216D/D218E/H273Q_f
H216D/D218E/H273Q_v
R287G_f
R287G_v
G289S_f
G289S_v
5´-TTGGATTGTCGGCTTGTAGTAGCCTCC-3´
5´-CCAACAAAACAAGACTGGAGTTGGACT-3´
5´-CAGTCTTGTTTTGTTGGTCCAGAGGTG-3´
5´-AACAAGACTAGAGTTAGTCTCTTATATTTC-3´
5´-ACTAACTCTAGTCTTGTTTTGTTGGTCCAG-3´
aMutated nucleotides are shown in boldface and magenta.
t1:4
ascorbic acid, 10% (v/v) glycerol and 0.5% Triton X-100. For cell lysis, 136
the suspension was sonicated on ice after 1 mM protease inhibitor 137
phenylmethanesulphonyl fluoride (PMSF) was added. The soluble 138
and insoluble fractions were separated by centrifugation at 10,000 g 139
for 10 min at 4 °C. The pellet containing the insoluble recombinant 140
pET3a-AbACO protein (inclusion bodies, IBs) was washed three times 141
with 50 mM Tris–HCl (pH 8.0), 100 mM NaCl, 1 M urea, and 1% Triton 142
X-100. Then, the pellet was resuspended in a buffer containing 8 M 143
urea and 10 mM Tris–HCl (pH 7.4), and the pellet was then solubilised 144
for 4 h with stirring at room temperature. The solubilised inclusion 145
body was centrifuged at 10,000 g for 15 min at 4 °C, and the superna- 146
100 characteristics. It is unknown whether Ab-ACO has catalytic residues
101 similar to plant ACOs.
102
To address this need, we purified and analysed the biochemical
103 properties of recombinant Ab-ACO. The residues that are involved in
104 catalysis were identified by site-directed mutagenesis guided by protein
105 structure homology modelling in combination with spectral analysis
106 to identify the reported Ab-ACO as the ethylene-forming enzyme in
107 A. bisporus.
tant was collected.
147
Denatured proteins were refolded as described by Wang et al. 148
[22] with minor modifications. The proteins were dialysed against 149
2 L of freshly made 6, 4, 2, 1.5, 1, 0.5, and 0 M urea, consecutively, 150
with 50 mM Tris (pH 7.4), 12 mM Hepes (pH 7.4), 60 mM KCl, 1 mM 151
EDTA and 1 mM DTT. For each concentration, the protein was dialysed 152
108 2. Materials and methods
for 12 h with stirring at 4 °C.
153
109 2.1. Cloning and expression of Ab-ACO proteins
The wild-type and mutant Ab-ACO proteins were both purified 154
according to the reported method [23]. The protein content was deter- 155
mined by the Bradford method with bovine serum albumin as standard 156
110
Total RNA was isolated from the fruiting bodies of A. bisporus
111 according to our optimised method [20] and was then stored at
112 −80 °C for further use. First strand cDNA was synthesised using oligo-
113 dT18 (Promega) and 2 μg total RNA treated with RNase-free DNase I
114 and M-MLV reverse transcriptase (Promega), according to the method
115 of Zhao et al. [21]. The coding sequence of ACO from A. bisporus was
116 amplified from the first strand cDNA with EasyPfu DNA Polymerase
117 (TransGen Biotech, Beijing, China) and specific primers, WT_for and
118 WT_rev (Table 1), which were designed based on the genome sequence
120 Agabi_varbur_1). The resulting PCR product was ligated and introduced
121 into the pET3a (Novagen Inc., Madison, WI, USA) expression plasmid,
122 which contains an isopropyl β-D-thiogalactoside-inducible promoter
123 and was used to express wild-type recombinant Ab-ACO.
[24]. SDS-PAGE was then performed to identify the proteins.
157
2.3. NH2-terminal amino sequence analysis
158
The amino acid sequence of the N-terminus was determined using 159
a Procise 491 protein sequencer (Applied Biosystems) by automated 160
Edman degradation as described by Li et al. [25].
161
2.4. Sequence alignment and molecular modelling
162
To select candidates for site-directed mutagenesis, the amino 163
acid sequence of ACO from A. bisporus was aligned with ACO1 from 164
Malus domestica (SWISSPROT Q00985), ACO4 from Solanum lycopersicum 165
(SWISSPROT P24157), ACO2 from Carica papaya (SWISSPROT Q9ZRC9), 166
ACO1 from Petunia hybrida (SWISSPROT Q08506), ACO4 from 167
Arabidopsis thaliana (SWISSPROT Q06588), and ACO4 from Vigna 168
radiata (SWISSPROT Q2KTE3) using DNAMAN (5.2.2) and EMBOSS 169
124 2.2. Preparation of wild-type and mutant Ab-ACO proteins
125
Both wild-type and mutant plasmids were transformed into
126 Escherichia coli BL21 (DE3) pLysS competent cells using conventional
127 methods. The transformed E. coli cells were grown in Luria–Bertani
128 broth with 50 μg/mL ampicillin, initially at 37 °C until the cultures
129 reached in logarithmic phase (OD600 ≈ 0.5). Thereafter, the cells
130 were cooled to 27 °C to induce protein production by the addition
131 of isopropyl-β-D-thiogalactoside (IPTG) at a final concentration of
132 0.5 mM. Induction was carried out at 27 °C for 8 h.
sequence alignment, respectively.
171
The most successful general approach for predicting the structure 172
of proteins involves the detection of homologs with known three- 173
dimensional (3D) structure–template-based homology modelling or 174
fold-recognition [26]; thus, several homologous proteins for the prelimi- 175
nary model of Ab-ACO were initially selected based on the GenTHREADER 176
scores. However, all homologous proteins from the database showed 177
b25% sequence identity. In general, alignment errors are relatively 178
133
The cell pellets were harvested by centrifugation and were
134 resuspended in a buffer containing 50 mM MOPS (3-(N-morpholino)
135 propanesulphonic acid, pH 7.4), 1 mM dithiothreitol (DTT), 3 mM
Please cite this article as: D. Meng, et al., Identification and active site analysis of the 1-aminocyclopropane-1-carboxylic acid oxidase catalysing
Meng, Demei
