Discovery, Characterization, and Kinetic Analysis of an Alditol Oxidase from Streptomyces coelicolor

Abstract

A gene encoding an alditol oxidase was found in the genome of Streptomyces coelicolor A3(2). This newly identified oxidase, AldO, was expressed at extremely high levels in Escherichia coli when fused to maltose-binding protein. AldO is a soluble monomeric flavoprotein with subunits of 45.1 kDa, each containing a covalently bound FAD cofactor. From sequence alignments with other flavoprotein oxidases, it was found that AldO contains a conserved histidine (His46) that is typically involved in covalent FAD attachment. Covalent FAD binding is not observed in the H46A AldO mutant, confirming its role in covalent attachment of the flavin cofactor. Steady-state kinetic analyses revealed that wild-type AldO is active with several polyols. The alditols xylitol (Km = 0.32 mm, kcat = 13 s–1) and sorbitol (Km = 1.4 mm, kcat = 17 s–1) are the preferred substrates. From pre-steady-state kinetic analyses, using xylitol as substrate, it can be concluded that AldO mainly follows a ternary complex kinetic mechanism. Reduction of the flavin cofactor by xylitol occurs at a relatively high rate (99 s–1), after which a second kinetic event is observed, which is proposed to represent ring closure of the formed aldehyde product, yielding the hemiacetal of d-xylose. Reduced AldO readily reacts with molecular oxygen (1.7 × 105 m–1 s–1), which confirms that the enzyme represents a true flavoprotein oxidase. A gene encoding an alditol oxidase was found in the genome of Streptomyces coelicolor A3(2). This newly identified oxidase, AldO, was expressed at extremely high levels in Escherichia coli when fused to maltose-binding protein. AldO is a soluble monomeric flavoprotein with subunits of 45.1 kDa, each containing a covalently bound FAD cofactor. From sequence alignments with other flavoprotein oxidases, it was found that AldO contains a conserved histidine (His46) that is typically involved in covalent FAD attachment. Covalent FAD binding is not observed in the H46A AldO mutant, confirming its role in covalent attachment of the flavin cofactor. Steady-state kinetic analyses revealed that wild-type AldO is active with several polyols. The alditols xylitol (Km = 0.32 mm, kcat = 13 s–1) and sorbitol (Km = 1.4 mm, kcat = 17 s–1) are the preferred substrates. From pre-steady-state kinetic analyses, using xylitol as substrate, it can be concluded that AldO mainly follows a ternary complex kinetic mechanism. Reduction of the flavin cofactor by xylitol occurs at a relatively high rate (99 s–1), after which a second kinetic event is observed, which is proposed to represent ring closure of the formed aldehyde product, yielding the hemiacetal of d-xylose. Reduced AldO readily reacts with molecular oxygen (1.7 × 105 m–1 s–1), which confirms that the enzyme represents a true flavoprotein oxidase. Carbohydrate oxidases are highly valuable biocatalysts for analytical and synthetic purposes. Chemical methods cannot compete with the exquisite regio- and/or enantioselectivity by which these enzymes oxidize polyols. Applications in which carbohydrate oxidases are used are, for example, biosensors for blood sugar, synthetic routes toward chiral building blocks, sweeteners, and flavors. Oxidase-mediated catalysis also leads to formation of hydrogen peroxide, a property that is used in a number of applications, such as bleaching processes and waste-water treatment (1van Hellemond E.W. Leferink N.G. Heuts D.P. Fraaije M.W. van Berkel W.J. Adv. Appl. Microbiol. 2006; 60: 17-54Crossref PubMed Scopus (61) Google Scholar, 2Xu F. Golightly E.J. Fuglsang C.C. Schneider P. Duke K.R. Lam L. Christensen S. Brown K.M. Jorgensen C.T. Brown S.H. Eur. J. Biochem. 2001; 268: 1136-1142Crossref PubMed Scopus (49) Google Scholar). At present, only a limited number of carbohydrate oxidases have been identified, which restricts the biocatalytic exploitation of this class of redox enzymes. The best known representative is glucose oxidase, which in fact is the most widely applied redox enzyme. Besides galactose oxidase, which contains copper as cofactor, all presently known oxidases acting on carbohydrates contain a flavin cofactor. Examples of such flavoprotein oxidases are glucose oxidase, l-gulono-γ-lactone oxidase, xylitol oxidase, hexose oxidase, lactose oxidase, glucooligosaccharide oxidase, and pyranose oxidase. Except for glucose oxidase and pyranose oxidase, all of these oxidases belong to a specific group of flavoproteins, the vanillyl-alcohol oxidase (VAO) 2The abbreviations used are: VAO, vanillyl-alcohol oxidase; MBP, maltosebinding protein; m-AldO, MBP fused to AldO; HPLC, high pressure liquid chromatography. family. Members of this family share a similar overall structure consisting of two domains (3Fraaije M.W. van Berkel W.J. Benen J.A. Visser J. Mattevi A. Trends Biochem. Sci. 1998; 23: 206-207Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar). One domain binds the adenine part of the FAD cofactor and is called the FAD-binding domain, whereas the other, called the cap domain, covers the isoalloxazine moiety of the cofactor and forms the major part of the active site around the isoalloxazine ring system. A special feature of this flavoprotein family is the fact that a relatively large number of VAO members bind the FAD cofactor in a covalent manner. This is also the case for all of the above mentioned VAO-type carbohydrate oxidases. In fact, the recent elucidation of the structure of glucooligosaccharide oxidase has revealed the first example where a flavin cofactor is covalently linked to two amino acid residues (4Huang C.H. Lai W.L. Lee M.H. Chen C.J. Vasella A. Tsai Y.C. Liaw S.H. J. Biol. Chem. 2005; 280: 38831-38838Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar). 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) (5Fraaije M.W. van den Heuvel R.H. van Berkel W.J. Mattevi A. J. Biol. Chem. 1999; 274: 35514-35520Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar). This is in line with the observation that most VAO-type covalent flavoproteins act as an oxidase (6Fraaije M.W. Mattevi A. Weber R. Flavins and Flavoproteins. Agency for Scientific Publications, Berlin1999: 881-886Google Scholar). In these cases, the FAD cofactor is typically tethered to a histidine residue, and this linking histidine can be readily identified by sequence motif recognition. Hence, the ability to identify covalent VAO homologs by sequence analysis can be used as a tool to find novel oxidase genes. Most of the characterized carbohydrate oxidases have been isolated from fungi, whereas only two from bacterial origin have been described (7Yamashita M. Omura H. Okamoto E. Furuya Y. Yabuuchi M. Fukahi K. Murooka Y. J. Biosci. Bioeng. 2000; 89: 350-360Crossref PubMed Scopus (18) Google Scholar, 8Hiraga K. Kitazawa M. Kaneko N. Oda K. Biosci. Biotechnol. Biochem. 1997; 61: 1699-1704Crossref PubMed Scopus (10) Google Scholar). Here we describe the discovery and characterization of an alditol oxidase (AldO) from the actinomycete 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 of AldO contains a histidine that is expected to form a covalent FAD-histidyl linkage. Therefore, it was anticipated that the protein would have a high probability to exhibit oxidase activity. In this report, we show that the enzyme is indeed a typical flavoprotein oxidase active on a number of alditols. By expressing the enzyme fused to maltose-binding protein (MBP), impressive amounts of alditol oxidase could be produced, enabling a detailed biochemical and kinetic characterization of this novel bacterial oxidase. Chemicals and Restriction Enzymes—Restriction enzymes were obtained from Roche Applied Science and New England Biolabs. Escherichia coli TOP10-competent cells, the TOPO TA cloning kit for sequencing, and the pBAD/Myc-His A vector were obtained from Invitrogen. VAO (EC 1.1.3.38) was a kind gift from Dr. W. J. H. van Berkel (Wageningen University, The Netherlands). Xanthine oxidase (EC 1.2.3.2) was obtained from Stratagene. Horseradish peroxidase (EC 1.11.1.7) was obtained from Fluka. All other chemicals were of analytical grade. Cloning and Expression of the aldo Gene—The aldo genes were cloned into pBADNdeI and pBAD-MBP for expression. The latter is a pBADNdeI-derived vector in which the malE gene, including a Factor Xa protease cleavage site from pMAL-c2x, is inserted (NdeI-HindIII). The aldo gene from S. coelicolor A3(2) was amplified from genomic using The were used for cloning into site is and site is The were used for cloning into site is and site is and the is in The aldo gene from was amplified from using The were used for cloning into site is and site is The were used for cloning into site is and site is and the is in TOPO were by the amplified aldo genes with of at for pBADNdeI the were with and after which the aldo was isolated from an The were into an and pBADNdeI yielding and pBAD-MBP the were with and after which the aldo genes were isolated from an The aldo genes were into an and pBAD-MBP yielding and E. coli were with the and at 17 for in with and of and AldO containing a acid consisting of a and a of and containing a Factor Xa protease cleavage of from were by at × and for and in of The were by and at × and for to The was into two to the the a the was the was was by a The were and using an and from the enzyme it was a were at and in a The in the is and kinetic were by the of by to a of and This in formation of a to product, which can be at = R. R. L. Mattevi A. P. Biochem. 1997; PubMed Scopus Google Scholar). The of and AldO The of was by the and after with it was that the flavin of the was to that of FAD of at E. van Berkel W.J. van J.A. Eur. J. Biochem. PubMed Scopus Google Scholar). the of xylitol by was by analysis using a × of the was by analysis using a and were by using the described by Flavins and Flavoproteins. New Scholar). A containing and redox was by with a of oxidase was and were using a were with an Applied were at of using a A was applied to in obtained by were using the of Applied From the several were to the observed The first two of the were by the at and after the enzyme with of The first was at the second and in whereas is an in the first enabling analysis of the second The was at The was at by a containing enzyme with a containing oxygen the of all and were by with VAO was to a of to obtained by were to an where A represents is a and is an observed rate The observed for of were using the was used to rate from the kinetic by the and New The kinetic and kcat as as the redox catalysis can be to the a and kcat can be using and a ternary complex and kcat can be using and and of a for putative oxidases using VAO-type flavoprotein a gene (SCO6147) from S. coelicolor A3(2) was identified that such a The protein of amino with a of and represents of the members in the VAO flavoprotein family. the known VAO-type it sequence with xylitol oxidase from Streptomyces sequence and l-gulono-γ-lactone oxidase sequence Except for sequence typical for a VAO other sequence can be identified, that it is a protein. of the protein sequence with known VAO-type protein revealed the of a histidine which is typically involved in covalent attachment of the FAD cofactor on VAO-type oxidases containing a covalently bound FAD to two amino (4Huang C.H. Lai W.L. Lee M.H. Chen C.J. Vasella A. Tsai Y.C. Liaw S.H. J. Biol. Chem. 2005; 280: 38831-38838Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar, A. F. A. P. J. Biol. Chem. 2006; Full Text Full Text PDF PubMed Scopus Google Scholar, C.H. J. 2006; PubMed Scopus Google Scholar). to an a second linkage to the isoalloxazine moiety of FAD is in these oxidases. alignments not such a conserved that would be involved in covalent FAD attachment. that the enzyme from S. coelicolor is as an oxidase and its the gene was amplified from genomic by and the gene was in a the active AldO was only expressed at a as observed by The protein is at of the of a covalently bound flavin cofactor A and M.W. M. van Berkel W. Appl. Microbiol. 1997; PubMed Google Scholar). the of the protein was expressed fused to MBP, it is known that to MBP of soluble protein 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). The linking MBP to the of AldO, the From of an impressive of of was by as a at kDa, which with a of The protein was found to be at of a FAD cofactor and The covalent binding of the flavin cofactor was also by the observation that of the the redox cofactor with the yielding a after the of the the H46A was and The H46A protein was after by The was with that of the part of the protein was The H46A was by in the of the H46A was found not to contain and also oxidase was This confirms that is involved in covalent FAD the fused protein similar to the AldO we also AldO the MBP was with activity. This the two MBP and MBP could be from this analysis that the cleavage at the expected site for in a of The of with the expected of AldO of which an of residues that were part of the and By it was that AldO and as The flavin of AldO by cleavage was to that of the This that the of the flavin cofactor is not by the MBP protein. it was found that isolated AldO was with the of as found for Hence, we all analyses with the to as The of two at and which are typical for an flavin cofactor. The relatively for the is a typical feature of flavin E. van Berkel W.J. van J.A. Eur. J. Biochem. PubMed Scopus Google Scholar). The of the covalently linked flavin was by the of and after treatment with E. van Berkel W.J. van J.A. Eur. J. Biochem. PubMed Scopus Google Scholar). From these an for was at A of flavoproteins acting as an oxidase is the flavin with F. R. M. J. Biol. Chem. Full Text PDF PubMed Google Scholar). also this it was with This revealed that the enzyme readily forms a with a of The of this in the of the in the which is of formation of a flavin It has been that is also a oxygen and the formation of a of the FAD cofactor (e.g. with a P. J. Biol. Chem. Full Text PDF PubMed Google Scholar). The FAD cofactor of also the ability to the when by The of the form of by an at was also observed redox The redox of the flavin in was by using the oxidase Flavins and Flavoproteins. New Scholar). the a redox of was by using as a whereas a redox of for the was by using The redox of is = and Steady-state to identify for this m-AldO, a large number of were The of a of and and a was in which hydrogen peroxide, formed oxidase is used by a peroxidase to form a The revealed that only a are readily as by The alditols sorbitol and xylitol are the best substrates. By it was found that on of the of analysis that only is formed of the with xylitol a similar kcat is observed with the with this is with Steady-state were also with which in a of kcat and The observed kcat for the alditols are in the as found for other oxidases and which that these are the for Besides only a rate s–1) was observed with other and It is that are preferred and that a role in recognition. to the of the is also for xylitol is the best with a and are all of relatively high on these we used xylitol as a to the kinetic behavior of in kinetic for in a we the redox of catalysis by flavin and AldO at we observed the first a of at to the The for which with the kcat and was by a and of the flavin to the of of the flavin was found to be in the that the and are the pre-steady-state of m-AldO, we the of the covalently linked flavin by using a with for and a for By the and was obtained the kinetic of The was by the enzyme with of were observed, from to flavin The in the that were obtained from the with the of the observed were for in such a that only the in the first the at was and the was at the at that The second was at which is the of the two for the in the first a rate the of the first The first the in flavin at that this kinetic event flavin A was observed the rate of this first and an increasing of that xylitol binding is the this redox The kinetic for the first could be by a that is a rate of and a of The formed flavin in not a typical flavin This that the formed product, the form of by a as is by the in S. PubMed Scopus Google Scholar). this first is a second to a flavin in This second also a the observed rate and xylitol to an observed rate of at xylitol A similar kinetic event has been observed in the of oxidase, from 2001; PubMed Scopus Google Scholar). The were by using a with a of This that this second kinetic event is By from the observed a of can be We that the observed second to the ring closure of which is formed in the first It is to form the hemiacetal of d-xylose. It be that the and are observed that are in fact a complex of the other of the the true kinetic for and is to the fact that the rate of and the observed are in the of The identified kinetic the were using the for the and this and the proposed It was shown that the observed are of the true and The observed is a relatively of and only This could represent The rate for this is relatively when with the kcat s–1), that from this complex is not part of the This that follows a kinetic in which the is from ternary The of was also by significant in was observed in for the a is proposed as in In this represents m-AldO, represents the substrate, represents the form of represents the form of and represents the form of The was by enzyme with of molecular the rate of with molecular was first with of of m-AldO, was with and was found to be a Reduced was with of molecular From the and a rate of × 105 m–1 was in the of a rate of 1.4 × 105 m–1 for are typical rate found for oxidases, that is a true oxidase A. Trends Biochem. Sci. 2006; Full Text Full Text PDF PubMed Scopus Google Scholar). In this we the discovery of AldO by genome AldO was identified searching for VAO homologs in the genome sequence on sequence with other flavoprotein oxidases, AldO was predicted to contain a covalent FAD-histidyl linkage and a high to represent an oxidase. This has shown that AldO is indeed a covalent flavoprotein oxidase acting on alditols. The of this oxidase is in is found with a xylitol oxidase isolated from Streptomyces (7Yamashita M. Omura H. Okamoto E. Furuya Y. Yabuuchi M. Fukahi K. Murooka Y. J. Biosci. Bioeng. 2000; 89: 350-360Crossref PubMed Scopus (18) Google Scholar). several homologs have been identified in other actinomycete for Streptomyces for Streptomyces for for and the gene from K. we have that the protein also as an alditol oxidase not the of the K. AldO was in E. coli and could not be by MBP it was found that the protein also contains covalent FAD and as an oxidase on the alditols xylitol and This that the above mentioned all AldO a number of alditol oxidases for a be involved in of a are known for ability to a of which contain a moiety Y. M. Chem. Biol. 1999; Full Text PDF PubMed Scopus Google Scholar). The enzyme could also be part of a for sorbitol xylitol of the genome of S. coelicolor that the aldo gene is not by genes that are to carbohydrate the in S. is of several genes to carbohydrate a a a and a S. coelicolor A3(2) is to on that AldO is involved in a R. M. K. H. M.H. F. J. PubMed Scopus Google Scholar). large amounts of AldO, the gene was in a pBAD-MBP vector in such a that AldO was expressed containing an maltose-binding protein. In a of of was From and analysis of and AldO obtained by it was that MBP has on the of Hence, all were with on sequence was expected to be involved in covalent FAD attachment. The of cofactor binding by the H46A that is for covalent FAD linkage. This is by the analysis of AldO which show that AldO contains FAD to F. S. Heuts D.P. Fraaije M.W. Mattevi A. Biol. 2006; PubMed Scopus Google Scholar). P. H. M. E. W. van and M. W. to the protein oxidase activity. From these it that the covalent FAD linkage purposes. In it is known from other flavoprotein oxidases that a covalently linked flavin can the redox and in that of molecular oxygen as (5Fraaije M.W. van den Heuvel R.H. van Berkel W.J. Mattevi A. J. Biol. Chem. 1999; 274: 35514-35520Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar). m-AldO, a redox of was which is the redox found for This is in line with the observation that all of a true oxidase. has been described for other flavoprotein oxidases, with = which forms a flavin by forms and the form of are typical for oxidases and the of a the flavin A high of the enzyme with molecular oxygen also is an for a protein to be a true oxidase A. Trends Biochem. Sci. 2006; Full Text Full Text PDF PubMed Scopus Google Scholar). This is the rate of × 105 m–1 and 1.4 × 105 m–1 were for and m-AldO, Steady-state analysis has shown that is active with a of alditols. The was found with xylitol (Km = 0.32 mm, kcat = 13 of the alditol by m-AldO, a is into the By and it was shown that as a oxidase, yielding as in the case of xylitol as This that has a high It also that the of the is for and as has a for and alditols. kinetic were by the and The from to The first to the of the flavin by the with a rate of A for the observed second is the the first of xylitol and the formed be in the it is for to the hemiacetal Therefore, it is to to that the second kinetic which is observed after flavin such a of the in the active the second in represents the complex of and the form of the The fact that this second is from the flavin can be by the of the formed aldehyde product, the form of to the FAD cofactor. the cofactor and the bound to of the flavin A significant in at in the second that the a complex S. PubMed Scopus Google Scholar). xylitol have an effect on the observed rate of the of the This that xylitol can bind to the complex in such a that it the proposed ring The most represents from the enzyme and has a rate that is to be of a rate of × 105 m–1 whereas a rate of 1.4 × 105 m–1 was found for are typical rate found for flavoprotein oxidases. other oxidases, it has been shown that the rate to on not the is bound to the enzyme A. Trends Biochem. Sci. 2006; Full Text Full Text PDF PubMed Scopus Google Scholar). The kinetic were used to the kinetic which could be a ternary complex mechanism. the kinetic of the and for most kinetic of the kinetic shown in From this which represents a and its kinetic for and kcat can be using and The for xylitol a of mm, whereas the kcat is are the and kcat obtained with kinetic it was found that the for the redox of AldO catalysis in not to the redox catalysis in we that the observed in the s–1) represents we also a in which is not we the rate of from to The kinetic were not with the kinetic and kinetic for represent the of that is in the represent the of that is in the in a The of the kinetic and the kinetic on a kinetic that kinetic is is in the in This a ternary complex in which the m-AldO, the is and the a rate of we a and redox of mm, 13 and in the from the ternary complex kinetic and the kinetic The above mentioned were by using the observed and it was shown by that these observed are the true the were also with and of to and not in significant of the and redox This that mainly follows a ternary complex This is by the of that were obtained from and kinetic this that a is on kinetic enzymes a ternary complex can also J. Biol. Chem. Full Text PDF PubMed Google Scholar, M.W. van Berkel W.J. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). By these using the pre-steady-state kinetic for m-AldO, a similar of was obtained From and all kinetic it is that the of kinetic on the of At the ternary complex would be whereas at relatively of a be This that is an oxidase that can a number of alditols to a other polyols. It is known that carbohydrate oxidases are widely used in applications, the and and carbohydrate on the in this is a for biocatalytic in the of and We Dr. R. H. H. van den Heuvel and Dr. H. F. M. from the of at the of for on with