Synthetic Biomimetic Coenzymes and Alcohol Dehydrogenases for Asymmetric Catalysis

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Communication Synthetic Biomimetic Coenzymes and Alcohol Dehydrogenases for Asymmetric Catalysis

Laia Josa-Culleré 1,2 , Antti S. K. Lahdenperä 1,2,†, Aubert Ribaucourt 1,2,†, Georg T. Höﬂer 1, Serena Gargiulo 1, Yuan-Yang Liu 3, Jian-He Xu 3, Jennifer Cassidy 4, Francesca Paradisi 5 , Diederik J. Opperman 6 , Frank Hollmann 1,* and Caroline E. Paul 1,*

1 Department of Biotechnology, Delft University of Technology, Van der Maasweg 9, 2629 HZ Delft, The Netherlands; [email protected] (L.J.-C.); [email protected] (A.S.K.L.); [email protected] (A.R.); G.T.Hofl[email protected] (G.T.H.); [email protected] (S.G.) 2 Chemistry Research Laboratory, University of Oxford, 12 Mansfield Road, Oxford OX1 3TA, UK 3 State Key Laboratory of Bioreactor Engineering and Shanghai Collaborative Innovation Centre for Biomanufacturing, East China University of Science and Technology, Shanghai 200237, China; [email protected] (Y.-Y.L.); [email protected] (J.-H.X.) 4 University College Dublin, Belfield, Dublin 4, Ireland; [email protected] 5 School of Chemistry, University of Nottingham, Nottingham NG7 2RD, UK; [email protected] 6 Department of Biotechnology, University of the Free State, 205 Nelson Mandela Drive, Bloemfontein 9300, South Africa; [email protected] * Correspondence: [email protected] (F.H.); [email protected] (C.E.P.); Tel.: +31-(0)1-5278-1957 (F.H.); +31-(0)1-5278-7995 (C.E.P.) † These authors contributed equally to this work.

Received: 1 February 2019; Accepted: 23 February 2019; Published: 26 February 2019

Abstract: Redox reactions catalyzed by highly selective nicotinamide-dependent oxidoreductases are rising to prominence in industry. The cost of nicotinamide adenine dinucleotide coenzymes has led to the use of well-established elaborate regeneration systems and more recently alternative synthetic biomimetic cofactors. These biomimetics are highly attractive to use with ketoreductases for asymmetric catalysis. In this work, we show that the commonly studied cofactor analogue 1-benzyl-1,4-dihydronicotinamide (BNAH) can be used with alcohol dehydrogenases (ADHs) under certain conditions. First, we carried out the rhodium-catalyzed recycling of BNAH with horse liver ADH (HLADH), observing enantioenriched product only with unpurified enzyme. Then, a series of cell-free extracts and purified ketoreductases were screened with BNAH. The use of unpurified enzyme led to product formation, whereas upon dialysis or further purification no product was observed. Several other biomimetics were screened with various ADHs and showed no or very low activity, but also no inhibition. BNAH as a hydride source was shown to directly reduce nicotinamide adenine dinucleotide (NAD) to NADH. A formate dehydrogenase could also mediate the reduction of NAD from BNAH. BNAH was established to show no or very low activity with ADHs and could be used as a hydride donor to recycle NADH.

Keywords: alcohol dehydrogenases; cofactor regeneration; formate dehydrogenase; nicotinamide coenzyme biomimetics; rhodium catalyst

1. Introduction The field of biocatalysis has flourished in the past few decades, with enzymes increasingly being used in industrial processes for fine chemical commodities. Alcohol dehydrogenases ADHs (EC 1.1.1.X, also ketoreductases KREDs) are one example, reversibly catalyzing the reduction of ketones

Catalysts 2019, 9, 207; doi:10.3390/catal9030207 www.mdpi.com/journal/catalysts Catalysts 2019, 9, 207 2 of 11

Catalysts 2019, 9 FOR PEER REVIEW 2 or aldehydes to the corresponding (enantioenriched) alcohols through the use of one equivalent of nicotinamideor aldehydes to adenine the corresponding dinucleotide (enantioenriched cofactor NAD(P).) alcohols ADHs are through known the to use be specificof one equivalent to either the of phosphorylatednicotinamide adenine NADP dinucleotide or non-phosphorylated cofactor NAD(P). NAD, ADHs although are known a few haveto be been specific found to either to accept the bothphosphorylated [1–3]. These NADP cofactors or non-phosphorylated act as hydride acceptor NAD, and although donor intermediatesa few have been between found to the accept substrate both and[1–3]. product. These cofactors act as hydride acceptor and donor intermediates between the substrate and product.Enzyme recognition of NAD(P), usually by a cofactor (binding) motif such as the Rossmann fold, is importantEnzyme forrecognition cellular processes,of NAD(P), however usually by when a cofactor using in(binding) vitro systems, motif such the as adenine the Rossmann dinucleotide fold, moietyis important of the for cofactor cellular becomesprocesses, obsolete however and when disadvantageous, using in vitro systems, being the prone adenine to hydrolysis dinucleotide [4]. Syntheticmoiety of nicotinamidethe cofactor coenzymebecomes obsolete biomimetics and (NCBs)disadvantageous, have been being shown pron to replacee to hydrolysis NAD(P)H [4]. in flavin-dependentSynthetic nicotinamide enzymes coenzyme such as biomimetics nitroreductase (NCBs) and have NAD(P)H been shown quinone to oxidoreductasereplace NAD(P)H [5– 7in], flavin-dependent enzymes such as nitroreductase and NAD(P)H quinone oxidoreductase [5–7], a a cytochrome P450 BM3 variant [8], ene-reductases [9–16], and styrene monooxygenase [17]. In all cytochrome P450 BM3 variant [8], ene-reductases [9–16], and styrene monooxygenase [17]. In all cases cases described, a flavin was involved as an electron mediator [18–22]. described, a flavin was involved as an electron mediator [18–22]. Jones pioneered the use of synthetic cofactor analogues with horse liver ADH (HLADH) [23–26], Jones pioneered the use of synthetic cofactor analogues with horse liver ADH (HLADH) [23– followed by Fish [27] and others [28,29]. A dehydrogenase from the aldo-reductase superfamily 26], followed by Fish [27] and others [28,29]. A dehydrogenase from the aldo-reductase superfamily from Pyrococcus furiosus (AdhD) was engineered to increase catalytic efficiency towards nicotinamide from Pyrococcus furiosus (AdhD) was engineered to increase catalytic efficiency towards nicotinamide mononucleotide (NMN, Figure1A) [ 30,31]. Acyclic analogues of NAD (Figure1B) were shown not only mononucleotide (NMN, Figure 1A) [30,31]. Acyclic analogues of NAD (Figure 1B) were shown not to be used with HLADH, but also to alter the substrate specificity of the enzyme [32]. More recently, only to be used with HLADH, but also to alter the substrate specificity of the enzyme [32]. More the group of Sieber showed the use of synthetic NCBs with an NADH oxidase [33,34], and a glucose recently, the group of Sieber showed the use of synthetic NCBs with an NADH oxidase [33,34], and dehydrogenase (GDH), producing variants displaying activity, albeit low, with 1-benzylnicotinamide a glucose dehydrogenase (GDH), producing variants displaying activity, albeit low, with 1- (BNA, Figure1C) [35]. benzylnicotinamide (BNA, Figure 1C) [35].

Figure 1. (A) Schematic representation of the natural NAD(P); (B) Naturally-based analogues; Figure 1. (A) Schematic representation of the natural NAD(P); (B) Naturally-based analogues; (C) (C) synthetic analogue. synthetic analogue. These examples are important steps towards the use of NCBs with dehydrogenases, and more speciﬁcallyThese examples ADHs, which are important would be advantageoussteps towards for the large use scaleof NCBs applications, with dehydrogenases, although NCB and recycling more andspecifically stability ADHs, is still inwhich its early would stages be [ 36advantageous]. In a previous for study, large cofactors scale applications, NR, NMN, andalthough carbocyclic NCB analoguesrecycling and showed stability no is observable still in its activityearly stages in the [36]. HLADH-catalyzed In a previous study, oxidation cofactors of NR, ethanol NMN, [37 ,and38], corroboratedcarbocyclic analogues by computational showed no studies observable on the activity mechanism in the for HLADH-catalyzed hydride transfer in oxidation HLADH of [39 ethanol]. [37,38],Here corroborated the research by was computational carried out to establishstudies on whether the mechanism the commonly for hydride used and transfer now commercially in HLADH available[39]. BNAH analogue could be used with ADHs and other ketoreductases for asymmetric catalysis. First,Here we explored the research the rhodium-catalyzed was carried out recyclingto establish of BNAH whether with the HLADH commonly [27], whichused seemedand now to showcommercially that BNAH available could replaceBNAH NADH,analogue contrary could be to recentused with attempts ADHs [16 and,35]. other This workketoreductases was followed for byasymmetric screening catalysis. series of ADHsFirst, we and explored KREDs to the assess rhodium-catalyzed biomimetic acceptance. recycling BNAH of BNAH was furtherwith HLADH used as a[27], hydride which source seemed in ato preliminary show that recyclingBNAH could system repl withace NADH, a formate contrary dehydrogenase to recent (FDH). attempts [16,35]. This work was followed by screening series of ADHs and KREDs to assess biomimetic acceptance. BNAH was further used as a hydride source in a preliminary recycling system with a formate dehydrogenase (FDH).

2. Results and Discussion

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2. Results and Discussion Catalysts 2019, 9 FOR PEER REVIEW 3 2.1. Rh-Catalyzed Cofactor Recycling 2.1.The Rh-Catalyzed group of Cofactor Fish reported Recycling activity with HLADH and cofactor analogues BNA and NMN using a Rh-catalyzedThe group recycling of Fish reported system [ 27activity]. For with this studyHLADH we and kept cofactor 4-phenyl-2-butanone analogues BNA as and a substrate, NMN using as the enantiomerica Rh-catalyzed excess recycling (ee) of system the reduced [27]. For product this study would we probekept 4-phenyl-2-butanone the enantioselectivity as ofa substrate, the enzymatic as reactionthe enantiomeric (Figure2A). excess Thesame (ee) of HLADH the reduced enzyme product was purchased, would probe although the enantioselectivity the provider and of purity the areenzymatic different, reaction as the enzyme(Figure is2A). now The being same produced HLADH enzyme by recombinant was purchased, expression although (see Supplementary the provider Informationand purity (SI)are Sectiondifferent, 1). as the enzyme is now being produced by recombinant expression (see Supplementary Information (SI) section 1).

(A)

(B)

FigureFigure 2. 2. ((AA)) Representative Representative scheme scheme of ofthe the HLADH-catalyzed HLADH-catalyzed reduction reduction of 4-phenyl-2-butanone of 4-phenyl-2-butanone to toenantioenriched enantioenriched 4-phenyl-2-butanol; 4-phenyl-2-butanol; (B ()B Time) Time course course of ofthe the reaction reaction (blue (blue diamonds, diamonds, ee eeorangeorange 2+ squares); NADH regeneration system with [Cp*Rh(bpy)(H O)] using2+ sodium formate as a hydride source. squares); NADH regeneration system with [Cp*Rh(bpy)(H2 2O)] using sodium formate as a hydride 2+ Reactionsource. conditions:Reaction 100conditions: mM KPi pH100 7.0, mM HLADH KPi (2pH U), [NAD]7.0, HLADH = 2.2 mM, (2 [Cp*Rh(bpy)(H U), [NAD]2 O)]= 2.2= 0.52mM, mM , ◦ [HCO[Cp*Rh(bpy)(H2Na] = 52 mM,2O)]2+ [4-phenyl-2-butanone] = 0.52 mM, [HCO2Na] = = 52 16.7 mM, mM, [4-phenyl-2-butanone] shaken at 30 C, 800 = rpm.16.7 mM, Conversion shaken at and 30 ee determined°C, 800 rpm. by Conversion chiral GC. and ee determined by chiral GC.

WeWe could could produce produce the the expected expected resultsresults with the Rh-catalyzed Rh-catalyzed regeneration regeneration of of NADH NADH under under the the samesame conditions conditions (Figure (Figure2B, 2B, 60% 60% conversion conversion 97% 97%ee after ee after 72 h)72 [ 27h)]. [27]. Next, Next, wecompared we compared reaction reaction results betweenresults between the commercially the commercially available available ‘crude’ (unpuriﬁed‘crude’ (unpurified cell-free cell-free extract) extract) HLADH HLADH and the and dialyzed the enzyme,dialyzed with enzyme, either with NAD, either nicotinamide NAD, nicotinamide mononucleotide mononucleotide (NMN) or(NMN) BNA or (Figure BNA3 (Figure and SI 3 Table and SI S3). WhenTable using S3). When either crudeusing oreither dialyzed crude HLADH or dialyzed with HLADH NAD, after with 18 NAD, h, conversions after 18 h, were conversions similar (38–42%) were withsimilar a good (38–42%) enantiomeric with a good excess enantiomeric (>90% ee). excess However, (>90% the ee). same However, reactions the same carried reactions out in carried the presence out ofin NMN, the presence BNA, or ofin NMN, the absence BNA, or of in a the cofactor absence showed of a cofactor 4–11% showed conversion 4–11% and conversion 4–37% ee comparedand 4–37% to 18–78%ee compared conversion to 18–78% and <1%conversionee with and the <1% dialyzed ee with enzyme the dialyzed (Figure enzyme3). (Figure 3).

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Figure 3. HLADH-catalyzed reduction of 4-phenyl-2-butan 4-phenyl-2-butanoneone with the Rh complex recycling system using either NAD, NMN, BNA, or nono cofactor.cofactor. HLADH was used as lyophilizedlyophilized cell-free extracts (crude)(crude) oror dialyzeddialyzed (conversion,(conversion, blue;blue; eeee,, orange).orange). ReactionReaction conditions:conditions: 100 mM KPi pH 7.0,7.0, HLADHHLADH 2+ (2(2 U),U), [cofactor][cofactor] == 2.22.2 mMmM (where(where applicable),applicable), [Cp*Rh(bpy)(H [Cp*Rh(bpy)(H22O)]O)]2+ = 0.52 mM, [HCO2Na] = 52 52 mM, ◦ [4-phenyl-2-butanone] == 16.7 mM, shaken at 30 °C,C, 800 rpm, 18 h.

A rationale for the higher conversions achieved with dialyzed HLADH with respect to the crude ee HLADH, with NMN, BNA or without cofactor, could be found by looking closer at the ee obtainedobtained ee ee with crude HLADHHLADH (4–37%(4–37% ee)) andand withwith dialyzeddialyzed HLADHHLADH (<1%(<1% ee), suggesting direct reduction of the ketone by the Rh complex. With the crude enzymeenzyme preparation, trace amounts of NAD could be reduced by the the Rh Rh complex, complex, hence hence showing showing some some enzymatic enzymatic reduction reduction of the of the ketone. ketone. In this In thiscase, case, Rh- Rh-catalyzedcatalyzed reduction reduction of the of the ketone ketone could could also also be be inhibited inhibited by by the the presence presence of of other other low molecular weight moleculesmolecules in in the the crude crude enzyme enzyme preparation preparation [40]. [40]. Removal Removal of these of lowthese molecular low molecular weight speciesweight (NADspecies or (NAD other) or by other) dialysis by dialysis (cut-off (cut-off at 12 kDa) at 12 would kDa) favorwould the favor non-enzymatic the non-enzymatic Rh-catalyzed Rh-catalyzed ketone reductionketone reduction and account and foraccount the higher for the conversions higher conversions and lack of enantioselectivityand lack of enantioselectivity of this transformation. of this transformation.The redox potentials of NAD and NMN are identical [41], therefore chemical reactivity cannot explainThe the redox difference potentials in the of enzyme-catalyzedNAD and NMN are reaction. identica Extensivel [41], therefore investigation chemical by Plappreactivity and cannot others throughexplain the analyses difference of high in the resolution enzyme-catalyzed X-ray structures reaction. of HLADHExtensive reveals investigation that the by binding Plapp ofand NAD others to thethrough coenzyme-binding analyses of high domain resolution induces X-ray a conformational structures of HLADH modiﬁcation reveals [42 that–44 ]the necessary binding for of catalyticNAD to activitythe coenzyme-binding of the enzyme domain [45]. With induces the truncated a conformational nicotinamide modification cofactors [42–44] NR, NMN necessary and the for synthetic catalytic BNA,activity the of conformationalthe enzyme [45]. change With the most truncat likelyed does nicotinamide not occur, cofactors indicating NR, the NMN complete and the coenzyme synthetic is requiredBNA, the [ 46conformational]. The importance change of speciﬁc most likely interactions does not between occur, coenzyme indicating and the enzyme complete (SI coenzyme Figure S2) is supportedrequired [46]. by molecularThe importance dynamics of specific simulations interact [47ions]. between coenzyme and enzyme (SI Figure S2) is supportedTherefore, by molecular we established dynamics that simulations trace amounts [47]. of NAD present in the commercial HLADH preparationTherefore, are responsiblewe established for the that low trace to modest amounts conversions of NAD andpresentee observed in the withcommercial NMN andHLADH BNA, andpreparation once removed are responsible only racemic for the product low to modest was observed conversions due to and non-enzymatic ee observed with reduction. NMN and HLADH BNA, showedand once no removed observable only or racemic very low product activity was with ob NMNserved and due BNA. to non-enzymatic reduction. HLADH 2.2.showed Screening no observable of ADHs andor very Ketoreductases low activity with NMN and BNA.

2.2. ScreeningLibraries of of ADHs ADHs and and ketoreductases KREDs were screened for activity with BNAH. A panel of KREDs from a Codexis kit was used for the reduction of cyclohexanone with BNAH as the sole cofactor (Figure4, Libraries of ADHs and KREDs were screened for activity with BNAH. A panel of KREDs from grey bars). Control reactions were run without cofactor (white bars). Product was observed in all cases a Codexis kit was used for the reduction of cyclohexanone with BNAH as the sole cofactor (Figure 4, in varying percent conversions, in the control reactions (no BNAH added) as well. Further screening grey bars). Control reactions were run without cofactor (white bars). Product was observed in all of various available ADHs and KREDs in the form of cell-free extracts (see SI Sections 3.3 and 3.4) cases in varying percent conversions, in the control reactions (no BNAH added) as well. Further showed similar activity. screening of various available ADHs and KREDs in the form of cell-free extracts (see SI sections 3.3 and 3.4) showed similar activity.

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FigureFigure 4. Screening 4. Screening of the of the ketoreductases ketoreductases (KREDs)(KREDs) Codexi Codexiss kit. kit. X-axis: X-axis: KREDs. KREDs. Y-axis: Y-axis: conversion conversion to to cyclohexanolcyclohexanol after after 24 h, h, measured measured by GC by analys GC analysis,is, with BNAH with (grey) BNAH and (grey) without and BNAH without (white). BNAH (white).Reactions Reactions conditions: conditions: 50 mM 50 KPi mM pH KPi 7.0, pH[cyclo 7.0,hexanone] [cyclohexanone] = 10 mM, [KRED] = 10 mM, = 10 mg/mL, [KRED] [BNAH] = 10 mg/mL, = 10 mM (where applicable). [BNAH] = 10 mM (where applicable). We hypothesizedWeFigure hypothesized 4. Screening that ofthat the the the conversionketoreductases conversion obtained(KREDs) obtained Codexi waswass due kit. X-axis: to to the the KREDs. presence presence Y-axis: of ofNAD(P)/NAD(P)Hconversion NAD(P)/NAD(P)H to from thefrom lyophilized thecyclohexanol lyophilized crudeafter crude 24 h, cell measuredcell extracts. extracts. by GC The The analys addi additionalis,tional with productBNAH product (grey) formation and formation without observed BNAH observed with (white). BNAH with can BNAH be attributedReactions to conditions: its ability 50 tomM reduce KPi pH trace 7.0, [cyclo amountshexanone] of NAD(P), = 10 mM, [KRED]through = 10a intermolecularmg/mL, [BNAH] hydride= can be attributed10 mM (where to its abilityapplicable). to reduce trace amounts of NAD(P), through a intermolecular hydride transfer, which was well established and demonstrated by Jones [23]. transfer, which was well established and demonstrated by Jones [23]. Using the heat purified TADH (Thermus sp. ATN1), no product was observed with BNAH as the UsingWe the hypothesized heat purified that TADH the conversion (Thermus obtainedsp. ATN1), was due no to product the presence was of observed NAD(P)/NAD(P)H with BNAH as sole cofactor. Further testing of other purified ADHs, HvADH2 (Haloferax volcanii ADH2) [48], the solefrom cofactor. the lyophilized Further crude testing cell ofextracts. other The purified additional ADHs, productHv formationADH2 ( Haloferaxobserved with volcanii BNAHADH2) can [48], HvbeADH2-F108G, attributed to itsHw abilityADH to(Haloquadratum reduce trace am walsbyiounts ofADH), NAD(P), and throughHLADH-EE a intermolecular [49] with the hydride natural HvADH2-F108G, HwADH (Haloquadratum walsbyi ADH), and HLADH-EE [49] with the natural cofactorstransfer, andwhich a series was well of synthetic established NCBs and (Figure demonstrated S1) showed by Jones the latter [23]. afforded no observable activity, cofactorsas well andUsing as a no series the significant heat of purified synthetic inhibitory TADH NCBs effects (Thermus (Figure (SI sp. section S1)ATN1), showed 3.3). no product the latter was observed afforded with no BNAH observable as the activity, as wellsole as noThus, cofactor. significant the conclusionFurther inhibitory testing from ofscreening effects other (SIpurified cell Section free ADHs, extracts 3.3). Hv andADH2 purified (Haloferax ADHs volcanii and KREDs ADH2) was [48], that Thus,BNAHHvADH2-F108G, the gave conclusion no detectable HwADH from activity (Haloquadratum screening but could cell walsbyi be free used extracts ADH), as a hydride and and HLADH-EE purified source. ADHs [49] with and the KREDs natural was that BNAHcofactors gave no and detectable a series of activity synthetic but NCBs could (Figure be usedS1) showed as ahydride the latter afforded source. no observable activity, 2.3.as wellBNAH as noas asignificant Hydride Donor inhibitory effects (SI section 3.3). 2.3. BNAH asThus, a Hydride the conclusion Donor from screening cell free extracts and purified ADHs and KREDs was that 2.3.1.BNAH Direct gave Hydride no detectable Transfer activity to NAD but could be used as a hydride source. 2.3.1. DirectDue Hydride to the lack Transfer of activity to NAD with purified enzyme, we investigated the use of BNAH as a hydride source2.3. BNAH to recycle as a Hydride NADH Donor for HLADH-catalyzed reactions (Figure 5 top scheme) [23,24,26]. Using the Duedialyzed to the HLADH lack offor activity the reduction with purifiedof 4-pheny enzyme,l-2-butanone, we investigatedwe could reach the 1.4 use mM of of BNAH the as 2.3.1. Direct Hydride Transfer to NAD a hydrideenantioenriched source to recycle alcohol NADH(98% ee)for product HLADH-catalyzed after 18 h (Figure reactions 5 bottom (Figuregraph), 5affording top scheme) a turnover [ 23,24 ,26]. Usingfrequency the dialyzedDue to(TOF) the HLADH lack of 7 of h activity−1 forwith the stoichiometricwith reduction purified enzy ofBNAH 4-phenyl-2-butanone,me, weand investigated 1 mM NAD, the compared use we of could BNAH to reacha asTOF a hydride 1.4of 23 mM h −1 of the enantioenrichedwithsource 1 equivalent to recycle alcohol NADH of (98%NADH, foree HLADH-catalyzed )and product 21 h−1 with after the reac 18 Rh htions (Figure recycling (Figure5 bottom 5system. top scheme) We graph), ascribe [23,24,26]. affording the Usingdecrease a the turnover in dialyzed HLADH for the reduction of 4-phenyl-2-butanone, we could reach 1.4 mM of the frequencyreaction (TOF) kinetics of 7 after h−1 with3 h to stoichiometricthe instability of BNAHBNAH over and time, 1 mM especially NAD, comparedin the phosphate to a TOFbuffer of as 23 h−1 previouslyenantioenriched described alcohol [34]. (98% ee) product after 18 h (Figure 5 bottom graph), affording a turnover with 1 equivalent of NADH, and 21 h−1 with the Rh recycling system. We ascribe the decrease in frequency (TOF) of 7 h−1 with stoichiometric BNAH and 1 mM NAD, comparedOH to a TOF of 23 h−1 reactionwith kinetics 1 equivalent after 3 of h NADH, to the instability and 21 h−1 with of BNAH the Rh over recycling time, system. especially We ascribe in the the phosphate decrease in buffer as previouslyreaction described kinetics [after34]. 3 hBNAH to the instabilityNAD of BNAH over time, especially in the phosphate buffer as previously described [34]. OH HLADH O BNAH NAD

BNA NADH

HLADH O (A) BNA NADH

(A)

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3 4 Product (mM) 2 3

2 1 1 0 024681012141618200 02468101214161820

Time (h) Time (h)

(B(B) )

Figure 5.Figure(A) Representative 5. (A) Representative scheme scheme of theof the HLADH-catalyzed HLADH-catalyzed reduction reduction of 4-phenyl-2-butanone of 4-phenyl-2-butanone to to Figure 5. (A) Representative scheme of the HLADH-catalyzed reduction of 4-phenyl-2-butanone to enantioenrichedenantioenriched 4-phenyl-2-butanol; 4-phenyl-2-butanol; (B) (B Time) Time course course ofof the reaction reaction with with NADH NADH (blue (blue diamonds) diamonds) or or enantioenriched 4-phenyl-2-butanol; (B) Time course of the reaction with NADH (blue diamonds) or BNAH +BNAH NAD + (green NAD (green squares). squares). Reaction Reaction conditions: conditions: 100100 mM KPi KPi pH pH 7.0, 7.0, 1 mL 1 mLfinal final volume, volume, HLADH HLADH BNAH + NAD (green squares). Reaction conditions: 100 mM KPi pH 7.0, 1 mL final volume, HLADH (2 U), [NADH](2 U), [NADH] = 16.7 = mM16.7 mM (blue (blue diamonds), diamonds), [BNAH][BNAH] = =16.7 16.7 mM mM + [NAD] + [NAD] = 1 mM = (green 1 mM squares), (green squares),[4- (2 U), [NADH]phenyl-2-butanone] = 16.7 mM =(blue 16.7 mM, diamonds), shaken at [BNAH]30 °C and 800= 16.7 rpm mM over + time. [NAD] = 1 mM (green squares), [4- [4-phenyl-2-butanone] = 16.7 mM, shaken at 30 ◦C and 800 rpm over time. phenyl-2-butanone] = 16.7 mM, shaken at 30 °C and 800 rpm over time. 2.3.2. Formate2.3.2. Formate Dehydrogenase Dehydrogenase with with BNAH BNAH 2.3.2. FormateTo Dehydrogenase improve the use of with BNAH BNAH as a hydride donor to reduce NAD, a formate dehydrogenase To improve(FDH)-catalyzed the use recycling of BNAH system as was a hydride established. donor The system to reduce shown NAD, in Figure a formate 6A uses dehydrogenasethe NAD- (FDH)-catalyzedTo improvedependent the recyclingFDH1 use β -subunitof systemBNAH from as was Methylobacterium a hydride established. donor extorquens The to reduce system AM1 NAD,( shownMeFDH) a formate inas a Figure relay dehydrogenase 6betweenA uses the NAD-dependent(FDH)-catalyzedBNAH and FDH1recycling NAD.β -subunitThe system iron-sulfur from was Methylobacteriumestablished. cluster contained The extorquenssystemin MeFDH shownAM1 can in (beMe Figure reducedFDH) 6A as by auses relayBNAH, the between NAD- BNAHdependent andsubsequently NAD.FDH1 Theβ -subunitreducing iron-sulfur NAD.from cluster This Methylobacterium new contained recyclingin system MeextorquensFDH was cancoupled AM1 be reduced to(Me a purifiedFDH) by BNAH, asADH a relay(ADH-A subsequently between reducingBNAH andfrom NAD. RhodococcusNAD. This newThe ruber recyclingiron-sulfur). The use system of MeclusterFDH was shows contained coupled catalytic to in abehavior purifiedMeFDH and ADH resultscan (ADH-Abe in 1.7reduced mM from of product Rhodococcusby BNAH, after 24 h (Figure 6B). The control reaction without MeFDH shows almost 5 times lower conversion rubersubsequently). The use reducing of MeFDH NAD. shows This new catalytic recycling behavior system and was results coupled in 1.7 to mM a purified of product ADH after (ADH-A 24 h with 0.3 mM of product. Catalytic turnover numbers of 17 (NAD), 338 (MeFDH) and 11 698 (ADH- from Rhodococcus ruber). The use of MeFDH shows catalytic behavior and results in 1.7 mM of product (Figure6B).A) were The controlobserved. reaction The significant without decreaseMeFDH of showsthe reaction almost kinetics 5 times after lower 3 h applying conversion the Me withFDH 0.3 mM ofafter product. 24 hcould (Figure Catalytic indicate 6B). turnoverstability The control problems numbers reaction regarding of 17 without (NAD), the enzyme. 338MeFDH (Me ImmobilizationFDH) shows and almost 11 of 698 the5 (ADH-A)times enzymes lower werecould conversion observed.be Thewith significant 0.3 envisagedmM of decrease product. to improve of Catalytic the the reaction system. turnover Further kinetics numbers optimiza after 3tions of h applying17 to (NAD), improve the 338 theMe (robustnessFDHMeFDH) could andof indicatethe 11 system 698 stability (ADH- problemsA) were couldobserved. regarding lead to Theeven the enzyme.significanthigher catalytic Immobilization decrease turnover. of the of reaction the enzymes kinetics could after be envisaged3 h applying to improvethe MeFDH the system.could indicate Further stability optimizations problems to regarding improve the the robustness enzyme. Immobilization of the system couldof the lead enzymes to even could higher be catalyticenvisaged turnover. to improve the system. Further optimizations to improve the robustness of the system could lead to even higher catalytic turnover.

(A)

Figure 6. Cont.

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Product (mM) 1

0 024681012141618202224 Time (h)

(B)

Figure 6. (A) Representative scheme of the ADH-A-catalyzed reduction of ethyl acetoacetate with Figure 6. (A) Representative scheme of the ADH-A-catalyzed reduction of ethyl acetoacetate with MeFDH; (B) Time course of the reaction (blue diamonds) or without MeFDH (orange squares). Reaction MeFDH; (B) Time course of the reaction (blue diamonds) or without MeFDH (orange squares). conditions: 50 mM NaPi pH 7.0, ADH-A (0.6 U), [MeFDH] = 5 µM (blue diamonds) or 0 µM (orange Reaction conditions: 50 mM NaPi pH 7.0, ADH-A (0.6 U), [MeFDH] = 5 µM (blue diamonds) or 0 µM squares), [NAD] = 0.1 mM, [BNAH] = 10 mM (solid), [ethyl acetoacetate] = 10 mM, shaken at 30 ◦C, (orange squares), [NAD] = 0.1 mM, [BNAH] = 10 mM (solid), [ethyl acetoacetate] = 10 mM, shaken at 800 rpm, anaerobic. 30 °C, 800 rpm, anaerobic. 3. Materials and Methods 3. Materials and Methods 3.1. Chemicals and Enzymes 3.1. Chemicals and Enzymes All commercially available reagents and solvents were purchased with the highest purity available and usedAll as commercially received. Enzymes available were reagents either commercially and solven availablets were orpurchased recombinantly with expressed.the highest Further purity detailsavailable are providedand used in as the received. Supplemental Enzymes Information were either (SI). commercially available or recombinantly expressed.The HLADH Further was details obtained are provided from Evocatal in the supplemental GmbH as ADH information 210 HLADH (SI). (≥0.5 U/mg, protein concentrationThe HLADH 0.22 mg/mg) was obtained and from from Sigma-Aldrich Evocatal GmbH (St. Louis, as ADH MO, 210 USA) HLADH as alcohol (≥0.5 dehydrogenase U/mg, protein equineconcentration (recombinant, 0.22 mg/mg) expressed and in fromE. coli Sigma-Aldrich, ≥0.5 U/mg). (St. When Louis, referring MO, USA) to the as ‘crude’alcoholpreparation dehydrogenase of HLADH,equine (recombinant, the enzyme was expressed used as in received. E. coli, ≥ When0.5 U/mg). referring When to referring the ‘dialyzed’ to the HLADH, ‘crude’ preparation the enzyme of wasHLADH, further the purified enzyme through was used dialysis. as received. The activity When ofreferring both the to crudethe ‘dialyzed’ and dialyzed HLADH, HLADH the enzyme was measuredwas further by UV purified spectroscopy through before dialysis. use. The 1-Benzyl-1,4-dihydronicotinamide activity of both the crude and BNAH, dialyzed other HLADH analogues was andmeasured rhodium by complexes UV spectroscopy were synthesized before as previouslyuse. 1-Benzyl-1,4-dihydronicotinamide described [7,9,40]. BNAH, other analogues and rhodium complexes were synthesized as previously described [7,9,40]. 3.2. Instrumentation 3.2.UV Instrumentation spectroscopy measurements were made using an Agilent Cary 60 UV-Vis spectrophotometer (SantaUV Clara, spectroscopy CA, USA) at themeasurements designated wavelength. were made using an Agilent Cary 60 UV-Vis spectrophotometerGas chromatography (Santa (GC) Clara, analyses CA, USA) were at carried the designated out on a Shimadzuwavelength. GC-2010 (Kyoto, Japan) gas chromatographGas chromatography equipped with (GC) a flameanalyses ionization were carried detector out (FID). on a Shimadzu GC-2010 (Kyoto, Japan) gas chromatographNuclear magnetic equipped resonance with a flame (NMR) ionization spectra detector were recorded(FID). on an Agilent Technologies 1 spectrometerNuclear (Santa magnetic Clara, resonance CA, USA) at(NMR) 400 MHz spectra ( H). were recorded on an Agilent Technologies spectrometer (Santa Clara, CA, USA) at 400 MHz (1H). 3.3. Experimental Setup 3.3.Screenings Experimental and Setup biocatalytic reactions were run in a microcentrifuge plastic tube (1.5 mL in volume). The ADHScreenings enzyme (10and mg biocatalytic of lyophilized reactions ADH perwere 1 mLrun reaction), in a microcentrifuge potassium phosphate plastic tube buffer (1.5 (50 mL mM, in pH 7.0; final volume of 1 mL), cofactor BNAH (10 mM, added in acetonitrile, 2% final volume: 20 µL) volume). The ADH enzyme (10 mg of lyophilized ADH per 1 mL reaction), potassium phosphate◦ andbuffer cyclohexanone (50 mM, pH (10 7.0; mM) final were volume added. of 1 ThemL), reaction cofactor mixture BNAH was(10 mM, placed added in a thermomixerin acetonitrile, at 2% 25 finalC µ andvolume: 800 rpm 20 forµL) 24 and h. The cyclohexanone reaction was stopped(10 mM) through were added. extraction The (500reactionL of mixture ethyl acetate was containingplaced in a thermomixer at 25 °C and 800 rpm for 24 h. The reaction was stopped through extraction (500 µL of

Catalysts 2019, 9, 207 8 of 11

5 mM dodecane as an internal standard). After centrifugation (13,000 rpm, 1 min) and separation of the two phases, the ethyl acetate layer was dried with anhydrous magnesium sulfate, centrifuged (12,000 rpm, 1 min), transferred to gas chromatography (GC) vials and analyzed by GC. Blank reactions were carried out in the absence of BNAH or any type of cofactor. For the biocatalytic Rh recycling experiments, stock solutions of NAD, NMN and BNA (10 mM), [Cp*Rh(bpy)(H2O)]Cl2, [Cp*Rh(bpy)(H2O)]OTf2 (5 mM), sodium formate (1 M) and HLADH (3 mg, 2 U/mg) in deoxygenated phosphate buffer (100 mM, pH 7.0) were freshly prepared before the experiments. In a microcentrifuge plastic tube (2 mL in volume), potassium phosphate buffer (100 mM, 2+ pH 7.0; ﬁnal volume of 1 mL), cofactor (2.2 mM or none), [Cp*Rh(bpy)(H2O)] (104 µL, 0.52 mM), sodium formate (52 µL, 52 mM), HLADH (200 µL, 2 U), and substrate 4-phenyl-2-butanone (2.5 µL, 16.7 mM) were added under nitrogen ﬂow. The reaction mixture was placed in an Eppendorf thermomixer at 30 ◦C and 600 rpm for 18 h, or other indicated times. The reaction was stopped at the indicated time intervals through extraction (500 µL of ethyl acetate containing 5 mM dodecane as an internal standard). After centrifugation (13,000 rpm, 1 min) and separation of the two phases, the ethyl acetate layer was dried with anhydrous magnesium sulfate, centrifuged (12,000 rpm, 1 min), transferred to GC vials and analyzed on a chiral GC column.

4. Conclusions From the Rh-catalyzed recycling of BNAH we could establish that unpurified ADH led to product formation via reduction of trace amounts of NAD. The screening of purified ADHs from various sources with biomimetic cofactors showed no detectable or very low activity. Additionally, no significant inhibition by these analogues was observed. BNAH can be used as a hydride donor to recycle NADH due to an intermolecular hydride transfer. For the first time, we show a regeneration system using formate dehydrogenase MeFDH and BNAH as a hydride donor, albeit with low catalytic turnover. Future prospects towards both coenzyme and protein engineering would be very promising for ADHs, based on encouraging results with other dehydrogenases AdhD and SsGDH from the enzyme perspective, and knowledge on the structure-activity relationship from the coenzyme point of view. With these prospects in progress, industrial applications of synthetic biomimetics would be of interest once their instability is overcome.

Supplementary Materials: The following are available online at http://www.mdpi.com/2073-4344/9/3/207/s1: the experiment details on enzymatic activity assays, syntheses of the rhodium complex and NCBs, biocatalytic reactions, analytical data and product characterization. Author Contributions: Conceptualization, F.H. and C.E.P.; validation, S.G., D.J.O., J.-H.X., F.P., F.H. and C.E.P.; investigation, L.J.-C., A.S.K.L., A.R., G.T.H., S.G., Y.Y.L., J.C. and C.E.P.; resources, J.-H.X., F.P., F.H. and C.E.P.; writing—original draft preparation, F.H. and C.E.P.; writing—review and editing, all authors; supervision, F.H. and C.E.P.; project administration, C.E.P.; funding acquisition, F.H. and C.E.P. Funding: This research was funded by the Netherlands Organization for Scientific Research (NWO), VENI grant number 722.015.011 (for C.E.P.); by the European Union through the Seventh Framework People Programme, grant number 316955 (for L.J.-C., A.L. and A.R.); and by the European Research Council ERC Consolidator, grant number 648026 (for G.T.H. and F.H.). Acknowledgments: We kindly thank Yong Hwan Kim at UNIST for the MeFDH. Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

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