UvA-DARE (Digital Academic Repository)

Oxidation reactions catalyzed by Vanadium peroxidases. ten Brink, H.B.

Publication date 2000

Link to publication

Citation for published version (APA): ten Brink, H. B. (2000). Oxidation reactions catalyzed by Vanadium peroxidases.

General rights It is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s) and/or copyright holder(s), other than for strictly personal, individual use, unless the work is under an open content license (like Creative Commons).

Disclaimer/Complaints regulations If you believe that digital publication of certain material infringes any of your rights or (privacy) interests, please let the Library know, stating your reasons. In case of a legitimate complaint, the Library will make the material inaccessible and/or remove it from the website. Please Ask the Library: https://uba.uva.nl/en/contact, or a letter to: Library of the University of Amsterdam, Secretariat, Singel 425, 1012 WP Amsterdam, The Netherlands. You will be contacted as soon as possible.

UvA-DARE is a service provided by the library of the University of Amsterdam (https://dare.uva.nl)

Download date:01 Oct 2021 ChapterChapter 3

Sulfoxidationn Activity of Vanadium Bromoperoxidase from AscophyllumAscophyllum nodosum: Evidence for Direct Oxygen Transfer Catalysis

J.J. Am. Chem. Soc. submitted for publication

Hildaa B. ten Brink,1 Hans E. Schoemaker and Ron Wever

11 E. C. Slater Institute, Biocentrum, University of Amsterdam, Plantage Muidergracht 12, 10188 TV Amsterdam, The Netherlands 22 DSM Research, Bio-organic Chemistry, P.O. Box 18, 6160 MD Geleen, The Netherlands ChapterChapter 3

Sulfoxidationn Activity of Vanadium Bromoperoxidase from As cop hyHum nodosum'.nodosum'. Evidence for Direct Oxygen Transfer Catalysis

Hildaa B. ten Brink,1 Hans E. Schoemaker2 and Ron Wever1

11 E.C. Slater Institute, BioCentrum, University of Amsterdam, Plantage Muidergracht 12, 10188 TV Amsterdam, The Netherlands. 22 DSM Research, Bio-Organic Chemistry, P.O. box 18, 6160 MD Geleen, The Netherlands.

Abstract t Wee have previously shown that the vanadium bromoperoxidase from AscophyllumAscophyllum nodosum mediates the production of the (R)-enantiomer of methyl phenyll sulfoxide with 91% enantiomeric excess. Investigation of the intrinsic selectivityy of the vanadium bromoperoxidase reveals that the enzyme catalyzes the sulfoxidationn of methyl phenyl sulfide in a purely enantioselective manner. The affinityy of the enzyme for methyl phenyl sulfide was determined to be approximatelyy 3.5 mM in the presence of 25% methanol or tert-butanol. The selectivityy of the sulfoxidation of methyl phenyl sulfide is optimal at a temperature rangee of 25-30°C and can be further optimized by increasing the enzyme concentration,, yielding selectivities with up to 96% enantiomeric excess. Further we establishedd for the first time that the vanadium bromoperoxidase is functional at temperaturess up to 70°C. A detailed investigation of the sulfoxidation activity of thiss enzyme using lsO-labeled hydrogen peroxide shows that the vanadium bromoperoxidasee mediates the direct transfer of the peroxide oxygen to the sulfide. AA schematic model of the vanadium haloperoxidase sulfoxidation mechanism is presented. .

Introduction n Thee development of catalytic methods for the production of enantiomerically puree sulfoxides has increased in response to scientific and pharmaceutical interest in thesee chiral synthons in asymmetric synthesis.1 Several different methods for the preparationn of chiral sulfoxides by enantioselective oxidation of the corresponding organicc sulfides have been found using both chemical2 and biological, whole-cell3 andd enzymatic, 4' 5' 6 approaches. In particular, the potential application of biocatalystss in the production of these chiral auxiliaries as an alternative to chemical proceduress has been studied in great detail. This is mainly due to the fact that the enzymaticc conversions generally exceed the chemical based reactions in selectivity.

52 2 Inorganicc vanadium (V) peroxo-complexes, some of which have been suggestedd to be functional models for the vanadium peroxidases,7 have been reportedd to mediate oxygen-transfer reactions to a variety of organic compounds includingg sulfides.8 Several chiral Schiff-base ligated vanadium(V) peroxo- complexess have been shown to catalyze the production of optically active sulfoxides,, yielding selectivities up to 78% ee in the conversion of methyl phenyl sulfidee in the presence of hydrogen peroxide to the (5)-enantiomer of the correspondingg sulfoxide.9 Inn line with these results we have recently demonstrated that the vanadium haloperoxidasess are capable of mediating selective sulfoxidation reactions in the presencee of hydrogen peroxide.10 The vanadium bromoperoxidase (VBPO) from the brownn seaweed Ascophyllum nodosum promotes the formation of the (R)- enantiomerr of the methyl phenyl sulfoxide with 91% ee under optimal reaction conditions.. The VBPO from the red seaweed Corallina pilulifera mediates the sulfoxidationn of methyl phenyl sulfide to the (5)-enantiomer of the sulfoxide with 55%% ee. The recombinant VCPO on the other hand produces a racemic mixture of thee sulfoxides, which appeared to be an intrinsic characteristic of the enzyme. In addition,, it has recently been reported that the VBPO from the red seaweed CorallinaCorallina officinalis catalyzes the selective sulfoxidation of small aromatic sulfides andd small sulfides possessing a cis-positioned carboxyl group to the (5)-enantiomer off the corresponding sulfoxides with selectivities exceeding 95% ee.1 however, this enzymee was observed to be incapable of converting methyl phenyl sulfide.113 Thee vanadium haloperoxidases form a group of enzymes that possess a single boundd vanadate ion as a prosthetic group. In the presence of hydrogen peroxide and halidess these enzymes produce hypohalous acid (HOX) as a reactive intermediate, whichh consequently reacts with an organic compound producing a halogenated component.122 The first isolated and characterized vanadium haloperoxidases are the bromoperoxidasess (VBPO's) present in marine algae.13 These organisms are thoughtt to be responsible for the production of large amounts of volatile halogenatedd organic compounds, which are troublesome pollutants contributing to thee destruction of the ozone layer.14 Thee first vanadium-containing bromoperoxidase was found in the brown seaweedd Ascophyllum nodosum and has been studied in great detail.15 Vanadium is presentt as ortho-vanadate in the active site of this peroxidase, one metal atom per enzymee molecule, and the metal was shown to reside in the highest oxidation state, V(V),, even during catalysis.150 Upon reduction to V(IV), which can be observed

53 3 usingg EPR, the enzyme is completely inactive.150 The VBPO from A. nodosum oxidizess bromide in the presence of hydrogen peroxide through a so called "bi bi pingg pong mechanism";15f first hydrogen peroxide binds to the vanadium metal formingg an activated peroxo-intermediate, which facilitates the attack of the halide too yield hypobromous acid, which can further react with either an organic compoundd or an additional equivalent of hydrogen peroxide to form the halogenated componentt or singlet molecular oxygen, respectively. Severall structural studies have been conducted to characterize the nature of the activee site using different techniques, including EPR, EXAFS and XANES,15d'16d'17 andd though the enzyme has been crystallized the 3-D structure this enzyme is not knownn at present.1S Also the complete primary structure of the enzyme has not been reportedd yet.19 Unfortunately no spectroscopic studies can be performed due to the naturee of the vanadate prosthetic group. Ann extraordinary feature of this enzyme from the brown seaweed, which is actuallyy shared by all vanadium peroxidases, is the remarkable stability. ' ' e' Thee VBPO retains complete functionality upon storage in up to 60% of methanol, ethanoll and isopropanol, remains active when exposed to temperatures up to 70 °C andd was observed to be unaffected by detergents and to withstand oxidative inactivationn in the presence of high concentrations of highly reactive oxidants, includingg hydrogen peroxide and hypohalous acid. These fascinating features are of considerablee interest for the potential application of this vanadium peroxidase as (industrial)) biocatalyst in organic synthesis. Moree recently a vanadium chloroperoxidase was discovered and studied in evenn greater detail.21 This enzyme was isolated from the fungus Curvularia inaequalis.inaequalis. In contrast to the bromoperoxidases, chloroperoxidases (VCPO's) mainlyy originate from terrestrial fungi.22 Kinetic studies revealed that the chloride oxidationn mechanism of this enzyme is similar to the bromide oxidation mechanism off the vanadium bromoperoxidase, although the kinetic parameters differ. Thee VCPO has been successfully expressed in the yeast Saccharomyces cerevisiae,cerevisiae, therefore the recombinant VCPO can easily be acquired in large quantities.233 In contrast to the vanadium bromoperoxidase, the primary structure and thee X-ray structure of this enzyme are known.24 Sequence comparisons have shown thatt the architecture of the active site in the VBPO and VCPO is very similar.234'24b Directt evidence for the formation of an active enzyme-peroxo-intermediate in VCPOO during catalysis has been obtained from X-ray crystallography.25 The side-on coordinationn of the peroxide to the vanadium metal in the peroxo-intermediate of

54 4 thee VCPO is similar to that found in the vanadium(V) peroxo-complexes and probablyy also occurs in the VBPO. Anotherr class of peroxidases, harboring a heme group in the active site, also catalyzee enantioselective oxygen-transfer reactions.26 High enantioselectivity (> 99%% ee) can be obtained using heme CPO from the fungus Caldariomyces fumago inn the oxidation of organic sulfides.6b Other heme peroxidases, including Coprinus cinereuscinereus peroxidase and lactoperoxidase mediate sulfoxidation reactions with a lowerr enantioselectivity (73% and 80% ee,27 respectively). Inn heme CPO all the oxygen in the sulfoxide is derived from the hydrogen peroxide,266 which was deduced from incorporation studies using O-labeled H2O2. 288 Incorporation of the peroxide oxygen into methyl phenyl sulfoxide was 93% and 85%% for horseradish peroxidase29 and lactoperoxidase,28 respectively, while also 18 I8 significantt incorporation of oxygen derived from H2 0 or 02 was observed in the sulfoxidess produced by these enzymes.28'29 Clearly, the heme peroxidases mediate enantioselectivee O-transfer by different mechanisms of oxidation. The high enantioselectivityy of the heme CPO can be accounted for by a direct oxygen- transferr mechanism, whereas an oxygen-rebound mechanism is proposed for horseradishh peroxidase and lactoperoxidase. ' Fromm all these previous studies it can be concluded that the production of enantiomericallyy pure methyl phenyl sulfoxide is still very difficult and that as of yett no completely reliable biocatalyst has been found to convert methyl phenyl sulfidee in to the corresponding optically pure sulfoxide. In view of this we investigatedd the sulfoxidation mechanism of vanadium haloperoxidases and the sulfoxidationn activity of VBPO from A. nodosum in particular in greater detail. Wee now present the first evidence that the VBPO from A. nodosum directly promotess the transfer of oxygen from the peroxide to methyl phenyl sulfide. The recombinantt VCPO, however, appears to mediate the sulfoxidation via a different, non-selectivee mechanism. A more detailed investigation of the intrinsic selectivity off the vanadium bromoperoxidase reveals that the enzyme catalyzes the sulfoxidationn of methyl phenyl sulfide in a purely enantioselective manner. Enantiomericallyy pure sulfoxides can be produced with the vanadium peroxidases providedd that the non-selective reaction between hydrogen peroxide and sulfide is prevented. .

55 5 Experimentall Section Vanadiumm bromoperoxidase from Ascophyllum nodosum was isolated as describedd previously, c'e including the additional purification procedure using a Monoo Q column on a FPLC system yielding an uncolored pure enzyme preparation.. The recombinant vanadium chloroperoxidase was obtained from the developedd Saccharomyces cerevisiae expression system.23 This enzyme was observedd to exhibit the same kinetic characteristics compared to the native enzyme fromm the fungus Curvularia inaequalis.21 '23 As this enzyme is purified in the apo- formm vanadate was added as described.22'23 Unless stated otherwise all chemicals weree obtained from Merck or Fluka. Inn order to investigate the origin of the oxygen in the methyl phenyl sulfoxide producedd by vanadium peroxidases, VBPO from A. nodosum (0.5 u,M) was incubatedd in 100 mM sodiumcitrate/NaOH buffer, pH 5.0, with methyl phenyl sulfidee (1.5 mM) and l80-labeled hydrogen peroxide (1.5 mM final concentration) forr three days at 25°C. The labeled peroxide (>90% enrichment, Icon) was added in fivefive sequential steps, distributed over the three days, in order to prevent significant contributionn of the direct reaction between sulfide and peroxide to the produced sulfoxide.100 The same reaction conditions were used for the uncatalyzed reaction, howeverr buffer was added instead of enzyme. In addition, both the catalyzed and uncatalyzedd experiments were conducted in the presence of either 25% of tert- butanoll or methanol. After three days the samples were quenched with sulfite, extractedd with CH2CI2 and the enantiomeric excess was analyzed by chiral HPLC analysiss as described previously.10 Thee products of both the catalyzed and uncatalyzed sulfoxidation reactions weree analyzed by mass spectrometry (142 m/z for 180-labeled sulfoxide and 140 m/zz for 160 sulfoxide). Electron Impact (EI) mass spectrometry was carried out usingg a JEOL JMS SX/SX102A four-sector mass spectrometer, coupled to a JEOL MS-MP70000 data system. The samples were introduced via the direct insertion probee into the ion source. Samples were measured at 15 eV. Forr the recombinant VCP021c' 23 similar reaction conditions were applied, howeverr the reaction mixture was incubated for 20 hours instead of three days, in whichh the ,80-labeled and unlabeled peroxide was added in five consecutive steps. Thee recombinant VCPO catalyzed sulfoxidation reaction was also performed in 50%% O-labeled water (purchased from Icon). The samples were recovered and analyzedd as previously described.10

56 6 Thee minimal turnover frequency (mol of formed sulfoxide/mol of enzyme min'1)) of the recombinant VCPO in the sulfoxidation of methyl phenyl sulfide was determinedd by following the conversion of the sulfide at 290 nm (Ae=0.44 mM"1 cm'1)100 in time with a Zeiss spectrophotometer for 2 hours, after which the reaction wass completed. In this particular experiment recombinant VCPO (1 uM) was incubatedd in 100 mM sodiumcitrate/NaOH buffer, pH 5.0, with methyl phenyl sulfidee (1.5 mM) and hydrogen peroxide (2 mM). Too determine the intrinsic selectivity of the VBPO from A. nodosum in the sulfoxidationn of methyl phenyl sulfide the experiments were conducted in the followingg way. The incubations were performed in cuvettes (1.7 ml) with a capillaryy opening and no headspace containing 100 mM sodiumcitrate/ NaOH bufferr (pH 5.3), methyl phenyl sulfide (1.5 mM), H202 (0.5 mM) and 0.1, 0.25, 0.5 orr 1 fiM of enzyme. To monitor the formation of the sulfoxide continuously the absorbancee decrease at 290 nm was followed with a HP diode array spectrophotometerr and an extinction coefficient of 0.44 mM"1 cm"1 was used to determinee the concentration of sulfoxide produced.10 After 3 hours the reaction was quenched,, the mixture extracted, evaporated and dissolved as described before and thee enantioselectivity of the reaction was determined by chiral HPLC analysis.10 Thee amount of the (R)-and (S)-enantiomer of methyl phenyl sulfoxide formed was calculatedd from these data.

Forr the determination of the Km of VBPO for methyl phenyl sulfide the enzymee (0.5 \iM) was incubated in 100 mM sodiumcitrate/NaOH buffer (pH 5.0) andd 25% of either terf-butanol or methanol (in order to dissolve the sulfide at relativelyy high concentrations) with 1.5 mM H202 and 0.5, 1, 1.5, 2.5, 4, 5, 7.5, 10, 12.5,, 15 or 20 mM of methyl phenyl sulfide at RT for 3 hours. In addition, to study thee uncatalyzed reaction in 100 mM sodiumcitrate/NaOH buffer (pH 5.0) and 25% off tert-butanol or methanol 5, 10 and 20 mM of methyl phenyl sulfide was incubatedd with 1.5 mM H202 at RT for 3 hours. The production of the sulfoxide was monitoredd at 290 nm on a Cary 50 spectrophotometer as mentioned above and after 33 hours the yield and selectivity of the reactions were determined using a diacel OD 10 chirall HPLC column as described before. In order to determine the Km of VBPO forr methyl phenyl sulfide the following procedure was used. The amount of (R)-md (S)-methyll phenyl sulfoxide was determined. Since the (SJ-enantiomer is only formedd due to the uncatalyzed sulfoxidation reaction, yielding both the (R)-and (S)- enantiomer,, subtraction of the amount of (J?)-and (iS)-sulfoxide gives the yield of the

57 7 (^)-enantiomerr produced enzymatically. The Km was calculated using Enzpack for Windowss version 1.4 (Biosoft). Thee temperature dependence of the selective sulfoxidation of methyl phenyl sulfidee by VBPO from A. nodosum was studied in 100 mM sodiumcitrate/NaOH bufferr (pH 5.3) using similar reaction conditions as described before,10 however the concentrationn of methyl phenyl sulfide and hydrogen peroxide was changed to 1.5 mM.. Buffer was added instead of enzyme in the control experiments. The reaction temperaturee dependence was studied using waterbaths to control the temperature in thee range of 0°C to 70°C during the incubation period of 20 hours.

Resultss and Discussion IntrinsicIntrinsic Selectivity of VBPO Thee VBPO from A. nodosum was shown to mediate the sulfoxidation of methyll phenyl sulfide with selectivities up to 91% ee for the (i?)-enantiomer of the sulfoxide.100 Clearly this VBPO exhibits high enantioselectivity, however enantioselectivityy and versatility of the heme chloroperoxidase from C. fumago in thee biotransformation of sulfides is still better.5'6 We believe this difference is due too the low turnover frequency of the vanadium peroxidase in comparison to the hemee CPO,5'6 resulting in a relatively large contribution of the uncatalyzed reaction andd subsequent decreased selectivity. In addition, for all the heme peroxidase catalyzedd sulfoxidation reactions the concentration of enzyme used is a ten-fold higherr than the concentrations used in our studies, which may also contribute to higherr selectivity of the heme CPO catalyzed reaction.

100 0 75 5 ££ 50 ao> > 25 5 0 0 00 0.25 0.5 0.75 1 VBPOO (nM)

Figuree 1. Influence of enzyme concentration on the selectivity of the sulfoxidation of methyl phenyll sulfide catalyzed by vanadium bromoperoxidase from A. nodosum at pH 5.3 for 3 at 25°C.

Inn view of this we have studied the dependence of the enantiomeric excess of thee catalyzed sulfoxidation reaction on the VBPO concentration. The results are presentedd in Figure 1. When increasing amounts of enzyme are used to produce

58 8 methyll phenyl sulfoxide, the selectivity of the reaction increases linearly at low concentrationss of enzyme, but gradually levels off to about 93% ee when 5 \iM of enzymee is used under these reaction conditions (results not shown). When the formationn in time of the enantiomers of methyl phenyl sulfoxide is followed separatelyy a different trend is observed (Figure 2).

100 0 ££ 75 c c o o 'raa 50 E E ££ 25 O O

Figuree 2. Effect of enzyme concentration on the formation of the (K)-enantiomer (-#-) and (S)- enantiomerr (-A-) of methyl phenyl sulfoxide in the sulfoxidation of methyl phenyl sulfide mediated byy vanadium bromoperoxidase form at pH 5.3 for 3 hours at 25 °C.

Thee formation of the (7?)-enantiomer of methyl phenyl sulfoxide in time is linear dependentt on the enzyme concentration in the reaction mixture and increases when largerr quantities of enzyme are used, whereas the production of the (5)-enantiomer off the sulfide gradually decreases. The formation of the (

concentrationss the concentration of H202 will be more rapidly reduced leading to a decreasee in the contribution of the uncatalyzed reaction. Consequently, we have establishedd that the vanadium bromoperoxidase from A. nodosum is absolutely enantioselectivee in the asymmetric conversion of methyl phenyl sulfide.30 It is likely thatt the selectivity of the catalyzed sulfoxidation reaction will improve by decreasingg and controlling the hydrogen peroxide concentration in the reaction, as hass been previously demonstrated.10' u'27

DeterminationDetermination of the Kmfor methyl phenyl sulfide Inn addition to the contribution of the uncatalyzed reaction between the sulfide andd hydrogen peroxide, which clearly decreases the selectivity of the enzymatic sulfoxidationn reaction, the low solubility of the sulfide in water remains a problem. Thee affinity of the vanadium enzyme for methyl phenyl sulfide was determined in

59 9 earlierr studies to be higher than 1.5 mM,10 which exceeds the maximal solubility of methyll phenyl sulfide in water (1.7 mM). Therefore the addition of an organic solventt is needed to increase the solubility of the sulfide. Ass has been shown previously the sulfoxidation activity of vanadium and hemee peroxidases is slightly affected by the presence of fórf-butanol, especially comparedd to other co-solvents.10' llb' 31 Moreover, it was established that the uncatalyzedd sulfoxidation reaction between hydrogen peroxide and methyl phenyl sulfidee can be prevented when the sulfoxidation is carried out in 50% terf-butanol.31 Methanoll has been used throughout our earlier studies and also in these studies to dilutee methyl phenyl sulfide prior to addition to the reaction mixture.

AA Km of VBPO for methyl phenyl sulfide of approximately 3.5 mM was found inn the presence of either 25% tert-butanol or methanol at pH 5.0 (not shown). The ratee of the enzymatic sulfoxidation at 1.5 mM methyl phenyl sulfide is therefore sub-optimal.. However, in 25% methanol there is a clear effect of the sulfide concentrationn on the selectivity of the sulfoxidation reaction. An optimal value of

48%% ee for the (/?)-sulfoxide was found near the Km for methyl phenyl sulfide, but at higherr sulfide concentrations the selectivity decreased to 28% ee. The decrease is probablyy due to a larger contribution of the non-enzymatic formation of the sulfoxidee at higher concentrations of sulfide (results not shown). A similar effect of thee sulfide concentration on the ee, but less profound, was observed in the presence off tert-butanol.

TemperatureTemperature dependence Previously,, it has been shown for several lipases and a secondary alcohol dehydrogenasee that the reaction temperature greatly affects the selectivity of certain reactionss and that altering the reaction temperature can even change the stereochemistryy of the reaction.32 Therefore the temperature-dependence of the VBPOO in the sulfoxidation of methyl phenyl sulfide was studied by conducting the reactionn at different temperatures ranging from 0°C to 70°C for 20 hours (see the Experimentall Section). The influence of different reaction temperatures on the selectivityy of the enzymatic conversion of methyl phenyl sulfide is shown in Figure 3.. As this figure shows the reaction temperature clearly affects the selectivity of the reactionn and a distinct temperature optimum is found over a narrow range from approximatelyy 25°C to 30°C. Within this temperature range the ee of the product reachess a maximum of 96%, which is higher than the selectivities found for the conversionn of methyl phenyl sulfide by vanadium peroxidases in a previous study.10

60 0 Furtherr increase of the reaction temperature results in the loss of selectivity presumablyy due to the increased non-stereoselective contribution of the uncatalyzed reaction,, which has an increased contribution at elevated temperatures (results not shown).. In addition, higher reaction temperatures, such as 60°C and 70°C, which approachh the melting temperature of 72°C,'6d may disturb the structure of the enzymee and reduce the selectivity of the conversion. Nevertheless, these results highlightt again the remarkable stability of the VBPO as it is active at these high temperatures. .

Figuree 3. Temperature dependence of the enantiomeric excess of the sulfoxidation of methyl phenyll sulfide by vanadium bromoperoxidase from A. nodosum (1 uM) at pH 5.3.

Decreasingg the reaction temperature to temperatures below 20°C does not inducee great loss of selectivity, as the enantiomeric excess is found to remain approximatelyy 73%. Low reaction temperatures decrease enzyme activity in general,, however constrain the enzyme structurally as well, which could explain the lackk of an effect on the selectivity of the catalyzed sulfoxidation at temperatures beloww 20°C. Thee rate of conversion of the enzyme catalyzed sulfoxidation reaction was alsoo strongly affected by the reaction temperature. At a reaction temperature of 40°CC or higher the conversion of methyl phenyl sulfide was now complete within 200 hours (results not shown). Similarly the rate of the non-enzymatic reaction betweenn sulfide and hydrogen peroxide also increased at higher temperatures but, as expected,, the product remained a racemic mixture of sulfoxide over the entire temperaturee range.

''ssO-labelingO-labeling studies Wee investigated the source of oxygen in methyl phenyl sulfoxide produced by VBPOO from A. nodosum from methyl phenyl sulfide using 180-labeled hydrogen peroxide.. As the reaction rate was found to be 1 min" under these reaction

61 1 conditionss (see Experimental Section),10 three days are needed for the VBPO to completee the conversion. Methyl phenyl sulfide was also incubated with lsO-labeled peroxidee in the absence of enzyme using the same reaction conditions. The incorporationn of l80 into methyl phenyl sulfide was determined by mass spectrometry.. Figure 4 represents the mass chromatograms of the sulfoxide producedd in the presence of VBPO and 180-labeled peroxide and that of unlabeled commerciallyy available methyl phenyl sulfoxide.

1000 -| 1 1 a a

II 50- c c 3 3 A A ii 25 - 00 -L, T I I ' ! r 1361381400 142144148 mass/charge e 1000 -| , 1 b b o o II 50 - c c 3 3 .Q Q "" 25 - 00 J-, 1 I. * , 1 , 1361381400 142144148 mass/charge e

Figuree 4. Mass chromatogram of a) methyl phenyl sulfoxide formed by VBPO from A. nodosum in 18 thee presence of H2 02 and b) methyl phenyl sulfoxide purchased from Sigma. The peak at m/z 140 inn panel a corresponds to enzymatically produced sulfoxide due to the presence of a small amount off unlabeled hydrogen peroxide in the labeled peroxide and to a minor amount of sulfoxide already presentt in the methyl phenyl sulfide used. The peaks with m/z of 141, 143 and 144 in panel a and thosee of 141 and 142 in panel b originate from 33S and 34S isotopes in the sulfoxide.

Thee chromatogram of the enzymatically produced sulfoxide shows that only 11 O-labeled sulfoxide is formed by the VBPO. In addition, the uncatalyzed sulfoxidationn reaction produces only '80-labeled methyl phenyl sulfoxide in the presencee of H2 02 (results not shown). Therefore no oxygen atoms are spontaneouslyy exchanged between peroxide and water or molecular oxygen during thee reaction. These experiments demonstrate that the VBPO from A. nodosum mediatess the conversion of the sulfide with essentially quantitative incorporation of oxygenn atoms derived from peroxide.

62 2 Althoughh both the catalyzed and uncatalyzed sulfoxidation reaction produce 18 18 0-labeledd methyl phenyl sulfoxide in the presence of H2 02, the VBPO catalyzes thee formation of the (i?)-sulfoxide with 63% ee, whereas a racemic mixture is producedd in the absence of the biocatalyst. The selectivity of the enzyme-catalyzed reactionn in this particular experiment is lower than may be expected from prior results,100 since a lower enzyme concentration was used. Thesee results indicate that VBPO from A. nodosum promotes the direct transferr of oxygen from the vanadium bound peroxide to the sulfide in a selective manner,, strongly suggesting that the aromatic sulfide binds near/in the active site withh a relatively low affinity (Figure 5). That a binding site in VBPO from A. nodosumnodosum is present for organic substrates is in agreement with an earlier study performedd by Tschirret-Guth and Butler.12" A direct oxygen-transfer pathway has beenn suggested for the enantioselective sulfoxidation catalyzed by heme CPO from C.fumago.C.fumago.261261*'*'2929 Due to the accessibility of the active site, substrates are presumed too bind near the activated oxygen of heme Compound I facilitating sulfoxidation via aa direct oxygen-transfer mechanism.

RVV R2 OO V Rii , S 5*1

^R22 Ri R2 s^R2 x 9 A-oo A-o Rl R2 55 c^U cn^ LL /-His U /^His

Figuree 5. Schematic models for the sulfoxidation mechanism of the VBPO from A. nodosum (left) andd the recombinant VCPO (right). The schematic representations of the active sites of the enzymes,, in the presence of peroxide, are based on the tertiary structure of the peroxo-intermediate 25 off the VCPO. The sulfide is shown as R,SR2. For methyl phenyl sulfide R, is the methyl and R2 thee phenyl group.

Thee addition of several solvents decreases the selectivity of the VBPO- catalyzedd sulfoxidation reaction.10'llb In order to study the origin of the suppressed selectivityy labeling studies were also carried out in the presence of methanol or tert- butanol.. The VBPO was incubated with methyl phenyl sulfide and 180-labeled hydrogenn peroxide in either 25% terf-butanol or methanol for three days as described.. In addition, these experiments were conducted in the absence of enzyme. Thee chromatograms of the methyl phenyl sulfoxides either produced in the presencee or absence of the biocatalyst show that only 180-labeled sulfoxide is

63 3 producedd (results obtained resemble Figure 4a and are therefore not shown). Consequently,, it is clear that also in the presence of 25% terf-butanol or methanol VBPOO still catalyzes the direct transfer of oxygen from hydrogen peroxide to methyll phenyl sulfide to form the corresponding sulfoxide as depicted in Figure 5 (left).. However, the selectivity of the conversion of methyl phenyl sulfide catalyzed byy the vanadium enzyme decreases due to the presence of these alcohols. A slight decreasee from 63% ee to 58% ee is found for the (7?)-enantiomer of the sulfoxide producedd by VBPO in the presence 25% terf-butanol and the ee decreases to 34% in thee presence of 25% methanol. Similar effects were observed in an earlier study.10 Thee origin of the decrease is probably due to a distortion of the three- dimensionall structure of the enzyme and penetration of the solvent in the active site off the enzyme affecting the intrinsic selectivity of the enzyme. These experiments showw that the presence of terf-butanol only slightly influences the selectivity of VBPOO and that terf-butanol is the solvent most likely to be used in further studies. Inn this context it should be noted that the presence of pure hydrophobic organic solventss greatly improves the selectivity of several lipases.33 Thee origin of the oxygen in methyl phenyl sulfoxide produced by recombinant VCPOO was also studied, since, as previously demonstrated, this enzyme produces a racemicc mixture.10 Methyl phenyl sulfide was incubated for 20 hours with lsO- labeledd hydrogen peroxide in the presence of recombinant VCPO. The minimal turnoverr frequency of the recombinant VCPO in the sulfoxidation of methyl phenyl sulfidee was determined to be 18 min"1, therefore the reaction was completed in 20 hours. . Thee mass chromatogram of the product (Figure 6a) clearly shows that less thann 5% of 180 is present in the sulfoxide. Similar results are obtained, when unlabeledd H202 is used (results not shown). Analysis of the products by chiral HPLCC analysis yielded racemic methyl phenyl sulfoxide as expected. In order to reveall the origin of the oxygen in the sulfoxide formed by the VCPO, the same experimentt was performed in lsO-labeled water (50%) using unlabeled hydrogen peroxide.. Now approximately 25% of the product contains the labeled oxygen atom (Figuree 6b) corresponding to 50% of the sulfoxide oxygen originating from water. Ass we have shown the VCPO, in contrast to the VBPO, is not capable of mediatingg the direct and selective transfer of peroxide oxygen to methyl phenyl sulfidee and a different oxidation pathway should be present in the VCPO. The sulfoxidationn activity of the recombinant VCPO seems to resemble the so-called oxygen-reboundd pathway proposed for the sulfoxidation mechanism of

64 4 lactoperoxidasee and Coprinus peroxidase.27 We suggest that the aromatic sulfide is oxidizedd by a 1 -electron transfer step by the peroxo-intermediate of the enzyme, formingg a positively charged sulfur radical, which migrates from the enzyme and is subsequentlyy converted to the product via chemical steps (Figure 5). ' ' The additionall electron still present in the enzyme can either reside on the vanadium- peroxo-intermediate255 or could be transferred to an aromatic amino acid34 in the vicinityy of the prosthetic group, until a second substrate molecule arrives to be oxidized. .

100 0

~ii r 1361381400 142144148 mass/charge e

100 0

TT r 1361381400 142144148 mass/charge e

Figuree 6. Mass chromatogram of methyl phenyl sulfoxide formed by recombinant VCPO in the 18 18 presencee of a) H2 02 and b) unlabeled H202 in 50% H2 0.

Inn contrast to the existing conception of the catalytic activity of vanadium haloperoxidase,, that only halides are oxidized by the enzymes in the presence of hydrogenn peroxide,35 we have recently found that the recombinant VCPO catalyzes thee oxidation of 2,2'-azino-di-(3-ethyl-benzthiazoline-6-sulphonic acid), ABTS, andd ortho-dianisidine, both classical heme peroxidase substrates (results not shown).. These findings support our hypothesis that this enzyme is also able to catalyzee 1-electron oxidation reactions. In line with our direct oxygen transfer modell proposed for the VBPO (Figure 5) we found that this enzyme is unable to

65 5 mediatee the 1 -electron oxidation of these organic substrates in the presence of hydrogenn peroxide.

Conclusions s Ourr labeling studies and investigations concerning the intrinsic selectivity of thee VBPO show that the VBPO from the brown seaweed A. nodosum converts methyll phenyl sulfide in a pure selective manner due to a direct oxygen transfer mechanism.. We propose that there is a specific substrate binding site in or near the activee site of the enzyme and we are presently investigating the presence and nature off such a binding site. The affinity of VBPO from A. nodosum for methyl phenyl sulfidee has been determined to be approximately 3.5 mM at pH 5 in the presence of eitherr 25% methanol or terf-butanol. Wee have established that the VBPO from A. nodosum only produces the (R)- enantiomerr of methyl phenyl sulfoxide in the enzymatic sulfoxidation of methyl phenyll sulfide and that the formation of the (S)-enantiomer of the sulfoxide should bee attributed to the uncatalyzed reaction between the sulfide and hydrogen peroxide. Clearly,, the main reason that it is difficult to obtain sulfoxides with very high enantiomericc purity using vanadium peroxidases is the significant contribution of thee non-specific uncatalyzed reaction between hydrogen peroxide and the organic sulfidee due to the slow turnover of the enzyme. Presently, the VBPO from A. nodosumnodosum mediates the biotransformation of methyl phenyl sulfide with enantioselectivitiess up to 96% ee for the (7?)-enantiomer. Severall mutants of the VCPO are now available3015' 37 and are being investigatedd to gain further insight into the role of the specific active site residues in thee halide and sulfide oxidation activity of the vanadium peroxidases. Attempts are beingg made to express the VBPO from A. nodosum and to perform site-directed mutagenesiss and/or directed evolution38 on this enzyme eventually.

Acknowledgment t Thee authors wish to thank J.W.H. Peeters and T.A. Molenaar-Langeveld from thee Institute for Mass Spectrometry at the University of Amsterdam. This work was supportedd by the Innovation Oriented Research Program Catalysis (IOPKatalyse) andd received financial support from the Council for Chemical Sciences (CW) of the Netherlandss Organization for Scientific Research (NWO) and the Netherlands Technologyy Foundation (STW). This work has been performed under the auspices off NIOK, The Netherlands Institute for Catalysis Research, Laboratory Report.

66 6 References s (1)) a) Carreno, M. C. Chem. Rev. 1995, 95, 1717-1760. b) Mikolajczyk, M.; Drabowitz, J.; Kielbasinski, P. Inn Chiral Sulfur Reagents; Rees, C. W. Ed.; CRC Press LLC, New York, 1997. (2)) a) Drabowicz, J.; Kielbasinski, P.; Mikolajczyk, M. In The Chemistry ofSulphones andSulphoxides; Patai,, S., Rappoport, Z., Stirling, C. J. M, Eds.; John Wiley & Sons Ltd., New York, 1988; pp 233-378. b)) Kagan, H. B. In Catalytic Assymetric Synthesis; Ojima, I. Ed.; VCH, New York, 1993; pp 203-226. (3)) a) Holland, H. L. Chem. Rev. 1988,88,473-485. b) Holland, H. L.; Brown, F. M.; Lakshmaiah, G.; Larsen,, B. G.; Patel, M. Tetrahedron: Assym. 1997, 8, 683-697. c) Holland, H. L.; Pöpperl, H.; Ninniss, R.. W.; Chenchaiah, P.C. Can. J. Chem. 1985, 63, 1118-1120. (4)(4) a) Holland, H.L. In Organic Synthesis with Oxidative Enzymes; VCH, New York, 1992; pp 255-311. b) Blée,, E.; Schuber, F. Biochemistry 1989, 28,4962-4967. c) Freutel, J.; Chang, Y. -T.; Collins, J.; Loew, G.;; Ortiz de Montellano, P. R. J. Am. Chem. Soc. 1994,116, 11643-11648. d) Carrea, G.; Redigolo, B.; Riva,, S.; Colonna, S.; Gaggero, N.; Battistel, E.; Bianchi, D. Tetrahedron: Assym. 1992, 3, 1063-1068. (5)(5) a) Colonna, S.; Gaggero, N.; Manfredi, A. Biochemistry 1990, 29, 10465-10468. b) Harris, R. Z.; Newmeyer,, S. L.; Ortiz de Montellano, P. R. J. Biol. Chem. 1993, 268, 1637-1645. c) Miller, V. P.; DePillis,, G. D.; Ferrer, J. C; Mauk, A. G.; Ortiz de Montellano, P. R. J. Biol. Chem. 1992,267, 8936- 8942. . (6)) a) Colonna, S.; Gaggero, N.; Richelmi, C; Pasta, P. TIBTECH1999, / 7, 163-168. b) Van Deurzen, M. P.. J.; van Rantwijk, F.; Sheldon, R. A. Tetrahedron 1997, 53, 13183-13220. (7)) a) Sakurai, H.; Tsuchiya, K. FEBS Lett. 1990,260,109-112. b) De la Rosa, R. I.; Clague, M. J.; Butler, A.. J. Am. Chem. Soc. 1992, 114, 760-761. c) Butler, A.; Baldwin, A. H. InMetal Sites in Proteins and Models,Models, Vol. 89; Sadler, P. J. Ed.; Springer-Verlag, Berlin, Germany, 1997; pp 109-132. (8)) Butler, A.; Clague, M. J.; Meister, G. E. Chem. Rev. 1994, 94, 625-638. (9)(9) a) Bolm, C; Bienewald, F. Angew. Chem. Int. Ed. Engl. 1995, 34, 2640-2642. b) Vetter, A. H.; Berkessel, A.. Tetrahedron Lett. 1998, 39, 1741-1744. (10)Tenn Brink, H. B.; Tuynman, A.; Dekker, H.L.; Hemrika, W.; Izumi, Y; Oshiro, T.; Schoemaker, H.E.; Wever,, R. Inorg. Chem. 1998, 37, 6780-6784. (11 l)a) Andersson, M.; Willets, A.; Allenmark, S. J. Org. Chem. 1997, 62, 8455-8458. b) Andersson, M. A.; Allenmark,, S. G. Tetrahedron 1998, 54, 15293-15304. (12)a)Tschirret-Guth,, R. A.; Butler, A. J. Am. Chem. Soc. 1994, 116, 411-412. b) Wever, R.; Krenn, B. E. In VanadiumVanadium in Biological Systems; Chasteen, N. D., Ed.; Kluwer Academic Publishers; Dordrecht, The Netherlands,, 1990; pp 81-98. c) Butler, A.; Walker, J. V. Chem. Rev. 1993, 93, 1937-1944. (13)) Wever, R.; Tromp, M. G. M.; Krenn, B. E.; Marjani, A.; van Tol, M. Environ. Sci. Technol. 1991, 25, 446-449. . (14)a)) Gschwend, P. M.; MacFarlane, J. K.; Newman, K. A. Science 1984, 227, 1033-1035. b) Wever, R. NatureNature 1988,335, 501. c) Butler, J. H. Nature 1995, 376, 469-470. (15)a)) Vilter, H. Phytochemistry 1984,23, 1387-1390. b) KrennB. E.; Tromp. M. G. M.; Wever, R. J. Biol. Chem.Chem. 1989, 264, 19287-19292. c) de Boer, E.; van Kooyk, Y.; Tromp, M. G. M.; Plat, H.; Wever, R. Biochim.Biochim. Biophys. Acta 1986,869,48-53. d) de Boer, E.; Boon, K.; Wever, R. Biochemistry 1988,27, 1629-1635.. e) Wever, R.; Plat, H.; de Boer, E. Biochim. Biophys. Acta 1985, 830, 181-186. f) Soedjak, H.. S.; Butler, A. Biochim. Biophys. Acta 1991,1079, 1-7. g) Soedjak, H. S.; Walker, J. V.; Butler, A. BiochemistryBiochemistry 1995, 34, 12698-12696. h) Meister-Winter, G. E.; Butler, A. Biochemistry 1996, 35, 11805-11811. . (16)) a) de Boer, E.; Wever, R. J. Biol. Chem. 1988, 263, 12326-12332. b) Everett, R. R.; Kanofsky, J. R.; Butler,, A. J. Biol. Chem. 1990, 265, 4908-4914. c) Everett, R. R.; Butler, A. Inorg. Chem. 1989, 28, 393- 395.. d) Tromp, M. G. M.; 'Olafsson, G.; Krenn, B. E.; Wever, R. Biochim. Biophys. Acta 1989,1040, 192-198. . (17)a)) de Boer, E.; Wever, R. FEBS Lettr. 1988, 235,93-97. b) Hormes, J.; Kuetgens U.; Chauvisrre, R.; Schreiber,, W.; Anders, K; Vilter, H.; Rehder, D.; Weidemann Biochim. Biophys. Acta 1988, 956, 293- 299.c)) Kusthardt, U.; Hedman B.; Hodgson, K. O.; Hahn, R.; Vilter, H. FEBS 1993, 329, 5-8. d) Arber, J.. M.; de Boer, E.; Gamer, C. D.; Hasnain, S. S.; Wever, R. Biochemistry 1989, 28, 7968-7973. (18)) Weyand, M.; Hecht, H.-J.; Vilter, H.; Schomburg, D. Acta Cryst. 1996, D52, 864-865. (19)) Vilter, H. In Metal Ions in Biological Chemistry Vol. 31; Sigel, H. S., Sigel A., Eds.; Dekker, New York, USA,, 1995; pp. 326-362. (20)a)) de Boer, E.; Plat, H.; Tromp, M.G.M.; Wever, R.; Franssen, M. C. R.; van der Plas, H. C; Meijer, E. M.;; Schoemaker, H. E. Biotechnol. Bioengin. 1987, 30,607-610. b) de Boer, E.; Plat, H.; Wever, R. In BiocatalysisBiocatalysis in Organic Media, Proceedings of an International International Symposium held in Wageningen; Laane, C,, Tramper, J., Lilly, M. D., Eds.; Elsevier Science Publishers B. V., Amsterdam, The Netherlands, 1987;; pp. 317-322.

67 7 (21)a)) Lieu, T.-N. E.; MTimkulu, T.; Geigert, J.; Wolf, B.; Neidleman, S. L.; Silva, D.; Hunter-Cevera, J. C. Biochim.Biochim. Biophys. Res. Comm. 1987,142, 329-333. b) Van Schijndel, J. W. P. M.; Vollenbroek, E. G. M.;; Wever, R. Biochim. Biophys. Acta 1993,1161,249-256. c) Van Schijndel, J. W. P. M.; Barnett, P.; Roelse,, J.; Vollenbroek, E. G. M; Wever, R. Eur. J. Biochem. 1994, 225, 151-157. (22)) Vollenbroek, E. G. M.; Simons, L. H.; Van Schijndel, J. W. P. M.; Bamett, P.; Balzar, M.; Dekker, H. L.;; Van der Linden, C; Wever, R. Biochem. Soc. Trans. 1995, 23, 267-271. (23)a)) Hemrika, W.; Renirie, R.; Dekker, H. L.; Barnett, P.; Wever, R. Proc. Natl. Acad.Sci. USA 1997, 94, 2145-2149.. b) Hemrika, W.; Renirie, R.; Macedo-Ribeiro, S.; Messerschmidt, A.; Wever, R. J. Biol. Chem.Chem. 1999,274,23820-23827. (24)a)) Simons, B. H.; Barnett, P.; Vollenbroek, E. G. M; Dekker, H. L.; Muijsers, A. O.; Messerschmidt, A.; Wever,, R. Eur. J. Biochem. 1995, 229, 566-574. b) Messerschmidt, A.; Wever, R. Proc. Natl. Acad. Sci. USAUSA 1996, 93, 392-396. (25)Messerschmidt,, A.; Prade, L.; Wever, R. Biol. Chem. 1997, 378, 309-315. (26)Kobayasi,, S.; Nakano, M.; Goto, T.; Kimura, T.; Schaap, P. A. Biochem. Biophys. Res. Comm. 1986 135,135, 166-171. (27)Tuynman,, A.; Vink, M. K. S.; Dekker, H. L.; Schoemaker, H. E.; Wever, R. Eur. J. Biochem 1998 258 906-913. . (28)Doerge,, D. R.; Cooray, N. M.; Brewster, M. E. Biochemistry 1991, 30, 8960-8964. (29)Baciocchi,, E.; Lanzalunga, O.; Malandrucco, S. J. Am. Chem. Soc. 1996, 118, 8973-8974. (30)Straathof,, A. J. J.; Jongejan, J. A. Enzyme Mkrop. Technol. 1997, 21, 559-571. (31)) Van Deurzen, M. P. J.; Remkes, I. J.; van Rantwijk, F.; Sheldon, R. A. J. Mol. Cat. A: Chemical 1997, /;; 7, 329-337. (32)a)) Holmberg, E.; Hult, K. Biotechnol Lett. 1991, 13, 323-326. b) Pham, V. T.; Phillips, R. S.; Lijungdahl,, L. G. J. Am. Chem. Soc. 1989, 111, 1935-1936. (33)a)) Wescott, C. R.; Klibanov, A. M. Biochim. Biophys. Acta 1994, 1206, 1-9. b) Khmemitsky, Y. L.; Welch,, S. H.; Clark, D. S.; Dordick, J. S. J. Am. Chem. Soc. 1994,116, 2647-2648. c) Ke, T.; Wescott, C.R.;; Klibanov, A. M. J. Am. Chem. Soc. 1996, 118, 3366-3374.) Ke, T.; Klibanov, A. M. J. Am. Chem. Soc.Soc. 1998,120,4259-4263. (34)) Ortiz de Montellano, P. R. Ace. Chem. Res. 1987, 20, 289-294. (35)) Wever, R.; Hemrika, W. In Vanadium in the Environment. Part 1: Chemistry and Biochemistry; Nriagu, J.. O., Ed.; John Wiley & Sons, Inc., 1998; pp. 285-305. (36)Childs,, R. E.; Bradsley, W. G. Biochem. J. 1975, 145, 93-103. (37)Macedo-Ribeiro,, S.; Hemrika, W.; Renirie, R.; Wever, R.; Messerschmidt, A. J. Biol. Inorg. Chem. 1999,4,209-219. . (38)Reetz,, M. T.; Zonta, A.; Schimossek, K.; Liebeton, K.; Jaeger, K.-E. Angew. Chem. Int. Ed. Engl. 1997, 36,36, 2830-2832.

68 8