University of Groningen

Discovery, characterization, and kinetic analysis of an alditol oxidase from streptomyces coelicolor Heuts, Dominic P. H. M.; van Hellemond, Erik W.; Janssen, Dick B.; Fraaije, Marco W.

Published in: The Journal of Biological Chemistry

DOI: 10.1074/jbc.M610849200

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Publication date: 2007

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Citation for published version (APA): Heuts, D. P. H. M., van Hellemond, E. W., Janssen, D. B., & Fraaije, M. W. (2007). Discovery, characterization, and kinetic analysis of an alditol oxidase from streptomyces coelicolor. The Journal of Biological Chemistry, 282(28), 20283-20291. https://doi.org/10.1074/jbc.M610849200

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THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 282, NO. 28, pp. 20283–20291, July 13, 2007 © 2007 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.

Discovery, Characterization, and Kinetic Analysis of an Alditol Oxidase from Streptomyces coelicolor*□S Received for publication, November 24, 2006, and in revised form, May 21, 2007 Published, JBC Papers in Press, May 21, 2007, DOI 10.1074/jbc.M610849200 Dominic P. H. M. Heuts, Erik W. van Hellemond, Dick B. Janssen, and Marco W. Fraaije1 From the Laboratory of Biochemistry, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands

A gene encoding an alditol oxidase was found in the genome best known representative is , which in fact is of Streptomyces coelicolor A3(2). This newly identified oxidase, the most widely applied redox . AldO, was expressed at extremely high levels in Escherichia coli Besides galactose oxidase, which contains copper as , when fused to maltose-binding protein. AldO is a soluble mono- all presently known oxidases acting on carbohydrates contain a meric flavoprotein with subunits of 45.1 kDa, each containing a flavin cofactor. Examples of such flavoprotein oxidases are glu- Downloaded from covalently bound FAD cofactor. From sequence alignments cose oxidase, L-gulono-␥-lactone oxidase, xylitol oxidase, hex- with other flavoprotein oxidases, it was found that AldO con- ose oxidase, lactose oxidase, glucooligosaccharide oxidase, and tains a conserved histidine (His46) that is typically involved in pyranose oxidase. Except for glucose oxidase and pyranose oxi- covalent FAD attachment. Covalent FAD binding is not dase, all of these oxidases belong to a specific group of flavopro- observed in the H46A AldO mutant, confirming its role in cova- teins, the vanillyl- (VAO)2 family. Members of lent attachment of the flavin cofactor. Steady-state kinetic anal- this family share a similar overall structure consisting of two www.jbc.org yses revealed that wild-type AldO is active with several polyols. domains (3). One domain binds the adenine part of the FAD ؊1 ؍ ؍ The alditols xylitol (Km 0.32 mM, kcat 13 s ) and sorbitol cofactor and is called the FAD-binding domain, whereas the ؊1 ؍ ؍ (Km 1.4 mM, kcat 17 s ) are the preferred substrates. From other, called the cap domain, covers the isoalloxazine moiety of pre-steady-state kinetic analyses, using xylitol as , it the cofactor and forms the major part of the around at University of Groningen on September 17, 2007 can be concluded that AldO mainly follows a ternary complex the isoalloxazine ring system. A special feature of this flavopro- kinetic mechanism. Reduction of the flavin cofactor by xylitol tein family is the fact that a relatively large number of VAO ؊ occurs at a relatively high rate (99 s 1), after which a second members bind the FAD cofactor in a covalent manner. This is kinetic event is observed, which is proposed to represent ring also the case for all of the above mentioned VAO-type carbo- closure of the formed aldehyde , yielding the hemiacetal hydrate oxidases. In fact, the recent elucidation of the structure of D-xylose. Reduced AldO readily reacts with molecular oxygen of glucooligosaccharide oxidase has revealed the first example ؊1 ؊1 5 ؋ 10 M s ), which confirms that the enzyme represents where a flavin cofactor is covalently linked to two amino acid 1.7) a true flavoprotein oxidase. residues (4). It has been shown that a covalent FAD-protein linkage can have a significant effect on the redox behavior of the flavin cofactor (e.g. increasing the redox potential) (5). This is in Carbohydrate oxidases are highly valuable biocatalysts for line with the observation that most VAO-type covalent fla- analytical and synthetic purposes. Chemical methods cannot voproteins act as an oxidase (6). In these cases, the FAD cofac- compete with the exquisite regio- and/or enantioselectivity by tor is typically tethered to a histidine residue, and this linking which these oxidize polyols. Applications in which histidine can be readily identified by sequence motif recogni- carbohydrate oxidases are used are, for example, biosensors for tion. Hence, the ability to identify covalent VAO homologs by blood sugar, synthetic routes toward chiral building blocks, sequence analysis can be used as a tool to find novel oxidase sweeteners, and flavors. Oxidase-mediated also leads genes. to formation of hydrogen peroxide, a property that is used in a Most of the characterized carbohydrate oxidases have been number of applications, such as bleaching processes and waste- isolated from fungi, whereas only two from bacterial origin have water treatment (1, 2). At present, only a limited number of been described (7, 8). Here we describe the discovery and char- carbohydrate oxidases have been identified, which restricts the acterization of an alditol oxidase (AldO) from the actinomycete biocatalytic exploitation of this class of redox enzymes. The Streptomyces coelicolor A3(2). We identified the putative aldo gene (SCO6147) in the genome sequence of this bacterium by searching for VAO homologs. The predicted protein sequence * This work was performed within the framework of Integration of Biosynthe- sis and Organic Synthesis. The costs of publication of this article were of AldO contains a histidine that is expected to form a covalent defrayed in part by the payment of page charges. This article must there- FAD-histidyl linkage. Therefore, it was anticipated that the fore be hereby marked “advertisement” in accordance with 18 U.S.C. Sec- protein would have a high probability to exhibit oxidase activ- tion 1734 solely to indicate this fact. □S The on-line version of this article (available at http://www.jbc.org) contains ity. In this report, we show that the enzyme is indeed a typical an appendix. 1 To whom correspondence should be addressed: Laboratory of Biochemis- try, Groningen Biomolecular Sciences and Biotechnology Institute, Univer- 2 The abbreviations used are: VAO, vanillyl-alcohol oxidase; MBP, maltose- sity of Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands. Tel.: binding protein; m-AldO, MBP fused to AldO; HPLC, high pressure liquid 31-50-36-34345; Fax: 31-50-36-34165; E-mail: [email protected]. chromatography.

JULY 13, 2007•VOLUME 282•NUMBER 28 JOURNAL OF BIOLOGICAL CHEMISTRY 20283 Alditol Oxidase from S. coelicolor flavoprotein oxidase active on a number of alditols. By express- 0.02% (w/v) L-arabinose. Induction of pBAD-aldo(krad) and ing the enzyme fused to maltose-binding protein (MBP), pBAD-aldo clones yielded AldO containing a 25-amino acid impressive amounts of alditol oxidase could be produced, ena- C-terminal extension consisting of a c-Myc epitope and a His6 bling a detailed biochemical and kinetic characterization of this tag. Induction of pBAD-MBP-aldo(krad) and pBAD-MBP-aldo novel bacterial oxidase. clones yielded MBP-AldO fusion proteins containing a Factor Xa protease cleavage site. EXPERIMENTAL PROCEDURES Purification of MBP-AldO—Cells from three 0.5-liter cul- Chemicals and Restriction Enzymes—Restriction enzymes tures were collected by centrifugation at 4,000 ϫ g and 4 °C for were obtained from Roche Applied Science and New England 15 min and resuspended in 30 ml of 50 mM KPi buffer (pH 7.5). Biolabs. Escherichia coli TOP10-competent cells, the TOPO The cells were disrupted by sonication and subsequently cen- TA cloning kit for sequencing, and the pBAD/Myc-His A vector trifuged at 23,000 ϫ g and 4 °C for 30 min to remove cell debris. were obtained from Invitrogen. VAO (EC 1.1.3.38) was a kind The supernatant was divided into two 20-ml aliquots to prevent gift from Dr. W. J. H. van Berkel (Wageningen University, The overloading during the purification procedure. After loading Netherlands). (EC 1.2.3.2) was obtained from the supernatant onto a Q-Sepharose column, the column was

Stratagene. Horseradish peroxidase (EC 1.11.1.7) was obtained washed until the A280 was lower than 0.05. MBP-AldO was from Fluka. All other chemicals were of analytical grade. eluted by applying a 0–1 M KCl linear gradient. The yellow Downloaded from Cloning and Expression of the aldo Gene—The aldo genes fractions were pooled and concentrated using an Amicon were cloned into pBADNdeI and pBAD-MBP for expression. stirred cell and YM-30 membrane (Millipore). To remove KCl The latter is a pBADNdeI-derived vector in which the malE from the concentrated enzyme solution, it was loaded onto a gene, including a Factor Xa protease cleavage site from pMAL- HiPrep 26/10 desalting column (Amersham Biosciences). c2x, is inserted (NdeI-HindIII). Analytical Methods—All experiments were performed at

The aldo gene from S. coelicolor A3(2) was amplified from 25 °C and in a 50 mM KPi buffer, pH 7.5. The S.D. value in the www.jbc.org genomic DNA using Pfu DNA polymerase. The following primers experiments is 5%, unless stated otherwise. Oxidase activity and were used for cloning into pBADNdeI: Scoel_fw (5Ј-CTC- steady-state kinetic parameters were determined by coupling

CATATGAGCGACATCACGGTCACCC; NdeI site is under- the production of H2O2 by (m-)AldO to a horseradish peroxi- lined) and Scoel_rv (5Ј-TATAAGCTTGCCCGCGAGCACC- dase-mediated oxidation of 4-aminoantipyrine and 3,5-di- at University of Groningen on September 17, 2007 CCGCGCAC; HindIII site is underlined). The following chloro-2-hydroxybenzenesulfonic acid. This results in forma- primers were used for cloning into pBAD-MBP: XOmbp_fw tion of a pink to purple colored product, which can be measured Ј ⑀ ϭ Ϫ1 Ϫ1 (5 -CTCGAATTCATGAGCGACATCACGGTC; EcoRI site is at 515 nm ( 515 26 mM cm ) (9). The reaction mixture Ј underlined) and XOmbp_rv (5 -TATCTGCAGTCAGC- contained 50 mM KPi buffer, pH 7.5, 0.1 mM 4-aminoantipyrine, CCGCGAGCACCCC; PstI site is underlined, and the stop 1mM 3,5-dichloro-2-hydroxybenzenesulfonic acid, 3 units of codon is in italic type). The aldo gene (KradDRAFT_2777) from horseradish peroxidase, and 15 nM AldO or m-AldO. Kineococcus radiotolerans was amplified from whole cells using The extinction coefficient of m-AldO was determined by Pfu DNA polymerase (Stratagene). The following primers were comparing the absorption spectra before and after incubation used for cloning into pBADNdeI: kinrad_fw (5Ј-CTCCATAT- with 0.1% SDS. For this, it was assumed that the flavin spectrum GAGCACCTCGACGACGTCGTCC; NdeI site is underlined) of the unfolded m-AldO was equal to that of free FAD (⑀ of 11.3 Ϫ1 Ϫ1 and kinrad_rv (5Ј-TATAAGCTTGGCGGTCAGCCCGAC- mM cm at 450 nm) (10). CCGGTC; HindIII site is underlined). The following primers For product identification, the conversion of xylitol by were used for cloning into pBAD-MBP: kinrad_fwmbp (5Ј- m-AldO was followed by HPLC analysis using a Shodex CTCGAATTCGTGAGCACCTCGACGACGTCG; EcoRI site SUGAR SP0810 (8.0-mm inner diameter ϫ 300 mm) col- is underlined) and kinrad_rvmbp (5Ј-TATCTGCAGTCAG- umn. Optical rotation of the product was determined by GCGGTCAGCCCGACCCG; PstI site is underlined, and the polarimetric analysis using a Schmidt and Haensch Polar- stop codon is in italic type). tronic MH8 apparatus. To facilitate TOPO cloning, 3ЈA-overhangs were generated Redox potentials were measured by using the method by incubating the amplified aldo genes with 1 ␮lofTaq DNA described by Massey (11). A cuvette containing m-AldO (5–10 polymerase at 72 °C for 15 min. For pBADNdeI cloning, the ␮M), xanthine (400 ␮M), KCl (107 ␮M), benzyl or methyl violo- TOPO-aldo clones were digested with NdeI and HindIII, after gen (2.0 ␮M), and redox dye (5–16 ␮M) was made anaerobic by which the aldo DNA was isolated from an agarose gel. The flushing with argon. Under a constant flow of argon, 68 nM fragments were subsequently ligated into an NdeI- and HindIII- xanthine oxidase was added, and spectra were collected every digested pBADNdeI vector, yielding pBAD-aldo(krad) and 2.5 min during reduction using a PerkinElmer Life Sciences pBAD-aldo. For pBAD-MBP cloning, the TOPO-aldo clones Lambda Bio 10 spectrophotometer. were digested with EcoRI and PstI, after which the aldo genes Kinetic Measurements—Pre-steady-state studies were per- were isolated from an agarose gel. The aldo genes were subse- formed with an Applied Photophysics stopped-flow apparatus, quently ligated into an EcoRI- and PstI-digested pBAD-MBP model SX17MV. Spectral data were collected at time intervals vector, yielding pBAD-MBP-aldo(krad) and pBAD-MBP-aldo. of 2.5 ms using a diode array detector. A photomultiplier detec- E. coli TOP10 cells were transformed with the constructed tor was applied to follow single wavelength traces in time. Spec- expression vectors and grown at 17 °C for 3 days in terrific tral data obtained by diode array measurements were deconvo- broth medium supplemented with 50 ␮g/ml ampicillin and luted using the program Pro-K of Applied Photophysics. From

20284 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 282•NUMBER 28•JULY 13, 2007 Alditol Oxidase from S. coelicolor the deconvoluted spectra, several wavelengths were selected to coelicolor A3(2) was identified that encodes such a flavoprotein. study the observed phases individually. The first two phases of The protein consists of 418 amino acids with a calculated mass the reductive half-reaction were monitored by following the of 44,347 Da (excluding FAD) and therefore represents one of absorption at 320 and 405 nm, respectively, after anaerobically the smallest members in the VAO flavoprotein family. Among mixing the enzyme solution with varying concentrations of the known VAO-type , it shows highest xylitol. The first phase was monitored at 320 nm, because here sequence similarity with xylitol oxidase from another Strepto- the second and third phase showed no change in absorbance, myces isolate (78% sequence identity) and pig L-gulono-␥-lac- whereas 405 nm is an isosbestic point in the first phase enabling tone oxidase (30% sequence identity). Except for sequence con- analysis of the second phase. The third phase was monitored at servation typical for a VAO homolog, no other sequence motifs 452 nm. The oxidative half-reaction was followed at 452 nm by can be identified, suggesting that it is a cytosolic protein. Align- mixing a solution containing reduced enzyme with a solution ment of the protein sequence with known VAO-type protein 46 containing either 0.25 or 1.25 mM oxygen (in the presence or sequences revealed the presence of a histidine residue (His ), absence of 100 mMD-xylose). For anoxic conditions, all solu- which is typically involved in covalent attachment of the FAD tions contained 1.0 mM 4- ethylphenol and were made anaero- cofactor (Fig. 1). Recent papers report on VAO-type oxidases bic by flushing with nitrogen. VAO was added to a final con- containing a covalently bound FAD attached to two amino centration of 100 nM to remove any residual oxygen. Traces acids (4, 13, 14). Next to an 8␣-N-histidyl link, a second 6-S- Downloaded from obtained by photomultiplier measurements were fitted to an cysteinyl linkage to the isoalloxazine moiety of FAD is present exponential function (Equation 1), in these oxidases. Sequence alignments did not reveal such a conserved cysteine that would be involved in covalent FAD ͑ ͒ ϭ ϩ ϫ ͑Ϫkt͒ A t A C e (Eq. 1) attachment. where A represents absorption, C is a constant, and k is an To verify that the enzyme from S. coelicolor is functional as observed rate constant. The observed rates for substrate con- an oxidase and explore its enzymatic properties, the corre- www.jbc.org centration dependent reduction of m-AldO were fitted using sponding gene was amplified from genomic DNA by PCR, the following equation (Equation 2). cloned, and expressed. For initial expression experiments, the (aldo) gene was subcloned in a pBAD vector. Using the pBAD- ϫ at University of Groningen on September 17, 2007 kred S aldo expression plasmid, recombinant active AldO was only kobs ϭ (Eq. 2) Kd ϩ S expressed at a very low level, as observed by SDS-PAGE analy- sis. The respective protein band is fluorescent at pH 4, indica- Mechanistic Calculations—Mathematica 5.2 software was tive of the presence of a covalently bound flavin cofactor (Fig. 2, used to derive rate equations from the kinetic scheme by the lanes A and B) (15). To increase the level of expression, the determinant method (12) (see supplemental materials). The protein was expressed fused to MBP, since it is known that kinetic parameters Km and kcat as well as the redox state during N-terminal fusion to MBP often boosts expression of soluble catalysis can be calculated according to the following equations. protein (16). The resulting pBAD-MBP-aldo vector, linking For a ping-pong mechanism, Km and kcat values can be calcu- MBP to the N terminus of AldO, drastically increased the lated using Equations 3 and 4. expression level (Fig. 2, lane D). From 1 liter of culture broth, an k k K k ͓O ͔ impressive amount of 350 mg of MBP-AldO was purified by one ϭ 3 4 d ox,1 2 KM anion exchange chromatography step. Purified MBP-AldO ͑k4 ϩ kϪ3͒kox,1kred͓O2͔ ϩ k3͑kox,1kred͓O2͔ ϩ k4͑kred ϩ kox,1͓O2͔͒͒ runs as a single band upon SDS-PAGE at about 87 kDa, which (Eq. 3) nicely agrees with a calculated mass of 88,068 Da (including k k k k ͓O ͔ FAD). The protein band was again found to be fluorescent at ϭ 3 4 ox,1 red 2 kcat pH 4, indicative of a histidyl-bound FAD cofactor (Fig. 2, lanes ͑k4 ϩ kϪ3͒kox,1kred͓O2͔ ϩ k3͑kox,1kred͓O2͔ ϩ k4͑kred ϩ kox,1͓O2͔͒͒ C and D). (Eq. 4) The covalent binding of the flavin cofactor was also con-

For a ternary complex mechanism, Km and kcat values can be firmed by the observation that upon denaturation of the calculated using Equations 5 and 6. enzyme, the redox cofactor coprecipitated with the protein, yielding a bright yellow pellet after centrifugation. To test the k k K k ͓O ͔ ϭ 3 7 d ox,2 2 location of the histidyl-FAD linkage, the H46A mutant was pre- KM ͑ ϩ ͓ ͔͒ ϩ ͑ ͓ ͔ ϩ ͑ ϩ ͓ ͔͒͒ k7kred kϪ3 kox,2 O2 k3 kredkox,2 O2 k7 kred kox,2 O2 pared and expressed. The H46A mutant protein was no longer (Eq. 5) fluorescent after separation by SDS-PAGE. The expression level was comparable with that of the wild type MBP-AldO; k k k k ͓O ͔ ϭ 3 7 red ox,2 2 however, part of the protein was insoluble. The H46A mutant kcat ͑ ϩ ͓ ͔͒ ϩ ͑ ͓ ͔ ϩ ͑ ϩ ͓ ͔͒͒ k7kred kϪ3 kox,2 O2 k3 kredkox,2 O2 k7 kred kox,2 O2 was purified by anion exchange chromatography in the pres- (Eq. 6) ence of 100 ␮M FAD. After purification, the H46A mutant was found not to contain FAD, and also no oxidase activity was RESULTS observed. This confirms that His46 is crucially involved in cova- Expression, Purification, and Biochemical Characterization lent FAD binding. of AldO—By a PSI-BLAST search for putative oxidases using To determine whether the fused MBP-AldO protein behaves VAO-type flavoprotein sequences, a gene (SCO6147) from S. similar to the native AldO protein, we also prepared AldO with-

JULY 13, 2007•VOLUME 282•NUMBER 28 JOURNAL OF BIOLOGICAL CHEMISTRY 20285 Alditol Oxidase from S. coelicolor

same range of polyols as found for MBP-AldO (see below). Hence, we conducted all further analyses with the MBP-AldO fusion protein, referred to here as m-AldO. The absorption spectrum of m-AldO shows two absorption FIGURE 1. Multiple sequence alignment of alditol oxidase and some VAO-type oxidases generated using ClustalW. Only the region surrounding the conserved FAD-linking histidine (*) is shown. The numbers refer to maxima at 348 and 452 nm, which the amino acid numbers, the asterisk indicates the conserved histidine residue, and the dashes correspond to are typical for an oxidized flavin gaps between the sequences. AldO, alditol oxidase from S. coelicolor A3(2) (Q9ZBU1); XylOx, xylitol oxidase from Streptomyces sp. IKD472 (Q9KX73); GulOx, L-gulono-␥-lactone oxidase from Rattus norvegicus (P10867); CholOx, cofactor. The relatively low wave- cholesterol oxidase from Brevibacterium sterolicum (gi:15825772); BBE, berberine-bridge enzyme from length for the 348 nm absorption Eschscholzia californica (AAC39358.1); HDNO, 6-hydroxy-D-nicotine oxidase from Arthrobacter nicotinovorans maximum is a typical feature of his- (gi:75766389); GoOx, glucooligosaccharide oxidase from Acremonium strictum (AAS79317.1). tidyl-bound flavin cofactors (10). The extinction coefficient of the covalently linked flavin was determined by comparing the absorption spectra of m-AldO before and after treatment with Downloaded from 0.1% SDS (10). From these spectra, an extinction coefficient for Ϫ1 Ϫ1 m-AldO was determined: 12.5 mM cm at 452 nm. A com- mon characteristic of flavoproteins acting as an oxidase is the flavin reactivity with sulfite (17). To test whether m-AldO also

exhibits this reactivity, it was titrated with Na2SO3 (Fig. 3,

inset). This revealed that the enzyme readily forms a sulfite www.jbc.org Ϯ ␮ adduct with a Kd of 59 5 M. The addition of this nucleophile resulted in the disappearance of the absorbance maxima in the visible area, which is indicative of formation of a flavin N-5 sulfite adduct. It has been suggested that there is also a correla- at University of Groningen on September 17, 2007 tion between oxygen reactivity and the formation of a red ani- onic semiquinone upon partial reduction of the FAD cofactor (e.g. with a strong white light source) (18). The FAD cofactor of m-AldO also shows the ability to stabilize the red anionic semiquinone when reduced by light. The appearance of the red anionic semiquinone form of FAD, typified by an intense absorbance band at 380 nm, was also observed during redox potential measurements (Fig. 3, spectrum 3). The redox potential of the flavin in m-AldO was determined by using the xanthine oxidase method (11). For the oxidized/semiqui- FIGURE 2. SDS-PAGE analysis of cell extracts from E. coli TOP10 with Ϫ none couple, a redox potential (E1)of 4 mV was deter- expressed AldO and MBP-AldO. Lane A, fluorescent band of AldO in E. coli ϩ TOP10 cell-free extract. Lane B, lane A stained with Coomassie Brilliant Blue. mined by using methylene blue ( 11 mV) as a reference dye, Ϫ Lane C, fluorescent band of MBP-AldO in E. coli TOP10 cell-free extract. Lane D, whereas a redox potential (E2)of 213 mV for the semiqui- lane C stained with Coomassie Brilliant Blue. none/reduced couple was determined by using anthraqui- none-2-sulfonate (Ϫ225 mV). The midpoint redox potential Ϫ ϭ ϩ out the MBP tag. For this, MBP-AldO was treated with modi- (Em) of m-AldO is 109 mV (Em (E1 E2)/2). fied trypsin lacking chymotrypsin activity. This yielded the two Substrate Identification and Steady-state Kinetic Analysis— separate proteins: MBP and AldO. Using amylose resin, MBP In order to identify substrates for this m-AldO, a large number could effectively be removed from this mixture. ESI-MS analy- of potential substrates were tested. The test set of 86 substrates sis showed that the cleavage had occurred at the expected target contained a wide range of aromatic and aliphatic amines and site for proteolysis resulting in a homogenous preparation of alcohols. For activity screening, a generic chromogenic assay AldO. The determined mass of 45,665 Ϯ 2 Da corresponds was employed in which hydrogen peroxide, inherently formed nicely with the expected mass of AldO of 45,663 Da, which upon oxidase activity, is used by a peroxidase to form a colored includes an N-terminal extension of 4 residues (Ile-Ser-Glu- product. The activity screening revealed that only a few polyols Phe) that were part of the MBP-AldO linker and one acetate are readily accepted as substrate by m-AldO. The alditols sor- molecule. By gel permeation chromatography, it was confirmed bitol and xylitol are the best substrates. By HPLC analysis, it was that AldO and MBP-AldO both behave as monomers. The fla- found that m-AldO performs selective oxidation on one of the vin absorbance spectrum (300–650 nm) of AldO prepared by primary hydroxyl groups of xylitol. Furthermore, polarimetric proteolytic cleavage was identical to that of the MBP-tagged analysis showed that only D-xylose is formed upon oxidation of AldO. This indicates that the microenvironment of the flavin xylitol. cofactor is not influenced by the MBP fusion protein. Further- m-AldO shows the highest catalytic efficiency with xylitol more, it was found that isolated AldO was reactive with the (Table 1). Although a similar kcat is observed with sorbitol, the

20286 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 282•NUMBER 28•JULY 13, 2007 Alditol Oxidase from S. coelicolor Downloaded from

FIGURE 4. Deconvoluted absorption spectra of m-AldO (28 ␮M) during FIGURE 3. Absorption spectra of m-AldO for redox potential measure- anaerobic reduction by xylitol (5 mM). Spectra were recorded every 2.5 ms ments during anaerobic reduction using the xanthine oxidase/xanthine for 1 s. Three phases can be distinguished in the deconvoluted spectra. The method. Reduction takes place in two 1-electron transfer steps, leading to first phase is the reduction of FAD, leading to an intermediate spectrum formation of the red anionic semiquinone of FAD (spectrum 3) after the first (spectrum 2); the second phase leads to appearance of fully reduced flavin electron transfer (from spectrum 1 to 3) and fully reduced FAD after the sec- (spectrum 3); and the third phase represents product release, leading to spec- ond electron transfer (from spectrum 3 to 5). The inset shows absorption www.jbc.org trum 4. The inset shows the relative concentrations of the four species during spectra of 12.4 ␮M m-AldO after titration with sulfite. After each addition, the anaerobic reduction. enzyme was incubated for 15 min before a spectrum was collected. Spectra are shown after the addition of 0 ␮M,39␮M,74␮M, 123 ␮M, and 110 mM Na2SO3, respectively. results, we used xylitol as a model substrate to explore the at University of Groningen on September 17, 2007 TABLE 1 kinetic behavior of m-AldO in more detail. Steady-state kinetic parameters for m-AldO Using stopped-flow absorbance spectroscopy, we deter-

Experiments were conducted in 50 mM KPi buffer, pH 7.5, containing 0.1 mM 4-ami- mined the redox state of m-AldO during steady-state catalysis noantipyrine, 1 mM 3,5-dichloro-2-hydroxybenzenesulfonic acid, 3 units of horse- by monitoring flavin absorbance. Upon mixing 0.25 mM O2, 5.0 radish peroxidase, 0.25 mM O , and 15 nM m-AldO. Change in absorbance was 2 ␮ measured at 515 nm. mM xylitol, and 11.6 M AldO at 25 °C, we observed within the first 30 ms a fast decrease of absorbance at 452 nm to reach the steady-state phase. The steady-state phase lasted for about 300

ms, which complies with the determined kcat value, and was followed by a rapid and full reduction of the flavin due to the excess of xylitol. During steady-state, 32% of the flavin was found to be in the oxidized form, indicating that the reductive and oxidative half-reactions are almost balanced. Pre-steady-state Kinetic Analysis—To study the pre-steady- state kinetics of m-AldO, we followed the spectral changes of the covalently linked flavin by using a stopped-flow instrument equipped with diode array detection for spectral scans and a photomultiplier for single wavelength measurements. By inves- tigating the reductive and oxidative half-reaction separately,

Km value with this polyol is almost 5 times higher than with information was obtained concerning the kinetic mechanism of xylitol. Steady-state experiments were also performed with 1.25 m-AldO. mM O2, which resulted in a 1.5-fold increase of both kcat and Km. The reductive half-reaction was monitored by anaerobically The observed kcat values for the alditols are in the same range as mixing the enzyme with varying concentrations of xylitol. After found for other oxidases and their substrates, which suggests mixing, three phases were observed, leading from oxidized to that these polyols or related polyols are the physiological sub- fully reduced flavin (Fig. 4). The inset in Fig. 4 shows the corre- strates for m-AldO. Besides alditols, only a low activity (conver- sponding concentration profiles that were obtained from the sion rate Ͻ1sϪ1) was observed with other aliphatic primary deconvolution with Pro-K. To study the rates of the observed alcohols, diols, and carbohydrates. It is clear that aliphatic poly- phases separately, strategic wavelengths were selected for every ols are preferred and that stereochemistry plays a role in sub- phase in such a way that only the desired phase showed changes strate recognition. Next to this, the chain length of the substrate in absorption. To follow the first phase, the absorption at 320 is also important for optimal reactivity. While xylitol is the best nm was monitored, and the third phase was studied at 452 nm, substrate with a 5-carbon skeleton, L-threitol (4 carbons), because the largest spectral difference occurred at that wave- D-sorbitol (6 carbons), and D-mannitol (6 carbons) are all worse length. The second phase was followed at 405 nm, which is the substrates because of relatively high Km values. Based on these isosbestic point of the two spectra for the transition in the first

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SCHEME 1. Proposed model for the reductive half-reaction of m-AldO. In this model, E represents oxidized m-AldO, S represents the substrate, PЈ ox SCHEME 2. Proposed models for the oxidative half-reaction of reduced represents the open form of D-xylose, P represents the closed form of D-xy- m-AldO with and without product bound. In this model, Ered represents the lose, and Ered represents the reduced form of m-AldO. reduced m-AldO, Eox represents oxidized m-AldO, and P represents the closed form of D-xylose. phase, allowing a rate determination without the influence of However, no significant change in fluorescence was observed in the first phase. time. The first phase showed the largest decrease in flavin absorp- Taken together, for the reductive half-reaction, a model is tion at 450 nm, indicating that this kinetic event reflects flavin proposed as depicted in Scheme 1. In this model, E represents reduction. A hyperbolic relation was observed between the rate ox oxidized m-AldO, S represents the substrate, PЈ represents the of this first phase and an increasing concentration of xylitol, open form of D-xylose, P represents the closed form of D-xylose, indicating that xylitol binding is the step preceding this redox and E represents the reduced form of m-AldO. reaction. The kinetic data for the first phase could be fitted by a red The oxidative half-reaction was monitored by mixing simple hyperbolic function, indicating that reduction is virtu- Downloaded from reduced enzyme with varying concentrations of molecular oxy- ally irreversible (Equation 2). Fitting yielded a reduction rate Ϫ1 gen. To determine the reoxidation rate constant of reduced constant (k )of99s and a K of 1.3 mM. The spectrum red d m-AldO with molecular oxygen, m-AldO was first anaerobi- formed upon flavin reduction (spectrum 2 in Fig. 4) does not cally reduced with 3 eq of xylitol. After full reduction of resemble a typical fully reduced flavin spectrum. This suggests m-AldO, reoxidation was followed with diode array detection that the initially formed oxidation product, the open form of and was found to be a monophasic process (Scheme 2). www.jbc.org xylose, causes some spectral perturbations, possibly by forming Reduced m-AldO was rapidly reoxidized with varying concen- a charge transfer complex, as is indicated by the increase in A520 trations of molecular oxygen. From the linear relation between (19). ϫ 5 kobs and [O2], a bimolecular rate constant (kox,1)of1.7 10 Following this first phase, there is a second fast phase leading Ϫ1 Ϫ1 at University of Groningen on September 17, 2007 M s was calculated. Reoxidation in the presence of 100 mM to a fully reduced flavin spectrum (spectrum 3 in Fig. 4). This ϫ 5 D-xylose yielded a bimolecular rate constant (kox,2)of1.4 10 second phase also displayed a hyperbolic relation between the Ϫ1 Ϫ1 M s for product-bound reduced m-AldO (Scheme 2). observed rate and xylitol concentration, leading to an observed These are typical rate constants found for oxidases, again indi- Ϫ1 rate of 51 s at saturating xylitol concentrations. A similar cating that m-AldO is a true oxidase (21). kinetic event has been observed in the reductive half-reaction of another polyol oxidase, amadoriase I from Aspergillus sp. (20). DISCUSSION The data were fitted by using a hyperbolic function with a non- In this paper, we report the discovery of AldO by genome Ϫ1 zero intercept of 7 s (kϪ3). This suggests that this second mining. AldO was identified while searching for VAO kinetic event is reversible. By subtracting kϪ3 from the maxi- homologs in the available genome sequence data bases. Based Ϫ1 mum observed rate, a k3 value of 44 s can be calculated. We on sequence homology with other sequence-related flavopro- propose that the observed second phase corresponds to the ring tein oxidases, AldO was predicted to contain a covalent FAD- closure of open D-xylose, which is formed in the first phase. It is histidyl linkage and therefore had a high chance to represent an energetically favorable to form the ring-closed hemiacetal of oxidase. This study has shown that AldO is indeed a covalent D-xylose. It should be noted that the k3 and kϪ3 are observed flavoprotein oxidase primarily acting on alditols. The physio- rates that are in fact a complex function of the other rates of the logical function of this oxidase is yet unknown. Overlap in sub- reductive half-reaction. Extracting the true kinetic values for k3 strate specificity is found with a xylitol oxidase isolated from and kϪ3 is complicated due to the fact that the rate of reduction another Streptomyces strain (7). Also several homologs have (kred) and the observed k3 are in the same order of magnitude. been identified in other actinomycete genomes (sequence iden- The identified kinetic events during the reductive half-reaction tity 81% for Streptomyces avermitilis; 78% for Streptomyces sca- were simulated using the determined values for the step pre- bies; 52% for Stigmatella aurantiaca; 49% for Arthrobacter sp. ceding and following this phase (k3/kϪ3) and the proposed FB24, Acidothermus cellulolyticus 11B, and Kineococcus mechanism (Scheme 1). It was shown that the observed rates radiotolerans SRS30216). For the gene from K. radiotolerans, are merely lower limits of the true k3 and kϪ3. we have demonstrated that the respective protein also acts as an The third observed phase is a relatively slow process of 3.5 alditol oxidase (data not shown). Although the expression of Ϫ1 s (k4) and involves only marginal spectral changes. This last the K. radiotolerans AldO was poor in E. coli and could not be phase could represent product release. The rate constant for boosted by MBP fusion, it was found that the protein also con- this process is relatively slow when compared with the kcat (13 tains covalent FAD and acts as an oxidase on the alditols xylitol Ϫ s 1), implying that product release from this complex is not and sorbitol. This indicates that the above mentioned bacteria part of the catalytic cycle. This suggests that m-AldO follows a (mainly actinomycetes) all harbor AldO orthologs. Apparently, kinetic route in which the product is released from another a selected number of bacteria employ alditol oxidases for a yet binary or ternary complex. The anaerobic reduction of m-AldO unknown catabolic or anabolic route. They may be involved in was also followed by monitoring tryptophan fluorescence. modification of a secondary metabolite. Actinomycetes are

20288 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 282•NUMBER 28•JULY 13, 2007 Alditol Oxidase from S. coelicolor

flavin N(1) locus. A high reactivity of the reduced enzyme with molecular oxygen also is an indica- tion for a protein to be a true oxi- dase (21). This is clearly the case, since bimolecular reoxidation rate 5 Ϫ1 Ϫ1 constants of 1.7 ϫ 10 M s and 5 Ϫ1 Ϫ1 1.4 ϫ 10 M s were measured for reduced and product-bound reduced m-AldO, respectively. SCHEME 3. Oxidation of xylitol by m-AldO, yielding D-xylose as a product, which adopts a closed hemi- acetal conformation. Steady-state analysis has shown that m-AldO is active with a narrow set of alditols. The highest catalytic known for their ability to produce a range of secondary metab- ϭ ϭ Ϫ1 efficiency was found with xylitol (Km 0.32 mM, kcat 13 s ). olites, which often contain a polyol moiety (22). The enzyme Upon oxidation of the alditol by m-AldO, a primary alcohol is could also be part of a catabolic route for sorbitol or xylitol converted into the corresponding aldehyde. By HPLC and Downloaded from degradation. Inspection of the genome of S. coelicolor showed polarimetric analysis, it was shown that m-AldO acts as a pri- that the aldo gene is not flanked by genes that are related to mary alcohol oxidase, yielding solely D-xylose as product in the carbohydrate modifications. However, the ortholog in S. aver- case of xylitol as substrate. This indicates that m-AldO has a mitilis is located upstream of several genes related to carbohy- high regioselectivity. It also appears that the chain length of the drate degradation (i.e. a xylose repressor, a sugar transporter, a substrate is important for optimal substrate recognition and ␤ ␤ -galactosidase, and a -1,4-xylanase). Nevertheless, S. coeli- reactivity as m-AldO has a preference for C5 and C6 alditols. www.jbc.org color A3(2) is unable to grow efficiently on sorbitol, suggesting Pre-steady-state kinetic experiments were conducted by that AldO is involved in a more dedicated route (23). studying the reductive and oxidative half-reactions separately. To obtain large amounts of AldO, the gene was subcloned in The reductive half-reaction showed three phases, leading from a pBAD-MBP vector in such a way that AldO was expressed oxidized to fully reduced flavin. The first phase corresponds to at University of Groningen on September 17, 2007 containing an N-terminal maltose-binding protein. In a single the irreversible reduction of the flavin by the substrate with a purification step, 350 mg of m-AldO per liter of culture was reduction rate constant of 99 sϪ1. A plausible explanation for obtained. From spectral, electrospray ionization-mass spec- the observed second phase is the following. During the first trometry, and gel permeation analysis of m-AldO and AldO phase, flavin-mediated oxidation of xylitol takes place, and the obtained by trypsin cleavage, it was clear that MBP has no influ- initially formed D-xylose product will be in the open form. ence on the properties of AldO. Hence, all experiments were However, it is energetically favorable for D-xylose to adopt the performed with m-AldO. closed hemiacetal conformation (Scheme 3). Therefore, it is 46 Based on sequence homology, His was expected to be tempting to assume to that the second kinetic phase, which is involved in covalent FAD attachment. The lack of cofactor observed after flavin reduction, reflects such a rearrangement 46 binding by the H46A mutant showed that His is required for (the cyclization) of the product in the active site. Consequently, covalent FAD linkage. This is supported by the preliminary the second deconvoluted spectrum (spectrum 2 in Fig. 4) rep- x-ray analysis of AldO crystals, which show that AldO contains resents the binary complex of reduced m-AldO and the open ␣ 1 46 3 8 -N -histidyl-bound FAD to His (24). Next to this, the form of the D-xylose product. The fact that this second spec- mutant protein no longer displayed oxidase activity. From these trum is different from the fully reduced flavin spectrum can be results, it appears that the covalent FAD linkage serves multiple explained by the close proximity of the formed aldehyde prod- purposes. In addition, it is known from other flavoprotein oxi- uct, the open form of D-xylose, to the FAD cofactor. Interac- dases that a covalently linked flavin can increase the redox tions between the reduced cofactor and the bound product lead potential and in that way facilitate usage of molecular oxygen as to perturbations of the flavin spectrum. A slight but significant electron acceptor (5). For m-AldO, a midpoint redox potential increase in absorption at 520 nm in the second spectrum sug- Ϫ of 109 mV was determined, which is higher than the average gests that the interaction involves a charge transfer complex midpoint redox potential found for flavin-dependent dehydro- (19). Increasing xylitol concentrations have an effect on the genases. This is in line with the observation that m-AldO shows observed rate of the rearrangement of the aforementioned all characteristics of a true oxidase. binary complex. This suggests that xylitol can bind to the As has been described for multiple other flavoprotein oxi- enzyme-product complex in such a way that it enhances the ϭ dases, m-AldO shows reversible reactivity with sulfite (Kd proposed ring closure. The third phase most likely represents Ϯ ␮ 59 5 M), which forms a flavin N-5 sulfite adduct. When product release from the reduced enzyme and has a rate con- reduced by light, m-AldO forms and stabilizes the red anionic stant that is too low to be catalytically relevant. semiquinone form of FAD. Both observations are typical for Reoxidation of reduced m-AldO yielded a bimolecular rate 5 Ϫ1 Ϫ1 oxidases and imply the presence of a positive charge near the constant of 1.7 ϫ 10 M s , whereas a bimolecular rate con- 5 Ϫ1 Ϫ1 stant of 1.4 ϫ 10 M s was found for product-bound 3 D. P. H. M. Heuts, E. W. van Hellemond, D. B. Janssen, and M. W. Fraaije, reduced m-AldO. These are typical rate constants found for unpublished data. flavoprotein oxidases. For other oxidases, it has been shown

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TABLE 2 Determined and calculated kinetic parameters for m-AldO The calculated values were obtained from the kinetic constants in Fig. 5 and the corresponding Equations 3–6. Calculated Calculated Experimental (ternary complex) (ping-pong) Ϯ KM (mM) 0.32 0.03 0.17 0.034 Ϫ1 Ϯ kcat (s ) 13 0.2 13 2.6 Redox state (%)a 32 Ϯ 4 29 2.6 a Numbers represent the percentage of m-AldO that is in the oxidized state.

by simulations that these observed rates are lower than the true rates, so the mechanistic calculations were also performed with Ϫ1 Ϫ1 k3 and kϪ3 values of up to 80 s and 21 s , respectively. These FIGURE 5. Proposed kinetic mechanism for m-AldO. Eox, oxidized m-AldO; Ј variations did not result in significant changes of the calculated S, substrate; P , product intermediate, P, product; Ered, reduced m-AldO. m-AldO follows mainly the upper cycle, which represents a ternary complex steady-state parameters and redox state. This suggests that mechanism, in contrast to the lower cycle, which stands for a ping-pong m-AldO mainly follows a ternary complex mechanism during mechanism. The following kinetic constants have been determined: Kd (kϪ1/ Ϫ1 Ϫ1 Ϫ1 Ϫ1 Downloaded from k ) ϭ 1.3 mM, k ϭ 99 s , k ϭ 44 s , kϪ ϭ 7s , k ϭ 3.5 s , k ϭ 1.7 ϫ steady-state catalysis. This is further supported by the set of 1 Ϫ Ϫ red 3 Ϫ Ϫ 3 4 ox,1 105 M 1 s 1, and k ϭ 1.4 ϫ 105 M 1 s 1. ox,2 parallel Lineweaver-Burk plots that were obtained from O2- and xylitol-dependent steady-state kinetic measurements (see supplemental materials). Normally, this implies that a ping- that the bimolecular reoxidation rate depends to some extent pong mechanism is operative. However, depending on some on whether or not the product is bound to the reduced enzyme individual kinetic parameters, enzymes obeying a ternary species (21). www.jbc.org complex mechanism can also yield parallel Lineweaver-Burk The kinetic measurements were used to analyze the kinetic plots (25, 26). By simulating these plots using the pre-steady- mechanism, which could be a ping-pong or ternary complex state kinetic parameters determined for m-AldO, a similar mechanism. Taking together the kinetic data of the reductive

set of parallel lines was obtained (see supplemental materi- at University of Groningen on September 17, 2007 and oxidative half-reaction yields values for most kinetic als). From Fig. 5 and all kinetic parameters, it is clear that the parameters of the lower kinetic cycle shown in Fig. 5. From this dominant type of kinetic mechanism depends on the con- cycle, which represents a ping-pong mechanism, and its asso- centration of O . At higher O concentrations, the ternary ciated kinetic constants, values for K , and k can be 2 2 m, xylitol cat complex mechanism would be dominant, whereas at rela- calculated using Equations 3 and 4. The calculated Km for xyli- tively low concentrations of O2, a ping-pong mechanism may tol yields a value of 0.034 mM, whereas the calculated kcat value Ϫ partly be operative. is 2.6 s 1. Both values are significantly lower than the K and m This study shows that m-AldO is an oxidase that can effi- k values obtained with steady-state kinetic experiments cat ciently convert a number of alditols and, to a lesser extent, other (Table 2). Furthermore, it was found that the calculated value polyols. It is known that carbohydrate oxidases are widely used for the redox state of AldO during steady-state catalysis (2.6% in in diagnostic applications, the food and drink industry, and car- oxidized state) did not correspond to the measured redox state bohydrate synthesis. Based on the findings in this paper, during steady-state catalysis (32% in oxidized state). So far, we m-AldO is a good candidate for further biocatalytic exploration assumed that the observed third phase in the reductive half- Ϫ1 in the field of biosynthesis and biosensors. reaction (3.5 s ) represents product release (k4). However, we also simulated a scenario in which product release is not spec- Acknowledgments—We thank Dr. R. H. H. van den Heuvel and Dr. trally observable. For this, we varied the rate of product release Ϫ1 Ϫ1 H. F. M. Mazon from the Department of Biomolecular Mass Spec- from 3.5 s up to 100 s . The corresponding calculated trometry at the University of Utrecht for performing electrospray ion- kinetic parameters were not consistent with the measured ization-mass spectrometry experiments on AldO. kinetic parameters. The incompatibility of the determined steady-state kinetic parameters and the calculated kinetic parameters based on a REFERENCES ping-pong kinetic mechanism suggests that another kinetic 1. van Hellemond, E. W., Leferink, N. G., Heuts, D. P., Fraaije, M. W., and van mechanism is operative. An alternative mechanism is repre- Berkel, W. J. (2006) Adv. Appl. Microbiol. 60, 17–54 sented in the upper cycle in Fig. 5. This upper cycle implies a 2. Xu, F., Golightly, E. J., Fuglsang, C. C., Schneider, P., Duke, K. R., Lam, L., Christensen, S., Brown, K. M., Jorgensen, C. T., and Brown, S. H. (2001) ternary complex mechanism in which the reduced m-AldO, Eur. J. Biochem. 268, 1136–1142 still having the product bound, is being oxidized and subse- 3. Fraaije, M. W., van Berkel, W. J., Benen, J. A., Visser, J., and Mattevi, A. quently releases the product. Assuming a product release rate (1998) Trends Biochem. Sci. 23, 206–207 Ϫ1 of 80 s , we calculated a Km, xylitol, kcat, and redox state of 0.17 4. Huang, C. H., Lai, W. L., Lee, M. H., Chen, C. J., Vasella, A., Tsai, Y. C., and Ϫ1 mM,13s , and 29% in the oxidized state, respectively, from the Liaw, S. H. (2005) J. Biol. Chem. 280, 38831–38838 ternary complex kinetic model (Equations 5 and 6). These val- 5. Fraaije, M. W., van den Heuvel, R. H., van Berkel, W. J., and Mattevi, A. (1999) J. Biol. Chem. 274, 35514–35520 ues approach the measured steady-state kinetic parameters 6. Fraaije, M. W., and Mattevi, A. (1999) in Flavins and Flavoproteins (Table 2). The above mentioned calculations were performed (Weber, R., ed) pp. 881–886, Agency for Scientific Publications, Berlin by using the observed k3 and kϪ3 values. However, it was shown 7. Yamashita, M., Omura, H., Okamoto, E., Furuya, Y., Yabuuchi, M., Fukahi,

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