stereochemistry of furfural reduction by a saccharomyces ... · aldehyde substrate (17), either of...

APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Aug. 2010, p. 4926–4932 Vol. 76, No. 150099-2240/10/$12.00 doi:10.1128/AEM.00542-10Copyright © 2010, American Society for Microbiology. All Rights Reserved.

Stereochemistry of Furfural Reduction by a Saccharomyces cerevisiaeAldehyde Reductase That Contributes to In Situ

Furfural Detoxification�

Michael J. Bowman,* Douglas B. Jordan, Karl E. Vermillion, Jay D. Braker,Jaewoong Moon, and Z. Lewis Liu

U.S. Department of Agriculture, Agricultural Research Service, National Center for Agricultural Utilization Research,1815 N. University Street, Peoria, Illinois 61604

Received 1 March 2010/Accepted 22 May 2010

Ari1p from Saccharomyces cerevisiae, recently identified as an intermediate-subclass short-chain dehydro-genase/reductase, contributes in situ to the detoxification of furfural. Furfural inhibits efficient ethanol pro-duction by yeast, particularly when the carbon source is acid-treated lignocellulose, which contains furfural ata relatively high concentration. NADPH is Ari1p’s best known hydride donor. Here we report the stereochem-istry of the hydride transfer step, determined by using (4R)-[4-2H]NADPD and (4S)-[4-2H]NADPD andunlabeled furfural in Ari1p-catalyzed reactions and following the deuterium atom into products 2-furanmetha-nol or NADP�. Analysis of the products demonstrates unambiguously that Ari1p directs hydride transfer fromthe si face of NADPH to the re face of furfural. The singular orientation of substrates enables construction ofa model of the Michaelis complex in the Ari1p active site. The model reveals hydrophobic residues near thefurfural binding site that, upon mutation, may increase specificity for furfural and enhance enzyme perfor-mance. Using (4S)-[4-2H]NADPD and NADPH as substrates, primary deuterium kinetic isotope effects of 2.2and 2.5 were determined for the steady-state parameters kcat

NADPH and kcat/KmNADPH, respectively, indicating

that hydride transfer is partially rate limiting to catalysis.

Increased energy demands and need for diversified and re-newable energy sources have strengthened interest in conver-sion of lignocellulosic biomass to monosaccharide intermedi-ates which can be converted by ethanologenic organisms toethanol for use as biofuel. As an essential step preceding de-construction of lignocellulosic biomass by enzymatic means,several different pretreatment strategies are employed. Dilute-acid pretreatment provides considerable benefits, including ex-posure of cellulose fibrils to enzymatic degradation; however,use of acidic pretreatment strategies leads to conditions whichgenerate furans from breakdown of monosaccharides (7, 23,29) via a dehydration mechanism. The presence of these sideproducts is problematic, as certain furans, e.g., furfural (2-furaldehyde) (Fig. 1), inhibit ethanol production in yeast (21,26, 34). An effective mechanism for microorganisms to miti-gate furfural toxicity during fermentation is to reduce the al-dehyde to 2-furanmethanol (FM) (10, 33, 37). Thus, in situdetoxification by reduction of furfural is an important means toaddress inhibitor challenges involved in achieving efficient bio-fuel production (28).

An NADPH-dependent aldehyde reductase (Ari1p) (ARI1,YGL157W), from Saccharomyces cerevisiae NRRL Y-12632has recently been identified as a new member of the interme-diate subclass of the short-chain dehydrogenase/reductase(SDR) superfamily (Saccharomyces Genome Database [http:

//www.yeastgenome.org/cache/genomeSnapshot.html]) (27).Ari1p was demonstrated to contribute in situ detoxification offurfural and other inhibitors of ethanol fermentation under5-hydroxymethyl furfural-furfural stress (26). Ari1p shows widesubstrate acceptance, as it is capable of reducing at least 14aldehyde substrates (16, 26, 27), including numerous inhibitorspresent in lignocellulosic biomass hydrolysates; however, thespecific reduction of furfural will likely provide the greatestbenefit to the fermenting organism due to its abundance andknown inhibitory effect on ethanol production (21, 26, 34).Without the availability of an X-ray structure of Ari1p incomplex with substrates, as only a few structures from thissubclass have been reported (32), our immediate interest isunderstanding the stereochemical binding characteristics ofthe Ari1p active site to aid construction of a model of theMichaelis complex. Such a model could be used to designmutagenesis studies for optimization of the catalyst for furfuralreduction. For example, a common feature of SDRs is aconserved hydrophobic channel that serves as a portal forsubstrate entry and a hydrophobic binding pocket for thealdehyde substrate (17), either of which could be modified atthe amino acid level to search for increased selectivity forfurfural.

An interesting aspect of the dehydrogenase/reductase en-zymes is their activity on prochiral ketone substrates. The useof SDRs as chiral induction agents for the generation of re-duced materials with high enantiomeric excess has beenthe subject of many studies (15, 31). Specifically, Ari1p(YGL157Wp) has previously been shown to be capable ofreducing �- and �-keto esters with high enantioselectivity (18,19) and t-butyl 6-chloro-3,5-dioxohexanoate with high enanti-oselectivity and regiospecificity (38). Within the data reported,

* Corresponding author. Mailing address: U.S. Department of Ag-riculture, Agricultural Research Service, National Center for Agricul-tural Utilization Research, 1815 N. University Street, Peoria, IL 61604.Phone: (309) 681-6327. Fax: (309) 681-6427. E-mail: [email protected].

� Published ahead of print on 4 June 2010.

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the Ari1p-catalyzed reduction of �-keto esters and �-chloro-�-keto esters with substituents ranging in size from methyl tobenzyl occurs from the re face regardless of substituent (18,19). Variations in the structure, including a �-branched sub-strate and a phenyl-substituted ketone, were poorly reduced,and no stereochemical data were reported (18, 19); however, inother yeast reductases the change in substrate size was suffi-cient to provide the opposite stereochemical product (19),suggesting that Ari1p may not tolerate certain bulky substitu-ents according to the large and small binding pocket model(36). In the case of �-keto esters, regardless of substituent size,the reported products of Ari1p-catalyzed reactions were re-duced from the si face (19). These data indicate that Ari1p iscapable of highly enantioselective reduction, but substrate vari-ation (i.e., carbonyl location) can lead to the opposite stereo-chemical products; therefore, determination of the stereo-chemical outputs of furfural reduction is a necessary step forconstructing an accurate binding model.

The determination of the putative substrate orientation inthe binding pockets of Ari1p for the two-component reactioncan be accomplished by tracking the substrates and productsthrough the reaction. In a previous report, a homology modelfor sn-glycerol-1-phosphate dehydrogenase, an archaeal mem-brane-synthesizing enzyme showing the stereochemical prod-uct opposite from those of bacterial and eukaryotic counter-parts, was used to predict the NADPH and glycerol phosphatebinding orientations based on the known difference in productformation (8). In a subsequent report, the stereochemistry oftransfer from NADP3H was shown to be stereospecific fromthe pro-R face (22), demonstrating the benefits of models thatincorporate the characterization of both substrate and productoutcomes. Similarly, Geissler et al. used homology modeling toidentify four critical active-site and nine binding pocket aminoacids of SDR salutaidine reductase, a potentially importantcomponent of the morphine biosynthetic pathway (13). Whenmutated, the identified residues followed the model’s pre-dicted outcome. This demonstrated the ability to increase thefidelity of a model by use of both substrate and product stereo-chemical analyses. In both of these cases the stereochemistryof the product was known, facilitating model construction.

For the current study, the use of stereo-defined NADPDsubstrates and furfural, as a prochiral substrate, should permitthe determination of the binding orientations of both NADPHand furfural substrates within the active site. The additionof hydride from NADPH can occur from the pro-S [(4S)-NADPH] or pro-R [(4R)-NADPH] face and may add to eitherthe si or re face of the aldehyde substrate (Fig. 1), thus providingthree possible alcohol products, i.e., unlabeled FM, (R)-[2-2H]FM, and (S)-[2-2H]FM, when reduced with NADPD. There-fore, determination of the chirality of Ari1p-catalyzed hydridetransfer from NADPH to the product, FM, will unequivocallydefine the binding mode within the Ari1p active site.

MATERIALS AND METHODS

Materials and general methods. NADP�, NADPH, glucose dehydrogenasefrom Pseudomonas sp., alcohol dehydrogenase from Thermoanaerobium brockii,D2O, furfural, FM, bovine serum albumin (BSA), methyl t-butyl ether (MTBE),and sodium borodeuteride were purchased from Sigma-Aldrich (Saint Louis,MO). Dimethylaminopyridine (DMAP) was from Acros (Morris Plains, NJ),(S)-(�)-�-methoxy-�-(trifluoromethyl)phenylacetyl chloride was from Alfa-Ae-sar (Ward Hill, MA), SylonBFT was from Supelco (Bellefonte, PA), and 1-d1-glucose (D-[1-2H]glucose) and d8-isopropanol were from Cambridge Isotopes(Cambridge, MA). Ari1p was purified as described using His tag affinity chro-matography (27). Ari1p concentrations were estimated from its absorbance at280 nm using the calculated extinction coefficient of 24,990 M�1 cm�1 (14). AllNADP�, NADPH(D), and FM ester nuclear magnetic resonance (NMR) spec-tra were collected using a Bruker Avance 500 MHz NMR spectrometer.

Preparation of (4S)-[4-2H]NADPD. The procedure for preparation of (4S)-[4-2H]NADPD is based on that of McCracken et al. (30). Briefly, NADP� (200�mol), D-[1-2H]glucose (400 �mol), and glucose dehydrogenase (60 units) weredissolved in 25 ml 20 mM Tris-HCl at pH 7.0 and 25°C. Aliquots of sodiumhydroxide (1 M) were added to maintain the pH during the course of thereaction. After 2 h, the reaction mixture was centrifuged through an AmiconUltra-15 3,000-molecular-weight (MW)-cutoff filter. The filtrate was loaded ontoa Q-Sepharose Fast Flow column (1.6 by 18 cm), equilibrated with water, andcontrolled by an AKTA 900 high-pressure liquid chromatography (HPLC) sys-tem (Amersham-Biosciences), which monitored absorbance of the eluent at 260and 340 nm. The column was washed with 40 ml water followed by a 200-mllinear gradient of 0 to 500 mM ammonium carbonate. The fraction containing(4S)-[4-2H]NADPD was lyophilized, followed by two repetitions of lyophilizationwith 10 ml D2O. The yield of (4S)-[4-2H]NADPD was 100% (150 mg, 200 �mol),as determined by UV absorbance. The identity of (4S)-[4-2H]NADPD was de-termined by 1H NMR, marked by the absence (100% deuterium incorporation)of the 4S hydrogen (� 2.67 ppm) (4) in the proton spectrum.

Preparation of (4R)-[4-2H]NADPD. The procedure for preparation of (4R)-[4-2H]NADPD is based on that reported by McCracken et al. (30). NADP� (280�mol), d8-isopropanol (1040 �mol), and alcohol dehydrogenase from Thermo-anaerobium brockii (100 units) were incubated in 25 ml 20 mM Tris-HCl at pH7.0 and 25°C; purification was as described above for (4S)-[4-2H]NADPD. Theyield of (4R)-[4-2H]NADPD was 40% (82 mg, 110 �mol), as determined by UVabsorbance. Identity of (4R)-[4-2H]NADPD was determined by 1H NMR,marked by the absence (100% deuterium incorporation) of the 4R hydrogen (�2.76 ppm) (4) in the proton spectrum.

Production of stereochemically defined (R)-[2-2H]FM. According to the pro-cedure of Yamada et al. (39), treatment of 30 �l (0.27 mmol) furfural withbaker’s yeast (2.5 g) in deuterium oxide (10 ml) provided (R)-[2-2H]FM as theonly deuterated stereoisomer. 2-Furanmethanol (0.26 mmol, 96% conversion,65% deuterium incorporation) was extracted from the reaction mixture by theaddition of methyl t-butyl ether (MTBE) (three times with 10 ml). MTBE wasremoved under a gentle nitrogen stream.

Synthesis of racemic [2-2H]FM. Furfural (100 �l, 0.89 mmol) was dissolved inchloroform (500 �l), followed by addition of sodium borodeuteride (42 �g, 1mmol) in methanol (100 �l). The reaction mixture was warmed to 35°C for 2 h,followed by neutralization with acetic acid, removal of solvent, dissolving inMilli-Q water (5 ml), and extraction with MTBE (three times with 5 ml). MTBEwas removed under a gentle nitrogen stream.

Preparation of products of Ari1p-catalyzed furfural reduction by NADPD.Ari1p-catalyzed reductions of furfural by NADPD were performed in 25 ml 5mM Tris-HCl at pH 7.0 and 25°C. Mixtures for reactions of 120 �mol (4S)-[4-

FIG. 1. Reactions involving furfural discussed in this work.(A) Generation of furfural from biomass derived pentosans and mit-igation by Ari1p-mediated reduction to FM. (B) Potential reactantorientations. The hydride can be transferred from either the pro-S orpro-R face of the nicotinamide ring to either the si or re face of furfural.

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2H]NADPD with 200 �mol furfural contained 25 units (�mol product/min)Ari1p. The pH of the reactions was maintained with HCl (1 M). When theoxidation of NADPD was complete (absorbance monitored at 340 nm), reactionmixtures were filtered through an Amicon Ultra-15 3,000-MW-cutoff filter. Thefiltrate was separated by Q-Sepharose Fast Flow chromatography as describedabove, with absorbance of the eluent monitored at 217, 260, and 277 nm. Un-reacted furfural and reaction product FM eluted in the void volume. Yields ofFM were 88% (105 �mol from 120 �mol (4S)-[4-2H]NADPD) and 93% (28�mol from 30 �mol (4R)-[4-2H]NADPD) as determined by UV absorbance at216 nm and 277 nm (12).

Analysis of products of Ari1p-catalyzed furfural reduction by NADPD. Theanion-exchange chromatography fractions containing oxidized NADP� werelyophilized, followed by two repetitions of lyophilization with 10 ml D2O. TheNADP� samples, dissolved in D2O at pD 6.8 or 4.5, were analyzed by 1H NMRfor comparison to purchased NADP� (4). The NADP� fractions also wereanalyzed by gas chromatography-mass spectrometry (GC-MS). Aliquots (100 �l)of the two fractions, after MTBE extraction of FM products, were dried in vacuo.The samples were then treated with SylonBFT in acetonitrile (1:1) (100 �l) at65°C for 10 min. After cooling, 100 �l of acetonitrile was added and 1 �l wasinjected for analysis by GC-MS.

The presence of [2-2H]FM or FM in chromatography fractions was deter-mined by GC-MS analysis. Each FM sample (1 �l) was dissolved in 500 �lCH3CN containing 2.5% SylonBFT{BSTFA [bis(trimethylsilyl)trifluoro-acetamide]/TMCS [trimethylchlorosilane]/} ratio of 99:1 and incubated at 60°Cfor 10 min. After cooling, 1 �l was injected onto a DB5XB column (30 m by 0.25�m; Shimadzu). After an initial 4 min of isothermal elution at 50°C, elution waseffected by use of a thermal gradient extending from 50°C to 300°C at a rate of15°C/min. [2-2H]FM and FM were identified by comparison to the mass spec-trum fragmentation pattern of FM. Quantification of the incorporation of deu-terium was determined by comparison to FM standards and quantification ofunreacted furfural. During GC analysis, there is a small isotope effect observedwhen deuterium is present in the analytes (approximately 0.1 min). The responsefactors were assumed to be the same for deuterated products and standards.

The Ari1p-catalyzed reduction of furfural by (4S)-[4-2H]NADPD was deter-mined to contain [2-2H]FM in its HPLC fraction. The HPLC fraction (28 ml) wasextracted thrice with MTBE (12.5 ml), and MTBE was removed under a gentlenitrogen stream. Following MTBE extraction and drying, the Ari1p reactionproduct [2-2H]FM and the two [2-2H]FM standards were converted to (R)-(�)-�-methoxy-�-(trifluoromethyl)phenylacetyl-[2-2H]FM esters for NMR analysis.In a typical esterification reaction, FM samples (30 �l, 0.27 mmol) were dissolvedin 1 ml CH2Cl2, followed by the addition of (S)-(�)-�-methoxy-�-(trifluoro-methyl)phenylacetyl chloride (Mosher’s chloride; 75 mg, 0.30 mmol), triethyl-amine (20 �l, 0.27 mmol), and catalytic DMAP. The reaction proceeded at roomtemperature for 48 h with monitoring of FM consumption and product esterformation by GC-MS. The reaction was stopped by addition of 1 ml Milli-Qwater and removal of the aqueous layer. The organic layer was dried under anitrogen stream. The residue was dissolved in hexane (1 ml) and filtered througha silica plug. Hexane-ethyl acetate (9:1) was used to elute the (�)-�-methoxy-�-(trifluoromethyl)phenylacetyl-(R)-[2-2H]FM ester derivatives from the silicaplug. The purity of (R) (�)-�-methoxy-�-(trifluoromethyl)phenylacetyl-(R)-[2-2H]FM ester derivatives was determined by GC-MS, followed by 1H NMR and2H NMR. 2H NMR spectra were acquired in CHCl3-CDCl3 (99:1), the instru-ment was shimmed to the CDCl3 signal, and all data are reported in ppm basedon the chemical shift of CDCl3.

KIE. Reaction mixtures (1.0 ml) contained 10 mM furfural, various con-centrations of NADPH or NADPD, 0.1 mg/ml BSA, and 100 mM sodiumphosphate (ionic strength of 0.3 M adjusted with NaCl) at pH 7.0 and 25°C.Reactions were initiated with Ari1p and monitored at 340 nm for 1 min.Initial rates were fit to the equation v � (kcat � S)/Km � S through thecomputer program Grafit (24) to determine kinetic parameters for enzymeacting on NADPH and NADPD. kcat and kcat/Km values were expressed inunits of s�1 and s�1 mM�1, respectively. Deuterium kinetic isotope effects(KIEs) were calculated from the ratio of parameters determined withNADPH versus NADPD.

Modeling the Michaelis complex of Ari1p. The amino acid sequence of Ari1pwas submitted, through the Internet, to the Swiss-Model Workspace (3) forautomated building of a three-dimensional homology model. Swiss-Model re-turned a homology model based on the X-ray structure of Sporidiobolus salmo-nicolor aldehyde reductase in complex with NADPH (Protein Data Bank [PDB]no. 1Y1P). Chimera software (35) was used for subsequent modifications of themodel. The template’s NADPH ligand was transferred to the homology model ofAri1p. Furfural was added to the homology model by hand, obeying the stereo-

chemistry of hydride addition to the aldehyde described in this study and keepingreasonable contact distances between furfural, NADPH, and the protein.

RESULTS AND DISCUSSION

Definition of the productive binding orientations ofNADPH and furfural within the active site of Ari1p (Fig. 1)can be determined from analysis of two Ari1p-catalyzed reac-tions: reduction of furfural by (4S)-[4-2H]NADPD and reduc-tion of furfural by (4R)-[4-2H]NADPD. In pursuing this strat-egy, the two stereochemically defined, deuterium-labeledNADPD substrates of Ari1p were prepared enzymatically. Theextent of deuterium incorporation in chromatographically pu-rified substrates (4R)-[4-2H]NADPD and (4S)-[4-2H]NADPDwas determined by 1H NMR to be quantitative (100%). Next,the two Ari1p-catalyzed reactions were allowed to proceeduntil there was complete conversion of NADPD (the limitingsubstrate) to NADP�, as determined by UV absorbance. FMand NADP� products of the reactions were purified by anion-exchange chromatography. The resultant NADP�s were ana-lyzed by gas chromatography-mass spectrometry (GC-MS) and1H NMR, and the resultant FMs were analyzed by GC-MS(Fig. 2).

Analysis of the data from the Ari1p-catalyzed reduction offurfural by (4S)-[4-2H]NADPD reveals that transfer of deuter-ide occurs quantitatively from (4S)-[4-2H]NADPD to generate[2-2H]FM and unlabeled NADP�: there is a 1-Da increase inthe mass spectrum of the FM reaction product {m/z 99 (M� � );82 [M(-OH)� � ]} shown in Fig. 2B in comparison to that of theunlabeled FM standard shown in Fig. 2A. The presence of anundeuterated NADP� reaction product is indicated by thesignal from the dissociated nicotinamide ring m/z 122 (M� � );106 [M(-NH2)� � ]; 78 [M(-CONH2)� � ] of Fig. 2E, which issimilar to that of the NADP� standard (Fig. 2D). The 1HNMR spectrum of the NADP� reaction product shows a pro-ton signal at � 8.87 ppm (Fig. 2H), similar to that of theNADP� standard (Fig. 2G), which confirms the presence ofhydrogen at the C-4 position of the nicotinamide ring (4).Analysis of the data from the Ari1p-catalyzed reduction of furfuralby (4R)-[4-2H]NADPD reveals that (4R)-[4-2H]NADPD transfersa hydride to furfural, generating an undeuterated FM reactionproduct with retention of the deuterium in the nicotinamidering of NADP�; the mass spectrum of the FM reaction product{m/z 98 (M� � ); 81 [M(-OH)� � ]} of Fig. 2C is similar to thatof the unlabeled FM standard (Fig. 2A). There is a 1-Daincrease in the mass spectrum of the NADP� reaction productm/z 123 (M� � ); 107 [M(-NH2)� � ]; 79 [M(-CONH2)� � ] of Fig.2F in comparison to that of the NADP� standard (Fig. 2D).The 1H NMR spectrum of the NADP� reaction productshows no proton signal at � 8.87 ppm (Fig. 2I), consistentwith deuterium occupation of the C-4 position of the nico-tinamide ring (4). This establishes that hydride transfer oc-curs from the pro-S position of NADPH, a universal featureof members of the SDR superfamily (1, 2, 4–6, 11, 25).

To determine the stereochemistry of deuterium addition tothe prochiral furfural (re or si face) (Fig. 1), the stereochem-istry of the deutero-FM reaction product of the Ari1p-cata-lyzed reduction of furfural by (4S)-[4-2H]NADPD was deter-mined. First, it was necessary to prepare deuterated FMstandards. Furfural was reduced with sodium borodeuteride to

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provide racemic [2-2H]FM. Furfural was reduced in a stereo-controlled manner by baker’s yeast incubated in D2O accord-ing to the method of Yamada et al. (39) to produce (R)-[2-2H]FM. Each method generated [2-2H]FM as the majorproduct; however, the yeast method produced 35% FM withno deuterium incorporation. The Ari1p-catalyzed reactionproduct [2-2H]FM and the two [2-2H]FM standards were de-rivatized with the chiral reagent (S)-(�)-�-methoxy-�-(triflu-

FIG. 2. GC-MS and 1H NMR data collected for FM and NADP�

samples. GC-MS of the FM standard (A), FM from Ari1p-mediatedreduction of furfural by (4S)-[4-2H]NADPD (B), FM from Ari1p-mediated reduction of furfural by (4R)-[4-2H]NADPD (C), theNADP� standard (D), NADP� from Ari1p-mediated reduction offurfural by (4S)-[4-2H]NADPD (E), and NADP� from Ari1p-medi-ated reduction of furfural by (4R)-[4-2H]NADPD (F) and 1H NMR ofthe NADP� standard at pD 6.0 (G) and of NADP� at pD 4.5 fromAri1p-mediated reduction of furfural by (4S)-[4-2H]NADPD (H) andat pD 6.8 NADP� from Ari1p-mediated reduction of furfural by (4R)-[4-2H]NADPD (I) are shown. � 7.95, 8.10 pD-dependent proton ofadenine C2; � 8.15, pyridinium C5; � 8.4, pD-dependent proton ofadenine C8; � 8.75, pyridinium C4; � 9.17, pyridinium C6; � 9.25,pyridinium C2. Proton NMR integrations are indicated.

FIG. 3. 2H NMR spectra of monodeutero furfuryl esters in CHCl3-CDCl3 (99:1). (A) Racemic standard (R)-(�)-�-methoxy-�-(triflu-oromethyl)phenylacetyl-[2-2H]FM ester. (B) Yeast reduced standard(R)-(�)-�-methoxy-�-(trifluoromethyl)phenylacetyl-(R)-[2-2H]FM es-ter. (C) Ari1p-mediated reduction of furfural by (4S)-[4-2H]NADPD:(R)-(�)-�-methoxy-�-(trifluoromethyl)phenylacetyl-(R)-[2-2H]FM es-ter. R and S peaks are indicated.



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oromethyl)phenylacetyl chloride (Mosher’s chloride) (9) togenerate diastereomeric products, which were analyzed by 1HNMR and 2H NMR (Fig. 3). The absolute stereochemicalconfiguration of Ari1p-generated [2-2H]FM was determined bycomparison to [2-2H]FM reduced nonstereoselectively by so-dium borodeuteride and (R)-[2-2H]FM generated by yeast re-duction. The 2H NMR spectrum (Fig. 3) clearly shows thatthe Ari1p-catalyzed reduction of furfural by (4S)-[4-2H]NADPD produces a single deuterated product that has achemical shift that matches that of the previously assigned(R)-[2-2H]FM generated by yeast reduction. Therefore, thepro-S deuteride was completely enantioselective in adding tothe re face of furfural. As the incoming deuteride nucleo-phile has higher Cahn-Ingold-Prelog priority than the alde-hyde hydrogen, the product alcohol has (R-) stereochemistrydespite constituting a canonical Prelog addition (36).

A homology model of the three-dimensional structure ofAri1p was automatically generated by the Swiss-Model Work-space (3). The model used the X-ray structure of aldehydereductase from S. salmonicolor, an intermediate-class SDR, incomplex with NADPH (PDB no. 1Y1P) (20) as the template.Transfer of NADPH from the template to the Ari1p homologymodel indicates that the pro-S hydrogen must be the hydridedonor, as the A (pro-R) face of NADPH is sterically unavail-able for binding furfural due to its proximity to protein resi-dues. Furfural was docked into the modeled active site ofAri1p, avoiding steric conflicts and with its re face in position toaccept the pro-S hydrogen of NADPH, thus generating the

model of the Ari1p Michaelis complex (Fig. 4). Notably, fur-fural fits into the Ari1p active site, without steric conflict, witheither its re face or si face toward the pro-S hydrogen ofNADPH, potentially indicating a binding pocket that is notcapable of stereoselectivity. With the re face orientation of themodel, however, the furfural ring oxygen can share an H bondwith the side chain hydroxyl group of the conserved Thr132.Conserved residues Lys173, Tyr169, and Ser131 are in properalignment for catalysis, with Ser131 and Tyr169 sharing Hbonds with the carbonyl oxygen of furfural and Lys173 andTyr169 serving as part of the proton delivery mechanism, com-mon features of SDRs (17). The hydrophobic channel is de-fined by conserved amino acids Phe91 and Val166, correspond-ing to Phe97 and Leu174 from the original structure. Thehydrophobic pocket is composed of residues Gly201, Phe202,and Phe246, with modeled carbon-to-carbon distances of 3.9 Å(�-CGly201 to C2furfural), 3.6 Å (�-CPhe202 to C3furfural), 3.8 Å(�-CPhe202 to C4furfural), and 3.0 Å (�-CGly201 to C5furfural).These residues serve as potential targets for modification togenerate Ari1p mutants that may express greater proficiencyfor catalyzing furfural reduction, which in turn could increasethe ability of Saccharomyces cerevisiae to withstand exposure tofurfural.

Deuterium kinetic isotope effects (KIEs) were determinedby direct comparison of initial-rate determinations of kineticparameters of Ari1p acting on furfural with (4S)-[4-2H]NADPD

FIG. 4. Model of the Michaelis complex of Ari1p. Addition of thepro-S hydrogen from NADPH to the re face of furfural is shown.Amino acid residues and NADPH orientations are from a homologymodel of Ari1p based on the X-ray structure of aldehyde reductasefrom Sporobolomyces salmonicolor in complex with NADPH (PDB no.1Y1P) (20). Dashed lines indicate H bonds and path of HS to thecarbonyl carbon of furfural.

FIG. 5. Determination of deuterium kinetic isotope effects frominitial-rate data by direct comparison. Reaction mixtures contained100 mM sodium phosphate adjusted to ionic strength 0.3 M with NaCl,10 mM furfural, and various concentrations of NADPD or NADPH atpH 7.0 and 25°C.



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or (4R)-[4-2H]NADPD in comparison to NADPH at pH 7.0and 25°C. NADPH or NADPD concentrations were varied,and furfural was held at a near-saturating concentration of 10mM for determinations of initial rates (Fig. 5). Using (4S)-[4-2H]NADPD and NADPH as substrates, primary deuteriumkinetic isotope effects of 2.21 � 0.08 and 2.51 � 0.20 weredetermined for the ratios of steady-state kinetic parameterskcat

NADPH/kcatNADPD and (kcat/Km)NADPH/(kcat/Km)NADPD, re-

spectively, indicating that hydride transfer is partially rate lim-iting to steady-state catalysis. Using (4R)-[2H]NADPD andNADPH as substrates, secondary deuterium kinetic isotopeeffects of 1.2 � 0.03 and 1.02 � 0.08 were determined for theratios of steady-state parameters kcat

NADPH/kcatNADPD and

(kcat/Km)NADPH/(kcat/Km)NADPD, respectively. The magnitudesof the KIEs further substantiate that the pro-S hydrogen ofNADPH constitutes the hydride of transfer.

Conclusions. Ari1p has been shown to catalyze transfer ofdeuterium from (4S)-[4-2H]NADPD to furfural, generating anenantiomerically pure (R)-[2-2H]FM product. This leads unam-biguously to the conclusion that Ari1p catalyzes a canonical Pre-log reduction reaction in transferring hydride from the pro-Shydrogen of NADPH to the re face of furfural, consistent withthat of previously reported members of the intermediate subclassof the SDR enzyme superfamily. The stereochemical input andoutput of deuterium addition to furfural were needed for gener-ation of an accurate working model of the Ari1p Michaelis com-plex. This knowledge will be useful in designing mutant enzymeshaving improved properties for furfural reduction.

ACKNOWLEDGMENTS

This work was supported in part by the National Research Initiativeof the USDA Cooperative State Research, Education and ExtensionService, grant number 2006-35504-17359.

Mention of trade names or commercial products in this article is solelyfor the purpose of providing scientific information and does not implyrecommendation or endorsement by the U.S. Department of Agriculture.

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stereochemistry of furfural reduction by a saccharomyces ... · aldehyde substrate (17), either of...

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