A New Synthetic Route to N-Benzyl Carboxamides through theReverse Reaction of N-Substituted Formamide Deformylase

Yoshiteru Hashimoto, Toshihide Sakashita, Hiroshi Fukatsu, Hiroyoshi Sato, Michihiko Kobayashi

‹Institute of Applied Biochemistry and Graduate School of Life and Environmental Sciences, The University of Tsukuba, Tennodai, Tsukuba, Ibaraki, Japan

Previously, we isolated a new enzyme, N-substituted formamide deformylase, that catalyzes the hydrolysis of N-substitutedformamide to the corresponding amine and formate (H. Fukatsu, Y. Hashimoto, M. Goda, H. Higashibata, and M. Kobayashi,Proc. Natl. Acad. Sci. U. S. A. 101:13726 –13731, 2004, doi:10.1073/pnas.0405082101). Here, we discovered that this enzyme cata-lyzed the reverse reaction, synthesizing N-benzylformamide (NBFA) from benzylamine and formate. The reverse reaction pro-ceeded only in the presence of high substrate concentrations. The effects of pH and inhibitors on the reverse reaction were al-most the same as those on the forward reaction, suggesting that the forward and reverse reactions are both catalyzed at the samecatalytic site. Bisubstrate kinetic analysis using formate and benzylamine and dead-end inhibition studies using a benzylamineanalogue, aniline, revealed that the reverse reaction of this enzyme proceeds via an ordered two-substrate, two-product (bi-bi)mechanism in which formate binds first to the enzyme active site, followed by benzylamine binding and the subsequent releaseof NBFA. To our knowledge, this is the first report of the reverse reaction of an amine-forming deformylase. Surprisingly, analy-sis of the substrate specificity for acids demonstrated that not only formate, but also acetate and propionate (namely, acids withnumbers of carbon atoms ranging from C1 to C3), were active as acid substrates for the reverse reaction. Through this reaction,N-substituted carboxamides, such as NBFA, N-benzylacetamide, and N-benzylpropionamide, were synthesized from benzyl-amine and the corresponding acid substrates.

We have extensively studied the biological metabolism ofcompounds with a triple bond between carbon and nitro-

gen, such as nitriles and isonitriles (1–4). Nitriles are highly toxicand generally nonbiodegradable organic compounds containing aC'N moiety. As well as nitriles, isonitriles (more generally calledisocyanides) containing an N'C functional group are generallyhighly toxic. The isocyano group, which possesses an unusual va-lence structure and reactivity, exhibits a dual nucleophilic/electro-philic character, which is often exploited for synthetic applica-tions, e.g., in the synthesis of peptides, coordination chemistry,organometallic reactions, and carbohydrate chemistry (5). On theother hand, many natural isonitriles have been isolated from var-ious organisms, including bacteria, fungi, and marine sponges (5–7). An isocyanide metabolite, xanthocillin, was first isolated fromPenicillium notatum (8). Most isonitriles show a wide antibioticactivity spectrum and exhibit potential as possible agents withpractical use (9, 10). Some metabolic intermediates of some iso-nitriles have been elucidated through incorporation experiments(11–15), and the synthetic pathway involved was determined veryrecently (16–19). However, information on the metabolism of anisonitrile at the protein and gene levels had been quite limiteduntil we discovered an isonitrile-degrading enzyme. We have ex-tensively studied the biological metabolism of isonitriles and re-vealed that it is quite different from nitrile metabolism (20–23).We initially found isonitrile hydratase, which catalyzes the hydra-tion of an isonitrile (R-N'C) to the corresponding N-substitutedformamide [R-NH-CH(AO)] (24, 25). We also discovered N-substituted formamide deformylase, which catalyzes the hydroly-sis of an N-substituted formamide to the corresponding amineand formate (26). Isonitrile hydratase and N-substituted formam-ide deformylase have been approved as new enzymes by the Inter-national Union of Biochemistry and Molecular Biology (IUBMB),and new EC numbers (EC 4.2.1.103 and EC 3.5.1.91, respectively)have been given to them. Very recently, we discovered the second

isonitrile hydratase, which cooperated with N-substituted form-amide deformylase in Arthrobacter pascens F164 (27). These twoisonitrile hydratases are biochemically, immunologically, andstructurally different from each other. However, there have beenonly three reports (24–27) on the enzymes involved in isonitrilemetabolism.

There are several types of amine-forming deformylases in-volved in the metabolism of N-substituted formamides, includingkynurenine formamidase (EC 3.5.1.9) (28–30), formylmethio-nine deformylase (EC 3.5.1.31) (31, 32), and peptide deformylase(EC 3.5.1.88) (33–35). Although N-substituted formamide de-formylase is one of the amine-forming deformylases, it shows noamino acid sequence similarity to any other deformylases knownso far. Contrary to expectation, the deduced amino acid sequenceof N-substituted formamide deformylase exhibited the highestoverall sequence identity (28%) to those of regulatory proteins.Only 15 amino acid residues in the N-terminal region (residues 58to 72) of N-substituted formamide deformylase showed signifi-cant sequence identity (27 to 73%) to those in each member of theamidohydrolase superfamily (36), including imidazolonepropio-nase (37), atrazine chlorohydrolase (38), cytosine deaminase (39),dihydroorotase (40), and urease (41); except for these 15 aminoacid residues in the N-terminal region, there was no similarity inthe overall sequences. Like the other enzymes in the amidohydro-lase superfamily, N-substituted formamide deformylase would

Received 19 July 2013 Accepted 6 October 2013

Published ahead of print 11 October 2013

Address correspondence to Michihiko Kobayashi, sec-mmb@agbi.tsukuba.ac.jp.

Y.H. and T.S. contributed equally to this work.

Copyright © 2014, American Society for Microbiology. All Rights Reserved.

doi:10.1128/AEM.02429-13

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have an (�/�)8 barrel structure in the N-terminal region that con-tains five conserved residues (four histidines and one asparticacid). Moreover, this enzyme contains three zinc ions per subunit(26). Based on these findings, N-substituted formamide deformy-lase was proposed to be a member of the amidohydrolase super-family (36).

Here, we discovered that N-substituted formamide deformy-lase involved in the degradation of toxic isonitriles is able to cata-lyze a unique reverse reaction: N-benzylformamide (NBFA) syn-thesis from formate and benzylamine. When acetate andpropionate were each used as an acid substrate instead of formate,N-benzylacetamide and N-benzylpropionamide, respectively,were also formed. To our knowledge, this is the first report of thereverse reaction of amine-forming deformylase and the enzymaticproduction of N-benzyl carboxamides through the reverse reac-tion of amine-forming deformylase.

MATERIALS AND METHODSMaterials. Benzylamine was purchased from Tokyo Kasei Kogyo Co., Ltd.(Tokyo, Japan). Formate was obtained from Kishida Chemical Co., Ltd.(Osaka, Japan). NBFA was purchased from Sigma. Propionic acid wasobtained from Nakalai Tesque (Kyoto, Japan). N-Benzylacetamide waspurchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan).N-Benzylpropionamide was obtained from Chemical Technologies & In-vestigations, Ltd. (Moscow, Russia). DEAE-Sephacel, Resource ISO, Su-perose 12 10/30, and a low-molecular-weight standard kit were obtainedfrom GE Healthcare UK Ltd. (Buckinghamshire, United Kingdom). Allother biochemicals were standard commercial preparations.

Purification of N-substituted formamide deformylase. All purifica-tion procedures were performed at 0 to 4°C. Potassium phosphate buffer(KPB) (pH 7.5) containing 10% (vol/vol) glycerol was used throughoutthe purification unless otherwise noted. Centrifugation was carried outfor 15 min at 13,000 � g.

Cells were harvested by centrifugation, washed twice with 10 mMKPB, and then disrupted by sonication at 180 W for 20 min with anInsonator model 201 M (Kubota, Tokyo, Japan) to prepare a cell extract.The cell debris was removed by centrifugation. The resultant supernatantwas fractionated with ammonium sulfate (40 to 45%), followed by dialysisagainst 10 mM KPB. The dialyzed solution was applied to a DEAE-Sep-hacel column (5 by 40 cm; GE Healthcare UK Ltd.) equilibrated with 10mM KPB containing 0.25 M KCl. Proteins were eluted by increasing theionic strength of KCl from 0.25 to 0.5 M in a linear manner in the sameKPB. The active fractions were collected, and then ammonium sulfate wasadded to give 70% saturation. After centrifugation of the suspension, theprecipitate was dissolved in 10 mM KPB, followed by dialysis against 10mM KPB. The enzyme solution obtained was brought to 25% ammoniumsulfate saturation and then placed on a Resource ISO column (1.6 by 3 cm;GE Healthcare UK Ltd.) equilibrated with 10 mM KPB containing 25%saturated ammonium sulfate. The enzyme was eluted with a linear gradi-ent of ammonium sulfate (from 25 to 15% saturation) in 10 mM KPB. Theactive fractions were combined and concentrated with a Centricon-10microconcentrator (Amicon Inc., Beverly, MA) in 50 mM KPB contain-ing 0.15 M NaCl. The enzyme solution obtained was then applied to aSuperose 12 10/30 column (GE Healthcare UK Ltd.) equilibrated with 10mM KPB containing 0.15 M NaCl. Protein was eluted from the columnwith the same buffer. The active fractions were pooled and then precipi-tated with ammonium sulfate at 70% saturation. After centrifugation ofthe suspension, the precipitate was dissolved in 10 mM KPB, followed bydialysis against 10 mM KPB. The homogeneity of the purified protein wasconfirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE).

Assay of the reverse reaction of N-substituted formamide deformy-lase. All of the reactions were performed under linear conditions for pro-tein (0.1 mg/ml) and time (5 min). The standard assay mixture comprised

0.1 M KPB (pH 7.5), 100 mM benzylamine, 2 M formate, and 0.1 mg/mlenzyme (N-substituted formamide deformylase) in a total volume of 100�l. The reaction was started by the addition of the enzyme and carried outat 25°C for 5 min. To stop the reaction, 10 �l of the reaction mixture wastaken and added to 190 �l of 1 M citrate-Na2PO4 buffer (pH 4.0). Asupernatant was obtained by centrifugation (15,000 � g; 5 min). Theamount of NBFA formed was determined by high-performance liquidchromatography (HPLC) with a Shimadzu (Kyoto, Japan) LC-10AD sys-tem equipped with a Cosmosil 5C18-AR-II column (reversed phase; 4.6 by150 mm; Nacalai Tesque, Kyoto, Japan). The following solvent system wasused at a flow rate of 1.0 ml/min and 40°C: 10 mM KH2PO4-H3PO4 buffer(pH 2.7)–acetonitrile, 2:1 (vol/vol). The absorbance was measured at198 nm.

One unit of reverse activity of N-substituted formamide deformylasewas defined as the amount of enzyme that catalyzed the formation of 1�mol NBFA per min from benzylamine and formate under the above-mentioned conditions. Various amines and acids were tested for substratespecificity. The assay was carried out in a reaction mixture (100 �l) con-sisting of 100 mM amine substrate, 2 M acid substrate, and 0.1 mg/mlenzyme at 25°C for an appropriate time. The amount of reaction productwas determined by monitoring the column effluent at 198 nm with au-thentic standards, using the HPLC system described above. One unit ofreverse activity of N-substituted formamide deformylase was defined asthe amount of enzyme that catalyzed the formation of 1 �mol product permin from the amine substrate and acid substrate under the above-men-tioned conditions. Protein concentrations were determined as describedby Bradford (42). Specific activity was expressed as units per mg of pro-tein. kcat values were calculated using an Mr value of 58,694 for the N-sub-stituted formamide deformylase monomer (26).

Kinetic analysis. Bisubstrate kinetic analysis was performed with for-mate concentrations in the range of 0.6 to 1.5 M and benzylamine con-centrations in the range of 10 to 50 mM. The equations used for kineticsanalysis were as described by Cleland (43–45) and Segel (46). To discrim-inate sequential and ping-pong mechanisms for two-substrate kinetics,data were fitted to the following two equations: v � Vmax/(Ka/[A] �Kb/[B] � Kia � Kia/[A][B] � 1) (sequential) and v � Vmax/(Ka/[A] �Kb/[B] � 1) (ping-pong).

Enzyme kinetic data conforming to linear inhibition, as determined bysecondary replotting of the slopes and/or intercepts of the initial double-reciprocal plots (1/� versus 1/A) versus inhibitor concentration, were fit-ted to the following three equations, which correspond to competitive,noncompetitive, and uncompetitive inhibition models (47, 48): v � Vmax �[S]/{Km(1 � [I]/Ki) � [S]} (competitive), v � Vmax � [S]/{Km(1 � [I]/Ki) �[S](1 � [I]/Ki)} (noncompetitive), and v � Vmax � [S]/{Km � [S](1 � [I]/Ki)} (uncompetitive).

Under these conditions, v represents the measured velocity; Vmax themaximum velocity; A, B, and S the substrates; I the inhibitor; Ka, Kb, andKm the Michaelis constants for A, B, and S; Kia the dissociation constantfor A; Kib the dissociation constant for B; and Ki the inhibitor constant.

LC-ESI-MS analysis. Because liquid chromatography-mass spec-trometry (LC-MS) analysis was prevented by phosphate in the solventused for the HPLC analysis, the peak fraction of the reaction productobtained on HPLC was taken and again applied to the above-describedHPLC system with 33% acetonitrile instead of 10 mM KH2PO4-H3PO4

buffer (pH 2.7)–acetonitrile, 2:1 (vol/vol), in order to exclude phosphatefrom the sample. The peak fraction was collected and concentrated byevaporation. In order to determine whether the concentrated productsremained stable during the second HPLC process and evaporation, thepurified products were applied to the above-described HPLC system andconfirmed to show the same retention times as authentic standards. Thefraction was then subjected to LC-MS analysis. LC-MS was performed ona Waters Micromass ZQ coupled to a Waters Alliance HPLC system (2690Separations Module and Waters 996 photodiodoarray detector) employ-ing a Symmetry C18 column (2.1 by 150 mm; 3.5 �m). The column waseluted at 30°C with 20% (vol/vol) acetonitrile in water at a flow rate of 0.2

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ml/min. The sample was ionized with an electrospray ionization (ESI)probe in the positive-ion mode under the following source conditions:source temperature, 120°C; desolvation temperature, 300°C; capillary po-tential, 3.75 kV; sampling-cone potential, 35 V; extractor, 2 V; and nitro-gen flow rate, 300 liters/h.

NMR analysis. The reaction mixture obtained at 25°C for 20 h waspurified with a Sep-Pak C18 cartridge (Waters). The reaction product waseluted with 10% (vol/vol) methanol in water and concentrated by evapo-ration. The reaction product dissolved in water was further purified byHPLC (TSK-gel ODS-80Ts [7.8 by 300 mm; Tosoh Co., Tokyo, Japan],25% [vol/vol] acetonitrile in water). The peak fraction was collected andconcentrated to dryness (19 mg). Nuclear magnetic resonance (NMR)spectra were measured with a DPX-300 (Bruker, Ettlingen, Germany).Samples were prepared by dissolving in CDCl3.

RESULTSReverse activity of N-substituted formamide deformylase. Theability of the enzyme to catalyze the reverse reaction was examined(Fig. 1). First, when low concentrations of substrates (10 mMformate and 10 mM benzylamine) and the purified enzyme (0.1mg/ml) were added to the reaction mixture, a new peak was notdetected on HPLC. However, when high concentrations of sub-strates (1 M formate and 1 M benzylamine) and the purified en-zyme (0.1 mg/ml) were added to the reaction mixture, a new peakwas detected on HPLC. The amount of product was proportionalto the reaction time and the enzyme concentration. Without theenzyme, the new product was not detected. The retention time(3.0 min under the experimental conditions used) of the producton HPLC agreed with that of authentic NBFA. Then, the newproduct was purified, and its molecular weight was determined byLC-ESI-MS. The mass spectrum of the product was consistentwith the corresponding estimated molecular weight of NBFA (135Mr) (Fig. 2A). Additionally, the NMR spectrum of the product [1HNMR (ppm): 4.46 (2H, d, CH2), 7.20 –7.40 (5H, m), 6.11 (1H, br,NH), 8.23 (1H, s, CHO)] was consistent with that of authenticNBFA. These findings clearly demonstrated that N-substitutedformamide deformylase catalyzed the reverse reaction, synthesiz-ing NBFA from benzylamine and formate (Fig. 1).

To determine the kinetic parameters of the reverse reaction,the reverse activity was measured with various concentrations ofone substrate with a fixed concentration of another one. Fromlinear Lineweaver-Burk plots, the Km and Vmax values for formatewere found to be 3,010 110 mM and 33.0 1.0 units/mg,respectively, and those for benzylamine were 53.6 0.5 mM and22.5 0.2 units/mg, respectively (Table 1). On the other hand,these values for NBFA for the forward reaction were 0.075 mMand 52.7 units/mg, respectively (26). The catalytic efficiencies(kcat/Km) of formate (0.011 s1 · mM1) and benzylamine (0.411s1 · mM1) for the reverse reaction were significantly lower thanthat of NBFA for the forward reaction (687 s1 · mM1) (Table 1).Judging from the kcat/Km ratio, the reverse reaction proceededonly slightly compared with the forward reaction.

Effects of temperature and pH on the activity and stability ofthe reverse reaction. The effects of pH on the enzyme activity andstability of the reverse reaction were examined. The enzyme ex-hibited maximum activity at pH 7.0 (Fig. 3A). The optimum tem-perature was 25°C. The stability of the enzyme was examined at

FIG 1 Reverse reaction of N-substituted formamide deformylase.

FIG 2 Mass spectra on LC-ESI-MS of the reverse-reaction products of N-sub-stituted formamide deformylase. The structural formulae show NBFA (A),N-benzylacetamide (B), and N-benzylpropionamide (C). The major masspeaks at m/z 136, 150, and 164 correspond to NBFA, N-benzylacetamide, andN-benzylpropionamide, respectively.

TABLE 1 Kinetic parameters of N-substituted formamide deformylase

Reactiona Km (mM)Vmax

(units/mg) kcat/Km (mM1 · s1)

Reverse (formate) 3,010 110 33.0 1.0 0.011Reverse (benzylamine) 53.6 0.5 22.5 0.2 0.411Forward (NBFA) 0.075 52.7 687a NBFA was used as the substrate for the forward reaction. For the reverse reaction,benzylamine and formate were used as the substrates.

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various temperatures. After the enzyme had been preincubatedfor 30 min in 10 mM KPB (pH 7.5) containing 10% (vol/vol)glycerol, an aliquot of the enzyme solution was taken and theenzyme activity was assayed. The enzyme solution exhibited thefollowing activities: 60°C, 0%; 55°C, 0%; 50°C, 78%; 45°C, 100%;40°C, 100%; 35°C, 100%; 30°C, 100%; 25°C, 99%; 20°C, 100%;15°C, 99%; and 10°C, 99%. The stability of the enzyme was alsoexamined at various pH values. After the enzyme had been incu-bated at 25°C for 30 min in four buffers at a concentration of 0.1 M(citrate-Na2HPO4 buffer, pH 4.0 to 8.0; potassium phosphate buf-fer, pH 6.0 to 8.0; Tris-HCl buffer, pH 7.5 to 9.0; and NH4OH-NH4Cl buffer, pH 8.5 to 10.0), an aliquot of the enzyme solutionwas taken, and then the enzyme activity was assayed under thestandard conditions. N-Substituted formamide deformylase wasmost stable in the pH range of 7.5 to 8.0 (Fig. 3B).

Effects of inhibitors on the reverse activity. Various com-pounds were investigated for their inhibitory effects on the en-zyme activity (Table 2). Each compound was added to the stan-dard reaction mixture without the substrate, and then the reactionwas started by adding the substrate. The final concentration ofeach tested compound was 1 mM unless otherwise stated. Theenzyme was very sensitive to HgCl2, CuCl (at 0.25 mM), CuCl2,and AgNO3; the inhibition was 100% in all cases. SnCl2 and CdCl2also showed inhibitory effects on the reverse enzymatic activity(44% and 57%, respectively). The enzyme was inhibited bythiol-specific reagents, such as N-ethylmaleimide and p-chlo-romercuribenzoate, whereas iodoacetate and 5,5=-dithio-bis-2-nitrobenzoate did not inhibit the reverse activity at all. Carbonyl-specific reagents, e.g., aminoguanidine and semicarbazide, hardlyinhibited the enzyme, but phenylhydrazine caused only partialinhibition (23%). Chelating agents, such as �,�=-dipyridyl,KCN, diethyldithiocarbamate, and EDTA, did not influencethe reverse activity at all, but o-phenanthroline and 8-hydroxy-quinoline caused appreciable inhibition (32% and 84%, re-spectively). The enzyme was unaffected by oxidizing reagentsand serine-modifying reagents, such as H2O2, ammonium per-sulfate, phenylmethanesulfonyl fluoride, and diisopropyl fluo-rophosphates. However, reducing reagents, such as dithiothre-itol and 2-mercaptoethanol, caused remarkable inhibition(100% and 64% inhibition, respectively).

Substrate specificity of the reverse activity. First, the ability ofthe enzyme to catalyze the condensation of various amines and

formate was examined in a reaction mixture that included thelatter as the acid substrate. All of the tested amines, i.e., aniline,phenethylamine, 3-phenyl-1-propylamine, ethylamine, propyla-mine, isopropylamine, allylamine, and butylamine, were inert assubstrates for the reverse reaction. Next, the ability of the enzymeto catalyze the condensation of various acids and benzylamine wasexamined in a reaction mixture that included the latter as theamine substrate. Among the tested acids, acetate and propionatewere also active as substrates for the reverse reaction (Table 3),whereas the other acids, such as butyrate and isobutyrate, wereinert. The compounds produced from acetate and propionatethrough the reverse reaction were examined by HPLC. The reten-

FIG 3 Effects of pH on the reverse activity and stability of N-substitutedformamide deformylase. (A) Reactions were carried out for 5 min at 25°C inthe following buffers (0.1 mM): citrate-Na2HPO4 buffer (Œ), potassium phos-phate buffer (�), Tris-HCl buffer (Œ), and NH4OH-NH4Cl buffer (�). (B)The reverse activity was assayed after the enzyme had been preincubated for 30min in the buffers described in the legend to panel A. Relative activity is ex-pressed as a percentage of the maximum activity attained under the experi-mental conditions used.

TABLE 2 Effects of various compounds on the activity of N-substitutedformamide deformylasea

InhibitorRelativeactivity (%)

None 100LiCl, NaCl, MgCl2, CaCl2, BaCl2, MnCl2, AlCl3, Pb(NO3)2,

FeSO4, FeCl3, RbCl, SrCl2, CsCl, Na2MoO4, CoCl2,NiCl2, and ZnCl2

87–111

SnCl2 56CdCl2 43CuCl (0.25 mM), CuCl2, AgNO3, and HgCl2 0Iodoacetate 995,5=-Dithio-bis-2-nitrobenzoateb 105N-Ethylmaleimideb 21p-Chloromercuribenzoate 0Semicarbazide 92Aminoguanidine 81Phenylhydrazine 77�,�=-Dipyridyl 85o-Phenanthroline 688-Hydroxyquinolineb 16EDTA 127Diethyldithiocarbamate 98NaN3 101KCN 108Dithiothreitol 02-Mercaptoethanol 36H2O2 91Ammonium persulfate 112Phenylmethanesulfonyl fluorideb 102Diisopropyl fluorophosphateb 117a Each compound was added to the standard reaction mixture without the substrate,and then assay of the enzyme was performed after adding the substrate. The finalconcentrations of the tested compounds were 1 mM unless otherwise stated.b Methanol was added to the reaction mixture to a final concentration of 5% (vol/vol)to enhance the solubility of the tested compound.

TABLE 3 Substrate specificity of the reverse reaction of N-substitutedformamide deformylasea

Substrate Product Km (mM)Vmax

(units/mg)

kcat/Km

(mM1

· s1)

Formate N-Benzylformamide 3,010 110 33.0 1.0 0.011Acetate N-Benzylacetamide 1,210 30 10.0 0.2 0.008Propionate N-Benzylpropionamide 3,850 100 13.6 0.5 0.003a To determine kinetic parameters, the reverse activity was measured with variousconcentrations of acids and a fixed concentration of benzylamine. Apparent Km andVmax values were obtained from Lineweaver-Burk plots.

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tion times of the products (3.5 min for the product derived fromacetate and benzylamine and 3.9 min for that from propionateand benzylamine) on HPLC agreed with those of authentic N-benzylacetamide and N-benzylpropionamide. Each of these reac-tion products was purified and concentrated, and their molecularweights were determined by LC-ESI-MS. The results for the prod-ucts were consistent with the corresponding estimated molecularweights (N-benzylacetamide, 149 Mr, and N-benzylpropi-onamide, 163 Mr) (Fig. 2B and C). These findings strongly dem-onstrated that the products were N-benzylacetamide and N-ben-zylpropionamide, both of which are nontoxic, unlike NBFA.From linear Lineweaver-Burk plots, the Km and Vmax values foracetate were found to be 1,210 30 mM and 10.0 0.2 units/mg,respectively, with the values for propionate being 3,850 100 mMand 13.6 0.5 units/mg, respectively. The catalytic efficiencies(kcat/Km) were 0.008 s1 · mM1 and 0.003 s1 · mM1 for acetateand propionate, respectively (Table 3).

Initial velocity patterns. Distinguishing of the kinetics mech-anisms was performed by analyzing the initial velocity patternswhen the concentration of one substrate was varied with severalfixed concentrations of the second substrate. A classical sequentialmechanism is indicated when a family of double-reciprocal plotsintersects to the left of the y axis and converges at the x axis,because both the slope and the intercept change as the concentra-tion of the fixed substrate changes. In the case of a ping-pongmechanism, the slope of a series of double-reciprocal plots re-mains unchanged, i.e., only the intercept changes as the concen-tration of the fixed substrate changes, giving rise to a series ofparallel lines (47). A double-reciprocal plot was constructed byplotting 1/velocity against 1/[benzylamine], with different fixedconcentrations of formate (Fig. 4). These data best fitted a classicalsequential mechanism, because the family of curves intersected onthe x axis. In a sequential mechanism, the enzyme binds to bothsubstrates, and a ternary complex is formed before the first prod-uct is released.

Dead-end inhibitors. A variety of compounds, comprising an-

alogues of benzylamine and formate, were investigated as possibleinhibitors of the reverse activity. Each potential inhibitor wastested over the concentration range of 1 mM to 0.5 M, and the50% inhibitory concentration (IC50) was determined (Table 4).Amines containing a benzene ring, such as aniline, phenethyl-amine, and 3-phenylpropylamine, inhibited the reverse reaction.The most potent inhibitor was 3-phenylpropylamine, with an IC50

of 13.3 mM. However, amines containing no benzene ring did notcause 50% inhibition at concentrations as high as 300 mM. On theother hand, acids such as butyrate and isobutyrate inhibited thereverse reaction, with IC50s of 275 mM and 280 mM, respectively.Valerate, isovalerate, and caproate could not be tested becausethey were insoluble in the reaction buffer.

Aniline and butyrate, which are analogues of benzylamine andformate substrates, respectively, were used as inhibitors of bothsubstrates in order to elucidate the substrate-binding order. Theresults are summarized in Table 5. Aniline was found to be a com-petitive inhibitor of benzylamine (Kis [the dissociation constantfor the enzyme-inhibitor complex] � 2.87 mM) and also an un-competitive inhibitor of formate (Kii [the dissociation constantfor the inhibitor from the enzyme-substrate-inhibitor com-plex] � 4.56 mM) (Fig. 5A and B and Table 5). Butyrate was foundto be not only a noncompetitive inhibitor of benzylamine (Kis �57.5 mM and Kii � 59.7 mM), but also a competitive inhibitor offormate (Kis � 273 mM) (Fig. 5C and D and Table 5). This isconsistent with ordered two-substrate, two-product (bi-bi) sub-

FIG 4 Two-substrate kinetic analysis of N-substituted formamide deformy-lase. Double-reciprocal plotting of 1/v versus 1/benzylamine was performedusing the data obtained in the initial velocity studies. Initial velocities weremeasured in the presence of 10 to 50 mM benzylamine and 0.6 to 1.5 Mformate.

TABLE 4 Effects of substrate analogues on the reverse activitya

Compound IC50 (mM)

AminesAniline 14.5Phenethylamine 27.83-Phenylpropylamine 13.3Ethylamine �300Propylamine �300Isopropylamine �300Allylamine �300Butylamine �300

AcidsButyrate 275Isobutyrate 280Valerate NAIsovalerate NACaproate NA

a The reverse activity was assayed as described in the text, and the IC50 for eachinhibitor was determined. NA, not applicable.

TABLE 5 Kinetic constants of N-substituted formamide deformylaseand inhibition patterns with aniline and butyrate

InhibitorVariablesubstrate Fixed substrate

Inhibitionpatterna

Kis

(mM)Kii

(mM)

Aniline Benzylamine Formate C 2.87Formate Benzylamine UC 4.56

Butyrate Benzylamine Formate NC 57.5 59.7Formate Benzylamine C 273

a C, competitive; NC, noncompetitive; UC, uncompetitive.

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strate addition, with formate being the obligate first substrate,followed by benzylamine addition (Fig. 6).

Enzymatic production of N-benzyl carboxamides. Variousreaction conditions for the efficient production of N-benzyl car-boxamides were studied using the purified N-substituted form-amide deformylase. The effects of the reaction time and substrateconcentration on the production of NBFA were investigated. Atfirst, the concentration of formate in the reaction mixture con-taining 0.2 M benzylamine was varied. The production of NBFAwith 1 M formate was high compared with that with 0.5 M, 1.5 M,and 2 M formate (Fig. 7A). Thereafter, the amounts of NBFA weremeasured in the presence of 1 M formate and various concentra-tions of benzylamine. The maximum production of NBFA in the24-h reaction was 16 mM when the concentration of benzylamineused was 0.2 M (Fig. 7B). With regard to the production of N-benzylacetamide and N-benzylpropionamide, the effects of the

reaction time and substrate concentration were also investigated.The maximum production of N-benzylacetamide and N-benzyl-propionamide in the 24-h reaction amounted to 22 mM and 18mM, respectively, when the concentrations of each acid and ben-zylamine initially used were 1.0 M and 0.2 M, respectively (Fig. 7Cto F).

DISCUSSION

We are interested in how C-N hydrolases evolved (27, 49, 50).Because N-substituted formamide contains a nitrogen-carbonbond, an N-substituted formamide deformylase belongs to theC-N hydrolases. Functional and kinetic analyses of N-substitutedformamide deformylase would contribute to clarification of themetabolism of isonitriles in nature and provide us with newknowledge about C-N hydrolases, which might facilitate elucida-tion of their functional and structural evolution. Previously thereverse activity of an N-substituted formamide deformylase hadnever been reported. In this work, we clarified the biochemicalcharacteristics of the reverse activity of the purified N-substitutedformamide deformylase. It is well known that some lipases (51,52), proteases (53), and glycosidases (54, 55) catalyze reverse-hy-drolysis (condensation) reactions or exchange reactions. How-ever, these reactions hardly occur in an aqueous solution and

FIG 5 Dead-end inhibition of N-substituted formamide deformylase by aniline or butyrate. Shown are double-reciprocal plots of 1/v versus 1/benzylamine with5 to 25 mM benzylamine and 2 M formate (A), 1/v versus 1/formate with 0.6 to 1.5 M formate and 0.1 M benzylamine (B), 1/v versus 1/benzylamine with 5 to25 mM benzylamine and 2 M formate (C), and 1/v versus 1/formate with 0.3 to 1.2 M formate and 0.1 M benzylamine (D). The aniline and butyrateconcentrations were as indicated in the insets.

FIG 6 Proposed kinetic mechanism of N-substituted formamide deformy-lase. F, formate; B, benzylamine; Enz, enzyme.

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require the presence of substrates at almost saturated concen-trations. N-Substituted formamide deformylase was able to cata-lyze a condensation reaction efficiently even in an aqueous solu-tion with high concentrations of substrates. To our knowledge,this is the first report of the reverse reaction of not only an amine-forming deformylase, but also an enzyme in the amidohydrolasesuperfamily, to which N-substituted formamide deformylase be-longs (36).

It has also been reported that the reverse-hydrolysis and trans-fer reactions of several lipases, proteases, and glycosidases proceedwhen water molecules in the reaction mixture are depleted on theaddition of an organic solvent to the reaction mixture (51, 53, 55).Therefore, we examined the effects of various organic solvents onreverse hydrolysis at 12°C. Contrary to our expectations, N,N-dimethyl formamide, dimethyl sulfoxide, ethanol, methanol, and1,4-butanediol inhibited the reverse reaction of N-substitutedformamide deformylase at a concentration of 30% (vol/vol) (datanot shown). Because organic solvents are in general known toabolish the activity of enzymes, N-substituted formamide de-formylase may also become inactive in the presence of organicsolvents.

Investigation of the substrate specificities for various aminesrevealed that only benzylamine was active as a substrate of thereverse reaction. This finding is consistent with the fact that N-substituted formamide deformylase exhibits a narrow substratespectrum for the forward reaction (26). When various N-substi-tuted formamides, amides, and other compounds were examinedas forward reaction substrates, NBFA was found to be the mostsuitable substrate for the enzyme. N-Butylformamide (3.4%) washydrolyzed to N-butylamine and formate at rates significantlylower than those of the activity toward NBFA (100%). However,the product of the forward reaction from butylformamide, N-bu-

tylamine, was inactive as an amine substrate for the reverse reac-tion. On the other hand, when acetate and propionate were eachused as the acid substrate instead of formate, the reverse reactions,surprisingly, proceeded (Table 3 and Fig. 1), and N-benzylacet-amide and N-benzylpropionamide were detected as reverse-reac-tion products, respectively (Fig. 2B and C). These findings dem-onstrated that acids with numbers of carbon atoms ranging fromC1 to C3 were active as acid substrates for the reverse reaction ofN-substituted formamide deformylase, which produces formate(a C1 acid) as one of the forward reaction products. This uniquesubstrate specificity of the reverse reaction has not previously beenreported for any other deformylases known so far. If the substratespecificity for an amine is improved so as to be broad, variousN-substituted acetamides and N-substituted propionamidescould be obtained enzymatically. The effects of pH (Fig. 3) andchemical modification (Table 2) on the reverse reaction were sim-ilar to those on the forward reaction, suggesting that the samecatalytic site on the enzyme is involved in both the reverse andforward reactions.

Elucidation of the kinetic mechanism of N-substituted form-amide deformylase required the combination of several tech-niques. The enzyme kinetic mechanism of the two substrate reac-tions can be directly proved by simultaneously varying bothsubstrates (46). It is well established that double-reciprocal plotanalysis that generates an intersecting-line pattern suggests a se-quential mechanism and that a parallel-line pattern is character-istic of a ping-pong mechanism (47). Here, the bisubstrate inter-secting-line pattern obtained with N-substituted formamidedeformylase (Fig. 4) is consistent with a sequential mechanism. Inorder to distinguish an ordered sequential mechanism from a ran-dom sequential mechanism, dead-end inhibition analyses werecarried out. Aniline acted as a competitive inhibitor for benzyl-

FIG 7 Effects of the concentrations of the substrates on N-benzyl carboxamide synthesis. The amounts of NBFA were measured in the presence of variousconcentrations of formate and benzylamine: 0.5 to 2 M formate and 0.2 M benzylamine (A) and 0.1 to 0.4 M benzylamine and 1 M formate (B). The amountsof N-benzylacetamide were measured in the presence of various concentrations of acetate and benzylamine: 0.5 to 2 M acetate and 0.2 M benzylamine (C) and0.1 to 0.4 M benzylamine and 1 M acetate (D). The amounts of N-benzylpropionamide were measured in the presence of various concentrations of propionateand benzylamine: 0.5 to 2 M propionate and 0.2 M benzylamine (E) and 0.1 to 0.4 M benzylamine and 1 M propionate (F).

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amine, suggesting that both compounds bind to the same enzymeform, but was uncompetitive when formate was the substrate (Fig.5), suggesting formate binding generates the enzyme form towhich aniline can bind. These data are consistent with an orderedsequential mechanism wherein formate binds to the enzyme first,followed by benzylamine association (Fig. 6).

In conclusion, we initially demonstrated the reverse activity ofN-substituted formamide deformylase: enzymatic NBFA synthe-sis from formate and benzylamine. We found this unique charac-teristic only when the substrate concentration was high, althoughno reverse reaction was observed with low substrate concentra-tions. Interestingly, when acetate and propionate were each usedas the acid substrate instead of formate, N-benzylacetamide andN-benzylpropionamide, respectively, were formed enzymatically.The maximum production of NBFA, N-benzylacetamide, and N-benzylpropionamide in the 24-h reaction amounted to 16 mM, 22mM, and 18 mM, respectively, when the concentrations of eachacid and benzylamine initially used were 1.0 M and 0.2 M, respec-tively. Kinetic studies revealed that the reverse reaction follows anordered sequential mechanism. Additional characterization, in-cluding mutant analysis and determination of the enzyme struc-ture, could provide information for further clarification of thereaction mechanism.

Our discovery of the reverse reaction of N-substituted form-amide deformylase raises the possibility that other deformylases,which play various physiologically important roles, can also cata-lyze a reverse reaction to produce N-substituted carboxamides.The active site of N-substituted formamide deformylase has neverbeen identified experimentally. Further studies on N-substitutedformamide deformylase from the standpoint of its three-dimen-sional structure could provide information about the evolution-ary relationship of this enzyme and other enzymes involved in thecleavage and synthesis of a C-N bond, such as nitrilase (56, 57),nitrile hydratase (58), amidase (49, 50), and aldoxime dehydratase(59).

ACKNOWLEDGMENTS

We thank K. Tomita-Yokotani for the LC-ESI-MS analysis and H. Asakofor the NMR analysis.

This work was supported in part by a Grant-in-Aid for Scientific Re-search from the Ministry of Education, Culture, Sports, Science and Tech-nology (MEXT).

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