Studies on the Reaction Mechanism of Rhodotorula gracilis d-Amino-acid Oxidase

We have studied d-amino-acid oxidase from Rhodo- torula gracilis by site-directed mutagenesis for the purpose of determining the presence or absence of residues having a possible role in acid/base catalysis. Tyr-223, one of the very few conserved residues among d-amino-acid oxidases, has been mutated to phenylalanine and to serine. Both mutants are active catalysts in turnover with d-alanine, and they are reduced by d-alanine slightly faster than wild-type enzyme. The Tyr-223 → Phe mutant is virtually identical to the wild-type enzyme, whereas the Tyr-223 → Ser mutant exhibits 60-fold slower substrate binding and at least 800-fold slower rate of product release relative to wild-type. These data eliminate Tyr-223 as an active-site acid/base catalyst. These results underline the importance of Tyr-223 for substrate binding and exemplify the importance of steric interactions in RgDAAO catalysis. We have studied d-amino-acid oxidase from Rhodo- torula gracilis by site-directed mutagenesis for the purpose of determining the presence or absence of residues having a possible role in acid/base catalysis. Tyr-223, one of the very few conserved residues among d-amino-acid oxidases, has been mutated to phenylalanine and to serine. Both mutants are active catalysts in turnover with d-alanine, and they are reduced by d-alanine slightly faster than wild-type enzyme. The Tyr-223 → Phe mutant is virtually identical to the wild-type enzyme, whereas the Tyr-223 → Ser mutant exhibits 60-fold slower substrate binding and at least 800-fold slower rate of product release relative to wild-type. These data eliminate Tyr-223 as an active-site acid/base catalyst. These results underline the importance of Tyr-223 for substrate binding and exemplify the importance of steric interactions in RgDAAO catalysis. d-amino-acid oxidase (EC 1.4.3.3) R. gracilis d-amino-acid oxidase xanthine oxidase imino acid 3,3,3-trifluoro-dl-alanine The enzyme d-amino-acid oxidase (EC 1.4.3.3, DAAO)1 from the yeastRhodotorula gracilis has been exploited as a potent industrial biocatalyst (for a recent review see Ref. 1Pilone M.S. Pollegioni L. Recent Res. Dev. Biotechnol. Bioeng. 1998; 1: 285-298Google Scholar) and as a model system for mechanistic studies (2Pollegioni L. Blodig W. Ghisla S. J. Biol. Chem. 1997; 272: 4924-4934Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). In contrast to the well known pig kidney DAAO (3Curti B. Ronchi S. Pilone Simonetta M. Muller F. Chemistry and Biochemistry of Flavoenzymes. CRC Press, Boca Raton, FL1992: 69-94Google Scholar), the enzyme from R. gracilis is characterized by a comparatively much higher turnover number, good stability under a wide range of reaction conditions, and an active site sufficiently large to accommodate different substrates, even of considerable size (4Pollegioni L. Falbo A. Pilone M.S. Biochim. Biophys. Acta. 1992; 1120: 11-16Crossref PubMed Scopus (78) Google Scholar, 5Pollegioni L. Langkau B. Tischer W. Ghisla S. Pilone M.S. J. Biol. Chem. 1993; 268: 13850-13857Abstract Full Text PDF PubMed Google Scholar). Due to these properties, we have utilized the yeast enzyme for the oxidation of cephalosporin C in the two-step formation of 7-aminocephalosporanic acid (6Pilone M.S. Butò S. Pollegioni L. Biotechnol. Lett. 1995; 17: 199-204Crossref Scopus (42) Google Scholar) and for a human gene therapy paradigm that involves oxidant-mediated tumor cell death (7Stegman L.D. Zheng H. Neal E.R. Ben-Yoseph O. Pollegioni L. Pilone M.S. Ross B.D. Hum. Gene Ther. 1998; 9: 185-193Crossref PubMed Scopus (68) Google Scholar). DAAO catalyzes the oxidation of d-amino acids to α-keto acids and ammonia with concomitant reduction of molecular oxygen to hydrogen peroxide. In the reductive half-reaction, the amino acid substrate reduces the enzyme-bound FAD cofactor producing reduced flavin and the imino acid. In the oxidative half-reaction, the reduced FAD·imino acid complex reacts with molecular oxygen to form oxidized FAD·imino acid. The catalytic cycle is completed when the imino acid dissociates from the re-oxidized enzyme. Solvolysis of the initial imino acid product rapidly yields the corresponding α-keto acid and ammonia. The enzyme has a broad substrate specificity, utilizing all of the naturally occurring amino acids efficiently, with the only exceptions being aspartate and glutamate (3Curti B. Ronchi S. Pilone Simonetta M. Muller F. Chemistry and Biochemistry of Flavoenzymes. CRC Press, Boca Raton, FL1992: 69-94Google Scholar, 4Pollegioni L. Falbo A. Pilone M.S. Biochim. Biophys. Acta. 1992; 1120: 11-16Crossref PubMed Scopus (78) Google Scholar). RgDAAO belongs to the large class of flavoprotein oxidases that catalyze the oxidation of amino or α-hydroxy acids. A fundamental question remains within this class of enzymes regarding the mechanism by which a proton and two electrons are transferred from the substrate to the flavin N-5 position during the reductive half-reaction. Substrate oxidation could be accomplished by hydride transfer from the α-carbon to the flavin N-5. Whereas hydride transfer certainly requires stringent orientation of the substrate and flavin cofactor, there is no theoretical argument against it (8Miura R. Miyake Y. Bioorg. Chem. 1988; 16: 97-110Crossref Scopus (30) Google Scholar). A carbanion mechanism has been proposed in which an enzyme base removes the α-proton (or possibly the amino proton) and so forms a carbanion intermediate (9Walsh C.T. Schonbrunn A. Abeles R. J. Biol. Chem. 1971; 248: 6855-6866Google Scholar) (see also Ref. 10Mattevi A. Vanoni M.A. Curti B. Curr. Opin. Struct. Biol. 1997; 7: 804-810Crossref PubMed Scopus (31) Google Scholar for a recent review). The resulting unstable carbanion would rapidly attack the N-5 locus of the flavin. Subsequent rapid rearrangement would result in reduced flavin and iminopyruvate. The most convincing evidence in favor of a carbanion mechanism is the observation of β-elimination when β-chloroalanine was used as the substrate of pig kidney DAAO (9Walsh C.T. Schonbrunn A. Abeles R. J. Biol. Chem. 1971; 248: 6855-6866Google Scholar). Deprotonation of the α-hydrogen of amino acids is highly unfavorable in aqueous solution. Thus, in order for an enzyme to deprotonate the α-proton, it must have some highly specific means of removing the proton and stabilizing the resulting carbanion. Hence, the presence of an enzyme base for α-proton abstraction is essential for the carbanion mechanism. In order to determine the mechanism of substrate dehydrogenation, we are currently studying the active site of RgDAAO using different experimental approaches. Site-directed mutagenesis of pig kidney DAAO (11Pollegioni L. Fukui K. Massey J. Biol. Chem. Full Text PDF PubMed Google Scholar) and of A. Vanoni M.A. F. M. A. A. M. Curti B. S. A. PubMed Scopus Google Scholar, H. K. Y. K. R. J. PubMed Scopus Google Scholar) have that there is no essential base in the of the flavin N-5. and Tyr-223 are the only residues conserved in the amino acid of from R. and L. Pollegioni L. F. Ronchi S. Pilone M.S. Biotechnol. Lett. 1995; 17: Scopus Google Scholar). of is in and that of has this to be essential for and the of Tyr-223 has been In this we the and of and mutants of The of the role of the active-site residues of RgDAAO and the of the are for the of the molecular of the higher catalytic substrate specificity, and flavin binding that RgDAAO from the enzyme. Site-directed mutagenesis has a with to the of RgDAAO as a biocatalyst (for a review see Ref. 1Pilone M.S. Pollegioni L. Recent Res. Dev. Biotechnol. Bioeng. 1998; 1: 285-298Google Scholar). of the substrate and of this enzyme could have in and in gene therapy an enzyme form with higher for the d-amino acid and enzymes and from and from Site-directed mutagenesis using the was from xanthine and all from was a of Ghisla of in and and at The mutant was by site-directed mutagenesis using the the for the wild-type RgDAAO was in The was as by in the and used as a for the in The mutant was using a to a site-directed the for phenylalanine is and In mutagenesis reaction used to and J. A Press, Scholar). mutagenesis was for the of a site by and by The mutant was by The the wild-type RgDAAO L. S. Pilone M.S. J. Biotechnol. 1997; PubMed Scopus Google Scholar) was used as the in the The was using the the for is which the and the The was to the of the The product was the and to the presence of the A and the mutant was with the corresponding of L. S. Pilone M.S. J. Biotechnol. 1997; PubMed Scopus Google Scholar). The and the site of the S. and the mutated was The and and used to and DAAO was with an oxygen at and with as substrate (4Pollegioni L. Falbo A. Pilone M.S. Biochim. Biophys. Acta. 1992; 1120: 11-16Crossref PubMed Scopus (78) Google Scholar). DAAO is as the of enzyme that of d-alanine at was as by PubMed Scopus Google Scholar). The and RgDAAO mutants in using the system in Pilone M.S. Pollegioni L. 1998; PubMed Scopus Google Scholar). A of and was with of an of or system at for and with at for and by and at of and mutants was by the identical as for wild-type RgDAAO Pilone M.S. Pollegioni L. 1998; PubMed Scopus Google Scholar). The for the mutant DAAO enzymes by the in release of the flavin. The enzymes by for in the an of at for FAD was enzyme, and was and with a with the in a of the reaction was the at was from a of the and the for to at for is oxidized is enzyme flavin and reduced of and mutants by the of K. Biochim. Biophys. Acta. PubMed Google Scholar) using the oxidase reduction at Curti B. Ronchi S. and Scholar). enzyme, and of the was of and the reaction was by of The reaction was as by K. Biochim. Biophys. Acta. PubMed Google Scholar). The of oxidized and reduced was at a the enzyme has no and the of oxidized and reduced enzyme was at an for the or by of the in the for by of of to of enzyme, at The in was as a of for used for the are for and for for and for for acids was by the in the of the amino acid to the enzyme solution. The at in a with a cell and that is with a as (2Pollegioni L. Blodig W. Ghisla S. J. Biol. Chem. 1997; 272: 4924-4934Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). was by the of turnover by enzyme, with of d-alanine at at as Massey J. Biol. Chem. Full Text PDF Google Scholar) using the The complex mechanism in the of be using the of K. J. PubMed Scopus Google Scholar) as in and d-amino reductive the was by with to the was well with to the The reaction was by with a from by at and and to a of using A in the of at the of Subsequent of with A by least means with the wild-type formation of the complex for of the oxidative could be by of in the presence of and ammonia. the system was A with an was with a DAAO in A and and (for or and (for The was and enzyme reduction was by of The for The enzyme was the under was with of A that been at with or and as for the reductive half-reaction. The and used to and and the with by and DAAO to the wild-type RgDAAO Pilone M.S. Pollegioni L. 1998; PubMed Scopus Google Scholar), the of DAAO and DAAO specific was when the the and for the and the with at and at for of enzyme was from of of or with for wild-type DAAO Pilone M.S. Pollegioni L. 1998; PubMed Scopus Google Scholar). Both and well enzymes that FAD and that when at for of and enzymes are in in to the wild-type Both in oxidized an at of and a of A of an of d-alanine in enzyme reduction of mutant with a that of the The of form by mutant was by 17: PubMed Scopus Google Scholar) the of the flavin a this formation The by is a of in to the oxidized and reduced forms a of with the the of and enzymes and of the flavin with for the of the is for d-amino-acid oxidases and for the of flavoprotein oxidases S. A. formation and and of and d-amino-acid at and using as and oxidase as the of at and using as and oxidase as the of at and using as and oxidase as the of at and using as and oxidase as the of at and using as and oxidase as the of at and using as and oxidase as the of The at and using as and oxidase as the of Curti B. Ronchi S. and The at and using as and oxidase as the of Curti B. Ronchi S. and The at and using as and oxidase as the of Curti B. Ronchi S. and Scholar). in a In order to in the of the flavin by at Tyr-223, the of the enzymes by the of K. Biochim. Biophys. Acta. PubMed Google Scholar) using the reduction system Curti B. Ronchi S. and Scholar). A is in in which the of of was in to A of the as a of flavin the of when the of the is known K. Biochim. Biophys. Acta. PubMed Google Scholar). The in of has a of in with the theoretical of for a to the of and the rate at which the reaction no the In the of the of are identical to of the wild-type enzyme, whereas the mutant has than the in the have the FAD of enzyme reduction for wild-type slightly of the oxidative is an the is so much at than the and no the of the enzyme-bound for in order to determine the of Tyr-223 to substrate In binding was by the of the of the FAD formation of the complex and with all the and for and the identical to for the binding to the wild-type of in binding was for mutants with the and is a substrate for the enzyme, as by of the to the enzyme of and and to and d-amino acid in a The higher for the results with and is to the in of the mutants (see of and to the mutants was reduced to higher for with the these have to amino acid Both mutants the of the wild-type they are reduced by and under The of the and mutants to catalyze was by of enzyme and in the and the in the range at the at an initial rapid of the oxidized flavin was by a and by a to the reduced that the and mutants are catalysts and as well as for the wild-type the enzyme is in the oxidized form during that the of of reduced DAAO with oxygen is faster than the reductive half-reaction. the is the of turnover well as with a of with DAAO mutants only for with of with a complex mechanism. wild-type DAAO with d-alanine as a in the was L. Langkau B. Tischer W. Ghisla S. Pilone M.S. J. Biol. Chem. 1993; 268: 13850-13857Abstract Full Text PDF PubMed Google Scholar), with a of a complex mechanism some specific rate the of the reduction are sufficiently is only reduced by and the and are is reduced for d-alanine is and for oxygen is The in of with d-alanine was by studying the reaction of using as substrate of the for and d-amino-acid oxidases with d-alanine as substrates, at and enzyme data are from Pollegioni in and at enzyme data are from Pollegioni in and at enzyme data are from Pollegioni in and at enzyme data are from Pollegioni in and at enzyme data are from Pollegioni in and at enzyme data are from Pollegioni in and at enzyme data are from Pollegioni L. Langkau B. Tischer W. Ghisla S. Pilone M.S. J. Biol. Chem. 1993; 268: 13850-13857Abstract Full Text PDF PubMed Google Scholar), in and at in a The reductive of and of with d-alanine was to the of rate of enzyme was with of d-alanine, that a was with to the substrate In the absence of the enzyme only of a catalytic the substrate reduces the enzyme, and the product dissociates in The are in the reductive of with d-alanine, the oxidized enzyme is rapidly to the as is in the of in These a very of to the of the is no with formation of the oxidized of the involves a large at and a at with formation of complex J. Biol. Chem. Full Text PDF PubMed Google Scholar). of the results in a with the presence of reduced enzyme L. Langkau B. Tischer W. Ghisla S. Pilone M.S. J. Biol. Chem. 1993; 268: 13850-13857Abstract Full Text PDF PubMed Google Scholar). at and the of the using to a two that the to enzyme reduction and the to product The mutant has only a slightly of the reduction as a The are so as to be at the of the A of these data a the has been by and S. Massey J. Biol. Chem. Full Text PDF PubMed Google Scholar) to a reaction of a that a complex using a the reduction for is and is the data are with a which the these data that the reduction is the is to S. Massey J. Biol. Chem. Full Text PDF PubMed Google Scholar). wild-type it has been that d-alanine is a substrate and the in for possibly the in than a in from the has a substrate of a the wild-type enzyme. of is and would a in the in it is that is for The and could be by the in this or in a in the rate for from the enzyme using the of and a for of be (see is very much slower than within the catalytic and the mechanism must be specific rate from of and and as substrates, at and enzyme data are from Ref. in and at enzyme data are from Ref. 5Pollegioni L. Langkau B. Tischer W. Ghisla S. Pilone M.S. J. Biol. Chem. 1993; 268: 13850-13857Abstract Full Text PDF PubMed Google Scholar, in and at in a the reductive of a as a of d-alanine is to the that for d-alanine is for this is a of a reaction with d-alanine when the binding of the substrate is slower than flavin reduction using the for a reaction of a a was by a of these data and involves a large of of at with a for wild-type enzyme of substrate L. Langkau B. Tischer W. Ghisla S. Pilone M.S. J. Biol. Chem. 1993; 268: 13850-13857Abstract Full Text PDF PubMed Google Scholar), that enzyme reduction is in the be The of is for than for wild-type is only the this involves a in The in has a d-alanine of than and for a mechanism in and by using and turnover is at most a complex mechanism the of as for is at least than there must be a in in the oxidative of be with the of the the binding of the reductive of mutant was also The a The rate of reduction is only slightly by the whereas the an to a large in the of the reaction at is in of the and reduction of is much faster than the oxidative of mutants of wild-type the complex reacts with oxygen to form the complex is oxidized of A catalytic cycle is completed with the of L. Langkau B. Tischer W. Ghisla S. Pilone M.S. J. Biol. Chem. 1993; 268: 13850-13857Abstract Full Text PDF PubMed Google Scholar). of the complex for of the oxidative is to the of to ammonia and In order to to the oxidation of the the position of the reaction was by in the presence of and this well for wild-type it is possible that the mutant enzymes have for the product and that the reaction reduced enzyme. and the substrate reduction of oxidized enzyme in the presence of and ammonia to an enzyme form having of this and reduced in the presence of and ammonia using the reduction as for the of (see The enzyme was in the with with different of at and during the reaction that reduced enzyme is to oxidized intermediate The oxygen are and at the that the are and with rate for and for to the rate of the wild-type enzyme L. Langkau B. Tischer W. Ghisla S. Pilone M.S. J. Biol. Chem. 1993; 268: 13850-13857Abstract Full Text PDF PubMed Google from the oxidative and for product binding for and in the presence of and at the of the oxidized enzyme form with in and The in is for the enzyme data are from Ref. in and at in the presence of and at the of the oxidized enzyme form with in and The in is for the enzyme data are from Ref. 5Pollegioni L. Langkau B. Tischer W. Ghisla S. Pilone M.S. J. Biol. Chem. 1993; 268: 13850-13857Abstract Full Text PDF PubMed Google Scholar, in and at in a These that Tyr-223 mutants as rapidly with oxygen as the wild-type enzyme. there is no with and it is The binding of to the oxidized form of DAAO was by using of in the presence of ammonia the In all the of to wild-type and mutant results in an in at and with a different in the and are the of in the is These of the in enzymes is The system to the of wild-type RgDAAO was also used to mutant The enzymes be to with a good to wild-type RgDAAO Pilone M.S. Pollegioni L. 1998; PubMed Scopus Google Scholar). of the and reduced forms of the and mutants are the as wild-type DAAO The flavin of are from the wild-type enzyme, whereas the of is than wild-type A in to a than the of a hydrogen The that the remains mutants the as to be no to in for these These result in no or of The we have in the of and are to be to only specific and The rate at which is reduced by substrate is faster than result Tyr-223 as a possible a role in acid/base in the abstraction of the α-carbon be to the in for the and a was also for the mutant of a at the the and the F. PubMed Scopus Google Scholar). data for these mutants to be at least as as the wild-type and that the of Tyr-223 is for substrate The for of to be a in the rate of product from oxidized enzyme using an of be very much slower than within the catalytic and the mechanism must be complex of and is by and (see is in for the wild-type enzyme, the reduction is at least the reduction or A. Vanoni M.A. Curti B. Curr. Opin. Struct. Biol. 1997; 7: 804-810Crossref PubMed Scopus (31) Google Scholar) wild-type d-alanine binding has been to be L. Langkau B. Tischer W. Ghisla S. Pilone M.S. J. Biol. Chem. 1993; 268: 13850-13857Abstract Full Text PDF PubMed Google Scholar). binding to the of substrate is so for substrate as the rate of substrate a the of a from the data in and reduces The of in this for is only the of wild-type enzyme, L. Langkau B. Tischer W. Ghisla S. Pilone M.S. J. Biol. Chem. 1993; 268: 13850-13857Abstract Full Text PDF PubMed Google Scholar). of the we have for the rate are within a of of the wild-type. there are some in these are by the reduction rate of the catalytic of is to the wild-type enzyme, L. Langkau B. Tischer W. Ghisla S. Pilone M.S. J. Biol. Chem. 1993; 268: 13850-13857Abstract Full Text PDF PubMed Google Scholar). The most in the mutant is the reduction in the rate of product from oxidized In the results in only very in of the enzyme. The of Tyr-223 is essential for catalytic it is for substrate of at amino acid The for is reduced and the reaction with d-alanine is with to the substrate are using as a has been at rate and the for a complex mechanism The catalytic mechanism be to be a complex for the mutant by of the of product from reduced enzyme, with the of flavin reduction and the of for the mechanism (see is also by the of a in the (see to from being at all to being the in the in must be at least the in is reduced at least A. Vanoni M.A. Curti B. Curr. Opin. Struct. Biol. 1997; 7: 804-810Crossref PubMed Scopus (31) Google Scholar) the rate of product release from the enzyme, it the (see and in binding are also in from the reductive of which is with and than for wild-type for the The is the of this is to the rate of substrate rate be by the which to when the substrate is and it is slower than the corresponding for the wild-type enzyme the for d-alanine is in the for the mutant is only that of is by the known (see and The of is the for d-alanine, a the in the The rate of product release from the reduced enzyme, is also in as for the mutant and for wild-type enzyme. The product dissociates slower from than reduced enzyme, this is large to a from a complex to a mechanism. the no reduction in the in substrate and product are the is for binding for the in catalysis. The in for the binding of the in to is possibly to the of a hydrogen the of Tyr-223 and the of the the an reaction as that studied and reduces wild-type enzyme, is to the of within experimental L. Langkau B. Tischer W. Ghisla S. Pilone M.S. J. Biol. Chem. 1993; 268: 13850-13857Abstract Full Text PDF PubMed Google Scholar). for is than and for is than In the of a the rate The and could be as to a to a mechanism. and from the reductive these mutants complex We of in the oxidative to be than from turnover data at oxygen no in oxygen for the mutant The from experimental for a in mechanism. In a Tyr-223 of RgDAAO was to of the enzyme L. Pollegioni L. F. Ronchi S. Pilone M.S. Biotechnol. Lett. 1995; 17: Scopus Google Scholar). A that Tyr-223 is to of pig kidney the the that the size of the F. Vanoni M.A. A. M. A. Curti B. A. 1997; PubMed Scopus Google Scholar). in RgDAAO is to to of the enzyme. The of Tyr-223 in the active site of RgDAAO is different from that of the residues and of pig kidney In mutants and of the DAAO a large in and than in the for d-alanine (11Pollegioni L. Fukui K. Massey J. Biol. Chem. Full Text PDF PubMed Google Scholar). These the of the reduced enzyme with the product as by the observation that the formation of the at during the reduction of the complex form and residues are also at the active site of F. S. A. PubMed Scopus Google Scholar), oxidase Y. J. Biol. Chem. Full Text PDF PubMed Google Scholar), and Massey J. Biol. Chem. Full Text PDF PubMed Google Scholar). These enzymes have been proposed to by a carbanion and in enzyme they different in substrate interactions and enzyme by site-directed mutagenesis only by the with a phenylalanine We also the in the active site using a serine. from with and RgDAAO mutants that Tyr-223 is for and interactions and that the is also for steric The in the two mutants that the at position by the substrate in the orientation for by and than by or Tyr-223 is essential for is the base by a carbanion it be possible to the substrate of this biocatalyst by site-directed mutagenesis or by a using These results for of R. gracilis DAAO as an industrial for the of and for the of as large as cephalosporin We are to Ghisla for the to the We also Massey of and Ghisla for the review and the