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Supplementary Table 1 Data collection and refinement statistics

FBPA form (DHAP complex)

Data collectionSpace group I422

Cell dimensionsa, b, c (Å) 112.5, 112.5, 153.6

90, 90, 90

Resolution (Å) 50.00–1.50 (1.55–1.50) *

Rsym 6.4 (53.1)

I/σI 51.5 (5.1)

Completeness (%) 99.9 (100)

Redundancy

, , γα β (°)

14.6 (14.6)

RefinementResolution (Å) 26.22–1.50

No. reflections

Rwork / Rfree 19.6 / 21.5

No. atoms

Protein 2,791

Ligand/ion

264

B-factors

Protein 18.1

Ligand/ion 29.3

31.0

R.m.s deviations

Bond lengths (Å) 0.012

Bond angles (º)

Water

Water

1.43

74,120

20

*Highest resolution shell is shown in parenthesis.

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Supplementary Table 2 Temperature factor analysisRegion Average B-factor (Å2)*

FBPA form FBPase form (1UMG) Protein Overall 18.1 (17.1, 19.3) 19.5 (17.6, 21.4) Lid loop (97–110) 31.7 (30.9, 32.8) 27.7 (25.7, 30.4) Schiff-base loop (219–233) 32.4 (32.0, 32.9) 36.6 (31.9, 41.0) Lys232 23.6 (21.1, 25.7) 24.5 (23.1, 25.5) C-terminal loop (346–364) 39.2 (38.2, 40.1) 31.3 (29.9, 32.5) Ligands and solvents DHAP 20.9 – FBP – 15.3 Mg2+ 14.8 13.3 Water 31.0 31.7

*B-factor values of the main-chain and side-chain atoms are in parentheses.

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ST0318 1 T S KAD G GH D GDD H G H AW K VI V Y L M.....MM T I I SLA HIVHPDTMAAANK LASAKEQGIIL Y ITHV LQ I T TR ELDTKV ET NMsed2259 1 T S KAD G GH D GDD H G H AW VI V Y L M......MRS V I SLA HVVHPDTMAAANR LAEAKRQGIIL Y ITNV LE I S TR ELDTKV ET DIgni0363 1 T S KAD G GH D GDD H G H AW K VI V Y L M....MAQ T I I SLA HTVHPDCMAAASR LAEAKKNGVIN F VTHV LI I T TK VDHPDV GL EPF0613 1 T S KAD G GH D GDD H G H AW K VI V Y L M..MAVGE I I V GWP SRVHPALIERAKE LSEAQKEGTLI F VTYA LQ I T KK VDSPEI GL ETK2164 1 T S KAD G GH D GDD H G H AW K VI V Y L M..MAVGD I I I GWP SRVHPQLVETAED LSKAVEDGTII F VATC LQ I T KR VDSPDI GL KMJ0299 1 T S KAD G GH D GDD H G H AW K VI V Y L MMSRMENN V I V GLC TLAPDELLEACEA LEEAVD.EIIL Y VTRC ID I S KL CDNEKV GL RAQ1790 1 T S KAD G GH D GDD H G H AW K VI M......M V L I GFV SSTHPDVVNAVREHVEKEVEKGNLI CDILTC IAIV T TH VDAELV KI DTTHA0980 1 T S KAD G GH D GDD H G H AW K V V Y L......M V L L I SVG TLPSRKVLAKVEE VREEVG.RLLL A VFHI IV LLS TR VRNQEV AL KCENSYa0564 130 T S KAD G GH D GDD H G H AW I Y M......VRI V A V GIG TLPSSGLLDAVRRKVSSSS...LLI H IGYC VHIV T TR TDNSDI KL D

ST0318 77 AF V K LY AGQDLL D F GN G GP AE R E DKT P A N P F D GL A LG S V ME E S P F E G L M A P NT KEAAK D A S S R L GV IE A IAI MA Y LYK F P VMsed2259 76 AF V K LY AGQDLL D F GN G GP AE R E DKT P A N P F D GL T LG S E S P F E G F L M A P NT KEA K S E A S S LK M GI LDIE P IAI MA LYK F A VIgni0363 78 AF V K LY AGQDLL D F GN G GP AE R E DKT P A N P F D GL A LG S V ME E S P F E G F L A P NT KKAAE E A S A R L AA IE P IVI AA LYKI F A VPF0613 80 AF V K LY AGQDLL D F GN G GP AE R E DKT P A N P F D GL T A LG S V ME S P F E G F L M A P NT KKA E E G K A R L SV ITL K IVT HL IFR F A VTK2164 80 AF V K LY AGQDLL D F GN G GP AE R E DKT P A N P F D GL T A LG S V ME S P F E G F L M A P NT EEA K E G K A R M GV ITL K VVT HM IFR F A IMJ0299 81 AF V K LY AGQDLL D F GN G GP AE R E DKT P A N P F D GL T A L S V ME E S P F F M A P NT EEA K E K G A S R M GC FV K IVV CC D T Y LFK F A VAQ1790 76 AF V K LY AGQDLL D F GN G GP AE R E DKT P A N P F D GL T A L S V ME E S P F L M A P NT MKG E K K G T T K M GV FE P VIV FA S S W LYE M A VTTHA0980 75 AF V K LY AGQDLL D F GN G GP AE R E DKT P A N P F D GL T A G ME E P E G F L A P REG E AE G K A T LH L QV FT PA FMVLAA LYLA MYSS LCENSYa0564 202 AF V K LY AGQDLL D F GN G GP AE R E DKT P A N P F D GL T A G S V E E F E G F M MEG R EE G R S K M GV L FE AN AFTV AA Y FYR V SLSNT I

... .....

ST0318 158 G D S L AG YV DP F FE V L P E Y LALIG RYVI V AV II TMHG K VL VYQGE...A M SA Q I DL TPA RR YRNEDNLLA..... V IER NL KMsed2259 157 G D S L AG YV DP F FE V L PEE Y L LIG RYVI V V II TMNE K VL VYEGQ...S V NS M SL G TPA RR YRKADNMIG.....S T IER NL KIgni0363 159 G D S L AG YV DP F FE V L PEE Y LALIG RYVI V AV II RLHD V VV VFEDK...G H NT L DL TPS RR FRK.DGKIA..... V VER NL KPF0613 161 G D S L AG YV DP F FE V PEE Y LALIG RYVI V AVI KMHM R IW ILEHK...R IMNS M DI AKS KR YPKEGHPIPKDEPV V TEK YEV ETK2164 161 G D S L AG YV DP F FE V L PEE Y LALIG RYVI V AVI KMHM R VW ILEHK...R I NT L DL AKS KR YPKPGHPIPENEPV V TEK YEV EMJ0299 162 G D S L AG YV DP F FE V L PEE Y LALIG Y I V AV IF SMIS K VH VVGHK...K F DT M ML DYEK A KR YRRRDNEIA..... V TEK NY EAQ1790 157 G D S L AG YV DP F FE V L P E Y LALIG YV V A II KMHD T VL VYTGK...A K NT A V DL SVGK VKN YRNYDGEIA..... TA TQR SL QTTHA0980 156 G D S L AG YV P F F L PE Y L R I AV ILS ELRP R RIM LAQTERDSYIE DA RL DIAT LRDSH FA ESIWSRKHGEQA..... V TTR RN RCENSYa0564 283 G D S L AG YV L E A R V V A IVNKSLAE VVINIM VSKAR...TAR VLW DKPTIE ALMYPG F VSS ETR.DGEPI..... SA TDR HN T

ST0318 231 GKDDP VR Q PA E F V G RGSH P MPV F P L P D V I G G LE P WM G L A TR DG R LG G G L M L H L L A A AF HL P Y SQRD KA P LLG FNVK.N R V TMsed2259 230 GKDDP VR Q PA E F V G RGSH P MPV F P L P D V I G G LE P WM G A TR DG R LG G G L L L H F L A A SF YL P Y I SQRD KA P LIG FNVK.N K T SIgni0363 231 GKDDP VR Q PA E F V G RGSH P MPV F P L P D V I G G LE P WM G L A TR DG R LG G L M A S F V V P TF HL A N PVRD RP P VIA FQVK.NAK V SPF0613 239 GKDDP VR Q PA E F V G RGSH P MPV F P L P D V I G G LE P WM G L A TR DG R LG G G L A A S L L V P AF HL S N PLKY TP P AVA WQISPE K I VTK2164 239 GKDDP VR Q PA E F V G RGSH P MPV F P L P D V I G G LE P WM G L A TR DG R LG G G L A A S L L V P AF HL S N PMHQ NP P VVA WQISPE K V VMJ0299 235 GKDDP VR Q PA E F V G RGSH P MPV F P L P D V I G G LE P WM G L A TR DG R LG G G L A A S F V V P AN HF P W GEED TP A IIA FQVC.D M I NAQ1790 230 GKDDP VR Q PA E F V G RGSH P MPV F P L P D V I G G LE P WM G L A R DG R G G G L A S F V I P AR WI E N SFED RPS P VIGA YQLA.N K I A MTTHA0980 232 GKDDP VR Q PA E F V G RGSH P MPV F P L P D V I L T L G L A T KIF TE FGPV ALAPY A DT HM RANTPAS F CV .MVCG AFSLR.E R SE VCENSYa0564 355 GKDDP VR Q PA E F V G RGSH P MPV F P L P D P L L G G ICL T KRF TE AGSC NN HY A NT HM RLNSPASIN CI .IVEA VFSMH.E R T F G

ST0318 311 F R RR G P L EY DP FD A F HR EM TT P L RF D A ET RL NIVADYM H P M EPT L LILEK KD K..KESDVYKAKESIYAKEESQGHDMsed2259 310 F R RR G P L EY DP FD A F HR EM TT P L RF D A ET RM SVITDYM H P M EPT L TLIEK KP K..KEEDVKKAKPSVYTSKDQGMD.Igni0363 311 F R RR G P L EY DP FD A F HR EM TT P L RF D A EA RT NKVADYI H P M DPS L QVLER KD KPVKDLPVPKVKHSEMLSGAEEAHDPF0613 320 F R RR G P L EY DP FD A F HR EM TT P L RF D A WA QK LEITDYM H P E PLE L GVLEK KD EPIE.....................TK2164 320 F R RR G P L EY DP FD A F HR EM TT P L RF D A YA QK LEITEYM H P E PLE L GVLKR TD EPIE.....................MJ0299 315 F R RR G P L EY D FD A F HR M TT P L RF A KG KA EK LEMADII M P Q PATM V KVLEA ED IPLEGLELIEEGGITRKDRGDVE..AQ1790 310 F R RR G P L EY DP FD A F HR EM TT P L RF K A KA QV QDMADIL Q I E PAE L KVLKK EN YDTEVEKLSEEQKIEKEDMD.....TTHA0980 311 F R RR G P L EY DP E T R A VWEAV AKVVEKAQEM Q FYG AM PME L G.IAERLKALE EFS.......................CENSYa0564 434 F R RR G P L EY P D A E ST DW DV RT TRRAHAM Q FVH AT VPD L AEGYRSRMDVLDSKMVPLKDSGPAGTGRAYEDPD.....

β1 α1 β2 β3

α2 β4 β5 η1 α3 α4

η2 β6 β7 α5 β8 β9 α6

β10 α7 β11 η3 β12 β13 η4 β14 β15

η5 α8 α9

D12 H19

C-terminal loop (346-361)

Lid loop (97-110)

Schiff-base loop(219-233)

Y91

K232 Q242D233

Y348

R266 D287

Y229

Supplementary Figure 1. Multiple amino acid sequence alignment of FBPA/Ps.The secondary structure of ST0318 in the FBPA form is indicated above the sequences. The three mobile loops are boxed. Residues important for catalysis and substrate (or intermediate) binding for FBPA (green), FBPase (cyan) and both (black) reactions are indicated by filled and open stars, respectively. The disordered regions are enclosed by dashed lines. The sequences are labeled with the genome locus tags, and their source organisms are as follows: ST0318, Sulfolobus tokodaii; Msed2259, Metallosphaera sedula; Igni0363, Ignicoccus hospitalis; PF0613, Pyrococcus furiosus; TK2164, Thermococcus kodakaraensis; MJ0299, Methanocaldococcus jannaschii; AQ1790, Aquifex aeolicus; TTHA0980, Thermus thermophilus; and CENSYa0564, Cenarchaeum symbiosum. This figure was prepared using the ClustalW2 server1 and ESPript2.



CH2OH

CH2O P

O

H

OHOH

HH

HO

CH2O P

CH2O P

O

H

OHOH

HH

HO

CH2O P

O

OHH

CH2OH

CH2O P

O

F6P

Pi

H2O

kcat = 0.62 (s–1)Km = 0.027 (mM)

kcat = 0.91 (s–1)Km = 0.19 (mM)(anabolic direction)

kcat = 0.027 (s–1)(5 mM FBP)(catabolic direction)

DHAPGA3P

FBP

Supplementary Figure 2. Reactions of the bifunctional FBPA/P.The kcat and Km of ST0318 at 48°C are shown.



a

b

Y348

R266

Q242'

H243'

Y91

H19D287

Y229

D233

D234

D234 D234

D234

K232

DHAP

C-terminalloop

Lid loop

Schiff-baseloop

Y348

R266

Q242'

H243'

Y91

H19D287

Y229

D233

K232

DHAP

C-terminalloop

Lid loop

Schiff-baseloop

Y348R266

Q242'

H243'

Y91

H19D287

Y229

D233

K232

FBP

C-terminalloop

Lid loop

Schiff-baseloop

Y348R266

Q242'

H243'

Y91

H19D287

Y229

D233

K232

FBP

C-terminalloop

Lid loop

Schiff-baseloop

Supplementary Figure 3. Active sites of ST0318 in the two forms (stereoview).a, b, DHAP-Schiff base complex (the FBPA form) (a) and FBP complex (the FBPase form, PDB code 1UMG)3 (b). DHAP and FBP are colored green and cyan, respectively. The neighboring protomer is colored dark gray, and its residues are labeled with prime symbols. The lid, Schiff-base, and C-terminal loops are colored blue, yellow and pink, respectively. In (a), Mg2+ ions (Mg2–4) and a water molecule are shown as green and red spheres, respectively. Hydrogen bonds with the DHAP 3-hydroxyl group are shown as black dashed lines. In (b), Mg2+ ions (Mg1 and Mg2–4) are shown as magenta and cyan spheres, respectively. The disordered region is indicated by a yellow dashed line.



Supplementary Figure 4. Overall structure of ST0318 in the FBPA form.Ribbon diagrams of the octamer structure along the crystallographic 4-fold axis (left) and the structure rotated by 90 ° around the horizontal axis (right). Each protomer in one tetramer is shown in different colors, and the protomers in the other tetramer are shown in white. DHAP is shown in a stick representation (carbon in green). Mg2+ ions are shown as green spheres. The disordered regions (residues 354–360) are represented by dashed lines.



Supplementary Figure 5. Active site of ST0318 in the FBPA form (stereoview). Simulated annealing |Fo| – |Fc| omit electron density map for Tyr229, Lys232, Mg2–4 and DHAP, contoured at 3σ, is shown as a blue mesh.

Mg2

Mg3

Mg4

K232

Y229

DHAP

Mg2

Mg3

Mg4

K232

Y229

DHAP



Supplementary Figure 6. Mg2+-binding site of ST0318 in the FBPA form (stereoview).Mg1 (magenta) and Asp233 (cyan) in the FBPase form are superimposed onto the FBPA form. The nucleophilic water molecule for the FBPase reaction is labeled NW. Metal coordination is shown by green dashed lines. The interactions between Tyr229 and DHAP are shown by orange dashed lines. The distance between Asp12 and NW is shown by a yellow dashed line (2.7 Å). The binding modes of Mg2–4 are virtually the same in the FBPA and FBPase forms3, except that Mg2 is coordinated by the main-chain carbonyl group of Lys232 in the FBPA form, and by the side chain of Asp233 in the FBPase form. In the FBPA form, the main-chain amide group of Lys232 interacts with the DHAP phosphate group (shown by a black dashed line).

H19

Mg1

D234

Mg2

NWMg3

D132

Mg4D54

D12

Q95D53

D233

Y229

K232

DHAP

H19

Mg1

D234

Mg2

NWMg3

D132

Mg4D54

D12

Q95D53

D233

Y229

K232

DHAP



Supplementary Figure 7. Catalytic components of FBPA (stereoview). a, Superimposition of the FBPA (green) and FBPase forms (cyan, PDB code 1UMG) of ST0318, and the DHAP-Schiff base (enamine) intermediate (black, PDB code 2QUT) and the FBP-Schiff base intermediate (pericyclic transition state) of wild-type rmFBPA (white, PDB code 1ZAI), and Tyr363 of the rmFBPA K146M mutant (semi-transparent gray, PDB code 2QUU). The residues of ST0318 and rmFBPA are labeled with green and black characters, respectively. The Mg2+ ion (Mg1) in the FBPase form is shown as a magenta sphere.b, Superimposition of the FBPA (green) and FBPase forms (semi-transparent cyan, PDB code 1UMG) of ST0318. Two water molecules in the FBPA form are shown as red spheres. The interactions in the FBPA and FBP forms are depicted by green and cyan dashed lines, respectively, and the distances (Å) between the protein atoms (Tyr229 or Asp287) and the DHAP-Schiff base are shown (green). The distances (Å) between Asp287 and the DHAP C2 and C3 atoms and between Tyr229 and the FBP O4 atom are also shown with black dashed lines.

D287

H19

Y229

K232Q242'

Y91

H243'

C3

C2

C42.6

4.93.92.8

3.23.33.0

C3

C2

C42.6

4.93.92.8

3.23.33.0

D287

H19

Y229

K232Q242'

Y91

H243'

a

b

E187

D33

Y363

D287

Y229

K229

K232

Mg4 Mg3

Mg2

Mg1

E187

D33

Y363

D287

Y229

K229

K232

Mg4 Mg3

Mg2

Mg1



Supplementary Figure 8. Comparison of the overall structures of ST0318 and class I FBPA.Monomer structures of ST0318 in the FBPA form (left) and the DHAP-Schiff base intermediate of rmFBPA (right, PDB code 2QUT) are shown. rmFBPA exists as a homotetramer in solution4. Disordered regions (residues 354–360 in ST0318 and 356–358 in rmFBPA) are represented by dashed lines. The covalently bound DHAP and the Schiff base-forming lysine residues are shown as stick models with carbon atoms in green and yellow, respectively. Mg2+ ions are shown as green spheres. ST0318 has a unique fold comprising a four-layer α-β-β-α sandwich, which is designated as the “Sulfolobus fructose-1,6-bisphosphatase-like fold” in the SCOP database5. Class I FBPAs have a typical TIM (α/β)8 barrel fold. The two β-sheets of ST0318 are antiparallel, whereas the β-strands in the TIM barrel fold are parallel.

ST0318 rmFBPA



Supplementary References

1. Chenna, R. et al. Multiple sequence alignment with the Clustal series of programs. Nucleic Acids Res. 31, 3497-3500 (2003).2. Gouet, P., Courcelle, E., Stuart, D. I. & Metoz, F. ESPript: analysis of multiple sequence alignments in PostScript. Bioinformatics 15, 305-308 (1999).3. Nishimasu, H., Fushinobu, S., Shoun, H. & Wakagi, T. The first crystal structure of the novel class of fructose-1,6-bisphosphatase present in thermophilic archaea. Structure 12, 949-959 (2004).4. St-Jean, M. & Sygusch, J. Stereospecific proton transfer by a mobile catalyst in mammalian fructose-1,6-bisphosphate aldolase. J. Biol. Chem. 282, 31028-31037 (2007).5. Murzin, A. G., Brenner, S. E., Hubbard, T. & Chothia, C. SCOP: a structural classification of proteins database for the investigation of sequences and structures. J. Mol. Biol. 247, 536-540 (1995).



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