1
2
(Submitted to Microbial and Enzyme Technology sector of Biotechnology Letters)
3
Supporting information for
4
Characterization of a novel Acinetobacter baumannii xanthine dehydrogenase
5
expressed in Escherichia coli
6
Cheng-Hua Wang, Tong-Xin Zhao, Mei Li, Chong Zhang, Xin-Hui Xing*
7
Key Laboratory for Industrial Biocatalysis, Ministry of Education of China, Institute of
8
Biochemical Engineering, Department of Chemical Engineering, Tsinghua University,
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Beijing 100084, People's Republic of China.
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C. H. Wang, e-mail: wangchenghua@mail.tsinghua.edu.cn
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T. X. Zhao, e-mail: txzhao2014@sina.com
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M. Li, e-mail: meili2811@163.com
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C. Zhang, e-mail: chongzhang@tsinghua.edu.cn
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X. H. Xing, e-mail: xhxing@mail.tsinghua.edu.cn
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*Corresponding author: xhxing@mail.tsinghua.edu.cn
17
Phone/Fax: +86-10-62794771/62787472
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19
20
21
Supplementary Table 1 Comparison between Acinetobacter baumannii xanthine dehydrogenase (XDH) identified in this study and previously characterized
enzymes
Optimum
No
1
Source
Acinetobacter
pH
8.5-9.0
Temp. (℃)
40-45
NAD+
Xanthine
Km
Vmax
kcat
kcat/ Km
Km
Vmax
kcat
kcat/ Km
Production
(μM)
(μmol.mg-1)
(s-1)
(μM-1.s-1)
(μM)
(μmol.mg-1)
(s-1)
(μM-1.s-1)
method
25.32±1.48
29.16±0.37
69.32±0.88
113.1±18.35
46.92±2.58
111.54±6.13
2.74
0.99
baumannii
2
Rhodobacter
Arabidopsis
This study
in E. coli
8.0
35
36.2±7.3
17.6
38.8±3.4
1.07
36.2±4.5
--
--
1.07
capsulates
3
Expression
Ref.
8.0
25
--
1.712±0.236
4.28±0.59
--
--
0.702±0.042
1.76
--
thalianas
Expression
(Schumann
in E. coli
et al. 2008)
Expression
(Hesberg et
in Pichia
al. 2004)
pastoris
4
Human liver
8.5
25
8.8±0.60
--
18.3±0.87
2.2±0.12
--
--
--
--
E.coli,
(Yamaguchi
et al. 2007)
5
6
Rat liver
Rat liver
7.8
7.8
25
25
---
3.2
3.3
8
8.25
--
--
--
--
2.3
3.2
5.75
8
---
Insect Cell
(Nishino
System
al. 2002)
Extraction
(Nishino
et
et
al. 2002)
7
Sheep milk
7.2
25
7.14±0.9
0.69±0.04
1.73±0.1
0.24
2.10±0.10
0.21±0.04
0.5±0.1
0.24
Extraction
(Benboubetr
a et al. 2004)
8
Human milk
7.2
25
7.74±0.06
0.06±0.01
1.5±0.02
0.19
2.52±0.08
0.29±0.03
0.73±0.0.08
0.29
Extraction
(Benboubetr
a et al. 2004)
9
Goat milk
7.2
25
6.33±0.08
0.03±0.01
0.07±0.03
0.01
2
4.12±0.06
0.27±0.04
0.68±0.10
0.17
Extraction
(Benboubetr
a et al. 2004)
10
Cow milk
7.2
25
2.15±0.05
1.83±0.02
4.58±0.05
2.13
2.74±0.12
0.25±0.05
0.63±0.13
0.23
Extraction
(Benboubetr
a et al. 2004)
11
Veillonella
7.5
37
136
30
64.5
0.47
--
--
--
-
Extraction
atypica
12
(Gremer and
Meyer 1996)
Micrococcus
6.5
Lactilytkicus
unstabl
37
17
0.08
2.5
5.38
0.32
-
-
-
Extraction
(Smith et al.
1967)
e (<7)
13
Streptomyces
8.7
cyanogenus
40,
150
10.6
22.08
0.15
110
--
--
-
Extraction
(Sin 1975)
0.3
0.16
0.4
1.33
6.7
--
--
-
Extraction
(Hunt
stable
(<55℃)
14
15
Bos taurus
Camel milk
7.5
8.3
25oC
unstable
Massey
(>38℃)
1992)
22
--
0.013
0.03
--
--
-
-
-
Extraction
and
(Baghiani et
al. 2003)
16
Chlamydomon
--
as reinhardtii
25,
--
5.69
31.67
--
--
--
--
--
Extraction
instable
(Perez-Vicen
te
et
al.
1992)
17
Clostridium
7.5
25
4.5
2.7
5.85
1.3
-
-
-
-
Extraction
purinilyticum
(Self
and
Stadtman
2000)
18
Colias
7.8
butterflies
stable
30
61/64.7
10.58/
26.45/
0.43/
11.1
27.75
0.43
(7-8.5)
3
124
7.6
19
0.15
Extraction
(Watt 1972)
19
Comamonas
7.9
25
80.1
80
86
1.07
113
--
--
--
Extraction
acidovorans
20
(Ivanov et al.
2003)
Drosophila
8.0
--
18(Yen and
12.4
31
1.72/2.01
25(Yen
and
--
--
--
Extraction
melanogaster
stable
Glassman
Glassman
al. 2002; Yen
(<9.2)
1967)
1967)
and
(Adams
et
Glassman
1967)
21
Eubacterium
7.8
37
67
164
77.4
1.16
38 (NADP)
--
--
--
Extraction
barkeri
22
Gallus gallus
al. 1999)
7.5
30
17
0.22
0.55
0.03
19
--
--
--
Extraction
stable
stable
al.
(7.2-8.7
(25-40℃)
(Khobragad
)
23
Soybean
(Schrader et
7.5
(Tramper et
1979)
e et al. 2008)
30
5
2.4
5.64
1.13
12.5
--
--
--
Extraction
nodule
(Boland
al.
et
1983;
Triplett et al.
1982)
24
Gottschalkia
7
55
1350
2.8
10.45
0.008
--
--
--
Extraction
acidurici
25
Human
(Wagner
et
al. 1984)
7.4
37
8.8
0.40
0.99
0.11
--
--
--
Extraction
(Benboubetr
6-9.2
a et al. 2004;
stable
Yamaguchi
et al. 2007)
26
Neurospora
8.0
--
8.2
0.15
0.39
0.05
4
28
--
--
--
Extraction
(Lyon
and
crassa
Garrett
1978)
27
Pisum sativum
8.5
--
17
--
--
--
25
--
--
--
Extraction
(Sauer et al.
2002)
28
Pseudomonas
--
--
52
26.7
125.61
2.42
64
--
--
--
Extraction
putida
(Kim
and
Schmid
1989;
Parschat
et
al. 2001)
29
Pseudomonas
8.2-8.8
40
--
20
43.33
--
--
--
--
--
Extraction
synxantha A3
30
Rattus sp.
(Sakai
and
Jun 1979)
--
--
--
2.6
2.47
6.18
2.38
--
--
--
Extraction
(Waud
and
Rajagopalan
1976)
31
Sus scrofa
7.4
37
--
21.69
7.74
--
--
--
--
Extraction
(Chen et al.
2013)
32
Triticum
8-8.6
25
8.2
0.23
--
--
171
aestivum
--
--
--
Extraction
(Montalbini
1998)
22
23
5
24
Supplementary Table 2 Amino acid sequence identity (homology) of A. baumannii
25
XDH identified in this study and other XDHs characterized previously
A. baumannii
No.
1
2
3
4
5
6
7
8
9
10
11
12
13
s14
Origin
Arabidopsis thaliana
Bos taurus (Bovine)
Drosophila
melanogaster (Fruit
fly)
Calliphora vicina
(Blue blowfly)
Gallus gallus
(Chicken)
Mus musculus
(Mouse)
Homo sapiens
(Human)
Sus scrofa (Pig)
Capra hircus (Goat)
Camelus ferus
(Camel)
Chlamydomonas
smithii
Glycine soja
(Soybean)
Neurospora crassa
Delftia acidovorans
XDHA
(Fe/S cluster
and FAD
binding subunit)
XDHB
(Moco-binding
subunit)
XDHC
(Accessory
protein)
UniProtKB
Entry
22.8% (37.4%)
22.8% (37.7%)
25.0% (
38.5%)
39.2% (51.6%)
38.6% (51.5%)
38.3% (53.4%)
----
Q8GUQ8
P80457
P10351
24.6% (37.1%)
39.1% (53.3%)
--
P08793
22.2% (35.8%)
39.6% (52.7%)
--
P47990
22.0% (37.4%)
39.6% (51.9%)
--
Q00519
22.5% (37.9%)
39.3% (51.3%)
--
P47989
23.3% (37.7%)
23.1% (38.1%)
13.0% (25.6%)
38.3% (51.0%)
38.2% (51.0%)
35.3% (47.4%)
----
R4HZ39
A1YZ34
S9Y4T1
26.2% (38.7%)
32.8% (45.4%)
--
A8IY70
23.0% (37.4%)
38.8% (52.9%)
--
A0A0B2RM17
26.4% (41.8%)
46.9% (58.1%)
39.5% (52.6%)
51.7% (63.2%)
-Q7RXE4
28.6% (39.7%) Q8RLC1 (XDHA),
Q8RLC0 (XDHB),
A0A080NKLS (XDHC)
15
Clostridium acidurici
a
25.9% (39.2%)
&16.1%
(32.3%)b
22.6% (37.1%)
16.4% (26.8%) K0B4E4 (XDHA
Fe/S subunit) a
K0B3H0 (XDHA
FAD subunit) b
K0B2W0 (XDHB)
K0B333 (XDHC)
16
Pseudomonas putida
44.7% (56.9%)
45.8% (59.2%)
17
Pseudomonas
synxantha
43.5% (56.9%)
45.6% (58.6%)
6
27.6% (40.3%) Q88F21 (XDHA)
Q88F20 (XDHB)
Q88F19 (XDHC)
28.5% (40.5%) I4L544 (XDHA)
I4L2J2 (XDHB)
I4L6Z0 (XDHC)
18
Rhodobacter
capsulatus
40.6% (53.1%)
49.0% (61.8%)
29.8% (41.6%) O54050 (XDHA)
O54051 (XDHB)
D5API3 (XDHC)
19
40.6% (53.1%)
49.0% (61.8%)
29.8% (41.6%) 2W3S
20
Rhodobacter
capsulatus B10
Bovine milk
22.8% (37.7%)
38.6% (51.5%)
--
26
All the amino acid sequence alignments of the A. baumannii XDH identified in this
27
study and other XDHs characterized previously were carried out by AlignX module in
28
the Vector NTI 10.0 software.
29
a
30
entry: K0B4E4) and the counterpart of A. baumannii XDHA subunit.
31
b
32
entry: K0B3H0) and the counterpart of A. baumannii XDHA subunit.
3AX9
indicated the sequence comparison between the Fe/S binding subunit (UniProtKB
indicated the sequence alignment between the FAD binding subunit (UniProtKB
33
7
34
35
36
Supplementary Table 3 Purification of recombinant A. baumannii XDH following
37
heterologous expression in E. coli
Step
Volume
Protein
Activity
Specific activity
Purification
(ml)
(mg) a
(U) b
(U/mg) c
(fold)
Crude extract
70.0
406
105.6
0.26
1
Ni-NTA agarose
21.6
21.8
56.7
2.6
10
Q-Sepharose
10.0
6.3
49.2
7.8
30
Phenyl-Sepharose 10.0
4.6
40.0
8.7
33
38
a
Total protein was quantified by Bradford method with BSA as standard.
39
b
Activity of A. baumannii XDH was assayed by monitoring the increase of uric acid
40
in absorption at 295 nm in the presence of 0.1 mM xanthine and 0.1 mM NAD at 25
41
℃ in 50 mM Tris/HCl buffer, pH 7.5
42
c
43
mg enzyme under assay conditions.
Specific activity (U/mg) was defined as the increase of μmol uric acid per min per
44
8
45
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46
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13