supplementary information lysine and arginine biosyntheses ...€¦ · supplementary information...

Supplementary information

Lysine and arginine biosyntheses mediated by a common carrier

protein in Sulfolobus

Takuya Ouchi1§, Takeo Tomita

1, Akira Horie

1† , Ayako Yoshida1, Kento

Takahashi1, Hiromi Nishida

2, Kerstin Lassak

3, Hikari Taka

4, Reiko Mineki

4,

Tsutomu Fujimura4, Saori Kosono

1, Chiharu Nishiyama

5, Ryoji Masui

6, 7, Seiki

Kuramitsu6, 7

, Sonja-Verena Albers3, Tomohisa Kuzuyama

1, and Makoto

Nishiyama1, 7*

1Biotechnology Research Center, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku,

Tokyo 113-8657, Japan. 2

Agricultural Bioinformatics Research Unit, Graduate School

of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku,

Tokyo 113-8657, Japan. 3

Max Planck Institute for terrestrial Microbiology, Karl von

Frisch Strasse 10 D-35043 Marburg, Germany. 4

Division of Biochemical Analysis,

Central Laboratory of Medical Sciences, Juntendo University School of Medicine, 2-1-1

Hongo, Bunkyo-ku, Tokyo 113-8421, Japan. 5Atopy (Allergy) Research Center,

Juntendo University School of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421,

Japan. 6

Department of Biological Sciences, Graduate School of Science, Osaka

University, Toyonaka, Osaka 560-0043, Japan. 7RIKEN SPring-8 Center, 1-1-1 Kouto,

Sayo-cho, Sayo-gun, Hyogo 679-5148, Japan. Present address: §Nihon Unisys, Ltd.,

Tokyo, Japan. †Central Laboratories for Frontier Technology, Kirin Holdings Co.,

Yokohama, Japan.

*To whom correspondence should be addressed: [email protected]

Nature Chemical Biology: doi:10.1038/nchembio.1200

1.0

0.1

0.010 20 40 60 80 100

1.0

0.1

0.010 20 40 60 80 100

Opt

ical

Den

sity

at 6

00 n

m

Opt

ical

Den

sity

at 6

00 n

m

a b

Time (h) Time (h)

Supplementary Figure 1. Effects of various concentrations of lysine on growth. (a) Parental MW001 strain. (b) Δ0754 strain. Brock’ s basal salt medium supplemented with 0.2% D-xylose and 20 μg/ml uracil was used as the basal culture medium. Additives are: white diamonds, 0.1 mM lysine plus 0.1 mM arginine; blue diamonds, none; red squares, 0.01 mM lysine; purple crosses, 0.05 mM lysine; light green triangles, 0.1 mM lysine; orange circles, 0.5 mM lysine. Each strain was cultured in triplicate. Bars indicate standard deviations.

SUPPLEMENTARY RESULTS


1 2 3 4 5

10

15

2025

37

75

6

50

100150250

M.W. (kDa)

Supplementary Figure 2. Tricine SDS-PAGE (full size). Purified LysW-AAA and LysW-Glu were incubated with or without Saci_0751 in the presence of 1 mM MgSO4, 160 mM KOH, 160 mM NH2OH-HCl, 10 mM ATP, 200 mM HEPES-NaOH, pH8.0, for 1 h at 60°C. Lanes 1 and 6, molecular size marker; lanes 2 and 3, LysW-AAA was used as the substrate; Lanes 4 and 5, LysW-Glu was used as the substrate. Lanes 2 and 4, with Saci_0751; Lanes 3 and 5, without Saci_0751.


Supplementary Figure 3. LC-MS/MS of C-terminal tryptic fragments of LysW derivatives. (a) C-terminal tryptic fragments of the band shown in an asterisk in lane 2 in Figure 3. (b) C-terminal tryptic fragments of the band shown in an asterisk in lane 3 in Figure 3. (c) C-terminal tryptic fragments of the band shown in an asterisk in lane 4 in Figure 3. (d) C-terminal tryptic fragments of the band shown in an asterisk in lane 5 in Figure 3. X denotes AAA residue.

c

%In

tens

ity

100806040200200 400 600 800 1000 1200 1400

m/z: 1376.67

348.89y3534.93y4

540.99b5586.94

b6

650.00y5

727.33b7

836.35y7

935.44y8

1028.52b9

1063.50y9

1085.57b10

1192.55y111214.63b11

1358.68b12

m/z

L A E Q V G E DW G E E NHL AA EE QQ VV GG W EW E G DG D EE Eb4 b5 b7 b10 b11 b12b1 b2 b3 b6 b9b8

y8 y7 y5 y2 y1y11 y10 y9 y6 y3y4

1358.61214.51085.51028.5727.4598.3541.3442.2 842.4

1263.5 1192.5 1063.5 935.4 836.3 779.3 650.2 535.2 349.1

OHH O

d

%In

tens

ity

100806040200200 400 600 800 1000 1200 1400

m/z: 1361.66

441.96b4

519.92y4 634.95y4

727.34b7

821.34y7

920.42y8

1028.54b9

1048.50y9

1085.56b10

1214.61b11

1343.66b12

m/z

L A E Q V G E DW G E E OHL AA EE QQ VV GG W EW E G DG D EE EE Ob4 b5 b7 b10 b11 b12b1 b2 b3 b6 b9b8

y8 y7 y5 y2 y1y11 y10 y9 y6 y3y4

1343.61214.51085.51028.5727.4598.3541.3442.2 842.4

1248.5 1177.5 1048.4 920.4 821.3 764.3 635.3 520.2 334.1

b

%In

tens

ity

100806040200200 400 600 800 1000 1200 1400

347.90441.94540.99

648.97727.33

835.36

934.451085.57

1214.68 1357.67y3 b4

b5y5

b7

y7

y8b10

b11b12

L A E Q V G E DW G E X OHL AA EE QQ VV GG W EW E G DG D EE XX Ob4 b5 b7 b10 b11 b12b1 b2 b3 b6 b9b8

y8 y7 y5 y2 y1y11 y10 y9 y6 y3y4

1357.91214.51085.51028.5727.4598.3541.3442.2 842.4

1262.5 1191.5 1062.4 934.4 835.3 778.3 649.3 534.2 348.1

m/z: 1375.68

m/z

a

%In

tens

ity

100806040200200 400 600 800 1000 1200 1400

362.90540.95

y3

b5

663.95y5

727.29b7 793.34y6

842.41b8

949.42y8

1028.52b91085.52

b101206.55y10

1372.63b12

m/z: 1390.66

m/z

L A E Q V G E DW G E X NHL AA EE QQ VV GG W EW E G DG D EE Xb4 b5 b7 b10 b11 b12b1 b2 b3 b6 b9b8

y8 y7 y5 y2 y1y11 y10 y9 y6 y3y4

1372.61214.51085.51028.5727.4598.3541.3442.2 842.4

1277.5 1206.5 1077.5 949.4 850.3 793.3 664.3 549.2 363.1

OHH O


Supplementary Figure 4. Analytical ultra centrifugation of StArgX and StLysW. (a) Data plot for StArgX. (b) Distribution plot of sedimentation coefficent of StArgX. (c) Data plot for StLysW. (d) Distribution plot of sedimentation coefficent of StLysW.

-1

1

0 5 10 15

c(s)

(S-1)

a

b

d

0 5 10 15

0

0.5

1

0

0.2

0.4

0.6

Sedimentation coefficient [S]

c(s)

(S-1)

6 6.5 7- 1

0 0

1 1

2 2

3 3A

bsor

banc

e at

280

nm

6 6.5 7

- 1

0 0

1 1

2 2

3 3

Abs

orba

nce

at 2

80 n

m

cSedimentation coefficient [S]

0

0.5

1

0

0.2

0.4

0.6

Radius [cm]

Radius [cm]

-


monomer

tetramer

Supplementary Figure 5. MS analysis of StArgX. (a) Spectrum deconvoluted from mass spectrum for StArgX in (b).

a

b

Inte

nsity

x 1

06In

tens

ity x

106

0

50

100

150

200

250

0

5

10

15

20

30 40 50 60 70 80 90 100 110 120 130

800600 1200 1600 20001000 1400 1800

Mass (kDa)

m/z

trimerdimer

31,532

63,06494,636

126,207

538.4 751.8770.1

789.3

809.5

830.3

853.2

876.9

901.9

928.4

956.5986.4 1018.2

1052.11088.3

1127.2

1168.91213.8

1262.31314.9

13721434.31502.5

1577.71660.6 1754 1857.8

1913.21974.61069.9

1147.71107.41190.9

1237.61288.11342.8 1467.7

1402.5 1539.31618.11632.9 1690.7

1705.6 1802.91721.71787.2

1820.9 1984.61894.3


a

b

ADP ADP ADP

AMP-PNP

StArgX(A-chain)

StArgX(B-chain)

StArgX(D-chain)

StArgX(C-chain)

TtLysX(A-chain)

TtlysX(B-chain)

TtLysX(D-chain)

TtlysX(C-chain)

AMP-PNP AMP-PNP

ADP

AMP-PNP

90°

90°

Supplementary Figure 6. Overall structures of StArgX/ADP and TtLysX/AMP-PNP complex. (a) StArgX/ADP complex. (b) TtLysX/AMP-PNP complex. A-, B-, C-, and D-chains of these tetramer are shown as green, cyan, magenta, and yellow figures, respectively. The bound ligands are shown as stick models.


a b

E52 D53

W54

G55E56

C-terminalextension

N-terminalglobular domain

C9C29

C6H27

Zn2+N-ter

C-ter

S235AR178A

W54E

Glu

M57A

D253A

N158A

E48E

R59A

S159B

D53A

R135A

E256A

W133A

E56E

G55EV254A

S132A

V55A

S56A

E52E

I9A

S235AR178A

W54E

Glu

M57A

D253A

N158A

E48E

R59A

S159B

D53A

R135A

E256A

W133A

E56E

G55EV254A

S132A

V55A

S56A

E52E

I9A

Supplementary Figure 7. Structure of StLysW and the recognition by StArgX. (a) Domain architecture of StLysW. N-terminal globular domain and C-terminal extension are shown as pink and purple models, respectively. The Zn2+ ion bound to N-terminal globular domain and the coordinating residues are shown as a sphere model and stick models, respectively. The residues of strictly conserved C-terminal ‘EDWGE’ motif are shown as stick models. (b) Stereo view of interaction between StArgX and C-terminal extension of StLysW. Another substrate glutmate molecule, the α-amino group of which is to be ligated with the γ-carboxyl group of Glu56 residue of StLysW, is also shown by orange stick model. The residues involved in the interactions are shown as stick models and hydrogen bonds and electrostatic interactions are shown by dotted lines.


Glu Glu

E56E E56EGSH GSH

a b

ADP

Glu

d

K87

N202R192

R178

E250

N252

ADP

Glu

K87

N202R192

R178

E250

N252

ADP

GSH

K125

N235R225

R210

E281

N283

ADP

GSH

K125

N235R225

R210

E281

N283SO4

2-

Zn2+

Mg2+

SO42-

Zn2+

Mg2+

E56 E56

SO42-

Mg2+

SO42-

Mg2+

Mg2+ Mg2+

c

V203AA204A

ADP

SO42-

E256A

Glu

F260A

G259A

Y190A

E56E

R192A

V203AA204A

ADP

SO42-

E256A

Glu

F260A

Y190A

E56E

G259A

R192A

A

A

AA

AA

E

A

A

A

AA

A

E

Supplementary Figure 8. Active site of StArgX. (a) Stereoview of the active site structure of StArgX binding StLysW, Glu, ADP, SO4

2-, and metal ions. Bound Mg2+ and Zn2+ are shown by green and gray sphere models. The Fo-Fc maps of ligands at the active site of StArgX contoured at 3.0 σ are shown as black mesh. (b) Stereoview of the active site structure of the pseudo-Michaelis complex of glutathione synthetase (GSHase) binding GSH, ADP, SO4

2-, and metal ions (PDB ID: 1gsa). (c) Superposition of (a) and (b). Colors used are the same as in (a) and (b). (d) Recognition of bound glutamate by StArgX.


Supplementary Figure 9. Structural model of the phosphoryl intermediate in the StArgX reaction. Substrate glutamate, ADP, Arg178 and the C-terminal Glu residue (phosphorylated) of StLysW are shown. Based on the crystal structure of the StArgX/StLysW complex, a model of reaction intermediate of StArgX was generated. In the determined structure, the γ-carboxyl group of Glu56 of StLysW was not present at the position suitable for acyltransfer due to charge repulsion against the bound sulfate ion that mimics the γ-phosphate group from ATP. Therefore, the sulfate ion was replaced with the phosphate moiety of acylphosphate intermediate of StLysW and the conformation of the Glu56 side chain of StLysW was manually adjusted to form the acylphosphate linkage with the phosphate group. In the intermediate state, Arg178 completely conserved among LysX-ArgX proteins may form ionic interactions with the side-chain carboxyl group of Glu56 and the phosphoryl group of the intermediate.

R178

Glu

Mg2+

Zn2+

ADP

E56E

R178

Glu

Mg2+

Zn2+

ADP

E56E


Signature motif

Supplementary Figure 10. Amino acid sequence alignment of LysX/ArgX family proteins. Structure-based alignment was performed by ClustalW and ESPript. Abbreviations are: StArgX, ArgX from S. tokodaii; SaArgX, ArgX from S. acidocaldarius; StLysX, LysX from S. tokodaii; SaLysX, LysX from S. acidocaldarius; TtLysX, LysX from T. thermophilus. Signature motif that determines the substrate specificity of this protein family is boxed.

β1 β2 β3 β4 β5α2α1 α3η1

β1 β2 β3 β4 β5α2α1 α3η2η1

β6α4 η2α5 β7 β8 β9α6

β6α4 η3α5 β7 β8 β9α6

β10 α7

η4

α8β11 β12 β13 β14

β10 α7 α8β11 β12 β13 β14

α9

α9

1 10 20 30 40 50 60 70 80 . . . . . . . . .StArgX MRVVLIVDIVRQEEKLIAKALEENKVQYDIINVAQEPLPFN....KALGRYDVAIIRPVSMYRALYSSAVLEAAGVHTINSSDVINVCGDSaArgX MRVALVVDIVRQEEKLIAKALEKFQLQYDVINVAQEPLPFN....KALGRYDVAIIRPISMYRALYASAVLESAGVHTINSSDTISLCGDStLysX MILGVIYDLLRWEEKNLIQEARKLGHTVIPIYTKDFYYFYNNDSNETLGDLDVVIQRNTSHARAVITSTIFENLSYKTINDSSTLIKCENSaLysX .........MRWEEKDIITEAKKSGFKAIPIFTKDFYSAIGVGENYSELEADVIIQRNTSHARALTTSLIFEGWNYNVVNDATSLFKCGNTtLysX .MLAILYDRIRPDERMLFERAEALGLPYKKVYVPALPMVLG.ERPEALEGVTVALERCVSQSRGLAAARYLTALGIPVVNRPEVIEACGD

90 100 110 120 130 140 150 160 170 . . . . . . . . .StArgX KILTYSKLYREGIPIPDSIIALSAEAALKAYEQRGFPLIDKPPIGSWGRLVSLIRDVFEGKTIIEHRELMGNSALKAHIVQEYIQYKGRDSaArgX KILTYSKLYREGIPIPDSIIAMSSDAALKAYEQKGFPLIDKPPIGSWGRLVSLIRDIFEGKTIIEHRELMGNSALKVHIVQEYINYKSRDStLysX KLYTLSLLSKHGIRVPKTIVAFSKEKALELANKLSYPVVIKPVEGSWGRMVARAIDEDTLRNFLEYQEYTTLQFRYIYLIQEFVKKPDRDSaLysX KLYTLSLLAKHNIKTPRTIVTFSKDKAVDLAKKIGFPAVIKPIEGSWGRMVAKAVDEDILYSFLEYQEYTTSQFRQIYLVQEFVKKPNRDTtLysX KWATSVALAKAGLPQPKTALATDREEALRLMEAFGYPVVLKPVIGSWGRLLAKVTDRAAAEALLEHKEVLGGFQHQLFYIQEYVEKPGRD

180 190 200 210 220 230 240 250 260 . . . . . . . . .StArgX IRCIAIGEELLGCYARNIPPNEWRANVALGGTPSNIEVDEKLKETVVKAVSIVHGEFVSIDILEHPNKGYVVNELNDVPEFKGFMVATNISaArgX IRCIVIGSELLGCYARNIPSNEWRANIALGGYPSQIEVDHKLKETVLKATSIIGGEFVSIDVMEHQSKNYVINEFNDVPEFKGFMLATNIStLysX IRIFTIGDEAPVGIYRVN.SRNWKTNTALGAKAEPLKIDEELQDLALKVKDIIGGFFLGIDVFEDPERGYIINEVNGVPEYKNTVRVNNFSaLysX IRIFVMGDEAPVGIYRVN.ERNWKTNTALGARALPLKIDDELRDLALKVRDIMGGFFLGIDIFEDPERGYLVNEVNGVPEYKNTVRVNNFTtLysX IRVFVVGERAIAAIYRR..SAHWITNTARGGQAENCPLTEEIARLSVGAAEAVGGGVVAVDLFES.ERGLLVNEVNHTMEFKNSVHTTGV

270 280 . .StArgX NVAQKLVEYIKENYSKSaArgX DVAEELVSYVKNNYLRStLysX NVSEYLIRKIEEWIKKSaLysX NVSSYLLNKLREWIKKTtLysX DIPGEILRYAWEVARG

StArgX

TtLysX

StArgX

TtLysX

StArgX

TtLysX

StArgX

TtLysX

TT

TT

TTTTTTT

TT TT


NS Chloroflexus sp. Y400fl Chy400 0359NS Chloroflexus aurantiacus Caur 0334NS Chloroflexus aggregans Cagg 3597NS Herpetosiphon aurantiacus Haur 3306NS Roseiflexus castenholzii DSM13941 Rcas 3469NS Roseiflexus sp. RS1 RoseRS 1059

NS Thermobaculum terrenum Tter 0316 NS Thermomicrobium roseum trd A0165NS Sphaerobacter thermophilus Sthe 2943NS Deinococcus deserti Deide 13430NS Deinococcus radiodurans DR 2194

NS Deinococcus geothermalis Dgeo 1151NS Truepera radiovictrix Trad 1392

NS Meiothermus ruber Mrub 2724 NS Meiothermus silvanus Mesil 0438 NS Thermanaerovibrio acidaminovorans Taci 0444NS Thermus thermophilus HB8 TTHA1907NS Thermus thermophilus HB27 TTC1543

GL Thermobaculum terrenum Tter 0318GL Thermomicrobium roseum trd 1519GL Sphaerobacter thermophilus Sthe 0215

NA Candidatus Korarchaeum cryptofilum Kcr 0812 LA Pyrococcus furiosus PF0209 NA Pyrococcus abyssi PAB0290 NA Thermococcus kodakarensis TK0278 NA Pyrococcus horikoshii PH1721 NA Pyrococcus furiosus PF1682 NT Nitrosopumilus maritimus Nmar 1295 NT Picrophilus torridus PTO1469 GL Nitrosopumilus maritimus Nmar 1288 AL Natronomonas pharaonis NP5258A AL Halalkalicoccus jeotgali HacjB3 00990 AL Halorubrum lacusprofundi Hlac 2616 AL Haloarcula marismortui rrnAC2679 AL Halomicrobium mukohataei Hmuk 0772 AL Haloferax volcanii HVO 0046 AL Natrialba magadii Nmag 1761 AL Haloterrigena turkmenica Htur 0328 AL Halorhabdus utahensis Huta 1502 AL Haloquadratum walsbyi HQ3714A GA Picrophilus torridus PTO1467 NT Ignicoccus hospitalis Igni 0057 NT Ignisphaera aggregans Igag 1752 NT Sulfolobus tokodaii ST0192 NT Sulfolobus acidocaldarius Saci 0754 NT Metallosphaera sedula Msed 0168 NT Sulfolobus solfataricus SSO0159 NT Sulfolobus islandicus M.14.25 M1425 1980 NT Sulfolobus islandicus Y.N.15.51 YN1551 0815 NT Sulfolobus islandicus M.16.27 M1627 2058 NT Sulfolobus islandicus L.D.8.5 LD85 2241 NT Sulfolobus islandicus Y.G.57.14 YG5714 2103 NT Sulfolobus islandicus M.16.4 M164 1986 NT Sulfolobus islandicus L.S.2.15 LS215 2145 NT Aeropyrum pernix APE 1463 NV Thermoproteus neutrophilus Tneu 0259 NV Pyrobaculum calidifontis Pcal 1379 NV Pyrobaculum islandicum Pisl 1291 NV Pyrobaculum arsenaticum Pars 0291 NV Pyrobaculum aerophilum PAE1240 NV Caldivirga maquilingensis Cmaq 1298 NV Vulcanisaeta distributa Vdis 0200 GL Vulcanisaeta distributa Vdis 0196 AI Caldivirga maquilingensis Cmaq 1297 AL Pyrobaculum calidifontis Pcal 0115 AL Pyrobaculum aerophilum PAE0948 AL Pyrobaculum arsenaticum Pars 0145 AL Pyrobaculum islandicum Pisl 1185 AL Thermoproteus neutrophilus Tneu 0136 GF Ignicoccus hospitalis Igni 1403 GF Sulfolobus tokodaii ST1505 GF Sulfolobus acidocaldarius Saci 1621 GF Metallosphaera sedula Msed 1991 GF Sulfolobus solfataricus SSO0645 GF Sulfolobus islandicus L.D.8.5 LD85 1699 GF Sulfolobus islandicus Y.N.15.51 YN1551 1340 GF Sulfolobus islandicus Y.G.57.14 YG5714 1496 GF Sulfolobus islandicus L.S.2.15 LS215 1600 GF Sulfolobus islandicus M.16.4 M164 1489 GF Sulfolobus islandicus M.16.27 M1627 1607 GF Sulfolobus islandicus M.14.25 M1425 1492 GI Dickeya dadantii Ech586 Dd586 2326 GV Serratia proteamaculans Spro 1643 GM Ferrimonas balearica Fbal 1405 GF Planctomyces limnophilus Plim 2585 NL Oceanobacillus iheyensis OB2059 GM Methanococcus vannielii Mevan 0912 AV Streptomyces griseus SGR 3478 HS Lactobacillus plantarum WCFS1 lp 0484 GF Catenulispora acidiphila Caci 6289

99

72

65

93

6079

99

94

83

5994

93

99

72

99

94

68

80

69

95

89

87

8899

82

88

99

99

9499

99

99

99

83

56

56

76

57

87

89

70

51

73

94

55

64

LysX

LysX/ArgX

LysX

LysX

LysX

ArgX

ArgX

ArgX

ArgX

DAP pathwaylacking LysW

Signaturemotif

LysX/ArgXArgX

Arc

haea

Bac

teria

Sul

Pyb

NPPyc

TTS

Supplementary Figure 11. 50% bootstrap consensus tree of 90 argX/lysX-like gene products based on maximum likelihood analysis (full version).


Supplementary Figure 12. Purification of proteins involved in lysine and arginine biosynthesis. (a) Saci_0754 (lanes 2-10) and Saci_1621 (lanes 11-19). Lanes 2 and 11, precipitate of cell lysate by centrifugation; lanes 3 and 12, supernatant of cell lysate by centrifugation; lanes 4 and 13, heat-stable franction of lanes 3 and 12; lanes 5 and 14, passing fraction through Ni2+-column; lanes 6-9 and 15-18, washing fraction; lanes 10 and 19, eluate by 500 mM imidazole (purified proteins) (b) Saci_0751. Lane 2, precipitate of cell lysate by centrifugation; lane 3, supernatant of cell lysate by centrifugation; lane 4, heat-stable franction of lane 3; lane 5, passing fraction through Ni2+-column; lane 6, washing fraction; lanes 7 and 8, eluate by 200 mM and 500 mM imidazole (purified protein). (c) Saci_0753. (d) TtLysX. (e) St1505 (StArgX). (f) STs023 (StLysW). Pane (c)-(f), gel filtration profiles. Purified fractins are shown by divergent arrows. In every panel, lane 1 contains molecular size markers.

a b

c d

e f

1 2 3 4 5 6 7 8 9 10 111213 141516171819 1 2 3 4 5 6 7 8

1 1

1 1

9766

45

30

20

14

9766

45

30

20

14

976645

30

20

14

976645

30

20

14

16.914.4

10.78.2

6.2

16.914.4

10.78.2

6.2

kDa kDa

kDa kDa

kDa kDa


Supplementary Table 1 Data collection and refinement statistics (molecular replacement) StArgX/ADP TtLysX/AMP-PNP StArgX/StLysW Data collection Space group P1 P3121 P21 Cell dimensions a, b, c (Å) 63.3, 67.8, 80.0 130.3, 130.3, 77.4 70.5, 114.0, 78.6 , , (°) 87.8, 75.3, 67.0 90.0, 90.0, 90.0 90.0, 102.4, 90.0 Resolution (Å) 1.87 (1.87-1.94) * 1.95 (1.95-1.98) * 1.80 (1.80-1.86) * Rsym or Rmerge 7.8 (30.1) 7.3 (47.9) 7.3 (58.1) I / I 14.7 (1.8) 24.2 (2.7) 12.3 (2.0) Completeness (%) 95.8 (87.3) 99.7 (99.1) 99.7 (100.0) Redundancy 1.3 (1.3) 4.9 (4.4) 1.8 (1.8) Refinement Resolution (Å) 35.0-1.87 50.0-1.95 28.9-1.80 No. reflections 98302 55332 112342 Rwork / Rfree 19.8 / 25.3 19.8 / 23.8 18.7 / 23.5 No. atoms Protein 8841 4236 9709 Ligand/ion 4 5 22 Water 956 418 1040 B-factors Protein 26.2 34.5 22.7 Ligand/ion 25.0 57.6 23.8 Water 36.7 46.3 38.7 R.m.s. deviations Bond lengths (Å) 0.012 0.013 0.013 Bond angles (°) 1.5 1.5 1.5

Each data set was collected from a single crystal. *Highest-resolution shell is shown in parentheses.


Supplementary Table 2 Interactions between StArgX and StLysW

residue on StLysW atom atom residue on StArgX distance (Å)

Globular domain

Asp18 O 1 N Lys15 3.2

O 2 3.0

Gly22 O N Val10 3.0

Glu23 O 2 N Lys15 3.1

Glu34 O 1 O Asp8 3.2

Arg45 N 2 O 1 Gln35 3.1

O 1 Glu111* 2.8

C-terminal extension

Glu48 O 1 N 1 Arg59 2.7

O 2 N 2 3.1

Glu52 O 1 O Ser159* 2.7

N 2.8

N Asn158 2.8

O N Arg135 2.9

Asp53 O O Ser56 2.6

N Met57 3.2

N Tyr58 2.9

O N Met57 3.0

Trp54 O N 1 Arg135 3.2

N 2 2.8

Gly55 N O Val55 2.8

Glu56 O 1 N 1 Arg178 2.9

N Glu256 3.1

N Glu 2.5

O3 SO42- 3.0

O 2 N 1 Arg178 3.4

O Ser235 3.0

O3 SO42- 3.4

O N Asp253 3.2

N Val254 3.0

N O Ser132 2.8

* indicates residues from another subunit.


Supplementary Table 3 Oligonucleotides used in this study

Primers Sequence Restriction site

0753-Fw 5’- GGGGTACCCATATGGTACTATTAAAATGT -3’ KpnI / NdeI

0753-Rv 5’- GGAATTCTTACTCTCCCAGTCCTC -3’ EcoRI

0754-Fw 5’- GGGGTACCCCATGGCATGATAATAGGGGTATCG -3’ KpnI / NcoI

0754-Rv 5’- GGAATTCTTATTTCTTAATCCACTC -3’ EcoRI

1621-Fw 5’- GGGGTACCCCATGGCATGAGAGTAGCACTTGTC -3’ KpnI / NcoI

1621-Rv 5’- GGAATTCTTACCTGAGGTAATTATT -3’ EcoRI

0751-Fw 5’- GGGGTACCCCATGGCATGATAGTAGTAAAAAC -3’ KpnI / NcoI

0751-Rv 5’- GGAATTCTTATTCAATCACCGTGCC -3’ EcoRI

TtLysX-Fw 5’- GGGGTACCCATATGCTGGCCATCCTCTAC -3’ KpnI / NdeI

TtLysX-Rv 5’- GGAATTCTAATCCACGGGCCACCT -3’ EcoRI

0753up-Fw 5’- GCTTCATATGGGCAGACGTTGATGATAGTGATATTA -3’ NdeI

0753up-Rv 5’- CTCTCCCCAGTCACATTTTAATAGTACCATGATGGATC -3’ -

0753ds-Fw 5’- ACTATTAAAATGTGACTGGGGAGAGTGATAATAGG -3’ -

0753ds-Rv 5’- GTGGGCCCCCTAAATTGACTTGTTGTATATTCCTG -3’ ApaI

0754up-Fw 5’- GCTTCATATGGGTTAAAACTAATCCTCAAATTCCTA -3’ NdeI

0754up-Rv 5’-GATCAATTTCATTTCTCTTCCCATCTCAACAGGTC -3’ -

0754ds -Fw 5’- GATGGGAAGAGAAATGAAATTGATCCAACTGTATGGA -3’ -

0754ds-Rv 5’- GTGGGCCCGCAGCCGTCTCATTATCTATC -3’ ApaI

1621up-Fw 5’- TCCCCGCGGTAATGTCCAATTCGTAGT -3’ SacII

1621up-Rv 5’- CGGGATCCTCATATACCTGCCCCATA -3’ BamHI

1621ds-Fw 5’- GGAATTCTTACCTCAGGTAAGCCAA -3’ EcoRI

1621ds-Rv 5’- CCGCTCGAGCTACATTTACTTGTCTCA -3’ XhoI

pyrEF-Fw 5’- CGGGATCCTTTGAGCAGTTCTAGTAC -3’ BamHI

pyrEF-Ev 5’- GGAATTCGACCGGCTATTTTTTCAC -3’ EcoRI

GF_NT-Fw 5’- GACGTTCCAGAGTTCAAAAATACTATGTTGGCTACCAATATT -3’

GF_NT-Rv 5’- AATATTGGTAGCCAACATAGTATTTTTGAACTCTGGAACGTC -3’

Y_I-Fw 5’- AGTGAGCTTCTAGGTTGTATCGCTAGGAATATACCTTCT -3’

Y_I-Rv 5’- AGAAGGTATATTCCTAGCGATACAACCTAGAAGCTCACT -3’

NT_GF-Fw 5’- GGTGTACCGGAATATAAAGGTTTTGTAAGAGTTAATAATTTC -3’

NT_GF-Rv 5’- GAAATTATTAACTCTTACAAAACCTTTATATTCCGGTACACC -3’

I_Y-Fw 5’- GATGAGGCACCAGTTGGATATTATAGAGTTAATGAACGT -3’

I_Y-Rv 5’-ACGTTCATTAACTCTATAATATCCAACTGGTGCCTCATC -3’


supplementary information lysine and arginine biosyntheses ...€¦ · supplementary information...

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