Posted on Authorea 4 May 2021 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.162013331.11755851/v1 — This a preprint and has not been peer reviewed. Data may be preliminary. eainwsfudbtenavr o ree obacigatvt ihteasneo eil op a loop, flexible a of absence Introduction cor- the with A activity branching GH57GBEs. words: two mutant no Key and 2,500 even and tip, or almost native under- loop low of several better the very analysis of at a a activity bioinformatics tyrosine between acquiring branching sequence-based found at the systematic was aimed determining a relation study by and This family sequences GH57GBE gene far. the so an of characterized of standing biochemically formation been the have catalyze (GH57GBE) GH57GBEs between enzymes branching bond glycogen glycosidic 57 family hydrolase Glycoside Maarel Abstract der van J.E.C. Marc author: Corresponding [email protected] Maarel: der van J.E.C. Marc [email protected] Leemhuis: Hans [email protected] Xiang: of Netherlands Gang the University address: AA, Email (ENTEG), 9747 Groningen, Groningen Center, Innovation institute Avebe Technology 2. Netherlands and the Engineering AG, 9747 Engineering, Groningen, Groningen, Bioproduct 1. family Xiang hydrolase Gang glycoside of enzymes branching activity flexible glycogen transglycosylating a 57 of the absence determining and the elements with native activity Structural several branching of no two activity even and or branching tip, low sequence- the loop very systematic the determining a a at between and by tyrosine found family a sequences was GH57GBE loop, gene the correlation of 2,500 A understanding almost GH57GBEs. better mutant of a acquiring an analysis at of bioinformatics aimed formation based study This the far. catalyze so (GH57GBE) characterized enzymes branching glycogen between 57 family hydrolase Glycoside Abstract 2021 4, May 2 1 Xiang enzymes Gang branching glycogen 57 family of hydrolase activity glycoside transglycosylating the determining elements Structural v b Group Avebe Groningen of University α- , ikdguolooacaie.A nayia aiy iie ubro H7Bshv enbiochemically been have GH57GBEs of number limited a family, atypical an As glucooliogosaccharides. linked 1,4 1 asLeemhuis Hans , lcgnbacigezms lcsd yrls aiy5,Gyoe,Amylose Glycogen, 57, Family hydrolase Glycoside enzymes, branching Glycogen 1 asLeemhuis Hans , α- , ikdguolooacaie.A nayia aiy iie ubrof number limited a family, atypical an As glucooliogosaccharides. linked 1,4 1,2 n acJEC a e Maarel der van J.E.C. Marc and , β- 2 β- sheets. n acvndrMaarel der van Marc and , sheets. 1 1 1 α- , lcsdcbond glycosidic 1,6 α- 1,6 Posted on Authorea 4 May 2021 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.162013331.11755851/v1 — This a preprint and has not been peer reviewed. Data may be preliminary. otiigpoen r“lcsd yrls aiy5 rti”wr sdi h euneainetand alignment MEGA-X package sequence software the the in and with used Partial conducted domain- were was “DUF1957 respectively. protein” alignment title residues, multiple 57 the acid ClustalW family with amino hydrolase sequences analysis. 400-1,200 “Glycoside phylogenetic 2,497 and Finally, or RefSeq protein” manually. Biotechnology at containing deleted for set Center were were National sequences length the hypothetical at sequence “DUF1957” the word and 04 key on the (NCBI) searching Information by collected were analysis Sequences phylogenetic yet and more alignment or collection, one Sequence sheets in 2.1 beta but two biosynthesis methods the glycogen and and in Materials loop role in 2. flexible a role play the key pathways. not the of a metabolic do devoid play Besides identified and to GBEs be amylose. enzymes identified GH57 to on branching was that activity glycogen sheets, proposed no not beta is in are to adjacent analyzed It two low sequences activity. element, very GH57GBE branching displayed structural the the of additional GBEs majority an loop-deficient vast loop these the flexible of a in absent Several of was characterization study. site biochemical this active basic the in a covering overexpressed and loop GBEs flexible sequences GH57 sequence-based selected GBE in-depth genomes carefully an GH57 the GBEs, of 2,500 puzzling in GH57 number more GBE almost of even GH57 role of GBEs physiological a GH57 analysis possible of encoding bioinformatics and role one activity physiological enzymatic and the the GBE makes into bacteria GH13 insight of a number encoding large gene a a of of the presence in simultaneous alanine The an in with loop tyrosine the the replacing shortening activity; branching or the GBE in role key a identified play was tyrosine tip the at tyrosine conserved catalytic a including domains three of consisting shape of three-dimensional biosynthesis triangular the a ( in have role few a GBEs plays a GH57 of and from glucosylglycerate range branches a ranging GBE on zero, this activity polysaccharides that polymethylated detectable almost assumed no to have is have it GBEs low to organization, GH57 reported relatively solved was five is been GBEs, only has amylose GH57 of GBEs on GH57 properties GBEs five biochemical kodakarensis these GH57 the of five four far, ( these only So mU/mg of of structure clear. activity crystal less The the ( much while adenylyltransferase is reported, GBEs glucose-1-phosphate been GH57 of of action ( role tandem synthase linear the either glycogen in typical by - categorized a pathway are amylose, biosynthetic GBEs substrate known glycogen 57 all model or motifs, the 13 conserved family on and (GH) sequence hydrolase acid glycoside amino the primary the on Based glucans cyclic branched in resulting an of hydrolysis den- the chain a different glycogen, a of or biosynthesis cleaved-off the fungi, a microorganisms, in prokaryotic sylating many role in animals key present molecule and a storage yeast, play energy and 2.4.1.18) carbon (EC polyglucose drimeric (GBE) enzymes branching Glycogen obedslcmn ecinmcaimrsligi eeto fthe the of of retention CSRIV structures crystal in in the resulting located mechanism catalyst reaction acid/base double-displacement the and CSRIII in catalyst β/α ) 7 arlcnann h w aayi eius ltmt ulohl n napraeacid/base aspartate an and nucleophile glutamate a residues, catalytic two the containing barrel ,1,1,20 19, 10, 6, hrooamaritima Thermotoga KOD1) l H5 Bshv v osre eunergos(S) ihtencepielocated nucleophile the with (CSR), regions sequence conserved five have GBEs 57 GH All . 1-4 ,5-7 1, α- ,21 8, B aayetefrainof formation the catalyze GBE . glgA α- - lcsdclnaeuigwtra nacceptors an as water using linkage glycosidic 1-4 lcncanfo growing a from chain glucan nadto otetaslcsltn rbacigrato,GE locatalyze also GBEs reaction, branching or transglycosylating the to addition In . h H7GEof GBE GH57 The . th lcgnbacigezm ( enzyme branching glycogen - ) .horikoshii P. eray22.T le eudn n hr eune,tesuc database source the sequences, short and redundant filter To 2020. February M8 oafwhnrdm/g( mU/mg hundred few a to SMB8) .horokoshii P. 22-24 9 . . , ,1,19 10, 6, .kodakarensis, T. 10-13 B eutdi oso h rnhn activity branching the of loss a in resulted GBE yoatru tuberculosis Mycobacterium uainlaayi hwdta h eil opadthe and loop flexible the that showed analysis Mutational . h Bsfo H3hv usatal ihactivity high substantially a have GH13 from GBEs The . α- glucan 2 α- , ikdahdolcprns hi otesame the to chain anhydroglucopyranose linked 1,4 α- 25 , lcsdclnae ngyoe ytransglyco- by glycogen in linkages glycosidic 1,6 iia oG1 Bs H7GE mlya employ GBEs GH57 GBEs, GH13 to Similar . shrci coli Escherichia 14-17 and glgB H3GE r novdi h classical the in involved are GBEs GH13 . .thermophilus T. hru thermophilus Thermus ) ,18 2, α- ncnrs oG1 Bs the GBEs, GH13 to contrast In . 8 α- ofiuaini h products the in configuration rpromacciainreaction cyclization a perform or , glucans a odce.Srrsnl,the Surprisingly, conducted. was aigaltekyfaue of features key the all having , B eil opwt a with loop flexible a GBE 22 ae ntegenomic the on Based . HB8, 21 .thermophilus T. ogi more gain To . ,19 6, Thermococcus ,1,1,20 19, 10, 6, . glgC 6 In . 26 ) . Posted on Authorea 4 May 2021 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.162013331.11755851/v1 — This a preprint and has not been peer reviewed. Data may be preliminary. nrdcn n rfie fsmlsbfr n fe ernhn.Oeui fatvt sdfie s1 as defined is 3 activity conditions. of discussion reaction unit and aforementioned One under Results debranching. minute 3. after per and transferred or before released samples ends of standard reducing profiles as end glucose the reducing using monitor on method to (BCA) intervals acid time 40 2,2’-bicinchoninic at at taken 100 Ireland) were at (Megazyme, samples mg/ml Samples 0.13 boiling enzyme. by to with of mg/ml stopped treated was 0.03 activity reaction from total The ranging the progress. enzyme reaction on of concentration depending final used a was and substrate, as 50 Netherlands) at The performed was reaction branching (Bio-Rad, of The Bradford Purity activity (pH7.0). by catalytic PBS assayed of mM was Determination standard. 5 concentration 2.4 as to Protein albumin buffer SDS-PAGE. serum storage imidazole by bovine a checked mM using using and was USA) 25 removed USA) was protein containing Imidazole Scientific, target buffer imidazole. Fisher mM washing the (Thermo 250 with a column protein applying desalting target the by a of protein elution and host USA) (Sigma-Aldrich, bound a and non-specifically bar of 8 removal 50 of disrupted at pressure were performed denature air Cells was to an (pH7.0). treatment with PBS Heat temperature, mM bar. silvanus room 5 6 at in of chaperone Canada) resuspended pressure the (Avestin, finally valve protein, homogenizer and target pressure 7.0), the high (pH by of buffer solubility (PBS) 8,000 the saline at enhance buffered centrifugation to by order harvested in In were co-expressed mM. was Japan) 0.2 (Takara, pG-KJE8 of plasmid concentration final -80 a at to stock glycerol 20 25% in 37 stored were mutant cultivation. or further gene native for target plate agar the kanamycin-containing or carrying ampicillin from selected was colony E 8()(h eann Bsue nti td)vco yGncit(S) lsiswr transformed were Plasmids (USA). Genscript by vector study) this in ( used into GBEs B remaining pRSET (the in 28a(+) the overexpression pET in for codons ligated optimized then with and Genes 2). (Table in overexpressed constructed were GBEs GH57 structures. putative crystal Various loop the flexible production on The enzyme based and Pymol. defined Overexpression in were 2.3 command regions measurement sheet the beta with two measured the was and residues two between distances maritima software Pymol the using mophilus visualized sheets were beta structures two Protein and loop previously sequence flexible a described conserved of and as within Identification alignment, off, residues 2.2 MEGA-X sequence at of package multiple (JTT) software possibilities matrix Jones-Taylor-Thornton the the calculating negative on the in by the based using setting analyzed regions 0.2, default constructed were in at was regions method penalty tree sequence likelihood conserved phylogenetic extension maximum A gap the the with cutoff. model 10, divergent at delay penalty % 30 opening gap setting by . o o eunebsdaayi fpttv H7GEsequences GBE GH57 putative of analysis Sequence-based 1 n 5 p.We h pia est ece .-. 60n) rti xrsinwsidcdat induced was expression protein nm), (600 0.6-0.8 reached Isopropyl density adding optical by hours the 12 When for C rpm. 150 and C .coli E. GBE, PBI:3P0B) ID: (PDB B Ay)(D D 2B5D) ID: (PDB (AmyC) GBE .coli E. L1(E)b ujcigcmeetclst etsoka 42 at shock heat a to cells competent subjecting by (DE3) BL21 Pseudomonas ertg mexicana Petrotoga gbe otpoen.Te h uentn a nuae ihnce ea ei,floe by followed resin, metal nickel with incubated was supernatant the Then proteins. host a rw nLraBrai(B eim(etn ikno n opn,UA at USA) Company, and Dickinson (Becton, medium (LB) Luria-Bertani in grown was 6 o , piomls 04Um)and U/ml) (0.4 isoamylase sp o 4hwt osatml hkn.Rdcn nswr unie ythe by quantified were ends Reducing shaking. mild constant with h 24 for C .kodakarensis T. .kodakarensis T. B)o 65 or GBE) β- --hoaatprnsd IT)(hroFse cetfi,USA) Scientific, Fisher (Thermo (IPTG) D-1-thiogalactopyranoside × 20 g o n5m B ue p70 sn .2%ayoeV(Avebe, V amylose 0.125% using (pH7.0) buffer PBS mM 5 in C o 0mna 4 at min 10 for PBI:3N8T) ID: (PDB eedwlae rmtePoenDt ak(D) The (PDB). Bank Data Protein the from downloaded were o termiigGE sdi hssuy o 5minutes 15 for study) this in used GBEs remaining (the C shrci coli Escherichia GBE, 3 .coli E. .thermophilus T. lbilaplanticola Klebsiella 29 o o yfloigtemnfcuespooo.Cells protocol. manufacture’s the following by ,wse el wc ih5 Mphosphate mM 50 with twice cells washed C, h he-iesoa tutrsof structures three-dimensional The . ( C 10 30 , aiihru timidus Calidithermus .horokoshii P. h ciiyui a acltdbased calculated was unit activity The . Tbe1,adsvrlmtnswr also were mutants several and 1), (Table o o C. o 0mn rnhdsmlswere samples Branched min. 10 for C .coli E. 28 shrci coli Escherichia B and GBE . o PBI:5WU7) ID: (PDB o 0scns single A seconds. 90 for C uulns 1(. U/ml) (1.4 M1 pulullanase L2(E)cryn the carrying 21(DE3) BL .maritima T. GBE, eesynthesized were .coli E. Meiothermus 6 27 26, 19 B)or GBE) .ther- T. and strains Five . μ mol T. Posted on Authorea 4 May 2021 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.162013331.11755851/v1 — This a preprint and has not been peer reviewed. Data may be preliminary. otmWfu te rmtcaioaisaefudi ls iiiyo h ciestsof sites active the of vicinity close in found are acids amino thermophilus aromatic T. other four activity of W of structure bottom loss crystal complete the almost maritima in in T. resulted position A same an the into at W this Changing predict recognition. to difficult is the In (W411) equivalent clear. the W413 not In the is (W404). of R265 bonding the position hydrogen the and while (W274) stacking occur aromatic by maritima binding substrate T. in of involved GBEs are the Both in threonine a is thermophilus which W28, except GBEs GH57 ( keepers sequences; (R265) gate all arginine in aromatic one conserved and the fully proteins (H146), be -like histidine to In and identified one were W413), enzymes numbering). sites and GH57 active W404 the other (W274, of the tryptophans vicinity the three all in of residues none five 16 addition, as Martinoviˇcov´aIn fingerprint position position. by a this at and noticed as at seen also C C be feature a the still a have can that position, C this reported conserved in fully was acid it amino different previously Janeˇcek a Whereas with have in GBEs study, Y. sequences is this GH57 (2.9%) an in which among conserved analyzed acid, F proteins is GH13. the amino GBE (CSRIII) in position, containing all D in third chain stabilizer conserved not the state side completely is transition of are it the functional sextet exception structures, role as a the crystal key the role to of GH57 a similar assigned available positions a play proteins all other plays D in GH57 The E catalyst all stabilizer this acid-base in state that the conserved transition assume to completely to a close is unlikely and positioned E water E, is CSRIV a and catalyst the through subfamily base As residue acid [32-34]. subsite catalysis general -1 in a the D, binding nucleophile in catalytic involved is and thermophilus 2A) (Fig. D molecule catalyst acid-base the from h rtista r aeoie s-iepoen.Teepoen isoeo oho h aayi residues second catalytic The the of GBEs. 4- both GH57 or the in for one conserved In case miss not the E, active. proteins is the is These E not fingerprint, This as likely proteins. this subfamily. enzymes P -like very enzyme of as GH57 a are GH57 position categorized other not and functional first are a all and a The that to A/M In in proteins CSRIV. assigned or M. the in proteins sequences; Q or all present in GBE among a I ELF(Y)GHW whereas all conserved by sextet an is 4 almost followed the by position is of is replaced at fingerprint this CSRI is L GBE but in 4 an 4 found position of position T, at instead published at or present L recently is S the logos Q A, sequences the a GBE with being of study x number this with of small HxHLP, GBEs combination GH57 the the with all active. sequences second GH57 for not the ( 1,602 logo sequence are on on the sequence conserved they domain containing the five that C A-domain comparing assumed the the the When in was of within located it positioned four is as are in 5 regions analysis variation that region sequence further large sequences Conserved conserved from were a four exception excluded showed first The were and The sequences 1). residues contained These (Fig. catalytic and members catalyst both regions. acid/base GH57 and or for 418 nucleophile between typical one the length had regions missed in sequences sequence varied all conserved sequences 50, five These for the Except database. acids. NCIB 2,497 amino the protein”, 1,184 from 57 and retrieved family hydrolase were “Glycoside sequences or acid protein” amino domain-containing “DUF1957 words key the Using .horokoshii P. 25 31 h aoiyo hs aea 5;22) iehv nS v nLadtoa .Ti almost This F. an two and L an five S, an have nine 2.2%), (56; M an have these of majority the ; nteohrG5 rsa tutrs h osre s4 is E conserved the structures, crystal GH57 other the In . B,teW7 qiaet(26 sbre uhta rmtcsakn svr nieyto unlikely very is stacking aromatic that such buried is (W246) equivalent W274 the GBE, , B h 24i oiindt h ieadteW0 ttebto ftepstv subsites positive the of bottom the at W404 the and side the to positioned is W274 the GBE B)fo h cdbs aayt(i.2 n C.I H3ezms aayi ra fa of triad catalytic a enzymes, GH13 In 2C). and 2B (Fig. catalyst acid-base the from GBE) .mexicana P. , .kodakarensis T. .horokoshii P. B,atytpa W2 ttebto fteatv iegov sivle nsubstrate in involved is groove site active the of bottom the at (W22) tryptophan a GBE, 25 8 hsgopo oraoai aekee rpohn shgl osre nall in conserved highly is tryptophans keeper gate aromatic four of group This . w B pcfi nepit eoecer h rti une faioacids amino of quintet a is first the clear; become fingerprints specific GBE two , , .mobilis P. or , h he rpohn n h n tpsto 8hv endfie as defined been have 28 position at one the and tryptophans three the , .maritima T. α- and 6 lcntaseaeof glucanotransferase hspsto sntasltl nain,a 2oto h 2,497 the of out 72 as invariant, absolutely not is position this , .pacifica K. .thermophilus T. 2,F8,W6 n 41( F461 and W360 F289, F23, ; 4 .pacifica K. α- hsWi elcdb rEo .Bsdsthe Besides P. or E or D by replaced is W this , aatsdssad-eae rtisteei nL an is there proteins -related and galactosidases α Thermus helix. hroocslitoralis Thermococcus and B elcdb nte hydrophobic another by replaced GBE .kodakarensis T. ( A ˚ and .maritima T. Meiothermus .thermophilus T. 6 h oeo h 16and H146 the of role The . 19 hsWi lofound also is W This . nG5GE from GH57GBEs In . h sol 5.1 only is E the mC o7 to AmyC) pce.I the In species. .kodakarensis T. .thermophilus T. β/α numbering). ) 7 barrel. ( A ˚ T. T. A ˚ 6 , . Posted on Authorea 4 May 2021 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.162013331.11755851/v1 — This a preprint and has not been peer reviewed. Data may be preliminary. tpsto 4 hc stre wyfo h lf en a wyfo h w ciest eius(E191 residues site active two the from away folds far rest being the cleft while 13 cleft the of The site from active loop 5). away the flexible turned (Fig. into a cleft is folds have the which loop covers (9%) 245 flexible that proteins the position part of 17 the at part proteins behind while of first sequences or the acids number the to 201 of significant amino only next still the models more that but of show The or smaller, resp., three A 24 loop acids. and 4). of amino Hundred loop (Fig. 15 acids activity. flexible amino to branching a 22 have the to 17 40%) to of (81; respect loop flexible with a the optimal have of (51%) be configuration to and activity residues seems 19 the loop about than of lower GBEs. length GH57 substantially The the being of 3), activity (Tables overall activity the hydrolytic determines both of type the that loop wild of confirming flexible the shown), increase of partial not or three-fold (data full a introduced the V A the and enzymes in amylose of 4). both tyrosine, with Introduction (Table activity, the including incubated study low activity. GBE, this when branching a have in having product clearly used Although final (92%) enzymes the 3). sequences in (Table 2,497 of branches respectively the GBE towards 6% mU/mg of the activity 9.1 2,296 for branching and found in activity reproducible was mU/mg also branching activity but but the branching AmyC low of low in reduction a comparable absent considerable has only a loop, not and activity flexible is total a in of increase lack twofold the of activity. of branching tip amylose spite the the in in at role AmyC, tyrosine a that the play only loop not flexible that the the the resulting of suggest to mU/mg, configuration results contrast 26.3 and These (426 In to size activity mU/mg the 16.2. branching 12.6 also of dominant from but ratio the doubled loop hydrolysis retains mutant the over mutant Y/A branching Y/A the a the of the in 41, activity activity. of of hydrolytic branching that The ratio the as type hydrolysis mU/mg). configuration in over wild and role branching the the size determining a in Whereas same a and mutation the play 3). and alanine not acids, does to Fig. amino loop tyrosine 19 On that flexible a of twice the by 3). loop of being (Table confirmed tip mU/mg, 38.1 was the the of 356.9 This by at activity of dominated tyrosine hydrolytic activity amylose, the over towards branching that alanine, branching activity a an of high has Instead relatively ratio tip, a 17.2%). a has (35; and the tip, tip contrary, mU/mg the the the 168 at at of alanine tyrosine activity an a branching have with not residues The activity do 22 branching found. of loop of loss are acids, flexible a threonine amino in a or resulted 13 with alanine serine, being GBEs an with shortest of of tyrosine the minority loop this Replacing flexible with A activity. the acids branching of amino the tip varying in 26 the of role is At loop flexible size acids. a horokoshii loop amino sequences, P. average 50 acid is The amino longest other acids 4). the 201 amino while In (Fig. 19 3). present of (Fig. is sequence cleft length catalytic a the was covering loop first flexible The ( 247 visible. of to became structures gaps 229 major position two from alignment, sequence the tyrosine From and loop flexible The conserved 3.2 functionally sequences. are 2,497 acids the amino ( all aromatic al. D463 in four et and these Zhang S462, of conserved. H11, three not site, study, is this F23 of the sequences while other the all In .maritima T. 8 hreigo h eil opi the in loop flexible the of Shortening . .thermophilus T. B,atrsn spresent is tyrosine a GBE, .kodakarensis T. B Fg n ) swsarayntdb hn n coworkers and Zhang by noted already was as 5), and 3 (Fig. GBE aiihru timidus Calidithermus .thermophilus T. .thermophilus T. .thermophilus T. 6 , .kodakarensis T. B.Teflxbelo per o ob h nysrcua lmn that element structural only the be to not appears loop flexible The GBE. .kodakarensis T. .maritima T. hroneoauu aquaticum Thermoanaerobaculum , .kodakarensis T. ,1,19 10, 6, .hugenholzii T. B ihaflxbelo f2 mn cd n yoiea the at tyrosine a and acids amino 23 of loop flexible a with GBE 8 ubrn) hs r l osre ttersetv positions respective the at conserved all are These numbering). B ubrn)i ewe S I n V ntecrystal the In IV. and III CSR between in numbering) GBE eotdaohrtreiprataioaisna h active the near acids amino important three another reported The . B eutdi w-odices ftebacigactivity branching the of increase two-fold a in resulted GBE 10 .horokoshii P. and , B a oiatbacigatvt f40mU/mg 480 of activity branching dominant a has GBE .thermophilus T. 5 and and .horokoshii P. .thermophilus T. .horokoshii P. .lipolytica T. .hugenholzii T. .mexicana P. B rm1 o9aioaisrsle na in resulted acids amino 9 to 19 from GBE .kodakarensis T. .aquaticum T. .thermophilus T. yoiewssont lyaprominent a play to shown was tyrosine B,wihhsamdu ie loop sized medium a has which GBE, 19 B,teflxbelo sasn in absent is loop flexible the GBE, B,wt 6ad2 mn acids amino 28 and 36 with GBE, B,teeaioaismk pa up make acids amino these GBE, and and .thermophilus T. eil opcnan tyrosine a contains loop flexible .kodakarensis T. omtg pacifica Kosmotoga B.Ti eutindicates result This GBE. B,wihhsaflexible a has which GBE, 8 , thsbe reported been has It . .kodakarensis T. 19 h eil loop flexible The . .kodakarensis T. B Tbe3; (Table GBE B flexible GBE f14.1 of , and , 6 . Posted on Authorea 4 May 2021 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.162013331.11755851/v1 — This a preprint and has not been peer reviewed. Data may be preliminary. 2. Um) lo h rnhn vrhdoyi ai a eysmlrt hs Bsta isdthe missed that short GBEs activity very those branching the a to low for and similar very found tyrosine very a was was had result only ratio similar it hydrolysis A sheets, over branching beta two the without β the Also, but without loop GBEs mU/mg). flexible other a (29.1 with as GBEs but those amylose Of towards mU/mg. 550 the to mU/mg and the the 168 possess loop from also flexible ranging the loop activity a have flexible branching not with a do have GBEs also that the sequences loop of 201 flexible the the of miss majority that the %) the (92 in GBEs is 2,296 all alignment sequence ( sheets the beta prominent in gap second loop growing The a sheets a towards or beta two activity loop show The flexible do the 3.3. GBEs of these towards absence GBEs activity that most The of of be loss deep substrate complete required. could to positioned is It reduction tyrosine significant D a a enzyme, established. and present, in branching the E results when glycogen What acids catalytic “true” and, amino a amylose. the cleft 17-22 as than active to the larger act the close while or to covering smaller site mU/mg, GBE acids 13.9 active GH57 amino a only the 17-22 for of into of Thus, activity loop branching 3). flexible (Table low activity a very any a show has not GBE did This D382). and hc a ogflxbelo f2 eiuswtotatrsn n no and tyrosine β a without the residues Interestingly, 28 of loop the from flexible region long a has which nulse eut) The results). unpublished the incubating h aata upr h nig fti td r vial rmtecrepnigato pnreasonable upon author corresponding the from available are study this of findings request. the support that data The interests. of statement conflict availability no Special Data have they that support. declare financial authors The for Groningen collection. interest of sequence of University with Conflict assistance and for (GX) Zhang Zhenhua Council to Scholarship appreciation China thank We flexible optimal as Acknowledgments assigned were true which direction. and explicit features feature an structural former two two in the two of latter by of absence presence the defined and and by be loop loop designated flexible can two no be sequences having GH57GBE of can (92%) putative GH57GBEs them presence/absence that of two and verified majority are data a loop that Biochemical with flexible sequences, sequences, 2,497 in GH57GBE in putative variations noted of logos, features sequence sequence-based GH57GBE-specific the reveals analysis Bioinformatics branching the on influence its Conclusions exerts 4. region sheet beta this unclear. exactly remains How activity activity. branching the in particular 5- 5- β β β ein e.g. region; 6 ein utatoeepesdthe overexpressed Gusthart region. 6 5- β ein n,ta from that one, region, 6 .tuberculosis M. .kodakarensis T. yoatru tuberculosis Mycobacterium .pacifica K. in-vivo β 1 5- β- . β β hes hs eut ud st ute netgt H7Bso lk sequences –like or GH57GBEs investigate further to us guide results These sheets. usrt o hs opdfiin n ogloe H7GE s ean obe to remains is, GBEs GH57 long-looped and loop-deficient these for substrate ;188-205, 6; 5- β 5- β n mCwt rnhn vrhdoyi ai f38ad57respectively. 5.7 and 3.8 of ratio hydrolysis over branching a with AmyC and H7B ihayoe oatvt a eetd(agadvndrMaarel, der van and (Gang detected was activity no amylose, with GH57GBE ein(al ) oatvt ol edtce o the for detected be could activity No 3). (Table region 6 β B eutdi opeels fatvt Tbe3). (Table activity of loss complete a in resulted GBE eintu ly eemnn oei h ciiyo H7GE,in GBEs, GH57 of activity the in role determining a plays thus region 6 Meiothermus .hugenholzii T. .thermophilus T. β .tuberculosis M. 5- β 3R H7GEas issteflxbelo swl sthe as well as loop flexible the misses also GBE GH57 H37Rv einwr nlddi hssuyadsoe considerable showed and study this in included were region 6 p N-s,wsoepoue n nlsd twsactive was It analysed. and overproduced was PNK-Is4, sp. B,hvn ogflxbelo f3 mn cd iha with acids amino 36 of loop flexible long a having GBE, 6 β 5- B ubrn) r localized are numbering), GBE β ein hr nthe in where region, 6 H7B u i o n n activity any find not did but GH57GBE β nor 5 β β 5- 5- β β β ein eoigthe Removing region. 6 he Tbe4.I contrast, In 4). (Table sheet 6 ein(5 r7.%.Four 79.1%). or (159 region 6 α- .thermophilus T. lcncan the chain, glucan 6 ihu exception, Without . .lipolytica T. .lypolytica T. β- hes were sheets, 22 B two GBE β- When . in-vivo sheets; GBE, β GBE 5- β 6 Posted on Authorea 4 May 2021 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.162013331.11755851/v1 — This a preprint and has not been peer reviewed. Data may be preliminary. 6 hn ,Lehi ,Mae JC ytei fhgl branched highly enzymes. of branching Synthesis glycogen MJEC. GH57 Maarel and H, GH13 Leemhuis using 2009; X, Microbiol. Zhang Environm of Appl 16. patterns domains. branching N-terminal unique The their L. by Dijkhuizen, determined 75:1355–1362. MJEC, are Maarel enzymes der branching van S, glycogen Kralj M, Palomo 15. glucan the of Properties bacterium L. 207. hyperthermophilic Dijkhuizen the P, of Sanders enzyme A, branching Vos MJEC, Maarel Der Van 14. specificities. enzyme reflecting Martinoviˇcov´a Janeˇcek M, 13. JaneˇcekGH57. K, hydrolase Blesak glycoside large 12. of the annotations Dividing B. functional Sel. improved Henrissat Des towards PM, Eng Coutinho subfamilies: C, into Rancurel 13 EG, family Danchin hyperthermophilic MR, the Stam from enzyme 11. glycoside branching of novel activity a branching-enzyme of for studies basis stability Structural hyperther- and archaeon al. Structure from et 57: enzyme DM, family Trindade branching hydrolase CCC, Tonoli of CR, Santos Properties 10. S. Okada T, Kuriki Glycosci. T, Appl Takaha K, bacterium, Ohdan mophilic H, Takata activity. hydrolytic 9. high with enzyme glycogen-branching 103:6141-6151. a Janeˇcek as H, AmyC Leemhuis XW, microbial Zhang with 8. modification Starch H. Leemhuis Pol. MJEC, Carb Maarel, formed. der products Van and 7. action, al. of et mechanism T, structure, Booiman crystal T, branching enzyme Pijning glycogen the M, of Palomo characterization 6. and expression Recombinant J. Wu S, 994. Chen F, Hu from L, enzyme Su glycogen novel L, a Li of 5. characterisation and expression functional cloning, Gene 159:85-94. Z from Jiang Q, enzyme Yan bacteria. branching Y, and Liu S, yeast Wu in 4. Fern´andez-Fern´andez Ameneiros- metabolism humans. C, glycogen Donapetry-Garc´ıa C, in Gonz´alez-Luc´an of metabolism M, MM, Glycogen Regulation Rodr´ıguez Adeva-Andany E. al. et 3. M, Montero PJ, Rev eukaryotes. Microbiol Roach in FEMS WA, enzymes Wilson branching of 2. function and Structure E. Suzuki cotechnol. R, Suzuki 1. References References Extremophiles hroocskodakaraensis Thermococcus 03 93:116-121. 2013; 00 32:21-30. 2020; irovulnificus Vibrio 06 19:555-562. 2006; 03 0 15-20. 50: 2003; qie aeolicus Aquifex 00 34:952-985. 2010; . 02 16:497–506. 2012; . .Sqec nepit fezm pcfiiisfo h lcsd yrls family hydrolase glycoside the from specificities enzyme of fingerprints Sequence S. ˇ hzmcrmiehei Rhizomucor Biotech. n t plcto nsac oicto.ItJBo arml 00 155:987- 2020; Macromol. Biol J Int modification. starch in application its and .I iioaayi fthe of analysis silico In S. ˇ ,e l dnicto of Identification al. et S, ˇ n t oeta o rdcino ihybace ylcdextrin. cyclic highly-branched of production for potential its and , KOD1. 08 8:307. 2018; hru thermophilus Thermus n t plcto nwetbreadmaking. wheat in application its and Proteins. B Clin. BBA 7 abPol. Carb qie aeolicus Aquifex 01 79:547-557. 2011; 06 5:85-100. 2016; α- hrooamaritima Thermotoga mls aiyG5:eeta subfamilies eventual GH57: family amylase 09 216:231-237. 2019; lcsd yrls aiy5 branching 57 family hydrolase glycoside ilChem. Biol J ictlBornfr 03 21:199– 2003; Biotransfor. Biocatal . α- α- plMcoilBiotechnol. Microbiol Appl mls-eae proteins. amylase-related lcn ihdffrn structures different with glucans α- lcntaseaeenzymes. glucanotransferase 01 286:3520-3530. 2011; S8GH57 MSB8 rnsGyoc Gly- Glycosci Trends odChem. Food Deinococcus α- amylase Protein 2019; 2014; J Posted on Authorea 4 May 2021 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.162013331.11755851/v1 — This a preprint and has not been peer reviewed. Data may be preliminary. 4 idha C oiR akK,e l -a tutrsaogterato aha fcyclodextrin of pathway reaction the along structures X-ray al. et 6:3909-3916. KH, 1987; the J. Kalk in EMBO catalysis R, activity. alpha-amylase elucidate Mosi and pancreatic glycosyltransferase JC, structure porcine Taka- Uitdehaag in of structure calcium 4- of dimensional 34. of residues Three Role of F. catalytic Payan resolution. possible structures R, A Haser and 2.9 E, Crystal at Structure Duee G, M Buisson Kakudo 33. al. W, Harada et M, Kusunoki M, A. amylase Y, Yamamoto Matsuura enzyme S, branching 32. Fushinobu starch for H, method J Imamura assay from glucans. Quantitative of terminals al. reducing 31. et the PB, measuring Francisco by crystallog- acid M, protein bicinchoninic Yoshida on with Y, Newsletter 2021 CCP4 Utsumi 28 April tool. 30. Accessed graphics newsletter40.pdf. molecular http://legacy.ccp4.ac.uk/newsletters/ open-source generator. 40:82-92. An logo 2002; sequence Pymol: raphy a WL. protein DeLano WebLogo: from SE. 29. matrices Brenner data J-M, mutation Chandonia G, of 14:1188-1190. Hon 2004; generation GE, rapid Crooks analysis The 28. genetics JM evolutionary Thornton molecular WR, X: Taylor MEGA DT, sequences. K. Jones Tamura C, 27. Knyaz methylglucose M, of Li platforms. biosynthesis G, computing Stecher the across S, for Kumar basis 26. Genetic al. methylglucose to related et mycobacterial M, Janeˇcek of Guerin 25. Biosynthesis T, N. Sambou Empadinhas in G, lipopolysaccharides S, Stadthagen Alarico 24. A, Maranha V, lipopolysaccharides. Mendes 23. 2021 28, April from Accessed enzyme 425868/. branching glycan branching unusual tuberculosis glycogen an Mycobacterium into novel GH57 investigation and An the JS GH13 Gusthart of the 22. of Structure Characterization R. MJEC Maarel Ficner e0219844 H, W, Leemhuis from X, enzymes GH57-type Liebl Zhang a M, of activity 21. Ballschmiter transglycosylase the A, for basis Dickmanns Structural from N-C. Ha J, 20. new Cha I, some from Jo enzyme metabolism: M, branching its Park glycogen and S, Glycogen Na VS. 19. Tagliabracci TD, themes. Hurley old AA, and Depaoli-Roach developments PJ, Roach 4- 1, 18. Bacterial al. et applications. CM, commercial Li AS, and Dhoble X, Ban 17. hroocslitoralis Thermococcus maritima Thermotoga Bioinform. α- Biochem. J lcnbacigezmsad4- and enzymes branching glucan ,MrioicvaM e ruso rti ooousi the in homologues protein of groups Martinoviˇcov´a New S, M. ˇ ertg mobilis Petrotoga a rdRep. Prod Nat 92 8:275-282. 1992; yoatru tuberculosis Mycobacterium 94 95:697-702. 1984; . h hssUiest fSuhmtn 08 https://eprints.soton.ac.uk/ 2018. Southampton. of University thesis PhD : n t ope iha inhibitor. an with complex its and caCytlorD. Crystallogr Acta o ilEvol. Biol Mol rtRvBiotechnol. Rev Crit ice J. Biochem yoocshorikoshii Pyrococcus J5adptnilrl ngyoe biosynthesis. glycogen in role potential and SJ95 02 29:834-844. 2012; 02 441:763-787. 2012; 08 35:1547-1549. 2018; α- α- mls family. amylase lcntaseae.Gntc.22;148:77-86. 2020; Genetica. glucanotransferases. 06 62:262-270. 2006; . α- 00 40:380-396. 2020; ilChem. Biol J lcnbacigezms hrceitc,preparation characteristics, enzymes: branching glucan 8 . ice ipy e Commun. Res Biophys Biochem aueSrc Biol. Struct Nature ilChem. Biol J 07 282:27270-27276. 2007; plGlycosci. Appl α- 03 278:19378-19386. 2003; mls aiyG5 closely GH57 family amylase 99 6:432-436. 1999; lSOne. PloS α- glucanotransferase 07 484:850-856. 2017; α- 09 56:215-222. 2009; mls AmyC amylase eoeRes. Genome 09 14: 2019; Posted on Authorea 4 May 2021 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.162013331.11755851/v1 — This a preprint and has not been peer reviewed. Data may be preliminary. ftefiebohmclycaatrzdgyoiehdoaefml 7gyoe rnhn enzymes. branching glycogen 57 family hydrolase glycoside characterized the biochemically containing five regions the the of of alignment Partial 3. Figure 4- GH57 A. maritima (red). IV CSR in in Distance 2. Figure branching glycogen 57 family hydrolase glycoside 2,447 among CSRs five of logos homologues symbol. Fingerprint enzyme star red a 1. by Figure indicated are residues catalytic Two * .G5 lcgnbacigezm of enzyme branching Glycogen GH57 C. ; ewe h osre fCRV(le n aayi eiusEi S I n D and III CSR in E residues catalytic and (blue) CSRIV of E conserved the between A ˚ α- lcntaseaeof glucanotransferase hru thermophilus Thermus hroocslitoralis Thermococcus 9 β and 5 β hesadteflxbelo lstyrosine plus loop flexible the and sheets 6 . .G5 mCof AmyC GH57 B. ; Thermotoga Posted on Authorea 4 May 2021 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.162013331.11755851/v1 — This a preprint and has not been peer reviewed. Data may be preliminary. iue4 eghdsrbto fteflxbelo mn h 0 opcnann lcsd yrls family hydrolase glycoside loop-containing 201 the among loop enzymes flexible branching the glycogen of 57 distribution Length 4. Figure 10 Posted on Authorea 4 May 2021 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.162013331.11755851/v1 — This a preprint and has not been peer reviewed. Data may be preliminary. aquaticum Thermoanaerobaculum KOD1 kodakaraensis Thermococcus JAD2 hugenholtzii Thermoflexus PNK-Is4 Meiothermus 17022 timidus Calidithermus al .Bacigadhdoyi ciiyo aiu lcsd yrls aiy5 lcgnbranching glycogen 57 family hydrolase substrate. glycoside as various amylose using of determined activity was hydrolytic activity The and enzymes. Branching 3. Table study this in used sheets beta two and loop flexible residue, tyrosine of Mutants 2. Table maritima Thermotoga 14811 mexicana Petrotoga pacifica Kosmotoga 11003 lipolytica Thermosyntropha (aa) Length number Accession Abbreviation thermophilus Thermus study. this Organism in used GH57GBEs putative various the of of similarity presence/absence sequence the and and Information loop, 1. flexible Table residues. the catalytic of two tip the the the and showing at enzyme loop tyrosine branching glycogen the 57 loop, family and flexible hydrolase the glycoside several of the position of Configuration 5. Figure mGEL opsa 0 SHAFW 208 FADEQPRY SHAFW 208 extension Loop swap Loop EGPASNV SRLID 221 Sequence mutation site Single GBE Tk LE type GBE Mutant Tm LS GBE Tm Y233A GBE Tk code enzyme Mutant β hes(upe.Teisr hw h oiinaddsac ftetrsn ttetpo h flexible the of tip the at tyrosine the of distance and position the shows insert The (purple). sheets 6 SLHLJ1 DSM DSM β SMB8 DSM MP-01 he deletion sheet HB8 sp. opTyrosine Loop element Structural aGEWP WP GBE Ta WP WP GBE Tk WP GBE Th GBE Ms GBE Ct mGEWP WP GBE Tm WP GBE Pme GBE Tl WP GBE Tt pGEWP GBE Kp β he eein12PECAY 182 deletion sheet element Structural σηεετς β element Structural 1208. 7 44.6 46.3 62.9 675 66.2 011250387.1 100.00 558 520 088572346.1 129865543.1 708 520 018467494.1 011228999.1 0010. 2 32.8 33.6 36.0 528 36.4 004081707.1 39.4 538 531 103077822.1 047754759.1 519 522 073092191.1 053334947.1 11 (mU/mg) Branching \ EGPASNVYGEVLIADTEKT otPGWLGRVRQGIE190 soutRPAGEWELPGGRKVKR A ELATK TLRP248 GEVLIADTEKT GEVLIADTEKT (mU/mg) Branching (%) identity Sequence (mU/mg) Hydrolytic GVYR236 GVYR236 β 5 B/H & Posted on Authorea 4 May 2021 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.162013331.11755851/v1 — This a preprint and has not been peer reviewed. Data may be preliminary. deletion sheet GBE Tk LE GBE Tm LS GBE Tm Y233A GBE Tk enzyme Mutant SMB8 maritima Thermotoga SLHLJ1 pacifica Kosmotoga 14811 DSM mexicana Petrotoga JAD2 holtzii hugen- Thermoflexus 11003 DSM lipolytica Thermosyntropha PNK-Is4 sp. Meiothermus MP-01 aquaticum Thermoanaerobaculum 17022 DSM timidus Calidithermus KOD1 sis dakaraen- ko- Thermococcus HB8 mophilus ther- Thermus enzyme type Wild- β - - + - + - + + - - + - - + + + + + + - + + + + + + + + + + 12 AN A/ NA 16.2 NA 26.3 NA 22.6 26.3 24.4 426.2 10.5 9.1 14.1 13.9 NA 29.1 9.8 168.0 10.6 356.9 5.5 550.0 3.5 480.0 # $ ± ± ± ± ± ± ± ± ± ± ± . 9.1 0.8 . 5.6 3.1 6.2 3.9 10.5 0.3 14.1 3.4 13.9 0.1 29.1 3.5 10.6 356.9 5.5 550.0 3.5 480.0 AN / NA NA 9.8 168.0 # ± ± ± ± ± ± ± ± ± ± ± . 2.4 0.8 . 5.6 6.2 0.4 0.1 . 1.8 0.3 1.4 3.4 0.5 0.1 6.5 3.5 15.3 12.6 11.7 4.4 ± ± ± ± ± ± ± ± ± ± ± . 3.8 0.1 38.3 0.2 . 5.7 0.5 10.0 0.3 30.6 0.1 4.7 1.5 . 4.0 4.0 0.4 0.1 . 24.1 2.5 43.6 1.7 41.0 1.5 ± ± ± ± ± ± ± ± ± ± ± 0.8 0.1 1.7 0.8 0.6 4.6 3.3 2.1 4.1 0.5 3.5 Posted on Authorea 4 May 2021 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.162013331.11755851/v1 — This a preprint and has not been peer reviewed. Data may be preliminary. al .Dsrbto fflxbelo and loop enzymes flexible branching of Distribution 4. Table $ # & A=n activity no = NA ai rnhn ciiyt yrlssactivity hydrolysis to activity branching ratio vrg ftreidpnetmaueet ihsadr deviation standard with measurements independent three of average tutrleeet eune total % sequences # no and loop No elements Structural opadno and Loop and Loop β 5- β β hes195.3 159 sheets 6 5- β β β 5- 5- hes4 2.7 42 sheets 6 β β hesaog247gyoiehdoaefml 7glycogen 57 family hydrolase glycoside 2.497 among sheets 6 hes22692 2,296 sheets 6 13