Selective oxidation of alkyl and aryl glyceryl monoethers catalysed by an engineered and immobilised glycerol dehydrogenase

Regis e ed cha i y numbe : 207890 As ea u ed in: See Elisabe Pi es, Vicen Moline , Fe nando López-Gallego e al., Chem. Sci., 2020, 11, 12009. Showcasing esea ch om P o esso López-Gallego’s labo a o y, He e ogeneous Bioca alysis G oup, Cen e o Coope a i e Resea ch in Bioma e ials (CIC biomaGUNE) Basque Resea ch and Technology Alliance (BRTA), Donos ia-San Sebas ián, Spain. Selec i e oxida ion o alkyl and a yl glyce yl monoe he s ca alysed by an enginee ed and immobilised glyce ol dehyd ogenase A new a ian o glyce ol dehyd ogenase om Bacillus s ea o he mophilus has been enginee ed and immobilised on po ous mic obeads o accep a ba e y o alkyl/a yl glyce yl monoe he s as subs a es. QM/MM compu a ional s udies e eal he excellen S-enano oselec i i y owa ds he glyce ol de i a i es and si e-di ec ed immobilisa ion echniques allow he pe o ming o kine ic esolu ion o acemic (R)-3-(alkoxy/ a yloxy)p opan-1,2-diols- This he e ogeneous bioca alys is e-usable and can be coupled wi h an NAD+ ecycling sys em o in ensi y he p ocess. sc.li/chemical-science Selec i e oxida ion o alkyl and a yl glyce yl monoe he s ca alysed by an enginee ed and immobilised glyce ol dehyd ogenase†‡ Susana Velasco-Lozano, a Mai e Roca, b Alejand o Leal-Duaso, a Jos´ e A. Mayo al, ac Elisabe Pi es, * ac Vicen Moline * b and Fe nando L´ opez- Gallego * ade Enzymes ac ing o e glyce yl e he s a e sca ce in li ing cells, and consequen ly bioca aly ic ans o ma ions o hese molecules a e a e despi e hei in e es o indus ial chemis y. In his wo k, we ha e enginee ed and immobilised a glyce ol dehyd ogenase om Bacillus s ea o he mophilus (BsGlyDH) o accep a ba e y o alkyl/a yl glyce yl monoe he s and ca alyse hei enan ioselec i e oxida ion o yield he co esponding 3- alkoxy/a yloxy-1-hyd oxyace ones. QM/MM compu a ional s udies deciphe he key ole o D123 in he oxida ion ca aly ic mechanism, and e eal ha his enzyme is highly enan ioselec i e owa ds S-isome s (ee > 99%). Th ough s uc u e-guided si e-selec i e mu agenesis, we find ha he mu a ion L252A sculp s he ac i e si e o accommoda e a p oduc i e configu a ion o 3-monoalkyl glyce ols. This mu a ion enhances he k ca 163- old owa ds 3-e hoxyp opan-1,2-diol, esul ing in a specific ac i i y simila o he one ound o he wild- ype owa ds glyce ol. Fu he mo e, we immobilised he L252A a ian o in ensi y he p ocess, demons a ing he eusabili y and inc easing he ope a ional s abili y o he esul ing he e ogeneous bioca alys . Finally, we manage o in eg a e his immobilised enzyme in o a one-po chemoenzyma ic p ocess o con e glycidol and e hanol in o 3-e hoxy-1-hyd oxyace one and (R)-3-e hoxyp opan-1,2-diol, wi hou affec ing he oxida ion ac i i y. These esul s hus expand he uses o enginee ed glyce ol dehyd ogenases in applied bioca alysis o he kine ic esolu ion o glyce ol e he s and he manu ac u ing o subs i u ed hyd oxyace ones. In oduc ion Enzymes ac ing o e glyce yl e he subs a es a e sca ce in cell me abolisms. Among he low di e si y o biochemical eac ions in ol ing glyce yl e he s, we highligh lignin biosyn hesis, 1 he me abolic syn hesis o e he lipids 2 and plasmalogens 3–5 ha play impo an physiological oles. 6 Despi e he low abundance o glyce ol de i ed alkyl e he s in na u e, hese molecules a e eno mously a ac i e o he chemical indus y o manu ac u e bo h commodi ies 7 (i.e. uels and sol en s) 8 and specialised p oduc s (i.e. cosme ics and d ugs). 9–11 In pa icula , monoalkyla ed glyce ol e he s a e conside ed among he mos p omising a ge s o alo ise he su plus o glyce ol om bio ene y ac i i ies. 12–14 The chemical s uc u e o many app o ed and comme cial d ugs inco po a es a glyce ol skele on wi h a leas one alkoxy o a yloxy subs i uen (Scheme 1A). Some o hese d ugs a e adminis e ed as pu e enan iome s which encou ages medical chemis s o de elop enan ioselec i e syn he ic schemes. He ein, bioca alysis bu s s as a key enabling echnology in chemical manu ac u ing due o he exquisi e egio- and enan ioselec i i y o enzymes. In he las decade, he pha maceu ical indus y is success- ully in eg a ing mo e bioca aly ic s eps in hei d ug syn he ic p ocesses; new enzyme ou es o he manu ac u ing o si a- glip in 15 and isla a i 16 illus a e his end. Syn hesis o monoalkyl/a yl glyce ols ha e been a ained h ough many diffe en chemical me hodologies, including bo h s oichiome ic and ca aly ic ones. 17 Recen ly, Leal-Duaso a Ca ´ alisis He e og´ enea en S´ ın esis O g´ anicas Selec i as, Ins i u o de S´ ın esis Qu´ ımica y Ca ´ alisis Homog´ enea (ISQCH-CSIC), Uni e si y o Za agoza, Ped o Ce buna, 12, 50009, Za agoza, Spain. E-mail: [email protected]; [email p o ec ed] b Depa amen de Qu´ ımica F´ ısica i Anal´ ı ica, Uni e si a Jaume I, 12071 Cas ell´ o, Spain. E-mail: moline @uji.es c Dep o. de Qu´ ımica O g´ anica, Facul ad de Ciencias, Uni e si y o Za agoza, Ped o Ce buna, 12, 50009, Za agoza, Spain d He e ogeneous Bioca alysis Labo a o y, Cen e o Coope a i e Resea ch in Bioma e ials (CIC biomaGUNE), Basque Resea ch and Technology Alliance (BRTA), Paseo de Mi am´ on 182, 20014, Donos ia San Sebas i´ an, Spain e IKERBASQUE, Basque Founda ion o Science, Ma ´ ıaD ´ ıaz de Ha o 3, 48013 Bilbao, Spain †We wan o dedica e his wo k o he memo y o P o . Jos´ e Ignacio Ga c´ ıa who ge mina ed his wo k, encou aged us o ace his challenge and ac ed as a p iceless ad iso , p o iding answe s and cons an ly suppo ing us. Res in peace. ‡Elec onic supplemen a y in o ma ion (ESI) a ailable. See DOI: 10.1039/d0sc04471g Ci e his: Chem. Sci., 2020, 11, 12009 All publica ion cha ges o his a icle ha e been paid o by he Royal Socie y o Chemis y Recei ed 14 h Augus 2020 Accep ed 5 h Oc obe 2020 DOI: 10.1039/d0sc04471g sc.li/chemical-science This jou nal is © The Royal Socie y o Chemis y 2020 Chem. Sci.,2020,11,12009–12020 | 12009 Chemical Science EDGE ARTICLE Open Access A icle. Published on 05 Oc obe 2020. Downloaded on 3/8/2021 9:50:12 AM. This a icle is licensed unde a C ea i e Commons A ibu ion-NonComme cial 3.0 Unpo ed Licence. View A icle Online View Jou nal | View Issue e al. epo ed a s aigh o wa d me hodology o syn hesise glyce yl monoe he s om glycidol and se e al alcohols using KOH as ca alys (Scheme 1B). 18 Ne e heless, he eac ion yields a acemic p oduc ha makes he isola ion o he a ge enan- iome a non- iable ask o p ac ical pu poses. To access one o he enan iome s, a kine ic esolu ion is demanded. In his con ex , lipases 19,20 a e efficien ca alys s o selec i ely ace yla e only one enan iome o glyce ol de i a i es, so he un eac i e enan iome is easily pu ied. Al hough alcohol dehyd ogenases ha e also been applied o kine ic esolu ion o sec-alcohols, 21 hey ha e ne e been applied wi h glyce ol de i a i es. The enan ioselec i e oxida ion o monoalkyl glyce ols will p o ide a double oppo uni y o access enan iome ically pu e alkyl glyce ols as building blocks o d ug syn hesis and manu ac u e o 3-alkoxy/a yloxy-1-hyd oxyace one as a new amily o bio- based sol en s o be explo ed (Scheme 1B). 8 Mo eo e , enzy- ma ic syn hesis o hese hyd oxyace one monoe he s is unp eceden ed bu highly in e es ing as key building blocks o he biosyn hesis o non-na u al deoxysuga s 22 and cosme ics. 23 The bes candida es o ca alyse such selec i e oxida ion would be he glyce ol dehyd ogenases (GlyDH) due o hei na u al subs a e specici y. Bio echnological applica ions o hese enzymes howe e a e mainly limi ed o he whole cell p oduc ion o dihyd oxyace one 24 and aluable diols h ough me abolic enginee ing. 25 Besides, his amily o enzymes has also been widely applied in biosensing as he ac i e phase o glyce ol senso s. 26 Ne e heless, hei subs a e scope is ex emely na ow, es ic ed mainly o glyce ol in he oxida ion di ec ion. Among he ew excep ions, he wild- ype GlyDHs om Klebsiella genus a e able o oxidise a subs a e bulkie han glyce ol, and ha p omiscui y inc eases when eplacing he ca aly ic Zn 2+ by o he di alen me als. 27 Rema kably, he GlyDH om En e obac e (Klebsiella)ae ogenes oxidises he R-phenyl- glycol o 2-hyd oxyace ophenone in a egio- and enan iose- lec i e manne . 28 Hund eds o examples endo se he imp o emen o enzyme p ope ies (ac i i y, s abili y and selec i i y) and he widening o hei subs a e scope h ough p o ein enginee ing. As sa e pa h o enzyme op imiza ion, he scien ic communi y exploi s, ei he sepa a ely o syne gis ically, andom and ex ensi e mu agenesis campaigns h ough di ec ed e olu ion 29 and minimalis mu a ions h ough si e-di ec ed mu agenesis guided by s uc u al knowledge. 22,30,31 In he pa icula case o GlyDH, one o he ew documen ed successes is he combina- ion o DNA-shuffling and si e-di ec ed mu agenesis o expand he GlyDH subs a e scope o o he diols. This a ian was 2.6 imes mo e ac i e owa ds 1,3-bu anediol han he wild- ype. 32 Despi e hese effo s, GlyDHs ha e ne e been challenged agains bulkie subs a es like glyce ol monoe he s. 27 Besides e-shaping he ac i e si e o enzymes o imp o e hei in insic unc ional ea u es, he enginee ing jou ney mus include he enzyme immobilisa ion o ab ica e obus bioca alys s ha a e easily o sepa a e om he eac ion media and eadily o in eg a e in o diffe en bio eac o congu a ions. In he las yea s, he immobiliza ion o enginee ed enzyme a ian s has allowed hei applica ion unde indus ially ele- an condi ions. 16,33–35 To ha aim, selec i e immobiliza ion p o ocols ha assu e he ac i i y and inc ease he s abili y o he enzymes upon he a achmen o he solid ca ie a e p e ended. In his wo k, we a ionally enginee GlyDH om Bacillus s ea o he mophilus (W -BsGlyDH) o selec i ely oxidise alkyl/a yl glyce ols o 3-alkoxy/a yloxy-1-hyd oxyace ones. Be o e s a ing he enginee ing campaign, we shined ligh on he unce ain ies ound in i s ca aly ic mechanism. Besides explaining how his enzyme posi ions he subs a e in o i s ac i e si e, Quan um Mechanics/Molecula Mechanics (QM/MM) compu a ional s udies also e eal i s enan iop e e ence owa ds he S-isome o he alkyla ed glyce ol. This in o ma ion was ins umen al o u he design mu a ions a he ac i e si e ha allowed he enzyme accommoda ing bulkie glyce ol monoe he s. F om he molecula design, we walked owa ds he p ocess de elopmen by immobilizing he mos ac i e enginee ed a ian . The esul ing he e ogeneous bioca alys eases he p oduc sepa a- ion and inc eases he enzyme ope a ional s abili y. Finally, he he e ogeneous bioca alys he ein p esen ed was coupled o a one-po sequen ial chemo-enzyma ic p ocess o syn hesise enan iopu e glyce ol monoe he s om glycidol and e hanol wi hou in e media e pu ica ion s eps (Scheme 1C). Ma e ials and me hods Chemicals Glyce ol, hyd ogen pe oxide, dihyd oxyace one (DHA), a in- adenine-dinucleo ide sodium sal (FAD + ), ace ic anhyd ide Scheme 1 (A) D ugs con aining alkyl/a yl-glyce ol scaffolds (in blue). (B) Re osyn hesis pa hway owa ds enan iopu e alkyl/a yl glyce yl monoe he s and subs i u ed hyd oxyke ones om enewable s a ing ma e ials. (C). Two-s ep chemo-enzyma ic ou e o access (R)-3- (alkoxy/a yloxy)p opan-1,2-diol and hei co esponding 3-alkoxy/ a yloxy-1-hyd oxyace one. 12010 |Chem. Sci.,2020,11,12009–12020 This jou nal is © The Royal Socie y o Chemis y 2020 Chemical Science Edge A icle Open Access A icle. Published on 05 Oc obe 2020. Downloaded on 3/8/2021 9:50:12 AM. This a icle is licensed unde a C ea i e Commons A ibu ion-NonComme cial 3.0 Unpo ed Licence. View A icle Online and N-me hylimidazole, kanamycin sul a e om S ep omyces kanamyce icus and ampicillin, ca alase om bo ine li e (Hl- CAT) we e acqui ed om Sigma-Ald ich (S . Louis, IL, USA). Nico inamide adenine dinucleo ide educed disodium sal (NADH), nico inamide adenine dinucleo ide sodium sal (NAD + ) and isop opyl b-D- hiogalac opy anoside (IPTG), we e pu chased om GERBU Bio echnik GmbH (Wieblingen, Ge - many). Glyce yl monoe he s and die he s we e syn hesised as p e iously desc ibed (Table S1‡). 18,36 Cobal -ac i a ed aga ose mic obeads 4BCL (AG-Co 2+ ) (pa icle size; 50–15 mm, po e size; 112 nm and 15 mmol o Co 2+ pe g ca ie ) we e pu chased om ABT echnologies (Mad id, Spain). P ecision plus p o ein™ s anda ds, mic o Bio-spin™ch oma og aphic columns and B ad o d eagen we e acqui ed om BIORAD. All o he eagen s and sol en s we e analy ical g ade o supe io . Me hods Si e-di ec ed mu agenesis o c ea e he a ian s o GlyDH. A si e-di ec ed mu agenesis p o ocol was used o cons uc se en GlyDH mu an s ( ee single mu an s, V131A, Y142A, L252A; h ee double mu an s, V131A/L252A, V131A/Y142A, Y142A/ L252A; and one iple mu an , V131A/Y142A/L252A). These mu an s we e cons uc ed by using he W -GlyDH gene as empla e. The gene ic cons uc pET28b(+)_his-glydh was de eloped in a p e ious wo k. 37 B iey, o in oduce he desi ed amino acid change, he co esponding pai s o oligonucleo ides (Table S2‡) we e used as p ime s in he polyme ase chain eac ion (PCR) using he abo e men ioned cons uc as empla e and high deli y NZYP oo DNA polyme ase. The p oduc o he PCR was diges ed wi h DpnI ha exclusi ely es ic s me hyla ed DNA. E. coli DH5acells we e ans o med di ec ly wi h he diges ion p oduc . The plasmids ha ha bou he mu a ed GlyDH genes we e iden ied by sequencing and hen ans o med in o E. coli BL21(DE3) cells o exp ess he co esponding enzyme a ian s. Cloning o his- agged wa e o ming NADH oxidase a ian s om Lac obacillus pen osus (Lp-NOX). The gene desc ibed by Nowak e al. 38 was op imised o E. coli codon usage and syn- hesised by Gensc ip Bio ech (Pisca away, NJ, USA). The syn he ic gen was cloned in o pET28b(+) using NdeI and XhoI es ic ion si es. DNA isola ion, plasmid pu ica ion, es ic ion analysis, plasmid cons uc ion and DNA sequencing we e ca ied ou by s anda d me hods. 39 Bac e ial s ains and g ow h condi ions. Glyce ol dehyd o- genase om Bacillus S ea o he mophilus (BsGlyDH), NADH oxidases om The mus he mophilus (T -NOX) and Lac obacillus pen osus (Lp-NOX) we e o e exp essed in E. coli BL21 cells as p e iously epo ed. 37,40 B iey, 1 mL o an o e nigh cul u e o E. coli BL21(DE3) ha bo ing each plasmid was used o inocula e 50 mL o Lu ia-Be ain (LB) medium con aining he co e- sponding an ibio ic; kanamycin (nal concen a ion 30 mg mL 1 ) was used o all enzymes bu T -NOX ha equi ed ampicillin (nal concen a ion 50 mgmL 1 ). The esul ing cul u e was ae obically incuba ed a 37 C wi h o bi al shaking a 250 pm un il he OD 600 nm eached 0.6. Ae wa ds, he cul u e was induced wi h 1 mM IPTG. All enzymes we e induced o 3 h a 37 C bu Lp-NOX was incuba ed a 21 C o 18 h. Ae he induc ion ime, cells we e ha es ed by cen i uga ion a 4211 g o 30 min a 4 C. Supe na an s we e disca ded and he pelle was esuspended in 5 mL o 25 mM sodium phospha e buffe solu ion a pH 7. Cells we e b oken by sonica ion using a Sonoplus Se ie 4200, Bandelin a 20% ampli ude (5 s ON/5 s OFF) o 20 min a 4 C. The suspension was hen cen i uged a 10 528 g o 30 min a 4 C and he pelle was disca ded. The supe na an con aining cell ex ac s wi h he His- agged p o eins (excep T -NOX) we e collec ed and employed o u he pu ica ion and/o immobiliza ion. Pu ica ion o enzymes. The pu ica ion o GlyDH and he a ian s, as well as he Lp-NOX we e done acco ding he ollowing p ocedu e: 10 olumes o c ude cell ex ac con aining he his- agged enzymes we e mixed wi h 1 olume o AG-Co 2+ mic obeads and incuba ed unde o bi al shaking o 1 o 2 h a 4C. La e , he suspension was l e ed and he mic obeads con aining he enzyme we e washed wi h 5 olumes o 25 mM phospha e buffe a pH 7. Ae wa ds, enzymes we e elu ed by he addi ion o 5 olumes o 300 mM imidazole in sodium phospha e buffe 25 mM pH 7 and incuba ed o 1 h a 4 C wi h o bi al shaking. The elu ed p o ein was subjec ed o dialysis o emo e he imidazole. Finally, SDS-PAGE and B ad o d p o ein assay 41 we e ca ied ou ae each p oduc ion o de e mine he pu i y, concen a ion and specic ac i i y o he enzymes (Fig. S1‡). T -NOX was pu ied as p e iously desc ibed h ough he mal shock. 40 Enzyma ic ac i i y measu emen s. Enzyme ac i i ies we e spec opho ome ically measu ed in anspa en 96-well mic opla es, employing a Mic opla e Reade Epoch 2, BioTek® wi h he sowa e Gen5. GlyDH ac i i y (colo ime ic assay). (1a, ac-1b, ac-1c, ac-1d, ac-1e, ac-1 , ac-1g, ac-1h,1b-bis and 1c-bis) and 1 mM NAD + in sodium phospha e o sodium bica bona e buffe 100 mM a pH 7 o 9, espec i ely; we e incuba ed wi h 5 mL o enzyma ic solu ion o suspension a 30 C. The inc ease o he abso bance was moni o ed a 340 nm. One uni o ac i i y was dened as he amoun o enzyme ha was equi ed o educe 1 mmol o NAD + pe minu e a he assayed condi ions. NOX ac i i y (colo ime ic assay). 200 mL o a eac ion mix u e con aining 0.2 mM NADH and 150 mM FAD + in phospha e buffe pH 7 we e incuba ed wi h 5 mL o enzyma ic solu ion o suspension a 30 C. The dec ease in he abso bance was moni o ed a 340 nm. One uni o ac i i y was dened as he amoun o enzyme ha was equi ed o oxidise 1 mmol o NADH pe minu e a he assayed condi ions. CAT ac i i y (colo ime ic assay). The ac i i y was de e mined by eco ding he dec ease in he abso bance a 240 nm o 200 mL o a eac ion mix u e con aining 35 mM hyd ogen pe oxide in sodium phospha e buffe 100 mM pH 7 a 30 C. The eac ion was ini ia ed by adding 5 mL o he enzyma ic solu ion o suspension o he eac ion mix u e. One uni o CAT ac i i y was dened as he amoun o enzyme equi ed o he disp opo - iona ion o one mmol o hyd ogen pe oxide pe minu e a he assessed condi ions. Co-immobiliza ion o Lp-NOX and BsGlyDH-L252A on AG- Co 2+ .The enzymes we e immobilised by mixing 10 olumes o This jou nal is © The Royal Socie y o Chemis y 2020 Chem. Sci.,2020,11,12009–12020 | 12011 Edge A icle Chemical Science Open Access A icle. Published on 05 Oc obe 2020. Downloaded on 3/8/2021 9:50:12 AM. This a icle is licensed unde a C ea i e Commons A ibu ion-NonComme cial 3.0 Unpo ed Licence. View A icle Online c ude cell ex ac con aining he His- agged p o ein wi h 1 olume o AG-Co 2+ mic obeads and incuba ed unde o bi al shaking o 1 o 2 h a 4 C. La e , he suspension was l e ed and he mic obeads con aining he enzyme we e washed wi h 5 olumes o 25 mM phospha e buffe a pH 7. The immobilised bioca alys was s o ed a 4 C. The co-immobilised bioca alys comp ising GlyDH-L252A and Lp-NOX whe e p epa ed ollowing he o me me hodology bu sequen ially immobiliz- ing one by one enzyme, s a ing by he GlyDH-L252A ollowed by he Lp-NOX. Oxida ion o glyce yl e he s by GlyDH-L252A bioca alys s. Fo ac-1c, immobilised GlyDH-L252A on AG-Co 2+ , T -NOX and Hl-CAT we e mixed wi h 0.3–50 mL o 25–100 mM subs a e, as indica ed, 1 mM NAD + , 150 mM FAD + in 100 mM sodium phospha e buffe a pH 7. Fo ac-1h, co-immobilised GlyDH- L252A and Lp-NOX on AG-Co 2+ was mixed wi h 1 mL o 2 mM subs a e, 4 mM NAD + in 100 mM sodium phospha e buffe a pH 7. Bo h mix u es we e incuba ed a 30 C in a e ical o a ing shake a 40 pm. Reac ion cu se was moni o ed by wi hd awing samples a pe iodic in e als ha we e analysed by ch oma og aphic me hods. Chemo-enzyma ic syn hesis. 0.87 mmol o KOH we e sol ed in 3.81 mL o d ied absolu e e hanol (15 : 1 mol a io in espec o glycidol). Then he mix u e was hea ed a 65 C and 0.3 mL o ac-glycidol (4.35 mmol) we e added d opwise o 15 min. Ae 2 hou s, when ac-glycidol has comple ely been con e ed o ac- 1c, he empe a u e was cooled down o 25 C, KOH neu alised wi h 0.3 M HCl and he sal s l e ed off. He e ogeneous bioca alys GlyDH-L252A co-immobilised wi h Lp-NOX on AG-Co 2+ (100 mg bioca alys ) was placed inside a 1.5 mL Bio-spin™ch oma og aphic column. Then, 500 mL o eac ion mix u e consis ed in 20 mM o he ac-1c subs a e (pu e, aw om chemosyn hesis wi h e hanol and aw om chemosyn hesis wi hou e hanol), 1 mM NAD + , 150 mM FAD + in 100 mM sodium phospha e buffe a pH 8 and we e added and incuba ed a 30 C in a e ical o a ing shake a 40 pm. Ae 24 and 48 h, columns we e spin down and he eco e ed eac ion mix u es we e analysed as desc ibed in he ch oma og aphic me hods. Ope a ional s abili y o bioca alys . Recycling o he e oge- neous bioca alys BsGlyDH-L252A immobilised on AG-Co 2+ (144 mU, 100 mg bioca alys ) was de e mined by placing he bioca alys inside a 1.5 mL Bio-spin™ch oma og aphic column wi h 300 mL o eac ion mix u e consis ed in 25 mM o ac-1c, 1 mM NAD + , 150 mM FAD + , 720 mU o soluble T -NOX and 36 000 mU o Hl-CAT in 100 mM sodium phospha e buffe a pH 7 and incuba ed a 30 C in a e ical o a ing shake a 40 pm. Ae each eac ion cycle (24 h), columns we e spin down and washed one ime wi h one olume o 25 mM sodium phospha e buffe a pH 7 be o e s a ing he nex eac ion cycle. Reco e ed eac ion mix u es we e analysed as desc ibed in he ch oma og aphic me hods. Ch oma og aphic me hods Gas ch oma og aphy (GC). P io GC analysis, samples we e de i a ised as desc ibed elsewhe e. 42 B iey, 30 mL o aqueous eac ion simple we e placed in a 1.5 mL Eppendo ube, ol- lowed by he addi ion o 30 mLo N-me hylimidazole and 225 mL o ace ic anhyd ide and incuba ed by 10 min a oom empe - a u e. Ae wa ds, 300 mL o dis illed wa e was added and allowed o cool down. La e , liquid–liquid ex ac ion o ace y- la ed compounds was done by he addi ion o 300 mLo dichlo ome hane con aining 2 mM eicosane as ex e nal s an- da d disca ding he aqueous phase. 30–50 mg o anhyd ous MgSO 4 we e added o d y samples be o e GC analysis. Gas ch oma og aphy analyses we e ca ied ou in a Hewle Packa d 7890 Se ies II gas ch oma og aph using a column o phenyl silicone 5.5% (Zeb on ZB-5HT In e no 30 m 0.25 mm 0.25 mm), helium as ca ie gas, and equipped wi h a ame ioniza- ion de ec o (FID). Injec o a 250 C, FID a 280 C. Sepa a ion o 1c de i a i es we e done by he ollowing empe a u e p og am: ini ial empe a u e a 60 C, main ained 2 min, amp o 240 C a a a e o 20 C min 1 and nally main ained 2 min. Re en ion imes o ace yla ed compounds we e: ac-1c:7.88min, 3-e hoxy-1-hyd oxyace one: 6.75 min and eicosane: 12.2 min. The samples we e addi ionally analysed using a Hewle Packa d 6890 Se ies II Gas ch oma og aph using a column o phenyl silicone 5.5% (Zeb on ZB-5HT In e no 30 m 0.25 mm 0.25 mm), coupled o an Agilen 5973 ine Mass Spec ome e wi h Elec- onic Impac ioniza ion. Chi al high pe o mance liquid ch oma og aphy (HPLC). Be o e hei analysis, samples we e l e ed and dilu ed in iso- p opanol as equi ed. P epa ed samples we e analysed by HPLC (Wa e s 2690) wi h a Lux 5 mcellulose-1 chi al column (250 mm  4.60 mm) Phenomenex, equipped wi h a PDA de ec o a 270 nm. Analy es we e elu ed a 1 mL min 1 cons an ow wi h a no mal mobile phase composed by hexane/isop opanol (85 : 15). 43 Re en- ion imes we e: R-1h: 11.9 min, S-1h:23.4minand1-phenoxy-3- hyd oxyace one (2h): 5.4 min. QM/MM compu a ional me hods. All he sys ems, wild- ype BsGlyDH wi h glyce ol, S-1c and R-1c enan iome s, BsGlyDH- L252A mu an wi h glyce ol, S-1c and R-1c enan iome s and BsGlyDH-D123N mu an wi h glyce ol we e sol a ed by a box o wa e molecules and we e neu alised adding coun e ions by LEaP module 44 om Ambe Tools17 package. 45 P elimina y minimiza ions and molecula mechanics (MM) and molecula dynamics (MD) simula ions we e pe o med in o de o equili- b a e he sys ems wi h he AMBER ff14SB and TIP3P o ce elds o desc ibe he p o ein and wa e molecules, espec i ely. Explo a ion o he QM/MM po en ial ene gy su aces (PESs), and localiza ion and cha ac e iza ion o ansi ion s a e (TS) s uc u es we e ca ied ou p io o he gene a ion o he QM/ MM ee ene gy su aces gene a ed in e ms o po en ials o mean o ce (PMFs) a M06-2X(6-31+G(d,p)):PM3/AMBER/TIP3P PMFs le el o heo y, including ze o poin ene gy (ZPE) co ec- ions. Full compu a ional de ails a e gi en in he ESI.‡ Resul s and discussion Enginee ing o GlyDH and mechanis ic insigh s Inspi ed by p e ious wo ks, 46,47 we ske ched a e osyn he ic pa hway o access enan iopu e alkyl/a yl glyce yl monoe he s s a ing om inexpensi e epoxides such as glycidol, which can be eadily syn hesized om glyce ol (Scheme 1B). Once acemic glyce ol e he s a e chemically syn hesized unde basic 12012 |Chem. Sci.,2020,11,12009–12020 This jou nal is © The Royal Socie y o Chemis y 2020 Chemical Science Edge A icle Open Access A icle. Published on 05 Oc obe 2020. Downloaded on 3/8/2021 9:50:12 AM. This a icle is licensed unde a C ea i e Commons A ibu ion-NonComme cial 3.0 Unpo ed Licence. View A icle Online condi ions, 18 we p opose hei sequen ial enzyma ic egio- and enan ioselec i e oxida ion. Un o una ely, none oxido educ ase has been desc ibed o efficien ly conduc he selec i e oxida ion owa ds his ype o monoglyce yl e he s. GlyDH is one o he po en ial enzymes o ca alyse ha eac ion, since i efficien ly oxidises glyce ol o dihyd oxyace one. 48 Ne e heless, i s oxida- ion ac i i y is p ac ically es ic ed o i s na u al subs a e: glyce ol. To go beyond glyce ol, we es ed wo GlyDHs om diffe en bac e ial sou ces o he selec i e oxida ion o mono- alkylglyce ols. We de ec ed some oxida i e ac i i y owa ds a ba e y o pu e monoalkylglyce yl e he s using bo h wild- ype GlyDH om Cellulomonas sp. (CsGlyDH) and om Bacillus s ea o he mophilus (BsGlyDH) (Table S3‡). These esul s ag ee wi h he esidual ac i i y p e iously epo ed o CsGlyDH owa ds 1b. 49 Rema kably, he ela i e ac i i y o he enzyme om Bacillus was highe han he one om Cellulomonas owa ds a wide scope o glyce ol e he s. These insigh s mo i- a ed us o enginee BsGlyDH h ough a s uc u e-guided app oach. The epo ed c ys al s uc u es (PDB ID: 1JQ5 and 1JQA) 48 o BsGlyDH show ha O1 and O2 a oms o glyce ol a e coo di- na ed o he ca aly ic Zn 2+ a om, which explains he exquisi e specici y o his enzyme o 1,2-diols as subs a es. In con as , he O3 seems o be neu al in he ca aly ic mechanism bu i is conned in o he ca i y o med by V131, Y142 and L252, sup- po ing he ac , ha glyce ol is he p e e en ial subs a e (Fig. 1). Despi e he a ailable s uc u al in o ma ion, he p ecise ca aly ic mechanism o hyd ide ans e om C2 a om o glyce ol o C4 a om in NAD + emains unclea (Fig. 2). Then, be o e sculp ing he ac i e si e o accommoda e bulkie subs i uen s in O3, we decided o explo e he ee ene gy p oles o un eil he BsGlyDH mechanism a a omis ic le el (see ESI‡). In he li e a u e, a wa e molecule is sugges ed o ac as a base o abs ac he p o on om he C2 hyd oxyl g oup and o m he alkoxide in e media e o subsequen ly p oceed wi h he hyd ide ans e om C2 a om o glyce ol o NAD + co ac o . 48 Howe e , his mechanism has been demons a ed nei he h ough expe imen al no compu a ional e idences. The e a e o he sho - chain alcohol dehyd ogenases/ educ ases (SDRs) om D osophila melanogas e and D osophila lebanonensis 50 ha con ain highly conse ed ac i e si e esidues (Se and Ty ) ha ac as s ong base o he p o on abs ac ion. Mo eo e , in manni ol 2-dehyd ogenase om Pseudomonas uo escens, 51 a Lys esidue unc ions as he ca aly ic base ha acili a es hyd ide ans e o NAD + by p o on abs ac ion om alcohol. Classical MD simula ions o he ully sol a ed subs a e–enzyme complex keep he Zn 2+ ion  e coo di- na e complex, as in he ini ial X- ay s uc u e o Ruzheiniko e al., 48 including he coo dina ion o O2 a om. Ne e heless, wo diffe en con o ma ions o glyce ol bound o he ac i e cen e a e iden ied (Fig. 2 and S2–S4‡). In one con o ma ion he p o on om O2 a om o glyce ol es ablishes hyd ogen bond in e ac ion wi h a wa e molecule while in he o he con o ma ion; his p o on es ablishes a hyd ogen bond wi h D123 esidue. This ac led us o p opose wo diffe en eac ion mechanisms (Fig. 2). Mechanism 1 in ol es he p o on ans e om O2 a om o glyce ol o a wa e molecule ol- lowed by a hyd ide ans e om C2 a om o glyce ol o C4 a om o NAD + as p oposed in he li e a u e. 48 Con a iwise, mechanism 2 leads o a p o on ans e be ween he subs a e and he acid esidue be o e he hyd ide ans e om glyce ol o NAD + .To deciphe he mos likely mechanism, we gene a ed he ull ee ene gy landscapes o he wo p oposed mechanisms by means o mul iscale QM/MM simula ions. In pa icula , ee ene gy su aces we e compu ed o e e y chemical s ep as po en ials o mean o ce (PMFs) a M06-2X/6-31+G(d,p):PM3/MM le el o heo y (see ESI Fig. S5 and S6‡). Fig. 1 O e lap o c ys al s uc u es o GlyDH om Bacillus s ea - o he mophilus binding i s na i e subs a e glyce ol (pink s icks/PDB ID: 1JQA), he ca aly ic Zn 2+ (blue sphe e/PDB ID: 1JQ5) and he NAD + (yellow s icks/PDB ID: 1JQ5). Residues V131, Y142 and L252 ha o m he subs a e ca i y a e highligh ed as g een sphe es. Black dash line ep esen s he hyd ide ans e om C2 a om o glyce ol o C4 a om o NAD + (see Fig. 2). Yellow dash lines ep esen he coo dina ion o O1 and O2 a oms om glyce ol wi h he Zn 2+ a om. Fig. 2 P oposed glyce ol oxida ion mechanisms o W -BsGlyDH. (1) P o on ans e h ough wa e molecule. (2) P o on ans e h ough he Asp123. R: glyce ol/TS: ansi ion s a e o mechanism 1/TS1 and TS2 ansi ion s a e o mechanism 2/I1: alkoxide in e media e/P: dihyd oxyace one. This jou nal is © The Royal Socie y o Chemis y 2020 Chem. Sci.,2020,11,12009–12020 | 12013 Edge A icle Chemical Science Open Access A icle. Published on 05 Oc obe 2020. Downloaded on 3/8/2021 9:50:12 AM. This a icle is licensed unde a C ea i e Commons A ibu ion-NonComme cial 3.0 Unpo ed Licence. View A icle Online Acco ding o he esul s, mechanism 1 can be disca ded due o he esul ing o e all high ee ene gy ba ie (57.6 kcal mol 1 ), indica ing ha wa e is unable o ac as base o he p o on sub ac ion om he O2 hyd oxyl g oup ( o u he de ails see ESI, Fig. S5 and S6A‡). Fu he mo e, ou M06-2X/6-31+G(d,p):PM3/MM ee ene gy su aces and M06-2X/ 6-31+G(d,p)/MM po en ial ene gy su aces con ms ha mechanism 2 akes place in a s epwise manne (Fig. S5 and S6B‡), consis ing in he p o on abs ac ion o he alcohol by D123 esidue ollowed by he hyd ide ans e om glyce ol o NAD + . This second s ep, ha is he a e-limi ing one, ende s an ac i a ion ee ene gy (17.1 kcal mol 1 ) ha ag ees wi h he alues ha can be de i ed om he expe imen al a e cons an s (16.9 kcal mol 1 ).§To expe imen ally con m he mechanism 2, we cons uc ed he mu an D123N ha keeps he hyd ogen bond in e ac ions be ween glyce ol and he amide g oup o D123N wi hou affec ing he size bu p ecluding he o ma ion o he alkoxy in e media e, as he amide g oup is unable o sub ac he p o on. D123N mu an indeed abolished he oxida i e ac i i y (Fig. S7‡), which ag ees wi h he analysis o he geome ies de i ed om classical MD simula ions ha show no al e na i e base in he ac i e si e. Hence, we p opose he ca boxyla e o D123 as he base needed o abs ac ing he p o on o o m he alkoxy in e media e ha p ecedes he hyd ide ans e o he nico inamide co ac o (Fig. 2). Fo he  s ime, we p esen bo h expe imen al and compu a ional da a ha unambiguously un eil he key ole o D123 in he ca aly ic mechanism o a glyce ol dehyd ogenase (Fig. 2, mechanism 2, Fig. S5‡). This esidue is highly conse ed in GlyDHs and glyce ol 1-phospha e dehyd ogenases (Fig. S8 and S9‡), which suppo s i s impo an ole in he ca aly ic mecha- nism. Unlike manni ol dehyd ogenases 52 (medium chain dehyd ogenases) and sho -chain dehyd ogenases, 50 whe e Lys and Ty esidues ac s as gene al base, espec i ely, BsGlyDH uses he ca boxyla e o D123 o sub ac he p o on om he seconda y alcohol o glyce ol. 53–56 This conse ed esidue is no ound in i on-con aining polyol dehyd ogenases, like 1,2-p op- anodiol dehyd ogenase. 57 Once he oxida ion mechanism has been elucida ed, we a ionally selec ed h ee posi ions o sculp he ac i e si e o BsGlyDH o be e accommoda e bulkie glyce ol de i a i es. We pe o med an alanine scanning in he posi ions V131, Y142 and L252 since hey s e ically conne he C3 hyd oxyl g oup wi hin he enzyme binding pocke acco ding o he X- ay s uc u e (Fig. 1). We also cons uc ed he h ee possible combina ions o double mu an s and he iple one. By eplac- ing hese bulkie amino acids by alanine, we we e able o expand he sol en accessible a ea o he ac i e si e up o 108 ˚ A 2 (Table S4‡). Ne e heless, he e was no co ela ion be ween he size o he binding pocke and he ac i i y owa ds he glyce yl e he s (Fig. S10‡). All mu a ions d ama ically educed he enzyme ac i i y (<10%) owa ds he na i e subs a e 1a (glyce ol) excep BsGlyDH-V131A (Fig. 3). On he o he hand, he mu a ion Y142A conduc ed o inac i e mu an s owa ds any es ed subs a e ega dless hey we e single, double o iple a ian s. Pleasan ly, he a ian BsGlyDH-L252A p esen ed a ema kable ac i i y owa ds 1c, e en 30% highe han he oxida i e ac i i y o he W -GlyDH owa ds glyce ol. Fo all hose ac i e mu an s, we s udied hei subs a e scope using a pale e o alkyl/a yl glyce yl monoe he s (Table S1‡). BsGlyDH-L252A was he mos ac i e a ian owa ds all alkyla ed glyce ol de i a i es, while negligible ac i i y was de ec ed o he 1,3-dime hoxy (1b-bis) and 1,3-die hoxy (1c-bis) glyce yl de i a i es. The absence o ac i i y owa ds he glyce yl 1,3-dialkyle he s ag ees wi h he ac ha GlyDH equi es one ee p ima y hyd oxyl g oup o be coo dina ed wi h he ca aly ic Zn 2+ . In iguingly, widening he ac i e si e did no enhance he oxida i e ac i i y owa ds a yl de i a i es. In ac , we ound ou ha bo h wild- ype and he L252A a ian we e simila ly ac i e owa ds 1h. The second bes subs a e was 1g bea ing a 2,2,2- iuo e hyl subs i uen , which suppo s ha he 22 ˚ A 2 la ge ac i e si e also possesses he sui able shape o accommoda e CH 2 CF 3 moie ies (Table S4‡). Su p isingly, when he alipha ic chain was ei he smalle o la ge han 2 ca bons, he enzyme ac i i y d ama i- cally decayed (Fig. 3). The specic ac i i y esul s we e sup- po ed by kine ic s udies (Table 1, Fig. S11 and S12‡), which addi ionally demons a e ha sho ening he apola side chain a posi ion 252 d ama ically inc eases he K M owa ds glyce ol wi hou signican ly affec ing he k ca . In con as , mu a ion L252A signican ly inc eases (6 imes) he BsGlyDH-L252A ca aly ic efficiency owa ds 1c due o he k ca enhancemen (163 imes). This a ian showed simila ends owa ds all alkyl glyce yl de i a i es excep 1e, o which he k ca was signican ly lowe compa ed o o he subs i uen s (Table 1). BsGlyDH-L252A p esen ed simila K M alues owa ds NAD + unde he same condi ions using bo h glyce ol and 1c as subs a es, which indica es ha he mu a ion clea ly affec s he binding o he polyol a he han he co ac o (Fig. S13C–F‡). Using glyce ol as subs a e a pH 7, we also obse ed ha bo h wild- ype and mu an enzymes we e inhibi ed by NAD + a high concen a ion (>10 mM) (Fig. S13A–D‡). In he specic case o BsGlyDH- L252A, inhibi ion negligibly occu ed when using 1c as subs a e unde neu al condi ions (Fig. S13E and F‡), bu i was no iceable a pH 9 (Fig. S13G and H‡). Rema kably, he ca aly ic efficiency o BsGlyDH-L252A was 40 imes highe unde alkaline condi ions han unde pH 7, and i s op imal pH was shied 1 uni owa ds basic alues compa ed o he wild- ype enzyme Fig. 3 Specific ac i i y o wild- ype and se e al mu an s o BsGlyDH owa ds alkyl/acylglyce yl e he s. In all cases, eac ion mix u es con- sis ed in 100 mM subs a e, 1 mM NAD + in 100 mM sodium phospha e buffe a pH 7 a 30 C, 1b-bis and 1c-bis a e he co esponding dia- lkyl-e he wi h me hyl and e hyl subs i uen s, espec i ely. 12014 |Chem. Sci.,2020,11,12009–12020 This jou nal is © The Royal Socie y o Chemis y 2020 Chemical Science Edge A icle Open Access A icle. Published on 05 Oc obe 2020. Downloaded on 3/8/2021 9:50:12 AM. This a icle is licensed unde a C ea i e Commons A ibu ion-NonComme cial 3.0 Unpo ed Licence. View A icle Online (Fig. S14‡). These kine ics s udies e eal an impo an ole o pH on he glyce ol dehyd ogenase pe o mance ha affec s bo h binding (o e all o he co ac o ) and eac ion a e cons an . 58 Fig. 4, S15 and S16‡show he ee ene gies p oles and he hyd ide ans e ansi ion s a es (named TS2, see Fig. 2) esul ed om QM/MM simula ions, o he oxida ion eac ion o diffe en subs a es ca alysed by ei he he W -BsGlyDH o he BsGlyDH-L252A mu an . Ene gy ba ie s ollow he same end obse ed o he expe imen al kine ics shown in Table 1. This mu an p esen s an ene gy ba ie o 16.2 and 16.5 kcal mol 1 o bo h 1c (Fig. 4A) and glyce ol (Fig. S16‡) oxida ions, espec i ely. These alues a e sligh ly smalle han he ba ie p e iously ob- ained wi h he wild- ype enzyme using glyce ol as subs a e (17.1 kcal mol 1 ) (Fig. S5‡). Mo e in e es ingly, he ee ene gy p ole o W -BsGlyDH shows an ene gy ba ie 2.6 kcal mol 1 highe han BsGlyDH-L252A owa ds he oxida ion o 1c unde he same condi ions (Fig. 4A), in acco dance wi h he expe imen al ee ene gy ba ie s de i ed om he k ca alues (Tables 1, S5‡). We sugges ha he highe efficiency o enginee ed BsGlyDH-L252A esul s om a less s e ically impeded 1c con o ma ion wi hin he mu a ed ac i e si e, which leads o ansi ion s a es wi h lowe ene gy ba ie s han wi hin na i e one. QM/MM MD simula ions e ealed weak bu s abilizing in e ac ions ene gies be ween 1c and he esidues a posi ions 131, 142 and 252 o BsGlyDH-L252A du ing he eac an s a e. Thei syne gis ic Lenna d-Jonnes in e - ac ions signican ly con ibu e o he 1c binding (Fig. S17 and S18‡). The e o e, he inse ion o Ala a posi ion 252 bu keeping he na i e esidues a posi ions V131 and Y142 seems o op imise he binding pocke o imp o e he s abili y o he ansi ion s a es wi h he e hyl glyce yl monoe he . Fig. 4B shows indeed a o a ion (125 deg ees) o he CH 3 - o e hoxy subs i uen o 1c wi hin he less hinde ed L252A ac i e si e. Simila esul s we e ound o he a ional si e-di ec ed mu agenesis o a shuffled GlyDH, whe e he eplacemen o one aspa ic ha clashed wi h 1,3-bu anodiol enhanced he oxida i e ac i i y up o 2.6 imes compa ed o he wild- ype a ian . 32 The excellen oxida i e ac i i y o BsGlyDH-L252A encou - aged us o ca y ou he comple e biocon e sion o 1c and cha ac e ise he esul ing p oduc . Since his bio ans o ma ion is NAD + -dependen , we mixed BsGlyDH-L252A wi h an NADH oxidase om The mus he mophilus HB27 (T -NOX) and he ho se li e ca alase (Hl-CAT) in one-po , aiming a bo h in si u ecycling he co ac o and emo ing he H 2 O 2 ; a oxic by- p oduc om he ecycling sys em. Using he soluble enzymes, he eac ion oughly eached 25% subs a e con e - sion in 72 hou s wi h enzyme and co ac o o al u no e numbe s o 3491 and 13, espec i ely (Table 2, en y 1). Despi e ha low con e sion, we cha ac e ised he o med p oduc h ough 13 C and 1 H NMR (Fig. S19–S22‡), and mass spec om- e y (Fig. S23–S29‡). The analyses con m ha BsGlyDH-L252A oxidises he C2 o 1c as he wild- ype GlyDH does o he glyc- e ol. Rema kably, we did no de ec he o ma ion o he alde- hyde, which indica es ha he mu a ion a he ac i e si e al e s he subs a e specici y bu no he enzyme egioselec i i y. Al hough p oduc yields we e signican ly lowe han hose ones ob ained h ough chemical me hods, 59 he egioselec i i y o his a ian owa ds he C2 o he alkylglyce yl subs a e hus enables he syn hesis o he co esponding 3-alkoxy-1- hyd oxyace ones unde mild condi ions using wa e as sol en . Mo e impo an ly, he enzyma ic ou e smoo hly uns wi hou p o ec ion/dep o ec ion s eps unde acidic condi ions ha would p o oke he decomposi ion o he p oduc o me hyl glyoxal, subs an ially dec easing he isola ed yields. 59 Bio ans o ma ion o alkyl/a yl glyce yl e he s using immobilised GlyDH-L252A In o de o in ensi y he p ocess and walk owa ds highe yields, we immobilised BsGlyDH-L252A on aga ose po ous mic obeads ac i a ed wi h cobal -chela es (AG-Co 2+ ) o enhance i s ope a ional s abili y and inc ease he bioca alys olume ic ac i i y. Fu he - mo e, he use o he immobilised enzyme will ease he p oduc eco e y and enable he enzyme eu iliza ion once he eac ion is comple ed. Since all BsGlyDH a ian s used in his wo k we e agged wi h 6His polypep ide a hei N- e minus o pu ica- ion pu poses, we ha nessed ha ag o si e-selec i ely immobilise he BsGlyDH-L252A on AG-Co 2+ . This a ian was quan i a i ely immobilisedon hisca ie ;howe e , hespecic ac i i y o he immobilised enzyme was educed o 19% compa ed o i s ee coun e pa unde colo ime ic assay condi ions (Table S6‡). The immobilised BsGlyDH-L252A pe o med he selec i e oxida ion o 1c wi h a TOF 2.4- old highe han he soluble enzyme, eaching amaximumyieldo 50%ae 72 h unde ba ch condi ions (Table Table 1 Michaelis–Men en s eady-s a e pa ame e s o W -BsGlyDH and BsGlyDH-L25A on diffe en glyce yl e he s a Subs a e K M (mM) V max (U mg 1 )k ca (s 1 )k ca /K M (M 1 s 1 ) W L252A W L252A W L252A W L252A 1a 50 3 1880 80 1.40 0.04 1.70 0.05 7.4 0.2 9.0 0.3 148 5 1b 45 4 977 131 0.160 0.003 1.25 0.06 0.84 0.02 6.6 0.3 19 7 1c 13.4 0.4 371 71 0.036 0.001 6.0 0.5 0.190 0.003 31 31485 1d 30 1 608 102 0.052 0.003 2.2 0.2 0.27 0.02 12 1919 1e n.a 162 24 n.a 0.24 0.02 n.a 1.3 0.1 n.a 8 1 31.2 0.3 707 77 0.057 0.001 2.4 0.1 0.300 0.001 13 11018 1g 78 8 281 24 0.06 0.02 2.8 0.1 0.32 0.01 14 1451 1h 72 n.a 0.14 0.01 n.a 0.7 0.1 n.a 112 n.a a Ac i i y assay: 1 mM NAD + in 100 mM sodium phospha e buffe pH 7 a 30 C. n.a: no assessed because i did no each he subs a e sa u a ion. This jou nal is © The Royal Socie y o Chemis y 2020 Chem. Sci.,2020,11,12009–12020 | 12015 Edge A icle Chemical Science Open Access A icle. Published on 05 Oc obe 2020. Downloaded on 3/8/2021 9:50:12 AM. This a icle is licensed unde a C ea i e Commons A ibu ion-NonComme cial 3.0 Unpo ed Licence. View A icle Online 2, en y 2). This he e ogeneous bioca alys enabled an enzyme and co ac o o al u no e numbe s o 64 10 3 and 25, espec i ely, unde he s udied condi ions. These da a demons a e ha immobilised BsGlyDH-L252A mixed wi h soluble T -NOX and Hl-CAT ou pe o ms he sys em wi h he h ee soluble enzymes. The appa en lowe ac i i y o he immobilised enzyme seems o be compensa ed by i s highe obus ness unde ope a ional condi ions. 60 The immobiliza ion o BsGlyDH-L252A allowed us inc easing 8 imes he olume ic ac i i y o he he e ogeneous bioca alys , eaching 50% con e sion in only 24 h (Table 2, en y 3). F om he eac ion ime cou ses, we obse ed how he oxida ion eac ion slows down when he con e sion app oaches o 50% (Fig. 5A). Since we used 25 mM acemic mix u e o 1c, ha asymp o ic beha io poin s ou ha BsGlyDH-L252A is ex ao dina ily selec i e o one o he wo enan iome s. To un eil he enan ioselec i i y o his a ian , we pe o med u he QM/MM compu a ional s udies on he wo isome s o 1c. The ee ene gy ba ie s o he oxida ion o S-1c (16.16 kcal mol 1 ) was signican ly lowe han ha o he R- enan iome (44.40 kcal mol 1 ) (Fig. 4A), indica ing ha he enginee ed BsGlyDH a ian p e e s oxidizing he S-enan- iome . Simila esul s we e obse ed o he wild- ype enzyme (Fig. S15 and Table S5‡), sugges ing ha he na i e ac i e si e also p e e s he S-congu a ion o he subs a e o he hyd ide ans e o he NAD + . Fig. 4C illus a es an unp oduc i e posi- ion o R-1c as he seconda y alcohol loses i s coo dina ion wi h he ca aly ic Zn 2+ . The same p edic ed enan iop e e ence o bo h enginee ed and na i e a ian s sugges s ha he L252A mu a ion aids he accommoda ion o la ge glyce ol-like subs a es wi hou affec ing he egio- and s e eoselec i i y o he enzyme. This S-enan iop e e ence o BsGlyDH ag ees wi h he enan ioselec i i y epo ed o a cheal glyce ol-1-phospha e dehyd ogenase, 61 sugges ing a p o-R posi ion o he NAD + wi hin he ac i e si e. GlyDHs om En e obac e ae ogenes and om Cellulomonas sp. selec i ely oxidise he R-isome o 1,2- diols, kine ically esol ing he co esponding S-enan iome s o phenylglycol (ee 99%, con . 50%) 58 and 1,2-p opanodiol (ee 36%, con . 26%), 49 espec i ely. The opposi e CIP p io i y be ween 1c and non alkoxyla ed 1,2-diols indica es ha he diols a e simila ly coo dina ed and posi ioned wi hin he enzyme ac i e si es o hose GlyDHs, and he hyd ide om he subs a es is also ans e ed o Re- ace o he NAD + . Embold- ened by hese compu a ional insigh s, we challenged he immobilised BsGlyDH-L252A o he kine ic esolu ion o ac-1h (Figs. S29–S33‡). Fig. 5B shows ha he eac ion ime cou se ollows he classical kine ic esolu ion pa e n whe e he S- enan iome is ully oxidised o 3-phenoxy-1-hyd oxyace one, while he R-enan iome emains non eac i e (Fig. S33‡). This esul con ms he compu a ional s udies and hus demon- s a es ha BsGlyDH-L252A is an S-s e eoselec i e polyol dehyd ogenase, en iching he eac ion c udes wi h R-1h (ee > 99%) ae 72 hou s. Since we exploi ed BsGlyDHA-L252A o selec i ely oxidise alkyl and a yl glyce yl monoe he s, we a e able o eadily p epa e he pu e R-enan iome o glyce ol monoe he s in one-po unde mild condi ions. To he bes o ou knowledge, he only epo ed bio ans o ma ions able o p oduce his enan iome is based on asymme ic educ ion o hyd oxyace one monoe he s h ough whole cells o diffe en yeas s, bu wi h signican ly lowe enan iopu i y (ee ¼73– Fig. 4 (A) F ee ene gy p ofiles (M06-2X/6-31+G(d,p):PM3/MM PMFs + ZPE co ec ion) ollowing mechanism 2 o he S-1c oxida ion ca a- lysed by W -BsGlyDH (dashed o ange line) and BsGlyDH-L252A (solid g een line), and o R-1c oxida ion ca alysed by BsGlyDH-L252A (solid pu ple line). (B) O e lapped snapsho o he hyd ide ans e ansi ion s a es (TS2) loca ed in he oxida ion o S-1c ca alysed by he W - BsGlyDH (o ange) and BsGlyDH-L252A (g een). NAD + bound o he complex o W and L252A mu an was colou ed in o ange and yellow, espec i ely. (C) O e lapped snapsho s o he hyd ide ans e ansi- ion s a e (TS2) loca ed in he oxida ion o S-1c (g een) and R-1c (pu ple) by BsGlyDH-L252A. NAD + bound o he complex wi h Sand R isome s was colou ed in yellow and pale g een, espec i ely. 12016 |Chem. Sci.,2020,11,12009–12020 This jou nal is © The Royal Socie y o Chemis y 2020 Chemical Science Edge A icle Open Access A icle. Published on 05 Oc obe 2020. Downloaded on 3/8/2021 9:50:12 AM. This a icle is licensed unde a C ea i e Commons A ibu ion-NonComme cial 3.0 Unpo ed Licence. View A icle Online