1
2 ( ⅰ ) Hydroquinone 1, 2-dioxygenase.
The enzyme activity assay was performed as
3 described [13, 21], and the activity of His
6
- PdcDE was determined
4 spectrophotometrically by monitoring the formation of 4-HS at 290 nm to 320 nm.
5 The reaction mixture contained 20 mM phosphate buffer (pH 6.0), 50 μl of purified
6 protein (0.75 mg/ml) and 0.5 mM HQ in a final volume of 1.0 ml. The reaction was
7 started by the addition of 50 μl of purified enzyme. Following incubation at 40°C for
8 1 min, the reaction was terminated through the addition of 1.0 ml of ethanol. The
9 mixtures were centrifuged, and the absorbance of the resulting supernatants was
10 measured at 320 nm by use of Hitachi 3100 spectrophotometer (Hitachi, Japan).
11 The absorbance changes at 320 nm were subsequently used to assay the enzyme's
12 other characteristics. Its optimal pH at 40°C was determined using buffers ranging in
13 pH from 3.0 to 12.0. The following buffers were used; 20 mM disodium hydrogen
14 phosphate-citric acid buffer (pH 3.0–8.0), 50 mM Tris–HCl buffer (pH 8.0–10.0), and
15 20 mM glycine–NaOH (pH 10.0–12.0). To test its pH stability, 100 μl of the PdcDE
16 (0.75 mg/ml) was pre-incubated without substrate in 900 μl of different buffers with
17 pH values from 3.0 to 12.0 for 0.5 h at 25°C, and then PdcDE activity was measured
18 under standard conditions.
19 The enzyme's optimal temperature was determined by measuring its activity in 20
20 mM phosphate buffer (pH 6.0) at temperatures ranging from 20°C to 70°C. Its
21 thermostability was subsequently monitored by pre-incubating the enzyme without
22 substrate in 20 mM phosphate buffer (pH 6.0) for 20 min at 50, 60, and 70°C. PdcDE

23 activity was then measured under standard conditions.
24 The effects of different metal ions and chemical reagents on enzymatic activity
25 were assessed in 20 mM phosphate buffer (pH 6.0) at 40°C. The reactions contained 5
26 mM NaCl, KCl, BaCl
2
, FeCl
2
, FeCl
3
, CoCl
2
, CuCl
2
, NiCl
2
, MgCl
2
, MnCl
2
, ZnCl
2
,
27 CdSO
4
, EDTA and 0.1% (v/v) sodium dodecyl sulphate (SDS).
28 ( ⅱ
) 4-hydroxymuconic semialdehyde dehydrogenase.
The activity of 4-HS
29 dehydrogenase was determined spectrophotometrically by monitoring the
30 deterioration of 4-HS at 290 nm to 320 nm. The reaction mixture contained 500 μl
31 solution, which was the product of degradation by the HQ 1,2-dioxygenase, as the
32 substrate, and 445 μl 20 mM phosphate buffer (pH 7.0), 5 μl 10 mM NAD + , and 50 μl
33 of purified protein (0.75 mg/ml) in a final volume of 1.0 ml. The assay was initiated
34 by the addition of purified protein. Following incubation at 50°C for 2 min, the
35 reaction was terminated through the addition of 1.0 ml of ethanol. The mixtures were
36 centrifuged, and the absorbance of the resulting supernatants was measured at 260 nm
37 by use of Hitachi 3100 spectrophotometer (Hitachi, Japan).
One unit (U) of enzyme
38 activity was defined as the amount of enzyme that decreases of 1 μmol of NAD
+
per
39 minute at 50°C.
40 The decrease of NAD
+
was subsequently used to assay the enzyme's other
41 characteristics. Its optimal pH at 50°C was determined using buffers ranging in pH
42 from 3.0 to 12.0. The following buffers were used; 20 mM disodium hydrogen
43 phosphate-citric acid buffer (pH 3.0–8.0), 50 mM Tris–HCl buffer (pH 8.0–10.0), and
44 20 mM glycine–NaOH (pH 10.0–12.0). To test its pH stability, 100 μl of the PdcG

45 (0.75 mg/ml) was pre-incubated without substrate in 900 μl of different buffers with
46 pH values from 3.0 to 12.0 for 0.5 h at 25°C, and then PdcG activity was measured
47 under standard conditions.
48 The enzyme's optimal temperature was determined by measuring its activity in 20
49 mM phosphate buffer (pH 8.0) at temperatures ranging from 20°C to 70°C. Its
50 thermostability was subsequently monitored by pre-incubating the enzyme without
51 substrate in 20 mM phosphate buffer (pH 8.0) for 20 min at 40, 50, and 60°C. PdcG
52 activity was then measured under standard conditions.
53 The effects of different metal ions and chemical reagents on enzymatic activity
54 were assessed in 20 mM phosphate buffer (pH 8.0) at 50°C. The reactions contained 5
55 mM NaCl, KCl, BaCl
2
, FeCl
2
, FeCl
3
, CoCl
2
, CuCl
2
, NiCl
2
, MgCl
2
, MnCl
2
, ZnCl
2
,
56 CdSO
4
, EDTA and 0.1% (v/v) sodium dodecyl sulphate (SDS).
57 ( ⅲ ) Hydroquinone 1,2-dioxygenase and 4-hydroxymuconic semialdehyde
58 dehydrogenase. To confirm the activity of HQ 1,2-dioxygenase and 4-HS
59 dehydrogenase, the two enzymes were assayed in the same reaction mixture. The
60 reaction mixture contained 885 μl 20 mM phosphate buffer (pH 7.0), 50 μl
61 His
6
-PdcDE (0.75 mg/ml), 50 μl His
6
-PdcG (0.75 mg/ml), and 0.5 mM HQ in a final
62 volume of 1.0 ml. The assay was initiated by addition of the substrate and changes
63 were monitored at 340 nm. When the change in absorbance leveled off, NAD
+ was
64 added to the reaction mixture to a final concentration of 0.05 mM, and the changes
65 were also monitored at 340 nm.
66 ( ⅳ
) Maleylacetate reductase . The activity of MA reductase was assayed according

67 to Dayna L [22] . The activity to metabolize MA was assayed by monitoring the content
68 change of NADH in the reaction mixture (absorbance changes at 340 nm). The
69 reaction mixture contained 680 μl solution, which was the product of degradation by
70 BT 1,2-dioxygenase , as the substrate, and 260 μl 20 mM phosphate buffer (pH 7.0),
71
10 μl 10 mg/ml NADH and 50 μl purified His
6
- PdcF (1.5 μg/ml) in a final volume of
72 1.0 ml. The assay was initiated by the addition of the purified protein. Following
73 incubation at 40°C for 2 min, the reaction was terminated through the addition of 1.0
74 ml of ethanol. Then the mixtures were centrifuged, and the absorbance of the resulting
75 supernatants was measured at 340 nm by use of Hitachi 3100 spectrophotometer
76 (Hitachi, Japan).
One unit (U) of enzyme activity was defined as the amount of
77 enzyme that decreases of 1 μmol of NADH per minute at 40°C.
78 The decrease of NADH was subsequently used to assay the enzyme's other
79 characteristics. Its optimal pH at 40°C was determined using buffers ranging in pH
80 from 3.0 to 12.0. The following buffers were used; 20 mM disodium hydrogen
81 phosphate-citric acid buffer (pH 3.0–8.0), 50 mM Tris–HCl buffer (pH 8.0–10.0), and
82 20 mM glycine–NaOH (pH 10.0–12.0). To test its pH stability, 100 μl of the PdcF
83 (1.5 μg/ml) was pre-incubated without substrate in 900 μl of different buffers with pH
84 values from 3.0 to 12.0 for 0.5 h at 25°C, and then PdcF activity was measured under
85 standard conditions.
86 The enzyme's optimal temperature was determined by measuring its activity in 20
87 mM phosphate buffer (pH 7.0) at temperatures ranging from 20°C to 70°C. Its
88 thermostability was subsequently monitored by pre-incubating the enzyme without

89 substrate in 20 mM phosphate buffer (pH 7.0) for 20 min at 40, 50, and 60°C. PdcF
90 activity was then measured under standard conditions.
91 The effects of different metal ions and chemical reagents on enzymatic activity were
92 assessed in 20 mM phosphate buffer (pH 6.0) at 40°C. The reactions contained 5 mM
93 NaCl, KCl, BaCl
2
, FeCl
2
, FeCl
3
, CoCl
2
, CuCl
2
, NiCl
2
, MgCl
2
, MnCl
2
, ZnCl
2
, CdSO
4
,
94 EDTA and 0.1% (v/v) sodium dodecyl sulphate (SDS).

95
96
Tables and Figures
97
98
99
100
101 Table S1. Primers used in this study
102
103
Primers
Sequences (5’-3’) *
Ps-F
Ps-R
GAGAGCACCGTGYTNGGBCCSTT
CGGGCCGTCGCTSGGDATNGGRTA
GCAGGCCTAACACATGCAA 16s-F
16s-R
Mal-F
Mal-R
Hys-F
GTTACGACTTCACCCCAGTCAT cgcggatccATGAATCCTTTCATTTACCAAAGCC cccaagcttTCAGGCCGGTGGCAACCCTTTC cgcggatccATGCAAAATCAGCTCTATATCGAT
Hys-R
Dio2A-F
Dio2A-R
Dio2B-F ccgctcgagTCAGCGCTTGAAGTGGGGAGCGAT cgcggatccATGGCCATGCTTGAATCCGCTGTC ccgctcgagCTAGAACTCGATCGGATAAGGCTT cgcggatccATGAGTAACGCTGCAGTCAATACG
Dio2B-R ccgctcgagTTACTTGAGACAGATCTCTGCCCA
*
Specified restriction sites are underlined.
Target
Small fragment of pdcC
16SrDNA of strain 1-7 pdcF pdcG pdcD pdcE

104
110
111
112
113
114
115
116
105
106
107
108
109
Figure S1. HPLC analyses specific absorb curved line of (a) standard peak and
(b) detected peak.

117

118 Figure S2. PNP and PNP degradation intermediates changes in different time
119
120
121 point of strain 1-7 grown on PNP. (a) PNP, (b) HQ, (c) 4-NC.
122
123

124
125
126
127
Figure S3. The temperature and pH properties of His
6
-PdcDE.
(a) Effects of pH on the activity of His
6
-PdcDE; (b) The pH stability of His
6
-PdcDE;
128 (c) Effects of temperature on the activity of His
6
-PdcDE; (d) The temperature stability
129 of His
6
-PdcDE.

130
131
132

133
134
135 Figure S4. The temperature and pH properties of His
6
-PdcG.
136 (a) Effects of pH on the activity of His
6
-PdcG; (b) The pH stability of His
6
-PdcG; (c)
137 Effects of temperature on the activity of His
6
-PdcG; (d) The temperature stability of
138 His
6
-PdcG.

139
140

141
142
143 Figure S5. The temperature and pH properties of His
6
-PdcF.
144 (a) Effects of pH on the activity of His
6
-PdcF; (b) The pH stability of His
6
-PdcF; (c)
145 Effects of temperature on the activity of His
6
-PdcF; (d) The temperature stability of
146 His
6
-PdcF.