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AEM00084-12 (Revised) 1
Iodide Oxidation by a Novel Multicopper Oxidase from 2
α-Proteobacterium Strain Q-1 3
4
Running title: Bacterial iodide-oxidizing enzyme 5
Mio Suzuki,1 Yoshifumi Eda,1 Shiaki Ohsawa,1 Yu Kanesaki,2 Hirofumi 6
Yoshikawa,2,3 Kan Tanaka,1,4 Yasuyuki Muramatsu,5 Jun Yoshikawa,1 Ikuo Sato,1 7
Takaaki Fujii,1 and Seigo Amachi1# 8
9
Graduate School of Horticulture, Chiba University, 648 Matsudo, Matsudo City, Chiba 10
271-85101, Genome Research Center, NODAI Research Institute, Tokyo University of 11
Agriculture, 1-1-1 Sakuragaoka, Setagaya-ku, Tokyo 156-85022, Department of 12
Bioscience, Tokyo University of Agriculture, 1-1-1 Sakuragaoka, Setagaya-ku, Tokyo 13
156-85023, Chemical Resources Laboratory, Tokyo Institute of Technology, 4259-R1-29 14
Nagatsuta, Midori-ku, Yokohama 226-85034, and Department of Chemistry, Gakushuin 15
University, Mejiro 1-5-1, Toshima-ku, Tokyo 171-8588, Japan.5 16
Copyright © 2012, American Society for Microbiology. All Rights Reserved.Appl. Environ. Microbiol. doi:10.1128/AEM.00084-12 AEM Accepts, published online ahead of print on 23 March 2012
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Address correspondence to Seigo Amachi, [email protected]
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ABSTRACT 19
α-Proteobacterium strain Q-1 is able to oxidize iodide (I-) to molecular iodine (I2) by 20
an oxidase-like enzyme. One of the two isoforms of the iodide-oxidizing enzyme 21
(IOE-II) produced by this strain was excised from a native polyacrylamide gel, 22
eluted, and purified. IOE-II appeared as a single band (51 kDa) and showed 23
significant in-gel iodide-oxidizing activity in sodium dodecyl sulfate polyacrylamide 24
gel electrophoresis without heat treatment. However, at least two bands with much 25
higher molecular masses (150 and 230 kDa) were observed with heat treatment 26
(95°C, 3 min). IOE-II was inhibited by NaN3, KCN, EDTA, and a copper chelator, 27
o-phenanthroline. In addition to iodide, IOE-II showed significant activities toward 28
phenolic compounds such as syringaldazine, 2,6-dimethoxy phenol, and 29
p-phenylenediamine. IOE-II contained copper atoms as prosthetic groups and had 30
UV/VIS absorption peaks at 320 and 590 nm. Comparison of several internal amino 31
acid sequences obtained from trypsin-digested IOE-II with a draft genome sequence 32
of strain Q-1 revealed that the products of two open reading frames (IoxA and IoxC), 33
with predicted molecular masses of 62 and 71 kDa, are involved in iodide oxidation. 34
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Furthermore, subsequent tandem mass spectrometric analysis repeatedly detected 35
peptides from IoxA and IoxC with high sequence coverage (32% to 40%). IoxA 36
showed homology with the family of multicopper oxidases, and included four 37
copper-binding regions that are highly conserved among various multicopper 38
oxidases. These results suggest that IOE-II is a multicopper oxidase, and that it may 39
occur as a multimeric complex in which at least two proteins (IoxA and IoxC) are 40
associated.41
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INTRODUCTION 42
Iodine is an essential trace element for humans and animals, and is a constituent of the 43
thyroid hormones thyroxine and triiodothyronine. Insufficient iodine in the diet can cause 44
iodine deficiency disorders such as endemic goiter and cretinism (19, 20). Iodine is 45
present in seawater at a concentration of 0.45 µM (50), and the predominant chemical 46
forms are iodide (I-, oxidation state: −1), iodate (IO3-, oxidation state: +5), and organic 47
iodine compounds (14, 35, 44, 50, 53). In the biogeochemical cycling of iodine, 48
microorganisms may play important roles, together with marine algae and phytoplankton, 49
through the production (1, 2) and remineralization (27, 28) of organic iodine compounds 50
and the redox changing of inorganic iodine species, i.e., iodide and iodate (3, 4). 51
Oxidation of iodide is an important process in the redox cycling of iodine, but little is 52
known about the mechanism of this reaction in nature. Wong (50, 51) suggested that 53
autooxidation of iodide to iodate does not occur in seawater, since the first step of iodide 54
oxidation, i.e., oxidation of iodide to molecular iodine (I2, oxidation state: 0), is 55
thermodynamically unfavorable. However, subsequent oxidation of I2 to form iodate may 56
occur spontaneously, suggesting that the oxidation of iodide to I2 is mediated biologically 57
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in nature (52). Although marine algae such as Laminaria are known to oxidize iodide 58
using a cell wall haloperoxidase (25), there are still many uncertainties surrounding iodide 59
oxidation by other organisms, especially by microorganisms. 60
Previously, we screened for the presence of iodide-oxidizing microorganisms in 61
different environments, and found that certain heterotrophic bacteria isolated from 62
iodide-rich natural gas brine water were able to oxidize iodide to I2 (4). They were 63
phylogenetically divided into two groups (groups A and B) within α-Proteobacteria. 64
Group A was most closely related to Roseovarius tolerans, with 16S rRNA gene sequence 65
similarities of 94%–98%, while group B was distantly related to Rhodothalassium 66
salexigens, with sequence similarities of 89%–91%. Such iodide-oxidizing bacteria were 67
repeatedly isolated from brine water, which is a unique environment containing a very 68
high level of iodide (0.06−1.2 mM), but soil and seawater samples did not yield any 69
isolates. Interestingly, iodide-oxidizing bacteria were successfully isolated from natural 70
seawater samples, if the samples had been incubated with 1 mM iodide for a week to 71
several months. After incubation, the population size of iodide-oxidizing bacteria in the 72
samples increased to 103−105 CFU·mL-1, while, before incubation, the population size 73
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was below the detection limit (less than 10 CFU·mL-1) (4). 74
Iodide-oxidizing activity was detected in the culture supernatant of iodide-oxidizing 75
bacteria (4). The activity was not dependent on H2O2, but was completely inhibited under 76
anaerobic conditions. These results suggest that an extracellular oxidase is involved in 77
iodide oxidation. In this study, we purified the iodide-oxidizing enzyme (IOE) from a 78
group B iodide-oxidizing bacterial strain, Q-1, and characterized its enzymatic properties. 79
In addition, the draft genome sequence of strain Q-1 was determined in an attempt to 80
identify putative IOE genes encoding peptide sequences that were recovered from 81
trypsin-digested IOE. 82
83
MATERIALS AND METHODS 84
Bacterial strain and culture conditions. α-Proteobacterium strain Q-1, previously 85
isolated from iodide-rich natural gas brine water in Miyazaki prefecture, Japan (4), was 86
used in this study. The strain was grown aerobically in Marine Broth 2216 (Becton 87
Dickinson, Sparks, MD, USA) at 30°C. In most cases, the medium was supplemented 88
with 40 µM CuCl2·2H2O. 89
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Enzyme assays. In the presence of high concentration of iodide, I2 forms triiodide (I3-), 91
which has a high extinction coefficient of 25.5 mM-1·cm-1 at 353 nm. In this study, IOE 92
activity was determined by measuring I3- formation (4). The reaction mixture contained 93
the enzyme preparation (culture supernatant or purified enzyme), 20 mM Na-acetate 94
buffer (pH 5.5), and 10 mM KI. The reaction was started by the addition of the enzyme 95
preparation at 30°C. After 20 min of incubation, I3- formation was measured 96
spectrophotometrically. One unit of IOE was defined as the amount of enzyme required to 97
form 1 µmol of I3- per minute. 98
All assays to determine the oxidation of various substrates were performed at 30°C in 99
20 mM Na-acetate buffer (pH 5.5). One unit was defined as the amount of enzyme that 100
catalyzed the formation of 1 µmol of the appropriate product per minute in all cases. The 101
molar absorption coefficients used for these substrates were as follows: syringaldazine 102
[N,N′-bis(3,5-dimethoxy-4-hydroxybenzylidene hydrazine)], ε525 = 65.0 mM-1·cm-1; 103
2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonate) (ABTS), ε420 = 36.0 mM-1·cm-1; 104
p-phenylenediamine (pPD), ε487 = 14.7 mM-1·cm-1; hydroquinone, ε247 = 21.0 mM-1·cm-1; 105
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o-dianisidine, ε460 = 27.5 mM-1·cm-1; catechol, ε392 = 1.46 mM-1·cm-1; 2,6-dimethoxy 106
phenol (2,6-DMP), ε468 = 27.5 mM-1·cm-1; gallic acid, ε266 = 7.25 mM-1·cm-1; pyrogallol, 107
ε450 = 4.40 mM-1·cm-1; and guaiacol, ε465 = 12.1 mM-1·cm-1. 108
The activity of tyrosinase, which catalyzes both the hydroxylation of L-tyrosine to 109
L-3,4-dihydroxyphenylalanine (L-DOPA) and the oxidation of L-DOPA to dopaquinone, 110
was determined on the basis of autooxidation of dopaquinone to the orange pigment 111
dopachrome (ε475 = 3.7 mM-1·cm-1). The reaction mixture contained 20 mM Na-acetate 112
buffer (pH 5.5) and either 1 mM L-tyrosine or 1 mM L-DOPA. Mn(II)-oxidizing activity 113
was qualitatively assayed by observing the formation of brown Mn oxides with 20 mM 114
MnCl2 or MnSO4 as a substrate (13). Peroxidase and haloperoxidase activities were 115
assayed as described previously (4). 116
117
Purification of IOE. Strain Q-1 was cultured in Marine Broth 2216 containing 40 µM 118
Cu2+ at 30°C. Culture broth after 48 h was centrifuged at 6,000 × g for 20 min at 4°C, and 119
the supernatant was concentrated and desalted by ultra filtration with an Amicon Ultra 120
centrifugal filter (50K; Millipore, Bedford, MA, USA). The concentrated supernatant was 121
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applied to a 10% polyacrylamide gel for electrophoresis under non-dissociating 122
conditions (native PAGE). Following electrophoresis the gel was incubated in 20 mM 123
Na-acetate buffer (pH 5.5) containing 100 mM KI and 1% soluble starch to visualize IOE 124
proteins. Following this, the bands corresponding to IOE-I and -II were excised, and each 125
band was eluted with 50 mM Tris-HCl buffer (pH 8.0) for 20 h at 4°C. Protein 126
concentrations were determined by BCA protein assay (Thermo Scientific, Rockford, IL, 127
USA) with bovine serum albumin as the standard. 128
129
Electrophoresis. Purity of the enzyme was confirmed by sodium dodecyl 130
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). After the samples were boiled 131
for denaturation with 2% SDS and 5% 2-mercaptoethanol for 3 min, electrophoresis was 132
performed using 7% polyacrylamide gel in 25 mM Tris-glycine buffer (pH 8.3) 133
containing 0.1% SDS by the method described by Laemmli (26). In some cases, 134
electrophoresis was performed without boiling, but with SDS and 2-mercaptoethanol. 135
Precision Plus Protein Dual Color standards (Bio-Rad, Hercules, CA, USA) were used as 136
standard marker proteins. Proteins were visualized by staining with Coomassie brilliant 137
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blue (CBB) R-250. Isoelectric focusing (IEF) was performed using gels with pH gradients 138
from 3 to pH 10 (80 mm × 80 mm, 1-mm thickness, IEF-PAGE Mini; Tefco, Tokyo, 139
Japan) and calibration marker proteins from the Broad pI kit (GE Healthcare, 140
Buckinghamshire, UK). 141
142
Molecular weight estimation. The apparent molecular weight of the native enzyme 143
was estimated by high-performance liquid chromatography (HPLC) through a TSK 144
G3000SW (7.5 mm × 60 cm; Tosoh, Tokyo, Japan) column equilibrated with 20 mM 145
Tris-HCl buffer (pH 7.0) containing 0.3% NaCl. Thyroglobulin (669 kDa), apoferritin 146
(443 kDa), β-amylase (200 kDa), bovine serum albumin (66 kDa), and carbonic 147
anhydrase (29 kDa) were used as the standard marker proteins. The molecular weight of 148
the denatured enzyme was estimated by SDS-PAGE as described above. 149
150
UV–visible absorbance spectra. Absorption spectra were determined between 250 151
and 650 nm at room temperature in 100 mM Tris-HCl buffer (pH 8.0) by using a 152
BioSpec-nano spectrophotometer (Shimadzu, Kyoto, Japan). The purified enzyme was 153
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adjusted to 1.5 mg protein·mL-1. 154
155
Kinetic constants. Kinetic constants (Vmax, Km, and kcat) of the purified enzyme were 156
determined for iodide and ABTS under the assay conditions described above. 157
Lineweaver–Burk plots were constructed from the initial rates obtained at various 158
substrate concentrations. The kcat was obtained with the equation: kcat = Vmax/[E], where 159
[E] is the enzyme concentration. 160
161
Cofactor analysis. For determination of cofactor composition, the purified enzyme 162
was hydrolyzed with sulfuric acid, and metal detection was performed by inductively 163
coupled plasma mass spectroscopy (ICP-MS, 7500 Series; Agilent, Santa Clara, CA, 164
USA). 165
166
Internal amino acid sequence. Internal amino acid sequence analysis was performed 167
as described by Rosenfeld et al. (38). Briefly, the active IOE-II band was excised from a 168
native PAGE gel, digested with trypsin for 20 h at 35°C, and the resulting peptide 169
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fragments were separated using reverse-phase HPLC. The fragments were analyzed by a 170
protein sequencer (Procise 494 HT Protein Sequencing System; Applied Biosystems, 171
Carlsbad, CA, USA). 172
173
Preparation of the DNA library for sequencing. Genomic DNA of strain Q-1 was 174
extracted and purified by DNeasy Blood & Tissue kit (Qiagen, Hilden, Germany), 175
according to the manufacturer's instructions. DNA was uniformly sheared to 200 bp by 176
using Adaptive Focused Acoustics (Covaris, Woburn, MA, USA). We constructed a DNA 177
library with a median insert size of 200 bp for a paired-end read format. The quality of the 178
DNA library was checked with the Sanger method by Escherichia coli transformation of 179
aliquots of the library solution. The library was sequenced on a Genome Analyzer II 180
(Illumina, San Diego, CA, USA). Sample preparation, cluster generation, and paired-end 181
sequencing were carried out according to the manufacturer’s protocols with minor 182
modifications (Illumina Paired-End Cluster Generation Kit GAII ver. 2, 36-cycle 183
Sequencing Kit ver. 3). Image analysis and ELAND alignment were performed with 184
Illumina Pipeline Analysis software ver. 1.6. Sequences passing standard Illumina GA 185
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pipeline filters were retained. We obtained 584 Mb of total read bases with 11,698,620 186
read sequences. 187
188
De novo assembly of the read data and annotation of the coding regions in contigs. 189
Read sequences were assembled de novo with the Velvet assembly program (55). For 190
optimization of the hash value of the assembly process, we used the N50 size. Finally, 191
306 contigs were assembled with 3.09 Mb of total bases in the contigs. Assembled contigs 192
were annotated by the bacterial annotation pipeline b-MiGAP (46). Each CDS (CoDing 193
Sequence) was annotated by BLAST search of the databases RefSeq, TrEMBL, and NR. 194
Finally, 2,554 CDSs longer than 100 amino acid residues were called by b-MiGAP from 195
the contigs. 196
197
LC-MS/MS analysis. Protein sequencing using mass spectrometry was carried out as 198
described by Shevchenko et al. (45). The tryptic digest of IOE-II, prepared as described 199
above, was directly analyzed by nanoscale HPLC (Paradigm MS2; Michrom 200
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Bioresources, Auburn, CA, USA) with an L-column ODS (0.1 × 50 mm; Chemical 201
Evaluation and Research Institute, Tokyo, Japan). LC was coupled to a tandem mass 202
(MS/MS) spectrometer (Q-Tof2; Waters Micromass, Manchester, UK) equipped with a 203
nano-electrospray ionization source. Positive ion tandem mass spectra were measured. 204
205
Phylogenetic analysis. The ioxA gene sequence was translated to the amino acid 206
sequence, which was aligned with reference sequences by using the CLUSTAL X 207
program, version 2.1. The phylogenetic tree was constructed by the neighbor-joining 208
method (40), and bootstrap values were obtained from data resampling of 1,000 209
replicates. 210
211
Nucleotide sequence accession numbers. The sequence of the gene cluster 212
surrounding ioxA and ioxF genes was deposited in GenBank with the accession number 213
AB693882. 214
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RESULTS 216
Enhanced production of IOE with Cu2+ ion. When strain Q-1 was cultured in Marine 217
Broth 2216, maximum production of IOE in the supernatant (0.15 U·mL-1) was observed 218
at 48 h (Fig. 1). IOE production was not induced by iodide, and addition of 1 mM iodide 219
inhibited both growth and enzyme production, probably because of the toxicity of I2 (data 220
not shown). Thus, strain Q-1 was cultured without iodide in the following experiments. 221
When the culture medium was supplemented with 40 µM Cu2+ ion, enzyme production 222
was 20 times higher (3.1 U·mL-1) than in the medium without Cu2+ ion (Fig. 1). More 223
than 80 µM Cu2+ ion inhibited enzyme production. No other metal ions (40 µM Fe2+, Ca2+, 224
Mg2+, and Zn2+ each) enhanced IOE production. 225
226
Purification of IOE-II. Native PAGE analysis of concentrated culture supernatant 227
revealed that the readily visible proteins secreted by strain Q-1 were IOE, and that these 228
IOE consisted of two isoforms, designated IOE-I and IOE-II (Fig. 2A). Occasionally, one 229
more band was observed near IOE-I, but it did not appear to have IOE activity (Fig. 2A). 230
Attempts to purify each isoform by (NH4)2SO4 precipitation followed by several 231
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chromatographic separations (anion exchange, gel filtration, and hydrophobic interaction 232
chromatography) were unsuccessful, since a significant increase in specific activity was 233
not observed, and the two isoforms were not separated. Therefore, we decided to isolate 234
each isoform from a native PAGE gel by cutting out the active regions. Culture 235
supernatant containing 2.85 U·mg-1 of IOE was concentrated 3 times by ultra filtration 236
(with cutoff size of 50 kDa) and applied to native PAGE. After activity staining with 237
iodide and starch, the purple bands corresponding to IOE-I and -II were excised, and each 238
was eluted. Although IOE-I did not show significant activity after elution, 0.284 mg of 239
IOE-II with a specific activity of 22.1 U·mg-1 was successfully obtained (Table 1). From 240
12.0 mL of culture supernatant containing 9.41 mg of protein, IOE-II was purified 241
7.75-fold with a final yield of 23.4%. Purified IOE-II showed a slightly different mobility 242
from crude IOE-II on native PAGE gel (Fig. 2A). 243
When purified IOE-II was analyzed by SDS-PAGE without heat treatment (but using 244
2-mercaptoethanol), a single band with a molecular mass of 51 kDa was observed (Fig. 245
2B). This band still showed in-gel iodide-oxidizing activity, indicating that IOE-II is 246
resistant to SDS and reducing reagent. On SDS-PAGE gel with heat treatment (95°C, 3 247
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min), however, two bands with much higher molecular masses of approximately 150 and 248
230 kDa were observed (Fig. 2B). Under this condition, faint bands of 39, 68, and 86 kDa 249
were also observed. The profile of the SDS-PAGE gel did not change if the denaturation 250
time was extended to 30 min or if the sample solution was boiled with excess urea (data 251
not shown). When the denaturation temperature was decreased to 65°C, three bands with 252
molecular masses of 51, 150, and 230 kDa were observed (Fig. 2B). This indicated that 253
the two bands of 150 and 230 kDa were denatured products of the 51-kDa band. IOE-II 254
also appeared as a single band on IEF-PAGE gels, and its isoelectric point was 3.6 (Fig. 255
2C). The apparent molecular weight of native IOE-II, determined by gel filtration 256
chromatography, was 155 kDa with a single peak. These results suggest that IOE-II may 257
be a high-molecular-mass protein. Since only a single band or peak was observed in 258
native PAGE, SDS-PAGE (without heat treatment), IEF-PAGE, and gel filtration 259
chromatography, we concluded that IOE-II was homogeneously purified. 260
261
Biochemical characterization of IOE-II. ICP-MS analysis revealed that IOE-II 262
contained copper and zinc atoms. Based on the estimated molecular mass of 155 kDa in 263
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gel filtration chromatography, the presence of 3.96 copper atoms and 4.90 zinc atoms per 264
protein molecule was estimated. The UV–visible spectrum of IOE-II displayed a 265
significant peak at 590 nm, and a shoulder at 320 nm (Fig. 3). These peaks inferred the 266
presence of types 1 and 3 copper centers, respectively (see below in detail). 267
The activity of IOE-II was significantly inhibited by sodium azide (90% and 98% 268
inhibition at 0.1 and 1 mM, respectively), EDTA (48% and 99% inhibition at 0.1 and 1 269
mM, respectively), potassium cyanide (15% and 66% inhibition at 1 and 10 mM, 270
respectively), and the copper chelator o-phenanthroline (17% and 61% inhibition at 1 and 271
10 mM, respectively). The activity was not affected by 1 mM each of Ca2+, Mg2+, Zn2+, 272
and Mn2+ ions, but 1 mM of Fe2+ and Cu2+ ions showed 47% and 17% inhibition, 273
respectively. 274
The effect of pH on the activity and stability of purified IOE-II was examined with 20 275
mM citrate-NaOH buffer (pH 3.0−5.0), 20 mM acetate-NaOH buffer (pH 4.0−5.5), 20 276
mM potassium phosphate buffer (pH 6.0−8.0), 20 mM Tris-HCl buffer (pH 7.0−9.0), and 277
20 mM glycine-NaOH buffer (pH 9.0−10.0)(Fig. S1A). The optimum pH was 5.5 in 278
acetate-NaOH buffer. The activity was relatively stable under alkaline conditions after 30 279
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min of incubation at 4°C, and was most stable at pH 6.0 in phosphate buffer (Fig. S1B). 280
The temperature stability was measured after 30-min treatment at various temperatures. 281
The activity of IOE-II gradually decreased at temperatures higher than 40°C, while the 282
optimum temperature was 30°C (Fig. S1C). 283
IOE-II had significant activities toward ABTS and pPD (Table 2), both of which are 284
common substrates for laccases. The activity with pPD was much higher than that with 285
ABTS. Activity stain with ABTS and pPD on native PAGE gels verified that IOE-II 286
possessed these activities (data not shown). Laccases are multicopper oxidases that 287
catalyze the oxidation of phenolic compounds using molecular oxygen (33, 41). Many 288
phenolic compounds were then examined as potential substrates for IOE-II (Table 2). The 289
enzyme showed significant activities for syringaldazine, 2,6-DMP, hydroquinone, and 290
o-dianisidine. IOE-II was not able to oxidize manganese ion, and showed neither 291
peroxidase nor haloperoxidase activity. 292
When iodide was used as the substrate, IOE-II showed Km and kcat values of 2.64 mM 293
and 2.48 × 103 min-1, respectively (Table 3). On the other hand, Km and kcat values of 3.85 294
µM and 8.96 × 102 min-1, respectively, were observed when ABTS was the substrate. 295
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296
Internal amino acid sequences of IOE-II. We excised two bands with high molecular 297
masses (150 and 230 kDa) from an SDS-PAGE gel with heat treatment and determined 298
their N-terminal amino acid sequences. However, repeated trials were unsuccessful, 299
probably because both of these bands had blocked N-termini, or included several proteins. 300
Therefore, we digested purified IOE-II with trypsin and analyzed its internal amino acid 301
sequences. Three distinct peptides (MPVAQSVXQ, KDVVELGPGDEV, and 302
LAAIGMIGDL) were successfully recovered, although X represents an unidentified 303
residue. A search of the BLASTP database indicated that the second and third peptides 304
shared homologies with a putative multicopper oxidase (NCBI accession number 305
ZP_01035800) and hypothetical protein ROS217_06439 (ZP_01035798) of Roseovarius 306
sp. 217, respectively. 307
308
Draft genome sequence analysis and identification of putative genes for IOE-II. To 309
further understand the molecular aspects of IOE-II, the draft genome sequence of strain 310
Q-1 was determined with an Illumina Genome Analyzer II. De novo assembly of 584 Mb 311
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of the total read data by the assemble program Velvet (55) resulted in 306 contigs (see 312
Materials and Methods). The results suggested that the strain Q-1 genome is 313
approximately 3.09 Mb with a G+C content of 56.1%. Using the bacterial annotation 314
pipeline MiGAP (46), we identified 2,554 putative CDSs in the draft genome. 315
Three peptide sequences obtained as described above were used to query a BLAST 316
database of the draft Q-1 genome. The first and second peptide sequences 317
(MPVAQSVXQ and KDVVELGPGDEV) were found within the gene product of an 318
open reading frame (ORF) encoding a putative multicopper oxidase. This ORF (1,608 bp, 319
536 aa) was designated ioxA (Fig. 4). The predicted molecular mass of the deduced 320
protein (IoxA) was 62 kDa. A BLASTP search of the GenBank database showed that 321
IoxA shared homologies with putative multicopper oxidases or bilirubin oxidases found 322
in the genome-sequencing projects of Roseovarius sp. 217 (ZP_01035800, 60% identity), 323
Methylomonas methanica MC09 (YP_004515128, 59% identity), and Stackebrandtia 324
nassauensis DSM44728 (YP_003515051, 44% identity). From TatP analysis 325
(http://www.cbs.dtu.dk/services/TatP-1.0/), IoxA seemed not to contain an N-terminal 326
signal sequence. 327
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Multicopper oxidases are characterized by three different types of copper-binding sites, 328
i.e., type 1, 2, and 3 copper centers (33, 41). These redox centers enable the protein to 329
couple four one-electron oxidation reactions to the four-electron reduction of oxygen to 330
water. As shown in Fig. 5, each of the cysteine and histidine residues predicted to bind 331
copper to each of the copper centers (types 1-3) were present in IoxA. Phylogenetic 332
analysis demonstrated that IoxA is distantly related to previously known bacterial 333
multicopper oxidases such as CotA, CueO, CumA, and CopA (Fig. 6). It formed a clade 334
with putative multicopper oxidases and bilirubin oxidases found in the 335
genome-sequencing projects of various bacteria. 336
Analysis of the flanking region of ioxA revealed that six possible ORFs (ioxA, B, C, D, 337
E, and F) were present with the same orientation and with a relatively close sequential 338
arrangement (Fig. 4). Whether these genes reside in an operon and are cotranscribed 339
remains unknown. The third peptide sequence recovered from IOE-II (LAAIGMIGDL) 340
was found within a deduced protein encoded by ioxC (1,932 bp, 644 aa), which was 341
located downstream of ioxA (Fig. 4). The predicted molecular mass of IoxC was 71 kDa, 342
and it exhibited homologies with the hypothetical proteins of Roseovarius sp. 217 343
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(ZP_01035798, 42% identity), M. methanica MC09 (YP_004515130, 43% identity), and 344
S. nassauensis DSM44728 (YP_003515052, 37% identity). TatP analysis predicted that 345
IoxC contained a potential twin-arginine translocational (TAT) signal peptide. 346
The proteins encoded by ioxB, ioxD, and ioxF all showed homologies with SCO1/SenC 347
family proteins, which are known to bind copper and be involved in the assembly of the 348
cytochrome c oxidase complex in yeast (43). In IoxD and IoxF, the conserved 349
copper-binding domains (CXXXC) were also found. The deduced protein encoded by 350
ioxE showed homology with a hypothetical protein of Roseovarius sp. 217 351
(ZP_01035796, 38% identity). It is noteworthy that iox gene cluster arrangement found in 352
strain Q-1 (ioxA, B, C, D and E) is very similar to that surrounding the IoxA homolog in 353
Roseovarius sp. 217 (ZP_01035800, ZP_01035799, ZP_01035798, ZP_01035797 and 354
ZP_01035796). 355
356
LC-MS/MS analysis of IOE-II. The peptides from the digestion of the IOE-II band 357
with trypsin were separated by nanoscale reverse-phase chromatography and directly 358
analyzed by nano-electrospray ionization-tandem mass spectrometry. A database search 359
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of tandem mass spectra with a Mascot Search Program revealed that 14 different IoxA 360
peptides and 16 different IoxC peptides were consistently recovered from the active 361
IOE-II band (Fig. 4 and Table S1). The overall coverage of the IoxA and IoxC protein 362
sequences with peptides identified by tandem mass spectrometry was 32% and 40%, 363
respectively. 364
365
DISCUSSION 366
Heme- or vanadate-containing haloperoxidases oxidize halide ions in the presence of 367
H2O2 (30, 47). Heme-containing haloperoxidases include chloroperoxidase from the 368
fungus Caldariomyces fumago, lactoperoxidase, and horseradish peroxidase; 369
vanadate-containing haloperoxidases are represented by vanadate-dependent chloro- and 370
bromoperoxidases found in various fungi and marine algae (49). Although 371
haloperoxidases are actually capable of I2 production from iodide in vitro, the final 372
product in vivo may not be I2, but iodinated organic compounds, since haloperoxidases 373
usually catalyze the halogenation reaction in the presence of appropriate organic 374
substrates such as tyrosine (47). In fact, studies of the biological production of I2 are very 375
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scarce, except for that by marine kelps (24, 25). Previously, we isolated iodide-oxidizing 376
bacteria, which produce I2 from iodide, from iodide-rich natural gas brine water and 377
iodide-enriched seawater (4). Preliminary characterization of the extracellular 378
iodide-oxidizing activity revealed that the activity was not due to haloperoxidase but due 379
to oxidase. Since there have been few reports on oxidases with iodide-oxidizing activity, 380
we purified one of the isozymes from the group B iodide-oxidizing bacterial strain Q-1 381
and characterized its biochemical properties. 382
Production of IOE by strain Q-1 was not induced by iodide, but by Cu2+ ion. In the 383
presence of 40 µM Cu2+ ion, enzyme production was enhanced 20-fold, while growth was 384
unaffected. ICP-MS analysis revealed that the enzyme contained copper atoms as 385
prosthetic groups. An important role for Cu2+ ion was also indicated by the fact that 386
o-phenanthroline, a copper chelator, inhibited the activity of IOE-II. Interestingly, IOE-II 387
showed significant activities toward common substrates for laccases, i.e., ABTS, 388
methoxyphenols (syringaldazine and 2,6-DMP), p-diphenol (hydroquinone), and aromatic 389
diamines (pPD and o-dianisidine). Laccases belong to the family of multicopper oxidases, 390
and they have three spectroscopically different copper centers, types 1, 2, and 3 (33, 41). 391
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Among these, types 1 and 3 copper centers are characterized by absorption at around 600 392
and 330 nm, respectively, and the purified IOE-II had absorption peaks around these 393
wavelengths (590 and 320 nm). These results strongly suggest that IOE-II is a 394
multicopper oxidase. Strong inhibition of IOE-II activity by sodium azide also supports 395
the involvement of a multicopper oxidase, since azide bridges types 2 and 3 copper 396
centers and inhibits other bacterial multicopper oxidases, including CueO of E. coli (16), 397
laccase of Azospirillum lipoferum (11), and various Mn2+ oxidases (7, 12, 34, 39). 398
Enhancement of enzyme activity by Cu2+ ion has been reported in CueO (16) and YacK 399
(23) of E. coli, and Mn2+ oxidases of Pseudomonas putida GB-1 (8) and Bacillus sp. 400
SG-1 (48). Similarly, inhibition by o-phenanthroline has been found in Mn2+ oxidases of P. 401
putida GB-1 (34) and Erythrobacter sp. SD-21 (12). Our result of cofactor analysis (3.96 402
copper atoms and 4.90 zinc atoms per protein molecule) also shows a good agreement 403
with the previous reports, in which 4 copper atoms were found in each multicopper 404
oxidase chain (17, 37). Although it is not common that zinc atoms are included in 405
multicopper oxidases, laccases from Pleurotus ostreatus (36) and Phellinus ribis (32) are 406
known to contain 2 zinc atoms. 407
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To date, there have been only two reports on multicopper oxidases with 408
iodide-oxidizing activity. Xu (54) first found that laccase from the fungus Myceliophthora 409
thermophila possessed iodide oxidase activity. Leitner et al. (29) also reported that 410
laccase purified from the white-rot fungus Trametes multicolor oxidized iodide as well as 411
a variety of phenolic compounds. However, iodide was a very poor substrate for T. 412
multicolor laccase because of a high Km value (483 mM) and a low kcat value (6.42 × 102 413
min-1) (29). As shown in Table 3, IOE-II showed a much lower Km value (2.64 mM), 414
much higher kcat value (2.48 × 103 min-1), and thus, the catalytic efficiency (kcat/Km) for 415
iodide was 3−5 orders higher than that for the fungal laccases. Therefore, IOE-II is 416
considered as the most efficient iodide-oxidizing enzyme among the multicopper oxidases 417
reported so far. Iodide-oxidizing bacteria have been isolated from iodide-rich 418
environments such as natural gas brine water (containing up to 1.2 mM iodide) and 419
seawater enriched with 1 mM iodide (4). Recently, Arakawa et al. (5) found that 420
iodide-oxidizing bacteria became predominant in microcosms comprising natural 421
seawater and 1 mM iodide, and suggested that iodide-oxidizing bacteria potentially attack 422
other marine bacteria with I2 to occupy their ecological niche. Therefore, it is possible that 423
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high catalytic efficiency of IOE contributes to predominant growth of iodide-oxidizing 424
bacteria in iodide-rich environments. 425
Comparison of several internal amino acid sequences of IOE-II with a draft genome 426
sequence of strain Q-1 revealed that IoxA and IoxC, with predicted molecular masses of 427
62 and 71 kDa, respectively, were included in IOE-II. In addition, subsequent LC-MS/MS 428
analysis of IOE-II repeatedly detected peptides from these two proteins with high 429
sequence coverage (32% to 40%). These results suggest that at least two proteins, IoxA 430
and IoxC, comprise IOE-II. Sequence analysis demonstrated that IoxA is a typical 431
multicopper oxidase with four copper-binding regions that are highly conserved among 432
various multicopper oxidases. Multicopper oxidases have been found in fungi, plants, and 433
animals, and include laccases, ferroxidases, ascorbate oxidases, and ceruloplasmin (33, 434
41). There is increasing evidence for the existence of multicopper oxidases in various 435
gram-positive and gram-negative bacteria (9, 22). Their possible functions include 436
pigment formation, utilization of plant phenolic compounds, electron transport, 437
sporulation, copper resistance, and oxidation of metals such as manganese, copper, and 438
iron. To the best of our knowledge, however, no studies have been conducted on bacterial 439
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multicopper oxidases capable of iodide oxidation. Furthermore, phylogenetic analysis 440
suggests that IoxA is distinct from other known bacterial multicopper oxidases (Fig. 6). 441
IoxA showed the highest sequence similarity with a multicopper oxidase of 442
Roseovarius sp. 217, a bacterium originally isolated as a marine methyl halide oxidizer 443
(42). Phylogenetically, Roseovarius sp. 217 is closely related to known group A 444
iodide-oxidizing bacteria (4) such as strains RB-2A, N213-3, A-6, YS-11, SE-1, and Ka-4, 445
with 16S rRNA gene sequence similarities of 95%−98% (data not shown). It is thus 446
probable that this strain is also capable of iodide oxidation. 447
In this study, a discrepancy between the results of several experiments that measured 448
the molecular mass of IOE-II was apparent. The enzyme appeared as a single 51-kDa 449
band in SDS-PAGE without boiling, while at least two bands with much higher molecular 450
masses (150 and 230 kDa) were observed in SDS-PAGE with boiling. In gel filtration 451
chromatography, the apparent molecular mass of IOE-II was estimated to be 155 kDa. 452
Furthermore, the molecular masses of IoxA and IoxC, both of which are proteins detected 453
in IOE-II, were found to be 62 and 71 kDa, respectively. Bacterial multicopper oxidases 454
are often found in high-molecular-mass complexes (9), and several studies have shown 455
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differences between the size of the gene and the molecular mass of the enzymatically 456
active product. In the spores of a marine Bacillus species, for example, the size of Mn2+ 457
oxidase dramatically varied (140−205 kDa) among different strains (10). Tandem mass 458
spectrometry of the Mn2+ oxidase recovered from an in-gel activity assay repeatedly 459
detected peptides unique to the multicopper oxidase MnxG. However, the length of the 460
mnxG gene is nearly identical among these strains, and the predicted molecular mass of 461
MnxG is 138 kDa. Dick et al. (10) suggested that the Mn2+ oxidase of Bacillus spores 462
may occur as a high-molecular-mass complex because of the association of additional 463
subunits and/or proteins. Since molecular masses greater than 150 kDa were observed 464
both in SDS-PAGE and gel filtration chromatography, IOE-II may also be a 465
high-molecular-mass protein. In addition, repeated detection of peptides from IoxA and 466
IoxC indicates that IOE-II exists as a protein complex in which at least two subunits are 467
associated. However, at present, the accurate molecular mass and the subunit composition 468
of IOE-II remain unclear. Indeed, it is an uncommon feature that 150- and 230-kDa bands 469
are detected in SDS-PAGE under fully denatured condition (with reducing reagent and 470
boiling), despite the predicted molecular masses of IOE-II subunits being 62 and 71 kDa. 471
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Considering the fact that IOE-II still maintained in-gel activity in the presence of SDS and 472
reducing reagent (Fig. 2B), it is possible that IOE-II is highly resistant to denaturation. 473
Therefore, we speculate that the 150- and 230-kDa bands appearing on fully denatured 474
SDS-PAGE gels possibly represent native IOE-II and its aggregated form, respectively. 475
From an applied point of view, IOE-II could be used as a novel antimicrobial enzyme 476
system. I2 has antimicrobial activity against a wide variety of microorganisms, including 477
gram-positive and gram-negative bacteria, filamentous fungi, yeasts, viruses, and certain 478
bacterial spores (31). Because of its broad spectrum of activity, I2 is an important 479
disinfectant in hospitals and food industries. Iodophors such as povidone-iodine solution 480
are commonly used as chemically synthesized iodine disinfectants (6). Recently, however, 481
research efforts to evaluate natural antimicrobial compounds, including enzymes with 482
antimicrobial activity, have increased (15). Hickey et al. (21) reported that horseradish 483
peroxidase could be used to generate enough I2 to kill Staphylococcus aureus and the 484
spores of B. subtilis. Hansen et al. (18) also found that haloperoxidase isolated from 485
Curvularia verruculosa had the potential to kill a variety of bacteria, yeasts, and fungi in 486
the presence of bromide or iodide. These haloperoxidase-based systems, however, have 487
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the disadvantage of requiring hazardous H2O2. As discussed above, IOE-II is an oxidase 488
and does not need H2O2 for I2 production. Therefore, it is possible that IOE-II/iodide can 489
be used as a simple, safe, and cost-effective antimicrobial enzyme system. Determination 490
of its antimicrobial spectrum and comparison of its antimicrobial capacity with other 491
disinfectants are underway in our laboratory. 492
493
ACKNOWLEDGMENTS 494
This work was partially supported by grants from the Institute of Fermentation, Osaka, 495
Japan, and the Japan Society for the Promotion of Science (No. 20780049) to S. A. 496
497
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TABLE 1 Summary of purification of IOE-II from strain Q-1a
Purification procedure Protein
(mg)
Total enzyme
activity (U)
Specific activity
(U mg-1)
Yield
(%)
Purification
(fold)
Culture supernatant 9.41 26.8 2.85 100 1.00
Concentration by ultra filtration 2.84 24.0 8.45 89.6 2.97
Elution of IOE-II from excised active band 0.284 6.26 22.1 23.4 7.75 a Representative data from five independent experiments are shown.
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TABLE 2 Substrate specificity of the purified IOE-IIa
Substrate Substrate concn (mM) Relative activity (%)
Iodide 10 100
ABTS 0.1 9.0±0.2
pPD 15 69.5±5.2
Syringaldazine 0.075 3.2±0.5
2,6-DMP 0.15 0.6±0.0
Hydroquinone 0.2 8.2±0.2
o-Dianisidine 0.15 8.4±1.1
Catechol 1 0
Galic acid 0.1 0
Pyrogallol 1 0
Guaiacol 1 0
K4[Fe(CN)6] 0.2 0
L-Tyrosine 1 0
L-Dopa 1 0
a Mean values±SD from triplicate determinations are shown.
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TABLE 3 Apparent kinetic constants of several multicopper oxidases for iodide and ABTS
Enzyme source Substrate Km (mM) kcat (min-1) kcat/Km (min-1 M-1) Reference
IOE-II (strain Q-1)a Iodide 2.64 2.48×103 9.41×105 This study
ABTS 3.85×10-3 8.96×102 2.32×108
Laccase (Trametes multicolor) Iodide 4.83×102 6.42×102 1.32×103 29
ABTS 1.41×10-2 3.06×104 2.17×109
Laccase (Myceliophthora thermophila) Iodide 1.60×102 2.70 1.69×10 54
ABTS 5.60×10-3 1.46×103 2.61×108 a Mean values from three independent experiments are shown.
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10
2 5
3.0
3.5
L-1 )
0.1
1
1.5
2.0
2.5
Gro
wth
(O
D66
0)
zym
e ac
tivity
(U
mL
0.01 0
0.5
1.0
0 24 48 72 96 120
Enz
Time (hours)
FIG 1 Enhanced production of IOE by Cu2+ ionStrain Q-1 was cultured aerobically in Marine Broth 2216 with(closed symbols) or without (open symbols) 40 µM of Cu2+ ion.( y ) ( p y ) µCell growth (squares) and IOE activity in the culturesupernatant (circles) are shown.
Fig. 1.
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A1 2 3 4 kDa
B C1 2pI1 2 3 4 5 6 7 8 9
4˚C 4˚C24 h
65˚C3 min
95˚C3 min
4˚C 4˚C24 h
65˚C3 min
95˚C3 min
250150
10075
3
8.158.458.659.30
50
375.85
6.556.85
7.35
IOE-IIOE-II
4.55
5.2025
20
3.503.75
20
FIG 2 Native PAGE (A) SDS-PAGE (B) and IEF-PAGE (C) analyses of IOE-IIFIG 2 Native PAGE (A), SDS PAGE (B), and IEF PAGE (C) analyses of IOE IIA: Concentrated culture supernatant (lanes 1 and 3) and purified IOE-II (lanes 2 and 4) were run on native PAGE gels, and the gelswere stained with CBB (left) for total proteins or with iodide and starch (right) for activity assay. B: Purified IOE-II was run on SDS-PAGE gels with or without heat treatment, and the gels were stained with iodide and starch (left) or with CBB (right). Lanes 1 and 5,IOE-II was mixed with SDS and 2-mercaptoethanol, and the mixture was immediately applied on the gels without heat treatment;lanes 2 and 6, the mixture was stored at 4°C for 24 h and was applied without heat treatment; lanes 3 and 7, the mixture was heated at
Fig. 2.
65°C for 3 min before application; lanes 4 and 8, the mixture was heated at 95°C for 3 min before application; lane 9, size standard. C:IOE-II was run on an IEF-PAGE gel (lane 2). Lane 1 represents the standard marker proteins.
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0 10
0.12
0.14
0.06
0.08
0.10
bsor
banc
e
0.02
0.04
Ab
0.00300 500 600
Wave length (nm)
400
FIG 3 UV–visible absorbance spectrum of IOE-II
Fig. 3.
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ioxA ioxB ioxC ioxD ioxE ioxFA B C D
MPVAQSVXQ KDVVELGPGDEV LAAIGMIGDL
1,000 bp
FIG 4 Schematic representation of iox genes of the strain Q-1The locations of putative copper-binding regions are indicated by white bars and labeled A to D according to the data in Figure5. The locations of the three peptides detected by internal amino acid sequence analysis are indicated by black bars with arrowsand corresponding sequences. The locations of peptides detected by LC-MS/MS analysis of IOE-II are shown with black dotsbelow the iox genes.
Fig. 4.
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AnLc 79 HWHGLEMR 118 GTFWYHSHT2 T3 T3 T3
A B
CsAO 95 HWHGILQR 134 GTYFYHGHCopA 100 HWHGIILP 137 GTYWYHSHCotA 105 HLHGGVTP 148 AILWYHDHCueO 101 HWHGLEVP 136 ATCWFHPHCueO 101 HWHGLEVP 136 ATCWFHPHRMCO 116 HLHNGHTA 164 NTLWYHDHIoxA 113 HLHNGHTA 159 TTLWYHDH
C D
AnLc 505 HPPHPIHK 585 HCHIASHQMGGMCsAO 480 HPWHL-HG 542 HCHIEPHLHMGMCopA 542 HPIHL-HG 590 HCHLLYHMEMGM
T2 T3T1 T3T1T3 T1C D
CopA 542 HPIHL HG 590 HCHLLYHMEMGMCotA 419 HPIHL-HL 491 HCHILEHEDYDMCueO 443 HPFHI-HG 499 HCHLLEHEDTGMRMCO 482 HPVHT-HF 536 HCHNVVHEDHAMI A 456 HPVHT HM 512 HCHNVVHEDHAMIoxA 456 HPVHT-HM 512 HCHNVVHEDHAM
FIG 5 Alignment of the putative copper-binding regions of IoxA and other multicopper oxidasesThe amino acid residues corresponding to potential copper-binding sites are indicated as follows: Type 1, T1; Type 2, T2; Type3, T3. Highly conserved amino acid residues that are common in more than five multicopper oxidases among the seven aligned
Fig. 5.
g y pp g genzymes are shaded. Abbreviations: AnLc, Aspergillus nidulans laccase (accession no. CAA36787); CsAO, Cucumis sativusascorbate oxidase (CBY84386); CopA, Pseudomonas syringae CopA (AAA25806); CotA, Bacillus subtilis CotA (BAA22774);CueO, Escherichia coli CueO (ZP_03003336); RMCO, Roseovarius sp. 217 multicopper oxidase (ZP_01035800).
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O ib ill ih i C tA (NP692267)Bacillus subtilis CotA (BAA22774)
Streptomyces lavendulae laccase (BAC16804)Marinomonas mediterranea PpoA (AAF75831)
1000
1000
Methylobacter tundripaludum billirubin oxidase (ZP_08780197)* Leptothrix cholodnii billirubin oxidase (YP_001789109)*
Acremonium murorum PpoA (CAB75422)Myrothecium verrucaria billirubin oxidase (Q12737)
Oceanibacillus iheyensis CotA (NP692267)1000
1000
727662
Roseovarius sp. 217 MCO (ZP_01035800)*Strain Q-1 IoxAMethylomonas methanica billirubin oxidase (YP_004515128)*
Stackebrandtia nassauensis billirubin oxidase (YP_003515051)* Nitrospira defluvii MCO (YP_003799121)*1000
999
1000
1000
589
971
Pseudomonas syringae CumA (AAO54977)Bacillus halodurans laccase (AAP57087)
Escherichia coli CueO (P36649)Salmonella enterica CueO (Q8ZRS2)Yersinia pestis CueO (Q8ZBK0)
p ( _ )
1000
1000
1000
E h i hi li P A (S52253)Pseudomonas syringae CopA (P12374)Bordetella pertussis CopA (CAE43580)Ralstonia solanacearum CopA (CAD17807)
Pseudomonas putida CumA (AAD24211)Pseudomonas syringae CumA (AAO54977)
995996
1000
1000
747
1000
Xanthomonas campestris CopA (A36868)Xanthomonas campestris CopA (AAM39893)Escherichia coli PcoA (S52253)0.05
940
976
FIG 6 Phylogenetic analysis of translated amino acid sequences of the ioxA gene by using the neighbor-joining method
Fig. 6.
y g y q g y g g j gSequences with an asterisk indicate those found in the genome-sequencing projects of various bacteria without functional analysis.GenBank accession numbers are shown in parentheses. Scale bar represents the number of amino acid changes per site.
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