Posted on Authorea 16 Apr 2020 — CC BY 4.0 — https://doi.org/10.22541/au.158705333.31754085 — This a preprint and has not been peer reviewed. Data may be preliminary. iiImaizumi Riki for soybean enzyme in key biosynthesis a isoflavonoid synthase, chalcone of structure Crystal aignn rdc smro h H-aaye ecin obte nesadtectltcmechanism catalytic the understand a better of to components bound reaction, key with CHS-catalyzed the GmCHS1 the of GmCHS1. of of structure weof one isomer complex issue, as product the this a GmCHS1 determined addressing also naringenin, expressed of We protein-protein step recombinantly metabolon. first for of flavonoid the information well-characterized structure in structural crystal Thus, atomic-level the no formation. other is determined metabolon and flavonoid there CHS mediate metabolons, among that interactions the interactions protein-protein in of proteins production flavonoid significance and of of functional enzymes efficiency variety the the wide indicate enhance observations a to these CHS in of protein, promiscuity isomerase-like catalytic intrinsic chalcone partner, interacting plants. land isoflavonoid nonenzymatic its soybean production soybean. efficient with the the the GmCHS1, of for interactions in component providing protein-protein 5-deoxyisoflavonoids the a isozymes, of synthase is of 2-hydroxyisoflavanone importance as GmCHS1) the such regarding (termed components evidence CHS mechanistic other with previously soybean have interacts We of and isozyme S1). metabolon an (Fig. the precursor that catalyzes flavonoid pathway, common reported one biosynthetic as a flavonoid into as such serves molecules the stresses. legumes which malonyl-CoA of 2’,4,4’,6’-tetrahydroxychalcone, various all three enzyme almost of against committed condensations by first mechanisms consecutive produced the defense groups (CHS), UV-screening or flavonoid or synthase antioxidant Chalcone the reproductions their to of their owing one only in for not plants are identified plants, also land land been of in have but survival roles structures the indispensable functions different in have with to roles flavonoids known pivotal 6900 are play and than that more metabolites Currently, plant environments. specialized terrestrial of class a are GmCHS1. Flavonoids of mechanism a catalytic as the GmCHS1 understand of better a INTRODUCTION structure to metabolon, crystal as isoflavonoid the well the as determined of structure, we component metabolon Here, key the a production. (CHS), understanding is isoflavonoid synthase of have GmCHS1) Chalcone of step We (termed efficiency first plants. precursor. CHS enhance isoflavonoid land soybean common to of a of survival complex of the isozyme protein formation in an the roles that catalyzes pathway, pivotal reported biosynthetic play isoflavonoid previously that the flavonoids of of enzyme first groups the the of one is Isoflavonoid Abstract 2020 28, April 3 2 1 Nakayama Toru and Nakata SPring-8 University Tohoku University Kanazawa 1 ej Takahashi Seiji , 4 neatoso hspoenwt H r ieycnevdaogln lnsadrdc the reduce and plants land among conserved widely are CHS with protein this of Interactions 1 y Mameda Ryo , 2 2 uihg Kataoka Kunishige , 2 oe Takeshita Kohei , 2,3 oercnl,w eotdteitrcin fCS including CHS, of interactions the reported we recently, More 1 1 aaiYamamoto Masaki , 3 ohyk Waki Toshiyuki , p cuaolCAmlcl oproduce to molecule -coumaroyl-CoA lcn max Glycine 2 3 ioiKubo Hiroki , aoh Yamashita Satoshi , nplanta in L)Mr (soybean). Merr (L.) 1 Isoflavonoids 1 . 4 Shun , Although 1 , Posted on Authorea 16 Apr 2020 — CC BY 4.0 — https://doi.org/10.22541/au.158705333.31754085 — This a preprint and has not been peer reviewed. Data may be preliminary. h vrl tutr fGCS xiisthe sequence exhibits original GmCHS1 of the structure from overall residues to The corresponding all densities included His electron the structure the However, including refined The 1-388). with (residues model. GmCHS1 method, search of replacement a as molecular identity) a Fig. sequence see quality, using P2 density group solved electron space 1; were Table in statistics, crystallized (crystallographic GmCHS1 resolution A S2). an 2.5 to with diffracted chromatography crystal affinity the nickel using purified His was (complex). GmCHS1 7BUR expressed and Recombinantly (apo) 7BUS the codes and DISCUSSION PDB program AND with server RESULTS Bank 3.0 Data CASTp program. Protein the the PyMOL by in were the performed deposited structures were by pockets both visualized surface of was refinement and result cavities and interior building of model REFMAC. Analysis Manual and unit. Coot asymmetric using the performed in molecules inte- two and with indexed with KAMO. were MOLREP in and implemented using scheme, rejections DIALS. collection outlier using data with pipeline, helical XSCALE KAMO a the using with Japan. collected Harima, grated were SPring-8, data system, 20 ZOO reflection installed at completed 1.00000 beamline hr of tesque, BL32XU 1 wavelengths on (Nacalai GmCHS1- Switzerland) for at naringenin Baden-Daettwil, week. concentration collected with 1 were final crystal within data mM average apo-GmCHS1 diffraction 2 in an length with incubating mm Japan) by 0.5 Kyoto, prepared with was structures needle-like complex to naringenin grew crystals typical The 20 at 1 grown were crystals GmCHS1 determination structure mM 10/300, and increase 200 collection 200 containing (Superdex data A column buffer Crystallization, size-exclusion with USA). a eluted IL, to were Tris-HCl applied Chicago, proteins mM then Healthcare, Then, 50 was GE with GmCHS1 NaCl. equilibrated eluted mM Germany) The 150 Hilden, containing Qiagen, imidazole. A) mL; (5 the (buffer agarose and 7.5) Ni-NTA disrupted (pH with were filled cells column harvested a into The 100,000 to introduced induction. at cold-shock was centrifuged by was plasmid induced lysate was resultant GmCHS1 The of expression GmCHS1. the of an Met with initiating coli GmCHS1 original the the express indicates to vector The I tag. previously. pCold reported into were introduced GmCHS1 was of purification and expression Heterologous GmCHS1 expressed recombinantly of Purification METHODS AND MATERIALS niae htteei ag aiyaon h ciest hri h odnainratossol occur should reactions tunnel, condensation binding the Co-A wherein the site active including active the cavity, the interior and When around the environment cavity CoA-binding circle). of large solvent the dotted a analysis external Further a is termed the 1B). there in been between that highlighted (Fig. has contains connection indicates is monomer which GmCHS1 a one each structure, homodimeric making 1A, in tunnel-like The site studies, (Figure a fold previous is S1). thiolase in there the (Fig. tunnel representation, of solution surface middle in in the viewed homodimer in a sites as active two exists mainly GmCHS1 that sativa Medicago 11 and helices μ elslto otiig3%PG00 .Mamnu ctt n .Msdu irt p 5.6). (pH citrate sodium 0.1M and acetate ammonium 0.2M PEG4000, 30% containing solution well L 6 tg n yuigsz-xlso hoaorpy(i.S) h uie mH1wscytlie,and crystallized, was GmCHS1 purified The S1). (Fig. chromatography size-exclusion using by and -tag, L1sri.Tetransformed The strain. BL21 N tria xeso euneicue MNHKVHHHHHHIEGRH includes sequence extension -terminal β- afla D D 1BI5). ID: PDB (alfalfa, tad Fg A e ooe,a rvosyrpre o h rsa tutr fCSfrom CHS of structure crystal the for reported previously as monomer, per 1A) (Fig. strands 6 tgwr o iil wn otehgl iodrdsrcue fthe of structures disordered highly the to owing visible not were -tag iu sylvestris Pinus × .coli E. ° o h t4C h uentn rmtecnrfgto a applied was centrifugation the from supernatant The 4@C. at 1hr for g yvprdffso,mxn 1 mixing diffusion, vapor by C 10,11 H PBI:6X)a erhmodel. search a as 6DXA) ID: (PDB CHS 14 a utrdi Bmdu otiig100 containing medium LB in cultured was Dmlclriae eecetduigtePMLprogram. PyMOL the using created were images molecular 3D nfc,orsz-xlso hoaorpyeprmn suggests experiment chromatography size-exclusion our fact, In 1 2 αβαβα 13 6,7 1 2 1 h tmccodntsadsrcua atr aebeen have factors structural and coordinates atomic The nerto neste n egn eepromdusing performed were merging and intensities Integration ihtomlclspraymti nt h structures The unit. asymmetric per molecules two with hoaefl ihaped-ymti ie ih12 with dimer pseudo-symmetric a with fold thiolase 2 8 sn nEGR9 eetr(ETI Ltd., (DECTRIS detector 9M EIGER an using A ˚ tutrswr ovdb oeua replacement molecular by solved were Structures iu sylvestris Pinus ° ro ob ahfoe nlqi N liquid in flash-frozen be to prior C μ N rti ouin(. gm)with mg/mL) (6.7 solution protein L tria xeso nldn His including extension -terminal H PBI:6X,85.9% 6DXA, ID: (PDB CHS M N- 2 hr h nelndMet underlined the where , rey the Briefly, emnletninresidues extension terminal 9 mH1crystallized GmCHS1 μ /Lapcli and ampicillin g/mL N tria regions. -terminal GmCHS1 N Escherichia -terminal 2 5 X-ray . These ORF 12 α- 6 - Posted on Authorea 16 Apr 2020 — CC BY 4.0 — https://doi.org/10.22541/au.158705333.31754085 — This a preprint and has not been peer reviewed. Data may be preliminary. aebe eoie ntePoenDt akudracsincds7U n BR respectively. 7BUR, and 7BUS codes accession of under GmCHS1/naringenin formation Bank and the Data apo-GmCHS1 in References Protein of role the factors distinct in the a structural deposited of has and been components Further GmCHS1 Coordinates have other how CHS. a and metabolon. understand of provide GmCHS1 isoflavonoid mechanism better GmCHS1 between the catalytic to complex of is the a metabolon structures 264 of of isoflavonoid crystal position characterization understanding soybean the of structural our conclusion, include bulkiness of In will the improvement efforts or further GmCHS1. flexibility for of the temperature A basis efficiency average whether (44.2 structural catalytic the question Phe215 of the intriguing that of to Superposition an fact that orientationrelated is the than site. its by It higher change active supported is respectively). to sidechain is the suggests Phe215, likely Phe264 This at structure is the 2C). Phe265 complex product for the (Fig. our the factor that reaction CHS, of shows CHS-catalyzed of GmCHS1 existence the inhibitor of the through potential structures acid. upon sulfonic naringenin-bound a binding di-ethane and as 1,4-piperazine by inhibitor apo- studied or of occupied CoA been either was possibility not of structure the has that our mimic acid vicinity potentially the of (C citric could in Although ion acid ion also citrate citrate sulfonic tunnel, the di-ethane the binding of charge CoA of 1,4-piperazine the the or position of of malonyl-CoA corresponding site. end structure of active The the the moiety at 2B). at located CoA 2A, intermediates was the ions reaction (Fig. citrate and previously Phe264 was the substrates Phe215 of of of conserved one the of orientation of that those the importance found to in The We equivalent involvement the naringenin. almost an of In be as vicinity to the proposed apo-structure. in found the located were are as (the molecules Moreover, Phe264 of acids well naringenin structure and sulfinic liquor. crystal as of cysteine the mother respectively positions catalytic for the the reported The 164, of previously in 2A). and vicinity contained the Fig. 70 are in 164, positions which located residue are the ions, molecules citrate for naringenin molecules, three modeled structure, naringenin and complex two are ions GmCHS1, that CSD of acetate to and dimer procedure of a SME similar three includes a as structure in naringenin- determined complex well The was refined as structure reaction. The the CHS-catalyzed and apo-structure. the resolution, the of A of 1.8 analog the to product and diffracted a crystallized, decipher crystals was naringenin, soaked GmCHS1 to with purified needed soaked newly GmCHS1, were be of of apo-crystals mechanism will oxidation catalytic ID: investigations the the (ligand understand physiological sulfoxide to better To or oxidation. methionine addition Met biochemical the In Further the modeled of 1D). Met we 70. these importance structures, Although the positions (Fig. CHS forms. other the respectively oxidized any for their unit, for in SME) asymmetric observed reported are N not the sidechains GmCHS1). are the Met70 in oxidations of in for and B densities generation (His302 CSD164 electron and the residue of the A during Cys164, atom His Cys molecule conserved sulfur around for the the at environment A between by densities reactive distances assisted highly electron acid structure, was the of S-cysteinesulfinic Weng GmCHS1 that to clarity modeled anion due the positions. we thiolate was euphyllophytes, of corresponding nucleophilic residues to Because the Cys belongs for catalytic species. soybean molecules of moss that plants), cysteine CSD) seed or fact catalytic and ID: lycophyte the (ferns oxidized (ligand either and euphyllophytes doubly from from 164 for CHS The CHS positions a of densities 1D). the the structures electron as crystal in the (Fig. functions the with not forms in tunnel structures, but observed oxidized and CHS additional widely His302 doubly other is (Cys164, this their acid of residues whether sulfinic in those catalytic evaluate the to being that orientations to sidechains completely showed similar not site Cys164 future in active is the the located CHS of of are in view export Asn335) interest close-up (Fig. product solvent A of the external for channel. be the mechanism metabolic to will the downward Although tunnel it another arrow). forms understood, dotted cavity a large by the indicated that noteworthy 1C, is It 1C). (Fig. Wk ,YoD uioN aeaR eesokK aaht ,MthsiR ksiT oiT, Aoki T, Akashi R, Motohashi S, Yamashita K, Denessiouk R, their Mameda to N, adaptation Fujino plants D, Yoo mediating network T, Waki metabolic a 2. Flavonoids: G. Spangenberg A, Mouradov 1. elett.FotPatSi 2014;5:620. Sci. Plant Front estate. real .sativa M. H y14l uat(i.S,PBI:1M) ugsigta negative a that suggesting 1CML), ID: PDB S3, (Fig. mutant Cys164Ala CHS .sativa M. 3 H Fg S3). (Fig. CHS tal. et 2 14 n 34A 33.4 and nadto oteCD6,Phe215 CSD164, the to addition In yohszdta h oxidation the that hypothesized 8 ε H fHs0 r .5ad3.69 and 3.55 are His302 of 18 2 N o h h24adthe and Phe264 the for 2 O 6 S 2 ntecomplex the in ) 15 nthe In 14 Posted on Authorea 16 Apr 2020 — CC BY 4.0 — https://doi.org/10.22541/au.158705333.31754085 — This a preprint and has not been peer reviewed. Data may be preliminary. fteatv ieo mH1wt on aignn(A)adctaein(I) h 2 The (CIT). ion citrate and naringenin. (NAR) naringenin with bound complexed with GmCHS1 GmCHS1 N of the of and site structure CSD164 active crystal of of the The site atom of line. active dotted sulfur the a the 2. of as between Figure view shown (A) close-up and active Distance A labeled the D: is Asn335. A. to His302 and CoA-binding panel of connecting His302 in the (CSD164), tunnel shown of acid is another entrance sulfinic as indicates 2 the site, The (red) active indicates the arrow arrow GmCHS1. highlights dotted Black circle whereas Dotted representation. above, site. surface described Surface the as B: in tunnel The D. shown of Right: in A. is site shown panel active program, be with the will tunnel. direction indicates as same CoA-binging circle from models putative view Dotted stick 30 The in of green. Left: represented view or are homodimer. cyan residues apo-GmCHS1 either of catalytic representations in the apo-GmCHS1. colored and of is monomer, the structure monomer each crystal with The modimer, 1. Figure legend Figure 2018;46:363-367. Res. Acids Nucleic proteins. of topography Ta ,Ce ,LiX hoJ in .CSp30 optdalso surface of atlas computed 3.0: CASTp J. Liang J, Zhao X, Lei Schr C, F, 2.0 Chen Version Long W, System, MD, Tian Graphics Winn 13. Molecular RA, PyMOL The Nicholls RA, 12. Steiner NS, D Pannu Crystallogr Acta AA, Coot. Lebedev of Skub´ak P, development GN, and Murshudov Features K. 11. Cowtan WG, Scott B, Lohkamp P, Emsley 10. Lo ,Cin C agY egJ.Mcaitcbssfrteeouino hloesynthase chalcone of evolution the for basis Mechanistic JK. Weng Y, Wang molecular the YC, and Chiang synthase chalcone G, of Structure Liou JP. Noel 2. RA, Dixon ME, Bowman JM, Jez JL, Ferrer 1. Crystallogr. Biol D Crystallogr Biol Acta MOLREP. D. with Cryst replacement Acta Molecular A. post-refinement. Teplyakov A, and Vagin assignment 9. space-group L, scaling, Fuentes-Montero M, Integration, Gerstel W. RJ, Kabsch, Gildea 8. AS, Brewster JM, Parkhurst DG, microcrystals. for Waterman processing G, data Winter automated towards 7. KAMO: au- M. an Yamamoto K, ZOO: Hirata M. Yamamoto K, Yamashita T, Kumasaka 6. K, Hasegawa K, Y, Kawano Uno G, N, Ueno Fujino K, Y, Yamashita Li K, N, Hirata Tenma 5. K, Ito M, Terashita S, Yamada T, the Nakano in R, reductase Mameda chalcone T, of Waki Involvement 4. T. Nakayama S. Takahashi Y, Kawai T, Waki R, Mameda 3. aayi ytieratvt nln lns ilCe.2018;293:18601-18612. Chem. Biol J plants. land in reactivity 1999;6:775-84. cysteine catalytic Biol. Struct Nat biosynthesis. polyketide plant of basis D Crystallogr Acta structures. crystal macromolecular of refinement 2011;67:355-67. the Crystallogr. Biol for REFMAC5 AA. Vagin 2010;66:486-501. Crystallogr. Biol 2010;66:22-25. 2018;74:85-97. Biol. 2010;66:125-132. Struct evaluation Crystallogr. D and implementation Crystallogr DIALS: Acta G. package. Evans integration NK, new Sauter a ID, of Young T, Michels-Clark M, Vollmar 2018;74:441-449. Biol. Struct D Crystallogr Acta 2019;75:138-150. microcrystallography. Biol. protein Struct in D analysis Crystallogr structure Acta high-throughput to for protein system isomerase-like data-collection chalcone tomatic 2020;11:870. of Morita Commun. strategy K, Nat conserved Yonekura-Sakakibara specificity. K, A synthase Saito T. chalcone Nakayama S, promiscuous Sugawara S, rectify Takahashi Y, Kawai A, K, Hoshino Denessiouk Y, Y, Aoki S, Yamashita 2-hydroxyisoflavanone with interacts 2018;96:86–74. which J. GmCHR5, Plant of synthase. identification Biochem metabolon: isoflavone Merr.). soybean (L.) max (Glycine 2016;469:546-51. soybean Commun. in Res synthase biosyn- 2-hydroxyisoflavanone Biophys isoflavonoid of with interactions protein-protein enzymes of thetic Identification T. Nakayama S, Takahashi S, Ayabe ° oae rudteyai.Tearwaddte ice(rne niae h nrneo the of entrance the indicates (orange) circle dotted and arrow The y-axis. the around rotated Fo - Fc lcrndniymp otue t1 at contoured maps density electron 14 :A neircvt faoGCS,wihi rdce yteCASTp the by predicted is which apo-GmCHS1, of cavity interior An C: 4 :Teoealsrcueo p-mH1ho- apo-GmCHS1 of structure overall The A: σ dne,LLC. ¨ odinger, r ersne o h aayi cysteine catalytic the for represented are :Acoeu view close-up A A: Fo - Fc electron ε Posted on Authorea 16 Apr 2020 — CC BY 4.0 — https://doi.org/10.22541/au.158705333.31754085 — This a preprint and has not been peer reviewed. Data may be preliminary. nteatv ieadtu o iil nti ae.Dte iceidctstepsto hr h 1,4- the where position the indicates model. stick representation circle in Surface buried Dotted ion (d) is citrate moiety coordinate. panel. bound the malonyl the this in showing its in with occupies although visible complex partially GmCHS1/naringenin model, molecule not of acid stick thus residues sulfonic in and The di-ethane malonyl-CoA site naringenin piperazine (a). of bound and active to representation residues the the corresponding Surface important showing in are of (c) with molecules position 1CML) GmCHS1. naringenin super The and for ID: (b) residues only (b). the labeled are in of sites are shown active Colors be both in will molecules. residues as (NAR) Important models molecules. stick naringenin in bound shown indicating and without (cyan) 1CGK), ID: protein complex from PDB the GmCHS1/naringenin CHS of behind and shown Superposition GmCHS1 is (a) of naringenin alignment bound Structure-based The (b). S3. (b). in Figure and model (a) stick both white in a with labeled structure are residues Phe264 tures. com- 2). GmCHS/nagingenin figure 1 and to at apo-GmCHS1 (related solution. for in structures densities volume homodimer elution plex electron a the Representative from as S2. kDa exists 80 Figure GmCHS1 approximately suggesting be peak, to fraction calculated estimated major major is a the The mass indicates of arrowhead molecular The The GmCHS1. GmCHS1. its GmCHS1. purified of containing including chromatography the size-exclusion GmCHS1 of of recombinant purification. chromatogram SDS-PAGE The the protein (b) of mass and naringenin. molecular formulas and Structural (THC) tetrahydroxychalcone S1. S2. 2 Figure Figure The also structure. See legend complex Phe264. figure the of Supplemental movement in possible ion and the citrate apo- indicates the the arrow indicating of double without Superposition active The shown C: are structure). GmCHS1. - complex structures of the site both cyan: active from apo-structure, the sites the in GmCHS1(green: ion of citrate structures the naringenin-bound and naringenin the residues, 1 at contoured maps density Fc σ lcrndniympfrnrnei ssona npnlAecp niaigtesikmdl Dotted model. stick the indicating except A panel in as shown is naringenin for map density electron o the for β- tad ein( region strands σ r hw o aignn h25adPe6.B trove fteimportant the of view Stereo B: Phe264. and Phe215 naringenin, for shown are β and 7 β )fr()aoGCS n b mH/aignncmlxstruc- complex GmCHS/nagingenin (b) and apo-GmCHS1 (a) for 8) troiae fte2 the of images Stereo 5 N .sativa M. tria a sapoiaey4 D.(b) kDa. 44 approximately is tag -terminal .sativa M. Fo - a tutrlfrua f2’,4,4’,6’- of formulas Structural (a) Fc H/aoy-o ope (PDB complex CHS/malonyl-CoA H/aignncmlx(wheat, complex CHS/naringenin lcrndniymp contoured maps density electron eiaosativa Medicago (alfalfa). Fo Posted on Authorea 16 Apr 2020 — CC BY 4.0 — https://doi.org/10.22541/au.158705333.31754085 — This a preprint and has not been peer reviewed. Data may be preliminary. 6 Posted on Authorea 16 Apr 2020 — CC BY 4.0 — https://doi.org/10.22541/au.158705333.31754085 — This a preprint and has not been peer reviewed. Data may be preliminary. 7 Posted on Authorea 16 Apr 2020 — CC BY 4.0 — https://doi.org/10.22541/au.158705333.31754085 — This a preprint and has not been peer reviewed. Data may be preliminary. 8 Posted on Authorea 16 Apr 2020 — CC BY 4.0 — https://doi.org/10.22541/au.158705333.31754085 — This a preprint and has not been peer reviewed. Data may be preliminary. 9