Supplementary Information
Functional characterization of a vanillin dehydrogenase in
Corynebacterium glutamicum
Wei Ding1#, Meiru Si1,2#, Weipeng Zhang1, Yaoling Zhang1, Can Chen1,2, Lei Zhang1,2,
Zhiqiang Lu3, Shaolin Chen2& Xihui Shen1, 2*
1
State Key Laboratory of Crop Stress Biology for Arid Areas and College of Life Sciences,
Northwest A&F University, Yangling, Shaanxi 712100, PR China;
2
Biomass Energy Center for Arid and Semi-Arid Lands, Northwest A&F University, Yangling,
Shaanxi 712100, PR China;
3
College of Plant Protection, Northwest A&F University, Yangling, Shaanxi 712100, PR China.
Running title: Vanillin dehydrogenase in Corynebacterium glutamicum
#
These authors contributed equally to this work.
* Corresponding author
E-mail address: xihuishen@nwsuaf.edu.cn (X.H. Shen)
Key Words: Corynebacterium glutamicum; Vanillin dehydrogenase; Site-directed mutagenesis;
Aromatic compound
Figure S1 Multiple sequence alignment of VDHATCC13032 with identified VDHs and aldehyde dehydrogenases
from other sources. The amino acid sequence of the VDH from C. glutamicum ATCC13032 was aligined to the amino
acid sequence of the VDHs from Amycolatopsis sp. ATCC 39117, R. jostii RHA1, S. paucimobilis SYK6, and the
amino acid sequence of the betaine aldehyde dehydrogenase from Pseudomonas aeruginosa DK2, the amino acid
sequence of the aldehyde dehydrogenase from hyperthermophilic archaeon Sulfolobus tokodaii. And the site-directed
residues were marked in black.
Table S1 Substrate specificity of VDH.
Substrates
Relative activity (%)
vanillin
100
3, 4-dihydroxybenzaldehyde
90.29
p-hydroxylbenzaldehyde
89.04
3-hydroxylbenzaldehyde
81.59
p-nitrobenzaldehyde
75.80
terephthalaldehyde
56.67
o-phthaldialdehyde
28.37
2,4-dichlorobenzaldehyde
55.93
cinnamaldehyde
24.40
syringaldehyde
23.31
benzaldehyde
37.93
phenylacetaldehyde
2.13
benzenepropanal
31.22
formaldehyde
4.27
aldehyde
3.11
VDH activity was determined according to the methods with substrate (1 mM), NAD+ (0.5 mM) and an appropriate
amount of enzyme in potassium phosphate buffer (100 mM, pH 7.0), respectively. The activity was expressed as the
percentage of the activity toward 1 mM vanillin.
Table S2 Kinetics parameters of VDH towards vanillin, 3,4-dihydroxybenzaldehyde and p-hydroxylbenzaldehyde.
Substrates
Km(μM)
kcat(s-1)
kcat/Km (s-1μM-1)
vanillin
26.31±3.09
21.03±1.22
0.80±0.04
3,4-dihydroxybenzaldehyde
30.44±4.11
23.42±1.78
0.77±0.04
p-hydroxylbenzaldehyde
39.11±4.89
22.92±1.80
0.59±0.02
The kinetics parameters were determined according to the standard enzyme assay procedure with 0.5 mM NAD+ and
different concentrations of 3,4-dihydroxybenzaldehyde, p-hydroxylbenzaldehyde and vanillin by means of respective
Lineweaver-Burk plots. The concentrations were five values ranging from 0.2 to 1 mM for substrates.
Table S3 Kinetics parameters of VDH toward NAD+ and NADP+.
NAD+
NADP+
Substrates
Km (μM)
kcat(s-1)
kcat/Km
(s-1μM -1)
Km(μM)
kcat(s-1)
kcat/Km
(s-1μM -1)
vanillin
26.25±3.22
17.88±1.92
0.68±0.01
28.31±3.43
0.765±2.22
1.62±0.11
3,4-dihydroxy
benzaldehyde
31.33±4.06
19.62±1.86
0.63±0.02
35.28±4.27
0.827±2.52
1.41±0.09
p-hydroxyl
benzaldehyde
33.41±4.15
20.46±2.16
0.61±0.01
37.40±4.36
0.906±3.00
1.45±0.08
The kinetics parameters were determined according to the standard enzyme assay procedure with 1 mM 3,4-dihydroxy
benzaldehyde, p-hydroxyl benzaldehyde and vanillin by means of respective Lineweaver-Burk plots. The
concentrations were five values ranging from 0.125 to 1 mM for NAD+ or 0.125 to 2.5 mM for NADP+.
Table S4 Relative activity of VDH and its mutants.
Relative activity (%)
NAD+
NADP+
100
100
N157A
48.02
9.11
K180A
35.15
9.96
E199A
49.12
78.01
E258A
44.06
23.97
C292A
33.79
6.53
WT
VDH activity was determined according to the methods with vanillin (3 mM), NAD+ (1 mM) (or NADP+ (2.5 mM)) and
an appropriathe amount of enzyme in potassium phosphate buffer (100 mM , pH 7.0). The activity was expressed as the
percentage of the activity toward wild type. (100%, 0. 689±0.026 U/mg).
Table S5 Kinetics parameters of VDH toward NAD+ and NADP+.
NAD+
Mutants
Km(μM)
kcat(s-1)
NADP+
kcat/Km
(s-1μM-1)
Km(μM)
kcat(s-1)
kcat/Km
(s-1μM-1)
WT
20.36±3.65
12.96±1.32
0.64±0.04
22.36±3.01
49.80±1.86
2.23±0.19
N157A
98.79±7.53
8.94±0.96
0.09±0.01
242.29±10.11
20.14±0.90
0.08±0.01
K180A
90.33±7.36
12.54±1.08
0.14±0.01
256.37±21.36
54.66±1.38
0.21±0.01
E199A
103.29±6.01
6.30±0.61
0.06±0.01
61.23±4.29
46.92±0.78
0.77±0.04
E258A
70.19±5.25
10.26±0.66
0.15±0.01
114.33±9.48
13.20±1.26
0.12±0.01
C292A
88.95±6.35
5.76±0.42
0.06±0.01
148.40±12.32
12.72±1.20
0.09±0.01
The kinetics parameters were determined according to the standard enzyme assay procedure with 3 mM vanillin by
means of respective Lineweaver-Burk plots. The concentrations were five values ranging from 0.125 to 1 mM for
NAD+ or 0.125 to 2.5 mM NADP +.
Table S6 Kinetics parameters of VDH toward vanillin.
Mutants
Km(μM)
kcat(s-1)
kcat/Km(s-1μM-1)
WT
31.36±5.56
44.04±2.34
1.40±0.15
N157A
73.28±10.38
24.30±1.68
0.33±0.02
K180A
157.46±35.15
21.66±1.50
0.14±0.02
E199A
45.89±4.48
39.72±2.34
0.87±0.03
E258A
114.21±25.01
15.90±1.26
0.12±0.02
C292A
273.88±41.33
16.68±1.50
0.06±0.01
The kinetics parameters were determined according to the standard enzyme assay procedure with 1 mM NAD+ by
means of respective Lineweaver-Burk plots. The concentrations were five values ranging from 0.2 to 3 mM for vanillin.
Table S7 Bacterial strains and plasmids used in this study.
Relevant characteristics
Sources
JM109
recAl supE44 endAI hsdR17 gyrA96 relA1 thi Δ(lac-proAB)
Stratagene
BL21(DE3)
hsdS gal (λcIts857 ind-l Sam7 nin-5 lacUV5-T7 gene 1)
Novagen
RES167
Restriction-deficient mutant of ATCC 13032;Δ(cglIM-cglIR-cglIIR)
This study
RES167Δncgl2578
ncgl2578 deleted in RES167
This study
Mobilizable vector, allows for selection of double crossover in C.
Schäfer et al.1
Strains and plasmids
Strains
Escherichia coli
Corynebacterium
glutamicum
Plasmids
pK18mobsacB
lutamicum
pK18mobsacBΔncgl2578
Construct used for in-frame deletion of ncgl2578
This study
pXMJ19
Shuttle vector (Ptac lacIq pBL1 oriVC. glutamicum pK18 oriVE. coli)
Jakoby
pXMJ19-ncgl2578
ncgl2578 cloned into pXMJ19 for complementation
This study
pET28a
Experssion vector with N-terminal hexahistidine affinity tag
Novagen
pET28a-ncgl2578
pET28a derivative for expression of ncgl2578
This study
Table S8 Bacterial strains, plasmids and primers used in this study.
Primers
Ncgl2578upFBamHI
CGCGGATCCCGCCACATCACCGACGGAC
Ncgl2578upR
GGATCGGCATCAAGCGCAGC
Ncgl2578dwF
GCTGCGCTTGATGCCGATCCCTCCACCCACTGACCTCCG
Ncgl2578dwRHindIII
CCCAAGCTTTGCACTTCCCGGAGGCTACC
Ncgl2578FBamHI
CGCGGATCCATGACTGCAACATTTGCTG
Ncgl2578RKpnI/SalI
CCAGGTACCGTCGACTTAGCTGCGCTTGATGCCG
Ncgl2578rbsFBamHI
CGCGGATCCAAAGGAGGACAACCATGACTGCAACATTTGCTG
N157AF
TGATTAGTCCATGGGCTTTCCCACTGAACCT
N157AR
AGGTTCAGTGGGAAAGCCCATGGACTAATCA
K180AF
CAACGCCGTAGTGATTGCGCCTGCGAGTGATACCCCAGTTAC
K180AR
GTAACTGGGGTATCACTCGCAGGCGCAATCACTACGGCGTTG
E199AF
GCACGAATCTTTGAGGCGGCCGGAGTTCCTGC
E199AR
GCAGGAACTCCGGCCGCCTCAAAGATTCGTGC
E258AF
CAATGAAAACTGTTGCACTAGCGCTCGGTGGCAACGCGCCG
E258AR
CGGCGCGTTGCCACCGAGCGCTAGTGCAACAGTTTTCATTG
C292AF
ACCAGGGACAGATTGCTATGTCAATCAACCG
C292AR
CGGTTGATTGACATAGCAATCTGTCCCTGGT
Underlined sites indicate restriction enzyme sites added for cloning. Mutated bases in the primers are shown in boldface
and the ribosome binding site (rbs) is given in italic.
References:
1.
Schäfer, A. et al. Small mobilizable multi-purpose cloning vectors derived from the Escherichia coli plasmids pK18
and pK19: selection of defined deletions in the chromosome of Corynebacterium glutamicum. Gene 145,69-73
(1994).