Structure, function and inhibition of ent-kaurene synthase from Bradyrhizobium
japonicum
Wenting Liua,+, Xinxin Fengb,+, Yingying Zhenga,+, Chun-Hsiang Huanga, Chiaki Nakanod, Tsutomu
Hoshinod, Shannon Bogueb, Tzu-Ping Koc, Chun-Chi Chena, Yunfeng Cuia, Jian Lia, Iren Wangc,
Shang-Te Danny Hsuc, Eric Oldfieldb,* and Rey-Ting Guoa,*
a
Industrial Enzymes National Engineering Laboratory, Tianjin Institute of Industrial Biotechnology,
Chinese Academy of Sciences, Tianjin 300308, China
b
Department of Chemistry, University of Illinois, Urbana, IL 61801, USA
c
Institute of Biological Chemistry, Academia Sinica, Taipei 115, Taiwan
d
Department of Applied Biological Chemistry, Niigata University, Niigata 950-2181, Japan
*
Correspondence to RTG ([email protected]) or EO ([email protected]).
These authors contributed equally to this work.
+
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Supplementary Table S1. Summary of Se-Met KS protein data collection and phasing statistics.
Name
Se-Met KS
peak
inflection
Data collection
Wavelength (Å)
0.9789
0.9790
Resolution (Å)
25.0 - 2.7 (2.8 - 2.7)
25.0 - 2.7 (2.8 - 2.7)
Space group
P41
P41
Unit-cell a, b, c (Å)
66.0, 66.0, 136.3
66.0, 66.0, 136.3
No. of unique reflections
31514 (3170)
31480 (3200)
Redundancy
4.1 (4.2)
4.2 (4.1)
Completeness (%)
99.6 (100.0)
99.8 (100.0)
16.1 (3.9)
15.6 (3.8)
Mean I/(I)
Rmerge (%)
9.7 (38.8)
10.1 (39.8)
Phasing
No. of sites
10
Z-score
82.58 - 90.28
Figure of merit
0.52
Values in parentheses are for the outermost resolution shells.
2
remote
0.9611
25.0 - 2.7 (2.8 - 2.7)
P41
66.0, 66.0 136.3
31668 (3210)
4.1 (4.0)
99.8 (99.9)
14.3 (3.0)
10.9 (48.4)
Supplementary Table S2. Summary of observed protein residues and ligands
Name
PDB ID
Chain ID
Residues
4KT9
A
3-290
Apo-1
B
3-281
C
2-290 (213-220 are missing)
D
2-280 (211-222 are missing)
3W3F
A
1-281 (211-221 are missing)
Apo-2
B
3-290 (211-222 are missing)
3WBV
A
1-275 (212-220 are missing)
D75C + CPP
B
2-277 (147-150 and 211-221 are missing)
3W3H
A
1-281 (211-221 are missing)
WT + BPH-629
B
3-290 (149-150 and 211-222 are missing)
3
Ligands
CPP
CPP
629
-
Supplementary Table S3. Q-score table for homology tree shown in Figure S1.
4
Supplementary Table S4. Q-score table for homology tree shown in Figure 3d.
5
Supplementary Table S5. Activities of BjKS wild-type and mutant proteins using ent-CPP substrate and a PPi-release
assay.
protein
specific activity, nmol min−1mg−1
WT
64±2.9
D75C
2.4±0.32
D75A
0.81±0.18
D79C
0.20±0.86
R204A
0.77±0.51
CPP only
-0.32±0.21
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Figure S1. Homology tree of α, β, γ, , ε, and ζ-fold proteins obtained by using the Q-score program.
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Figure S2. Sequence alignment of Bradyrhizobium japonicum ent-kaurene synthase (BjKS) with abietadiene synthase
(AgABS), taxadiene synthase (TbTXS), 1, 8-cineole synthase (SfCinS1), ent-kaurene synthase (AtKS) and (+)-bornyl
diphosphate synthase (BPPS). Sequence alignments were performed with ClustalX1. Strictly conserved residues are
highlighted in red, conservatively substituted residues are boxed. The conserved DDXXD and
RL(N,D)DXX(S,T,G)XXX(E,D) motifs are boxed. Mg2+-binding residues are indicated by an asterisk. The Figure was
produced by using ESPript2.
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REFERENCES
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J. D. Thompson, T. J. Gibson, F. Plewniak, F. Jeanmougin and D. G. Higgins, Nucleic Acids Res., 1997, 25, 48764882.
P. Gouet, X. Robert and E. Courcelle, Nucleic Acids Res., 2003, 31, 3320-3323.
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