Insights into the Catalytic Mechanism of Tyrosine Phenol-lyase from X-ray Structures of Quinonoid Intermediates

Abstract

Amino acid transformations catalyzed by a number of pyridoxal 5′-phosphate (PLP)-dependent enzymes involve abstraction of the Cα proton from an external aldimine formed between a substrate and the cofactor leading to the formation of a quinonoid intermediate. Despite the key role played by the quinonoid intermediates in the catalysis by PLP-dependent enzymes, limited accurate information is available about their structures. We trapped the quinonoid intermediates of Citrobacter freundii tyrosine phenol-lyase with l-alanine and l-methionine in the crystalline state and determined their structures at 1.9- and 1.95-Å resolution, respectively, by cryo-crystallography. The data reveal a network of protein-PLP-substrate interactions that stabilize the planar geometry of the quinonoid intermediate. In both structures the protein subunits are found in two conformations, open and closed, uncovering the mechanism by which binding of the substrate and restructuring of the active site during its closure protect the quinonoid intermediate from the solvent and bring catalytically important residues into positions suitable for the abstraction of phenol during the β-elimination of l-tyrosine. In addition, the structural data indicate a mechanism for alanine racemization involving two bases, Lys-257 and a water molecule. These two bases are connected by a hydrogen bonding system allowing internal transfer of the Cα proton. Amino acid transformations catalyzed by a number of pyridoxal 5′-phosphate (PLP)-dependent enzymes involve abstraction of the Cα proton from an external aldimine formed between a substrate and the cofactor leading to the formation of a quinonoid intermediate. Despite the key role played by the quinonoid intermediates in the catalysis by PLP-dependent enzymes, limited accurate information is available about their structures. We trapped the quinonoid intermediates of Citrobacter freundii tyrosine phenol-lyase with l-alanine and l-methionine in the crystalline state and determined their structures at 1.9- and 1.95-Å resolution, respectively, by cryo-crystallography. The data reveal a network of protein-PLP-substrate interactions that stabilize the planar geometry of the quinonoid intermediate. In both structures the protein subunits are found in two conformations, open and closed, uncovering the mechanism by which binding of the substrate and restructuring of the active site during its closure protect the quinonoid intermediate from the solvent and bring catalytically important residues into positions suitable for the abstraction of phenol during the β-elimination of l-tyrosine. In addition, the structural data indicate a mechanism for alanine racemization involving two bases, Lys-257 and a water molecule. These two bases are connected by a hydrogen bonding system allowing internal transfer of the Cα proton. IntroductionTyrosine phenol-lyase (TPL) 2The abbreviations used are: TPL, tyrosine phenol-lyase, EC 4.1.99.2; HPPA, 3-(4′-hydroxyphenyl)propanoate; MME, monomethyl ether; PEG, poly-(ethyleneglycol); PLP, pyridoxal 5′-phosphate; PPT, N-(5′-phosphopyridoxyl)-l-tyrosine; r.m.s., root-mean-square; TPL-Ala, complex of tyrosine phenol-lyase and quinonoid intermediate formed with alanine; TPL-Met, complex of tyrosine phenol-lyase and quinonoid intermediate formed with methionine. is a pyridoxal 5′-phosphate (PLP)-dependent enzyme that catalyzes at physiological conditions the reversible hydrolytic cleavage (the β-elimination reaction) of l-tyrosine to give phenol and ammonium pyruvate (Scheme 1). In vitro, this protein can catalyze a variety of reactions. In addition to the β-elimination of several β-substituted l- and d-amino acids (e.g. l-Cys, l- and d-Ser (1Kumagai H. Yamada H. Matsui H. Ohkishi H. Ogata K. J. Biol. Chem. 1970; 245: 1767-1772Abstract Full Text PDF PubMed Google Scholar)), their derivatives (e.g. S-methyl-l-cysteine, β-chloroalanine (1Kumagai H. Yamada H. Matsui H. Ohkishi H. Ogata K. J. Biol. Chem. 1970; 245: 1767-1772Abstract Full Text PDF PubMed Google Scholar), and S-(o-nitrophenyl)-l-cysteine (2Phillips R.S. Arch. Biochem. Biophys. 1987; 256: 302-310Crossref PubMed Scopus (47) Google Scholar)), and compounds structurally analogous to l-Tyr (e.g. 1-amino-2-(4-hydroxyphenyl)ethyl phosphinic acid (3Faleev N.G. Zhukov Y.N. Khurs E.N. Gogoleva O.I. Barbolina M.V. Bazhulina N.P. Belikov V.M. Demidkina T.V. Khomutov R.M. Eur. J. Biochem. 2000; 267: 6897-6902Crossref PubMed Scopus (16) Google Scholar)), it also catalyzes the reversal of the β-elimination reaction (4Enei H. Nakazawa H. Matsui H. Okumura S. Yamada H. FEBS Lett. 1972; 21: 39-41Crossref PubMed Scopus (32) Google Scholar, 5Yamada H. Kumagai H. Adv. Appl. Microbiol. 1975; 19: 249-288Crossref PubMed Scopus (84) Google Scholar, 6Yamada H. Kumagai H. Kashima N. Torii H. Enei H. Okumura S. Biochem. Biophys. Res. Commun. 1972; 46: 370-374Crossref PubMed Scopus (79) Google Scholar), the β-substitution by phenol derivatives (7Kumagai H. Matsui H. Ohgishi H. Ogata K. Yamada H. Ueno T. Fukami H. Biochem. Biophys. Res. Commun. 1969; 34: 266-270Crossref PubMed Scopus (62) Google Scholar, 8Ueno T. Fukami H. Ohkishi H. Kumagai H. Yamada H. Biochim. Biophys. 1970; PubMed Scopus Google Scholar, R.S. K. Scopus Google Scholar), the racemization of l- and H. Kashima N. Yamada H. Biochem. Biophys. Res. Commun. 1970; PubMed Scopus Google Scholar), and the to an of l-Tyr and T.V. Eur. J. Biochem. 1987; PubMed Scopus Google of is both in and in the structures Demidkina T.V. J. R.S. PubMed Scopus Google Scholar, R.S. Demidkina T.V. Barbolina M.V. 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PubMed Scopus Google Scholar), in the quinonoid intermediates of both and of the by is found in the the in the open between the and that the closure of the active site during the quinonoid complex in formation of with in the of the in both are to in the of the Demidkina T.V. PubMed Scopus Google with the Cα of for the and for the open of and respectively, in the of the The between structures of the and quinonoid is in the of the the active site in the open residues with the Cα of for the and for the The that the and the of are in the with of found in the open of its and the in the the open of for the of two subunits that open and The is by the of the by about a it with the The subunits Cα The quinonoid are The and of the are in and respectively, and the are in The and of the open are in and respectively, the are in of the that a quinonoid complex in with T.V. Eur. J. Biochem. 1987; PubMed Scopus Google Scholar, T. H. K. Kumagai H. Yamada H. J. Biochem. PubMed Scopus Google Scholar, H. 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PubMed Scopus Google These by interactions in the internal aldimine with to the in the internal aldimine Lys-257 is to the PLP, in intermediates Lys-257 hydrogen with the and and the of the pyridoxal in the internal aldimine of and which in from the active site and the and the and We that the active of in the crystalline state in the open the of the in the the into the open active site is a by solvent and it is by protein binding of the substrate can The quinonoid intermediate in the active site is by interactions of the with and of residues to the and the active site to with the quinonoid intermediate. is that binding of the substrate with a of the and We that are by in the of binding in this of the two in the and internal with N.G. Demidkina T.V. Belikov V.M. Eur. J. Biochem. PubMed Scopus Google The of both internal aldimine and quinonoid intermediate in the of indicate between the two active of the is active site is of the residues from both subunits of the is that active site is and by the quinonoid the active site in a that and of the Cα in that racemization of catalyzed by TPL, is by the internal of the Cα hydrogen Kumagai H. S. Yamada H. 1987; PubMed Scopus Google of the Cα hydrogen to the phenol also in the β-elimination reaction with the l-Tyr N.G. Belikov V.M. Scopus Google The structural data that in both the internal the system of hydrogen the active site residues and water The of the internal during alanine racemization and β-elimination reaction are with the number of hydrogen the of Lys-257 with the water and and with We that in the residues and water are from the in the open of are solvent and to the hydrogen with the The of to the are for racemization β-elimination H. R.S. PubMed Scopus Google Scholar, H. R.S. Eur. J. Biochem. PubMed Scopus (47) Google the in the open the of the internal the N.G. Belikov V.M. Scopus Google Scholar, Kumagai H. S. Yamada H. 1987; PubMed Scopus Google also catalyzes the of the Cα hydrogen of several which with the of the acid data that the Cα hydrogen for a quinonoid intermediate N.G. Demidkina T.V. R.S. Eur. J. Biochem. PubMed Scopus Google In in the of an mechanism of involving the quinonoid is the quinonoid intermediate with is a complex by the of the reaction of the Cα proton abstraction from the external aldimine and of the quinonoid with the of the Cα hydrogen N.G. Demidkina T.V. R.S. Eur. J. Biochem. PubMed Scopus Google to this the Cα hydrogen for by a mechanism (Scheme involving a which the of Lys-257 and a water to it N.G. Demidkina T.V. R.S. Eur. J. Biochem. PubMed Scopus Google of the of the active site of that the active site a water which this this water is to the of Lys-257 in the of the quinonoid in the external mechanism of Cα hydrogen in the external aldimine of IntroductionTyrosine phenol-lyase (TPL) 2The abbreviations used are: TPL, tyrosine phenol-lyase, EC 4.1.99.2; HPPA, 3-(4′-hydroxyphenyl)propanoate; MME, monomethyl ether; PEG, poly-(ethyleneglycol); PLP, pyridoxal 5′-phosphate; PPT, N-(5′-phosphopyridoxyl)-l-tyrosine; r.m.s., root-mean-square; TPL-Ala, complex of tyrosine phenol-lyase and quinonoid intermediate formed with alanine; TPL-Met, complex of tyrosine phenol-lyase and quinonoid intermediate formed with methionine. is a pyridoxal 5′-phosphate (PLP)-dependent enzyme that catalyzes at physiological conditions the reversible hydrolytic cleavage (the β-elimination reaction) of l-tyrosine to give phenol and ammonium pyruvate (Scheme 1). In vitro, this protein can catalyze a variety of reactions. In addition to the β-elimination of several β-substituted l- and d-amino acids (e.g. l-Cys, l- and d-Ser (1Kumagai H. Yamada H. Matsui H. Ohkishi H. Ogata K. J. Biol. Chem. 1970; 245: 1767-1772Abstract Full Text PDF PubMed Google Scholar)), their derivatives (e.g. S-methyl-l-cysteine, β-chloroalanine (1Kumagai H. Yamada H. Matsui H. Ohkishi H. Ogata K. J. Biol. Chem. 1970; 245: 1767-1772Abstract Full Text PDF PubMed Google Scholar), and S-(o-nitrophenyl)-l-cysteine (2Phillips R.S. Arch. Biochem. Biophys. 1987; 256: 302-310Crossref PubMed Scopus (47) Google Scholar)), and compounds structurally analogous to l-Tyr (e.g. 1-amino-2-(4-hydroxyphenyl)ethyl phosphinic acid (3Faleev N.G. Zhukov Y.N. Khurs E.N. Gogoleva O.I. Barbolina M.V. Bazhulina N.P. Belikov V.M. Demidkina T.V. Khomutov R.M. Eur. J. Biochem. 2000; 267: 6897-6902Crossref PubMed Scopus (16) Google Scholar)), it also catalyzes the reversal of the β-elimination reaction (4Enei H. Nakazawa H. Matsui H. Okumura S. Yamada H. FEBS Lett. 1972; 21: 39-41Crossref PubMed Scopus (32) Google Scholar, 5Yamada H. Kumagai H. Adv. Appl. Microbiol. 1975; 19: 249-288Crossref PubMed Scopus (84) Google Scholar, 6Yamada H. Kumagai H. Kashima N. Torii H. Enei H. Okumura S. Biochem. Biophys. Res. Commun. 1972; 46: 370-374Crossref PubMed Scopus (79) Google Scholar), the β-substitution by phenol derivatives (7Kumagai H. Matsui H. Ohgishi H. Ogata K. Yamada H. Ueno T. Fukami H. Biochem. Biophys. Res. Commun. 1969; 34: 266-270Crossref PubMed Scopus (62) Google Scholar, 8Ueno T. Fukami H. Ohkishi H. Kumagai H. Yamada H. Biochim. Biophys. 1970; PubMed Scopus Google Scholar, R.S. K. Scopus Google Scholar), the racemization of l- and H. Kashima N. Yamada H. Biochem. Biophys. Res. Commun. 1970; PubMed Scopus Google Scholar), and the to an of l-Tyr and T.V. Eur. J. Biochem. 1987; PubMed Scopus Google of is both in and in the structures Demidkina T.V. J. R.S. PubMed Scopus Google Scholar, R.S. Demidkina T.V. Barbolina M.V. PubMed Scopus Google Scholar, E.N. N.G. Demidkina T.V. Scholar, Demidkina T.V. PubMed Scopus Google is of two which are by and a formed in the of the 1). can into two connected a The the the and of the the is of the of the The active site is from residues to the and of and the of the of the in the of the Demidkina T.V. PubMed Scopus Google Scholar), the is by of the and in a closure of the active mechanism of the β-elimination reaction R.S. Demidkina T.V. N.G. Biochim. Biophys. PubMed Scopus Google several key (Scheme formation of the external aldimine in the reaction of the internal aldimine with the Cα proton abstraction by the Lys-257 in formation of the quinonoid by by cleavage of the to phenol and the intermediate. and structural data the role of residues in the the of their interactions with the substrate and interactions their active during the quinonoid intermediate formation is the formation of a quinonoid intermediate with an acid is an in the reaction mechanism of PLP-dependent enzymes, this intermediate is its by structural data the quinonoid intermediate available for 2000; PubMed Scopus Google and T. PubMed Scopus Google reaction mechanism of β-elimination of l-tyrosine catalyzed by structures of two quinonoid intermediates of Citrobacter freundii formed with acids and and determined at 1.9- and 1.95-Å resolution, The the mechanism of the racemization of The interactions with the substrate in the In addition, the data reveal the network of protein-PLP-substrate interactions that stabilize the planar geometry of the quinonoid and the role of the active site closure during the physiological β-elimination of