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1

Supplementary Information

Molecular insights into the enzyme promiscuity of an aromatic

prenyltransferase

Ridao Chen1,#, Bingquan Gao2,3,#, Xiao Liu1,#,§, Feiying Ruan1, Yong Zhang4, Jizhong

Lou4, Keping Feng1, Carsten Wunsch5, Shu-Ming Li5, Jungui Dai1,*, Fei Sun2,3,*

1 State Key Laboratory of Bioactive Substance and Function of Natural Medicines,

Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union

Medical College, 1 Xian Nong Tan Street, Beijing 100050, China.

2 National Laboratory of Biomacromolecules, CAS Center for Excellence in

Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, 15 Datun

Road, Beijing 100101, China.

3 University of Chinese Academy of Sciences, 19 Yuquan Road, Beijing 100049, China.

4 Key Laboratory of RNA Biology, Institute of Biophysics, Chinese Academy of Sciences,

15 Datun Road, Beijing 100101, China.

5 Institut für Pharmazeutische Biologie und Biotechnologie, Philipps-Universität Marburg,

Deutschhausstraße 17A, 35037 Marburg, Germany.

§Present address: Modern Research Center for Traditional Chinese Medicine, Beijing

University of Chinese Medicine, Beijing 100029, China.

#These authors contributed equally to this work.

*Correspondence: [email protected] (Jungui Dai) or [email protected] (Fei Sun)

Nature Chemical Biology: doi:10.1038/nchembio.2263

mailto:[email protected]

2

Supplementary Results

Supplementary Table 1. A. terreus strains used in this study.

Fungal strain or transformants Gene mutation(s) Genotype

A. terreus A8-4 - wildtype

A. terreus NIH2624 - wildtype

D_04999 ATEG_04999 ATEG_04999 :: hph


3

Supplementary Table 2. Primers used in this study a.

Primer Sequence (5'→3')

CDS-F CGCCATATGCTCCCCCCATCAGACAGCAAAGATC

CDS-R CCCTCGAGTCACACACGTGCGACATTTCCCGCAAC

TC-F GCCATATGCCCTGGCAGATCCTGAGC

TC-R CTCGAGTCACACACGTGCGACATTTC

KO-LF GTCTGCGAGATGCGTTCATTGAAGAG

KO-LR GGGAGTCGATGGGATGTCAAGTACTG

KO-RF AAGAAGCCGCTCTATGCTAACCAG

KO-RR CTGAGAATGGCAGGTCGATCCTAT

KO-HF ATCGACTCCCCGACAGAAGATGATATTGAAGGAGC

KO-HR GCGGCTTCTTGATCCCGGTCGGCATCTACTCTATT

Dia-F1 GAGTTCATCGAGCTCCTCGTTGTCAT

Dia-F2 CGATTACAGCGTGCATCTGCAGTACA

Dia-R1 CGCCATGTAGTGTATTGACCGATTCC

Dia-R2 ATGCGATTAGTTCCTGTGCCGAGGTA

H88A-F GGCCTCTCCCACGGCGGCGCGCCGCTGGAGATCAGC

H88A-R CGCGCCGCCGTGGGAGAGGCCGCTGATGTACTCGGG

E91A-F CCACGGCGGCCATCCGCTGGCGATCAGCGTCAAGATC

E91A-R GCCAGCGGATGGCCGCCGTGGGAGAGGCCGCTGAT

E91D-F CCACGGCGGCCATCCGCTGGACATCAGCGTCAAGATC

E91D-R GTCCAGCGGATGGCCGCCGTGGGAGAGGCCGCTGAT

E91Q-F CCACGGCGGCCATCCGCTGCAGATCAGCGTCAAGATC

E91Q-R GCAGCGGATGGCCGCCGTGGGAGAGGCCGCTGATGTA

S177A-F GCCACAACATCGTCTGCACGGCGCTCGACCTCAAAG

S177A-R CCGTGCAGACGATGTTGTGGCTCTCCTTGATGAGC

G326M-F CGCGGAGCGTCTGCCGTTTATGATCAACTATGCGATG

G326M-R CATAAACGGCAGACGCTCCGCGTCCAGCGTCTCGCAG

R396A-F CGCAGACCAAGAACGTCCATGCCTGGCTGGGAGTGGC

R396A-R GCATGGACGTTCTTGGTCTGCGAGATATCCACATTG

W397A-F GACCAAGAACGTCCATCGCGCGCTGGGAGTGGCGTAC

W397A-R GCGCGATGGACGTTCTTGGTCTGCGAGATATCCAC


4

a Primers CDS-F/R were involved in the amplification of the entire coding sequence of

AtaPT by RT-PCR. Primers TC-F/R were used to obtain the truncated form AtaPT11-424.

The three primer pairs of KO-LF/LR, KO-RF/RR, and KO-HF/HR were used for the

construction of knockout cassette for gene deletion. Primers Dia-F1, Dia-F2, Dia-R1, and

Dia-R2 were used in screening of transformants performed by diagnostic PCR. The eight

primer pairs of H88A-F/R, E91A-F/R, E91D-F/R, E91Q-F/R, S177A-F/R, G326M-F/R

R396A-F/R, and W397A-F/R were used for constructing mutants of H88A, E91A, E91D,

E91Q, S177A, G326M, R396A, and W397A, respectively.


5

Supplementary Table 3. Kinetic parameters of AtaPT with various aromatic

acceptors.

Aromatic acceptor KM (M) k

cat (min

-1) k

cat/K

M(M

-1min

-1)

()-Butyrolactone II (1)* 31331 0.0010.000 3

()-Butyrolactone II (1)*∆ 2619 0.0230.001 88 ()-Butyrolactone II (1)** 892 0.0480.001 539 ()-Butyrolactone II (1)*** 66894 0.6610.083 990 ()-Butyrolactone II (2)** 11010 0.2780.010 2527 ()-cyclo-L-Trp-L-Trp (3)** 993 0.1630.002 1647 9-Aminocamptothecin (5)**, § 21722 0.2750.021 1267

Norathyriol (11)** 1343 0.1890.004 1410 4,4'-Dihydroxybenzophenone (13)** 3235 0.4520.005 1399 Genistein (21)* 28833 0.0550.004 191 Genistein (21)** 26129 0.4950.033 1897 Genistein (21)*** 29239 0.6180.063 2116 Sophoricoside (22)** 805 0.0830.004 1038 Resveratrol (41)** 2779 0.5590.010 2018 7-Hydroxycoumarin (42)** 1422 0.0720.001 507

* Using DMAPP as prenyl donor; ** Using GPP as prenyl donor; *** Using FPP as

prenyl donor; ∆ Kinetic parameters for G326M; § Compound 5 exhibited poor aqueous

solubility in reaction buffer. In addition, partially denaturation of AtaPT was observed for

the duration of incubation with 5.


6

Supplementary Table 4. Anti-proliferation activities of genistein and its prenylated

products against three cancer cell linesa.

Compounds IC50 (M)

HCT-8 Bel7402 BGC823

genistein (21) 133.1 103.9 146.7

50a 81.6 71.6 27.7

50b 36.0 24.6 76.9

50c 30.2 85.9 38.1

51a 78.8 38.9 31.4

51b 69.9 55.3 51.9

51c 75.4 34.0 32.4

51d 45.7 74.0 75.3

52a >100 >100 >100

52b >100 >100 >100

52c >100 >100 >100

52d >100 >100 >100

a The HCT-8 human colorectal adenocarcinoma cell line, the Bel-7402 human liver

cancer cell line, and the BGC-823 human gastric cancer cell line were purchased from

the Institute of Cell Biology (Shanghai, China). All three tumor cell lines were maintained

in RRMI 1640 medium supplemented with 10% (v/v) fetal bovine serum (FBS), 100

units/ml penicillin, and 100 g/ml streptomycin. Cultures were incubated at 37 C in a

humidified atmosphere of 5% CO2. Tumor cells were seeded in 96-well microtiter plates

at 1,200 cells/well. After 24 h, compounds were added to the wells. After incubation for

96 h, cell viability was determined by measuring the metabolic conversion of

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) into purple formazan

crystals by viable cells. The MTT assay results were read using an MK3 Wellscan

(Labsystem Dragon, Helsinki, Finland) plate reader at 570 nm. All compounds were

tested at five concentrations (103, 104, 105, 106, and 107 M) and were dissolved in

100% DMSO with a final concentration of DMSO of 0.1% (v/v) in each well. Each

concentration of the compounds was tested in three parallels. IC50 values were

calculated using Microsoft Excel software.


7

Supplementary Table 5. Data collection and refinement statistics.

AtaPT11-424

AtaPT11-424

+DMSPP AtaPT11-424

+GSPP AtaPT11-424

+GSPP

+DBu

AtaPT11-424

+DMSPP

+LBu

AtaPT11-424

+DMSPP

+Gen

AtaPT11-424

(E91A)

+GSPP+DBu

Data collection

Space group P21212 P21212 P21212 P21212 P21212 P21212 P21212

a, b, c (Å) 96.2, 135.8, 69.5 96.3, 133.0, 68.5 96.3, 135.7, 68.6 97.0, 134.9, 68.7 96.1, 134.1, 68.6 96.7, 136.1, 68.8 96.7, 139.2, 68.8

() 90.0, 90.0, 90.0 90.0, 90.0, 90.0 90.0, 90.0, 90.0 90.0, 90.0, 90.0 90.0, 90.0, 90.0 90.0, 90.0, 90.0 90.0, 90.0, 90.0

Resolution (Å) 48.58-1.90

(2.00-1.90) *

51.46-2.10

(2.21-2.10) *

48.24-2.00

(2.11-2.00) *

56.05-2.30

(2.42-2.30) *

47.96-1.84

(1.94-1.84) *

78.80-2.00

(2.11-2.00) *

56.50-2.82

(2.97-2.82) *

Rsym or Rmerge 0.088 (0.416) 0.079 (0.417) 0.106 (0.308) 0.083 (0.288) 0.077 (0.366) 0.078 (0.357) 0.093 (0.381)

I / I 5.4 (1.7) 7.0 (1.8) 4.5 (2.2) 5.5 (2.6) 5.7 (2.1) 7.0 (2.1) 7.0 (2.0)

Completeness (%) 99.1 (100.0) 98.6 (93.0) 91.3 (85.8) 97.0 (93.2) 99.4 (99.5) 88.9 (76.9) 98.8 (98.4)

Redundancy 4.9 (5.0) 4.9 (4.2) 4.3 (4.5) 3.7 (3.2) 4.6 (4.6) 4.2 (4.2) 8.1 (7.7)

Refinement

Resolution (Å) 48.6-1.9 51.4-2.1 48.2-2.0 56.1-2.3 47.96-1.84 78.8-2.0 56.02-2.82

No. reflections 68,155 48,717 52,816 37,460 73,216 52,312 23,126

Rwork / Rfree 19.2 (23.6) 19.4 (23.3) 18.6 (22.3) 20.4 (24.6) 18.2 (23.0) 21.5 (25.4) 20.4 (25.9)

No. atoms

Protein 6314 6347 6301 6082 6343 6335 6293

Ligand/ion 28 38 90 54 48 64

Water 433 215 330 81 583 410

B-factors

Protein 30.22 30.98 22.74 38.86 25.47 20.02 41.67

Ligand/ion 47.32 60.23 46.21 44.68 57.40 66.79

Water 32.43 30.04 23.12 28.10 34.83 31.78

R.m.s. deviations

Bond lengths (Å) 0.020 0.016 0.022 0.016 0.018 0.019 0.011

Bond angles () 1.88 1.76 1.91 1.79 1.87 1.93 1.67


8

Ramachandran

statistics

Favoured (%) 97.4 98.1 98.2 97.2 97.5 96.9 95.7

Allowed (%) 2.4 1.8 1.5 2.7 2.1 3.0 3.8

Outliers (%) 0.1 0.1 0.3 0.1 0.4 0.1 0.5

* Values in parentheses are for highest-resolution shell.


9

Supplementary Table 6. Solvent accessible surface area and volume of the

acceptor binding pocket of each aromatic prenyltransferase a.

a We utilized HOLLOW1 to generate a set of points (water molecules) filling in the

acceptor binding pocket and this set of points was used to generate a mask fitting with

the acceptor binding pocket by NCSMASK2. Then the surface area and volume of the

mask computed by using Chimera3 represent the solvent accessible surface area and

volume of the corresponding acceptor binding pocket.

Solvent accessible surface

area (Å2)

Solvent accessible volume

(Å3)

AtaPT 1583 3396

AnaPT 1362 2814

FgaPT2 1013 1750

FtmPT1 925 1621

CdpNPT 1047 2119


10

AtaPT

ATEG_10306

AnaPT

FgaPT2

MaPT

FtmPT2

FtmPT1

CTrpPT

CdpC3PT

CdpNPT

ATEG_02823

ATEG_04218

CdpC2PT

BrePT

NotF

7-DMATS

ATEG_08428

SirD

ATEG_00702

ATEG_09980

AO090102000322

VrtC

NphB

ATEG_00821

NovQ

CloQ

Supplementary Figure 1. The phylogenetic relationships between AtaPT and

known aromatic prenyltransferases. The sequence alignments were performed using

Clustal X4 and the molecular evolutionary genetics analysis was calculated using MEGA

6.05 with the method of neighbor-joining and bootstap of 1000. The branch lengths

represent relative genetic distances. The protein sequences and corresponding

accession numbers that were used here are as follows: 7-DMATS (Aspergillus fumigatus,

ABS89001); AnaPT (Neosartorya fischeri, EAW16181); AO090102000322 (A. oryzae

RIB40, BAE61387); AtaPT(A. terreus, KP893683); ATEG_00702 (A. terreus, EAU39348);

ATEG_00821(A. terreus, EAU39467); ATEG_02823 (A. terreus, EAU36097);

ATEG_04218 (A. terreus, EAU36020); ATEG_08428 (A. terreus, EAU31601);

ATEG_09980 (A. terreus, EAU29429); ATEG_10306 (A. terreus, EAU29303); BrePT (A.

versicolor, AFM09725); CdpC2PT (N. fischeri, AGR03830); CdpC3PT (N. fischeri,

EAW17508); CdpNPT (A. fumigatus, ABR14712); CloQ (Streptomyces

roseochromogenes, AAN65239); CTrpPT (A. oryzae, ADI60056); FgaPT2 (A. fumigatus,

AAX08549); FtmPT1 (A. fumigatus, AAX56314); FtmPT2 (A. fumigatus, ACF22981);

MaPT (A. fumigatus, ABZ80612); NotF (A. sp. MF297-2, ADM34132); NovQ (S.

caeruleus, AAF67510); NphB (S. sp. CL190, BAE00106); SirD (Leptosphaeria maculans,

AAS92554); VrtC (Penicillium aethiopicum, ADI24928).


11

H3C

OH

O

O

OH

OH

O

OOH

HO

OCH3

OHO

O

HO

HO

OH

O

O

HO

OH

OH

OH

OH

OH

O

O

H3CO

OH

OCH3

OH

O

O

H3CO

OH

O

O

HO

OCH3

OH

O

OH

O

OH

OH3CO

O

OH

OCH3

H3CO

OHO

O

OH

OCH3

H3CO

O

OOH

O

O

OH

OH

OH

OH

HO

NH

OH

O

NH2

H3C

NH

OH

O

NH2

H3CO

NH

OH

O

NH2

HO

NH

OCH3

O

NH2NH

NH2

NH

OH

O

HN

H3CO

CH3NH

O

NH

O

NN

O

HO

O

OHO

N

HO

H3CO

OCH3

OCH3

O

OH

O

O

OHO

HO

HO

HO O

O

OH

OHHO

H3C

O

HO

NN

O

O

OHO

NN

O

O

OHO

NN

O

O

OHO

NO2

N

H3CO

H3CO

OCH3

OCH3

H3C

OH

O

O

OCH3

OH OH

O

O

COOH

OH OH

O

O

CH3

OHOH

O

O OH

OH

O

OOH OH

OH

O

HO

OH

O

OHHO

OH

O

OH

HOOH

O

OH

OH

HOHO

OH

O

OH

OH

O

OH

OH

HO

OH

O

O

OH

OH

OH

HO

OHHO

NH

OH

O

NH2NH

OH

O

NH2

OH

O

NH2

HO

OH

O

NH2

HO

H3CO

O

O OO OOO OHO

O

O

HO

OH

HO

OH

O

OHH

O

O

O

O

O O

H

NHHO

HO

OH

HO

HO

OH O OH

OH O OH

OH

O

O

HO

HO

OH

O

HO O

H

HO

OH

COOH

# ## ## ## # ##

## ## ## ## ## ##

# ## ## ## ## ##

## # # # #

# # # ## # #

# # # # #

# ## ## # # ##

## ## ## # ## #

# # # ## ## ## #

## ## ## ## ## ## ##

O O

HO

HO

Supplementary Figure 2. Structures of tested aromatics with low ( 5%) or no

conversions. # conversion 5% in red color; ## no conversion in black color.


12

Supplementary Figure 3. Percent conversion of each aromatic when using

different prenyl donors. (a) Using DMAPP as prenyl donor. (b) Using FPP as prenyl

donor. The colors in the bar graphs represent different ratios of various prenylated

products in the total product yield of each aromatic compound, and the trace products

(yield 5%) are not counted. Experiments were performed in triplicate, and the standard

deviation is noted. The prenylated products marked with were isolated and further

characterized by HRMS and NMR spectroscopic analyses.

a

b


13

1

control reaction

negative ion MS spectrum for 1a

m/z 627, [356+272H]

negative ion MS spectrum for 1c

m/z 627, [356+272H]

negative ion MS spectrum for 1d

m/z 627, [356+272H]

HO

OOH

OH

O

OH3CO

negative ion MS spectrum for 1b

m/z 627, [356+272H]

Butyrolactone II (1) Geranylgeranyl

Ab

so

rba

nce

/ m

AU

Retention time / min

1a

1c

1d

enzymatic reaction

1

1b

400 5 10 15 20 25 30 35

300

0

50

100

150

200

250

405 10 15 20 25 30 35

0

50

100

150

200

250

300

350

400

450

0


Ab

so

rba

nce

/ m

AU

627.03

295.37 354.04204.13 786.41583.91 739.14

100 200 300 400 500 600 700 800 900

m/z

0

20

40

60

80

100

Rela

tive A

bundance

/ %

627.05

295.44 689.40143.05 226.08 368.20 795.54533.32

100 200 300 400 500 600 700 800 900

m/z

0

20

40

60

80

100

Rela

tive A

bundance

/ %

100 200 300 400 500 600 700 800 900

m/z

0

20

40

60

80

100

626.94

711.61662.67 734.02583.39 851.15800.25355.18133.03 447.12253.34

627.18

662.61711.45249.49 787.62440.35 555.99352.01146.19

100 200 300 400 500 600 700 800 900

m/z

0

20

40

60

80

100

Rela

tive A

bundance

/ %

Rela

tive A

bundance

/ %

Supplementary Figure 4. HPLC-MS analysis of catalytic products by AtaPT using

butyrolactone II (1) and geranylgeranyl pyrophosphate (GGPP) as the substrates.

(a) Molecular structures of 1 (m/z 356) and geranylgeranyl. (b) HPLC chromatogram of

control assay with boiled recombinant enzyme. (c) HPLC chromatogram of enzymatic

assay. (d–g) Negative ion MS spectra reveal that 1a, 1b, 1c, and 1d in (c) are four

prenylated products of 1 with one geranylgeranyl substitution.

a

b

c

d

e

f

g


14

1e1h

1g

1f

Ab

so

rba

nce

/ m

AU


enzymatic reaction

1

0 5 10 15 20 25 30 35

0

50

100

150

200

250

300

35

350

1

control reaction

negative ion MS spectrum for 1e

m/z 633, [356+278H]

negative ion MS spectrum for 1g

m/z 633, [356+278H]

negative ion MS spectrum for 1h

m/z 633, [356+278H]

negative ion MS spectrum for 1f

m/z 633, [356+278H]

HO

OOH

OH

O

OH3CO

Butyrolactone II (1) Phytyl

Ab

so

rba

nce

/ m

AU

5 10 15 20 25 30 35

0

50

100

150

200

250

300

350

400

0 40

450


632.89

695.61846.22

100 200 300 400 500 600 700 800 900

m/z

0

20

40

60

80

100

632.90

695.57593.09 732.47501.19

100 200 300 400 500 600 700 800 900

m/z

0

20

40

60

80

100

632.80

695.49295.24 588.89189.17

100 200 300 400 500 600 700 800 900

m/z

0

20

40

60

80

100

632.90

295.19353.94 526.27 671.48 733.60

100 200 300 400 500 600 700 800 900

m/z

0

20

40

60

80

100

Rela

tive A

bundance

/ %

Rela

tive A

bundance

/ %

Rela

tive A

bundance

/ %

Rela

tive A

bundance

/ %

Supplementary Figure 5. HPLC-MS analysis of catalytic products by AtaPT using

butyrolactone II (1) and phytyl pyrophosphate (PPP) as the substrates. (a)

Molecular structures of 1 ( m/z 356) and phytyl. (b) HPLC chromatogram of control assay

with boiled recombinant enzyme. (c) HPLC chromatogram of enzymatic assay. (d–g)

Negative ion MS spectra reveal that 1e, 1f, 1g, and 1h in (c) are four prenylated products

of 1 with one phytyl substitution.

a

b

c

d

e

f

g


15

H O

OO H

O H

O

OH 3C O

H O

OO H

O H

O

OH 3C O

ATEG_04999

hphtrpC

F1

R1

R2

F2

5000

3000

2000

1500

1000800

500

300

(bp)

Mark

er

WT KO

i ii iii

i, F1+R1: WT, no band

KO, 1734 bp

ii, F1+R2: WT, 2620 bp

KO, 2454 bp

iii, F2+R2: WT, 1546 bp

KO, no band

WT KO WT KO

20 30 40 50t / min

Butyrolactone II (1) Butyrolactone I (47a)

1

47a

1

47a

WT

KO

309 nm

309 nm

Supplementary Figure 6. Knockout of ATEG_04999 in A. terreus NIH2624. (a)

Schematic of the gene deletion and the diagnostic PCR screening strategy. Gene was

deleted using double homologous gene replacement with hygromycin resistance gene

(hph) as a selective marker. The hph gene with the trpC promoter was amplified from the

plasmid pCSN44, which was obtained from the Fungal Genetics Stock Center (FGSC).

The knockout cassette containing the hph gene was constructed by fusion PCR and

sequenced before using for transformation (see primers in Supplementary Table 2).

Polyethylene glycol-mediated transformation of A. terreus NIH2624 was done as

described previously for A. clavatus6. The screening of transformants was performed by

diagnostic PCR. Four primers (see Supplementary Table 2) are used for confirming

homologous integration of the knockout construct at the target locus. Primer F1 (Dia-F1

in Supplementary Table 2) anneals beyond the 5' homologous flank of the transforming

DNA construct, F2 (Dia-F2 in Supplementary Table 2) anneals within the ATEG_04999

sequence, R1 (Dia-R1 in Supplementary Table 2) anneals within the hph gene, and R2

(Dia-R2 in Supplementary Table 2) anneals within the 3' homologous flank of the

transforming DNA construct. (b) Agarose gel electrophoresis of diagnostic PCR for wild

type (WT) and gene knock out (KO) strains. (c) HPLC analyses of butyrolactone II (1)

and butyrolactone I (47a) in the extracts from WT and KO strains.

a

b

c


16

()-cyclo-L-Trp-L-Trp (3)

NH

HN

NH

HN

O

OH

H

NH

HN

NH

HN

O

OH

HR1

R2

53a (R1=geranyl; R2=H)

53b (R1, 2=geranyl) 54a (R1=geranyl; R2=H)

54b (R1=H; R2=geranyl)

54c (R1, 2=geranyl)

9-aminocamptothecin (5)

NN

O

O

OHO

NH2

R1

R2

sophoricoside (22)

57a (R1=H; R2=geranyl)

57b (R1=geranyl; R2=H)

OH

HO

OH

OH

O

OH

R3

R2 R1

R4

58a (R1, 2, 3=H; R4=geranyl)

58b (R1, 3=H; R2, 4=geranyl)

58c (R1, 4=geranyl; R2, 3=H)

58d (R1, 2=H; R3, 4=geranyl)

59a (R1=H; R2=geranyl)

59b (R1, 2=geranyl)

7-hydroxycoumarin (42)

O OHOO OHO

R2 R1

56a (R1, 2=geranyl; R3=H)

56b (R1, 3=geranyl; R2=H)

O

OHHO

O

OHO

R1 R2

R3

55a (R1, 2, 4=H; R3=geranyl)

55b (R1, 2, 3=H; R4=geranyl)

55c (R1, 3=geranyl; R2, 4=H)

55d (R1, 4=H; R2, 3=geranyl)

norathyriol (11)

O

O

HO

OH

OH

OH

O

O

O

OH

O

OR1

R2 R3

R4

NN

O

O

OHO

NH2

6

8

resveratrol (41)

4 2

3'

36

4,4'-dihydroxybenzophenone (13)

3 3'

4'

2

3 6

7

10

12

4

4′

GPP

PPi

AtaPT

OHO

OH OO

O

OH

OH

OH

OH

OHO

OH OO

O

OH

OH

OH

OH

R2

R1

GPP

PPi

AtaPT

GPP

PPi

AtaPT

GPP

PPi

AtaPT

GPP

PPi

AtaPT

GPP

PPi

AtaPT

GPP

PPi

AtaPT

Supplementary Figure 7. Identified products from enzymatic reactions by AtaPT

with various aromatic substrates.


17

Supplementary Figure 8. Crystal packing and sequence conservative surface

maping of AtaPT. (a) The interface of dimeric AtaPT in the asymmetric unit of the

crystal. (b) The biological assembly of AtaPT is a tetramer that is formed from two

asymmetric units (mageta and green) in the crystal. (c, d) Surface resprentation of

AtaPT colored with the sequence conservatives of AtaPT with AnaPT, FgaPT2, FtmPT1,

and CdpNPT that are derived from structure-based sequence alignments. Top view (c)

showing the prenyl donor binding pocket and the bottom view (d) showing the aromatic

acceptor binding pocket. Red, the sequence conservative high than 85%; Pink, the

sequence conservative between 35% and 85%; White, the sequence conservative less

than 35%. See also Supplementary Fig. 9.

c

d

b

a


18

Supplementary Figure 9. Structure-guided sequence alignments of AtaPT with

AnaPT, FgaPT2, FtmPT1, and CdpNPT. The residues for prenyl donor binding,

tyrosine shield and acceptor binding are indicated, respectively.


19

Supplementary Figure 10. Comparison of substrate-binding pockets between

AtaPT and four reported PTases. In the top panel, the substrate-binding pockets were

computed using HOLLOW1 and generated in PyMOL (DeLano, 2002,

http://www.pymol.org); the surrounding residues are shown in stick form and labeled

accordingly. In the bottom panel, the electrostatic surface of the protein is sliced to show

the internal surface of the substrate-binding pocket.

a b c d e


http://www.pymol.org/

20

Supplementary Figure 11. Substrates indentification of the AtaPT complex

structures. The substrates are shown in stick and its corresponding 2mFo-DFc

maximum-likelihood omit map shown in mesh with the countor levels of 1.0 in (a, c–f)

and 0.8 in (b). The 2mFo-DFc maximum-likelihood omit map was calculated by

REFMAC57. The residues coordinating the substrates are shown in stick and labeled

accordingly. The real-space correlation coefficients (RSCC) between the substrate and

the corresponding density map are calculated using Phenix8 and plotted below,

respectively.

b c

e f

a

d


21

Supplementary Figure 12. Isothermal titration calorimetry (ITC) measurements. (a)

Titration of butyrolactone II (DBu) into AtaPT. (b) Titration of DBu into AtaPT with the

mixture of GSPP. The profiles are subtracted from the baseline. (c) Plot of the

peak-integrated and concentration-normalized enthalpy changes versus the molar ratios

of DBu over AtaPT according to the profile in b. The calculated binding affinity of DBu for

AtaPT in the presence of GSPP is shown in the inset.

a b

c


22

Supplementary Figure 13. Molecular dynamic simulation of AtaPT in apo state

(a–d) and in DMSPP/GSPP bound state (e–h). (a, e) R.M.S.D (root mean of standand

deviation) of overall structure of AtaPT vesus the simulation time. (b, f) R.M.S.F (root

mean of structural fluctuation) of residues during the simulation process. The relatively

high dynamic segments are indicated. (c, g) R.M.S.F of the four residues of the tyrosine

shield. (d, h) R.M.S.D of the four residues of tyrosine shield. MD, molecular simulation.

a

b

e

f

c

d

g

h


23

Supplementary Figure 14. Diverse aromatic acceptors binding pocket of AtaPT.

The substrate-binding pockets are shown in electrostatic surface with the aromatic

acceptors shown in stick. The detailed interactions between the substrates and AtaPT

are analyzed and plotted below using Ligplot9. For the structure of AtaPT-DMSPP-LBu,

the dual conformations of Y190 are plotted in the same figure (b).

a b c


24

Supplementary Figure 15. Comparison of the substrate-binding pocket between

AtaPT-GSPP and FgaPT2-DMSPP-L-Trp. (a) The substrate-binding pocket of the

binary complex AtaPT-GSPP showing its surrounding small hydrophobic residues. (b)

Substrate-binding pocket of the ternary complex FgaPT2-DMSPP-L-Trp (PDB code:

3I4X) showing its surrounding residues at the equivalent positions in a.

a b


25

Supplementary Figure 16. Structural comparison of DBu binding to the wild type

AtaPT and the mutant E91A. (a) The interactions of GSPP and DBu with wild type

AtaPT. (b) The interactions of GSPP and DBu with the mutant E91A. The detailed

interactions between the substrates and AtaPT are analyzed and plotted using Ligplot9.

a b


26

Supplementary Figure 17. Comparison of the prenylation activities and product

diversities of wild-type AtaPT and different mutants. (a) Using genistein (21) as

acceptor with GPP as the prenyl donor. (b) Using sophoricoside (22) as acceptor with

GPP as the prenyl donor. Experiments were performed in triplicate with standard

deviation noted.

Supplementary Notes. The spectroscopic data of identified catalytic products by

AtaPT and the corresponding spectra.

a

b


27

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