Structure, Function and Inhibition of Ent-Kaurene Synthase from Bradyrhizobium Japonicum

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,*

aIndustrial Enzymes National Engineering Laboratory, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, China

bDepartment of Chemistry, University of Illinois, Urbana, IL 61801, USA

cInstitute of Biological Chemistry, Academia Sinica, Taipei 115, Taiwan

dDepartment of Applied Biological Chemistry, Niigata University, Niigata 950-2181, Japan

*Correspondence to RTG () or EO ().

+These authors contributed equally to this work.

Supplementary Table S1. Summary of Se-Met KS protein data collection and phasing statistics.

Name / Se-Met KS
peak / inflection / remote
Data collection
Wavelength (Å) / 0.9789 / 0.9790 / 0.9611
Resolution (Å) / 25.0 - 2.7 (2.8 - 2.7) / 25.0 - 2.7 (2.8 - 2.7) / 25.0 - 2.7 (2.8 - 2.7)
Space group / P41 / P41 / P41
Unit-cell a, b, c (Å) / 66.0, 66.0, 136.3 / 66.0, 66.0, 136.3 / 66.0, 66.0 136.3
No. of unique reflections / 31514 (3170) / 31480 (3200) / 31668 (3210)
Redundancy / 4.1 (4.2) / 4.2 (4.1) / 4.1 (4.0)
Completeness (%) / 99.6 (100.0) / 99.8 (100.0) / 99.8 (99.9)
Mean I/s(I) / 16.1 (3.9) / 15.6 (3.8) / 14.3 (3.0)
Rmerge (%) / 9.7 (38.8) / 10.1 (39.8) / 10.9 (48.4)
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.

Supplementary Table S2. Summary of observed protein residues and ligands

Name / PDB ID / Chain ID / Residues / Ligands
Apo-1 / 4KT9 / A / 3-290 / -
B / 3-281 / -
C / 2-290 (213-220 are missing) / -
D / 2-280 (211-222 are missing) / -
Apo-2 / 3W3F / A / 1-281 (211-221 are missing) / -
B / 3-290 (211-222 are missing) / -
D75C + CPP / 3WBV / A / 1-275 (212-220 are missing) / CPP
B / 2-277 (147-150 and 211-221 are missing) / CPP
WT + BPH-629 / 3W3H / A / 1-281 (211-221 are missing) / 629
B / 3-290 (149-150 and 211-222 are missing) / -

Supplementary Table S3. Q-score table for homology tree shown in Figure S1.

Supplementary Table S4. Q-score table for homology tree shown in Figure 3d.

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

Figure S1. Homology tree of α, β, γ, d, ε, and ζ-fold proteins obtained by using the Q-score program.

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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