(Submitted to Microbial and Enzyme Technology sector of Biotechnology Letters)

Supporting information for Characterization of a novel Acinetobacter baumannii xanthine dehydrogenase

expressed in Escherichia coli

Cheng-Hua Wang, Tong-Xin Zhao, Mei Li, Chong Zhang, Xin-Hui Xing*

Key Laboratory for Industrial Biocatalysis, Ministry of Education of China, Institute of

Biochemical Engineering, Department of Chemical Engineering, Tsinghua University,

Beijing 100084, People's Republic of China.

C. H. Wang, e-mail: wangchenghua@mail.tsinghua.edu.cn

T. X. Zhao, e-mail: txzhao2014@sina.com

M. Li, e-mail: meili2811@163.com

C. Zhang, e-mail: chongzhang@tsinghua.edu.cn

X. H. Xing, e-mail: xhxing@mail.tsinghua.edu.cn

*Corresponding author: xhxing@mail.tsinghua.edu.cn

Phone/Fax: +86-10-62794771/62787472

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Supplementary Table 1 Comparison between Acinetobacter baumannii xanthine dehydrogenase (XDH) identified in this study and previously characterized enzymes

Optimum Xanthine NAD+

No Source pH Temp. (℃)

Km

(μM)

Vmax

(μmol.mg-1)

kcat

(s-1)

kcat/ Km

(μM-1.s-1)

Km

(μM)

Vmax

(μmol.mg-1)

kcat

(s-1)

kcat/ Km

(μM-1.s-1)

Production

method

Ref.

1 Acinetobacter

baumannii

8.5-9.0 40-45 25.32±1.48 29.16±0.37 69.32±0.88 2.74 113.1±18.35 46.92±2.58 111.54±6.13 0.99 Expression

in E. coli

This study

2 Rhodobacter

capsulates

8.0 35 36.2±7.3 17.6 38.8±3.4 1.07 36.2±4.5 -- -- 1.07 Expression

in E. coli

(Schumann et al. 2008)

3 Arabidopsis

thalianas

8.0 25 -- 1.712±0.236 4.28±0.59 -- -- 0.702±0.042 1.76 -- Expression

in Pichia

pastoris

(Hesberg et al. 2004)

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 Rat liver 7.8 25 -- 3.2 8 -- -- 2.3 5.75 -- Insect Cell

System

(Nishino et al. 2002)

6 Rat liver 7.8 25 -- 3.3 8.25 -- -- 3.2 8 -- Extraction (Nishino 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

(Benboubetra 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

2

18192021

(Benboubetra et al. 2004)

9 Goat milk 7.2 25 6.33±0.08 0.03±0.01 0.07±0.03 0.01 4.12±0.06 0.27±0.04 0.68±0.10 0.17 Extraction

(Benboubetra 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

(Benboubetra et al. 2004)

11 Veillonella

atypica

7.5 37 136 30 64.5 0.47 -- -- -- - Extraction (Gremer and Meyer 1996)

12 Micrococcus

Lactilytkicus

6.5

unstabl

e (<7)

37 17 0.08 2.5 5.38 0.32 - - - Extraction (Smith et al. 1967)

13 Streptomyces

cyanogenus

8.7 40,

stable (<55

℃)

150 10.6 22.08 0.15 110 -- -- - Extraction (Sin 1975)

14 Bos taurus 7.5 25oC

unstable

(>38℃)

0.3 0.16 0.4 1.33 6.7 -- -- - Extraction (Hunt and Massey 1992)

15 Camel milk 8.3 22 -- 0.013 0.03 -- -- - - - Extraction (Baghiani et al. 2003)

16 Chlamydomon

as reinhardtii

-- 25,

instable

-- 5.69 31.67 -- -- -- -- -- Extraction (Perez-Vicente et al. 1992)

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17 Clostridium

purinilyticum

7.5 25 4.5 2.7 5.85 1.3 - - - - Extraction (Self and Stadtman 2000)

18 Colias

butterflies

7.8

stable

(7-8.5)

30 61/64.7 10.58/

11.1

26.45/

27.75

0.43/

0.43

124 7.6 19 0.15 Extraction (Watt 1972)

19 Comamonas

acidovorans

7.9 25 80.1 80 86 1.07 113 -- -- -- Extraction (Ivanov et al. 2003)

20 Drosophila

melanogaster

8.0

stable

(<9.2)

-- 18(Yen and

Glassman

1967)

12.4 31 1.72/2.01 25(Yen and

Glassman

1967)

-- -- -- Extraction (Adams et al. 2002; Yen and Glassman 1967)

21 Eubacterium

barkeri

7.8 37 67 164 77.4 1.16 38 (NADP) -- -- -- Extraction (Schrader et al. 1999)

22 Gallus gallus 7.5

stable

(7.2-

8.7)

30

stable(25-40℃)

17 0.22 0.55 0.03 19 -- -- -- Extraction (Tramper et al. 1979)

(Khobragade et al. 2008)

23 Soybean

nodule

7.5 30 5 2.4 5.64 1.13 12.5 -- -- -- Extraction (Boland et al. 1983; Triplett et al. 1982)

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24 Gottschalkia

acidurici

7 55 1350 2.8 10.45 0.008 -- -- -- Extraction (Wagner et al. 1984)

25 Human 7.4

6-9.2

stable

37 8.8 0.40 0.99 0.11 -- -- -- Extraction

(Benboubetra et al. 2004; Yamaguchi et al. 2007)

26 Neurospora

crassa

8.0 -- 8.2 0.15 0.39 0.05 28 -- -- -- Extraction (Lyon and Garrett 1978)

27 Pisum sativum 8.5 -- 17 -- -- -- 25 -- -- -- Extraction (Sauer et al. 2002)

28 Pseudomonas

putida

-- -- 52 26.7 125.61 2.42 64 -- -- -- Extraction (Kim and Schmid 1989; Parschat et al. 2001)

29 Pseudomonas

synxantha A3

8.2-8.8 40 -- 20 43.33 -- -- -- -- -- Extraction (Sakai and Jun 1979)

30 Rattus sp. -- -- -- 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)

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32 Triticum

aestivum

8-8.6 25 8.2 0.23 -- -- 171 -- -- -- Extraction (Montalbini 1998)

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Supplementary Table 2 Amino acid sequence identity (homology) of A. baumannii

XDH identified in this study and other XDHs characterized previously

A. baumanniiUniProtKB

EntryNo. OriginXDHA

(Fe/S cluster and FAD

binding subunit)

XDHB(Moco-binding

subunit)

XDHC(Accessory

protein)

1 Arabidopsis thaliana 22.8% (37.4%) 39.2% (51.6%) -- Q8GUQ82 Bos taurus (Bovine) 22.8% (37.7%) 38.6% (51.5%) -- P804573 Drosophila

melanogaster (Fruit fly)

25.0% (38.5%)

38.3% (53.4%) -- P10351

4 Calliphora vicina (Blue blowfly)

24.6% (37.1%) 39.1% (53.3%) -- P08793

5 Gallus gallus (Chicken)

22.2% (35.8%) 39.6% (52.7%) -- P47990

6 Mus musculus (Mouse)

22.0% (37.4%) 39.6% (51.9%) -- Q00519

7 Homo sapiens (Human)

22.5% (37.9%) 39.3% (51.3%) -- P47989

8 Sus scrofa (Pig) 23.3% (37.7%) 38.3% (51.0%) -- R4HZ399 Capra hircus (Goat) 23.1% (38.1%) 38.2% (51.0%) -- A1YZ3410 Camelus ferus

(Camel)13.0% (25.6%) 35.3% (47.4%) -- S9Y4T1

11 Chlamydomonas smithii

26.2% (38.7%) 32.8% (45.4%) -- A8IY70

12 Glycine soja (Soybean)

23.0% (37.4%) 38.8% (52.9%) -- A0A0B2RM17

13 Neurospora crassa 26.4% (41.8%) 39.5% (52.6%) -- Q7RXE4s14 Delftia acidovorans 46.9% (58.1%) 51.7% (63.2%) 28.6% (39.7%) Q8RLC1 (XDHA),

Q8RLC0 (XDHB),A0A080NKLS (XDHC)

15 Clostridium acidurici 25.9% (39.2%)a

&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%) 27.6% (40.3%) Q88F21 (XDHA)Q88F20 (XDHB)Q88F19 (XDHC)

17 Pseudomonas synxantha

43.5% (56.9%) 45.6% (58.6%) 28.5% (40.5%) I4L544 (XDHA)I4L2J2 (XDHB)I4L6Z0 (XDHC)

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18 Rhodobacter capsulatus

40.6% (53.1%) 49.0% (61.8%) 29.8% (41.6%) O54050 (XDHA)O54051 (XDHB)D5API3 (XDHC)

19 Rhodobacter capsulatus B10

40.6% (53.1%) 49.0% (61.8%) 29.8% (41.6%) 2W3S

20 Bovine milk 22.8% (37.7%) 38.6% (51.5%) -- 3AX9All the amino acid sequence alignments of the A. baumannii XDH identified in this

study and other XDHs characterized previously were carried out by AlignX module in

the Vector NTI 10.0 software.

a indicated the sequence comparison between the Fe/S binding subunit (UniProtKB

entry: K0B4E4) and the counterpart of A. baumannii XDHA subunit.

b indicated the sequence alignment between the FAD binding subunit (UniProtKB

entry: K0B3H0) and the counterpart of A. baumannii XDHA subunit.

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Supplementary Table 3 Purification of recombinant A. baumannii XDH following

heterologous expression in E. coli

Step Volume

(ml)

Protein

(mg) a

Activity

(U) b

Specific activity

(U/mg) c

Purification

(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

a Total protein was quantified by Bradford method with BSA as standard.

b Activity of A. baumannii XDH was assayed by monitoring the increase of uric acid

in absorption at 295 nm in the presence of 0.1 mM xanthine and 0.1 mM NAD at 25

℃ in 50 mM Tris/HCl buffer, pH 7.5

c Specific activity (U/mg) was defined as the increase of μmol uric acid per min per

mg enzyme under assay conditions.

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