Structures of Apo- and Holo-Tyrosine Phenol-lyase Reveal a Catalytically Critical Closed Conformation and Suggest a Mechanism for Activation by K⁺ Ions^,

Abstract

Tyrosine phenol-lyase, a tetrameric pyridoxal-5′-phosphate dependent enzyme, catalyses the reversible hydrolytic cleavage of L-tyrosine to phenol and ammonium pyruvate. Here we describe the crystal structure of the Citrobacter freundii holoenzyme at 1.9 Å resolution. The structure reveals a network of protein interactions with the cofactor, pyridoxal-5′-phosphate, and details of coordination of the catalytically important K + ion. We also present the structure of the apoenzyme at 1.85 Å resolution. Both structures were determined using crystals grown at pH 8.0, which is close to the pH of the maximal enzymatic activity (8.2). Comparison of the apoenzyme structure with the one previously determined at pH 6.0 reveals significant differences. The data suggest that the decrease of the enzymatic activity at pH 6.0 may be caused by conformational changes in the active site residues Tyr71, Tyr291, Arg381 and in the monovalent cation binding residue Glu69. Moreover, at pH 8.0 we observe two different active-site conformations: open -which was characterised before, and closed -which is observed for the first time in β-eliminating lyases. In the closed conformation a significant part of the small domain undergoes an extraordinary motion of up to 12 Å towards the large domain, closing the active site cleft and bringing the catalytically important Arg381 and Phe448 into the active site. The closed conformation allows rationalisation of the results of previous mutational studies and suggests that the observed active-site closure is critical for the course of the enzymatic reaction and for the enzyme's specificity towards its physiological substrate. Finally, the closed conformation allows us to model keto(imino)quinonoid, the key transition intermediate.Tyrosine phenol-lyase (TPL, EC 4.1.99.2) 1 belongs to the group of pyridoxal-5′-phosphate (PLP) dependent enzymes which catalyse a variety of reactions during metabolic † This work was supported by a grant from the Ministry of Science, Education and Sports of the Republic of Croatia (0119632) to DM and DMČ; by Wellcome Trust fellowship to AAA (ref. 067416), by International Fogarty Foundation grant (1 R03 TW006045-01A2) and by a grant from Russian Foundation for Basic Researchers (# 05-04-48521) to TVD, VVK and NIS. ‡ Coordinates and structure factors of holoTPL and apoTPL were deposited with the RCSB Protein Data Bank with accession numbers 2EZ1 and 2EZ2, respectively. * To whom correspondence should be addressed. Telephone: +385 1 460 6377. Fax: +385 1 460 6341. E-mail: dmilic@chem.pmf.hr.. 1 Abbreviations: apoTPL, apoform of tyrosine phenol-lyase in complex with phosphate at pH 8.0; AspAT, aspartate aminotransferase; holoTPL, holoform of tyrosine phenol-lyase in complex with pyridoxal-5′-phosphate at pH 8.0; r.m.s., root-mean-square; PLP, pyridoxal-5′-phosphate; s.u., standard uncertainty; TPL, tyrosine phenol-lyase [EC 4.1.99.2]; Trpase, tryptophan indole-lyase (tryptophanase) [EC 4.1.99.1]. Europe PMC Funders Group Europe PMC Funders Author ManuscriptsEurope PMC...

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“…The three structures hold significant deviations in the relative orientation of the 'large' and 'small' domains and, as a consequence, show variations in the width of the catalytic site cleft and the geometry of the cofactor binding site. The conformational changes of Tnase molecule are supposed to be of functional importance [26], in a way similar to tyrosine-phenol lyase [29]. The flexibility of Tnase makes a structure based design of specific inhibitors a challenging task.…”

C-S bond cleavage by a polyketide synthase domain

“…S32). TPL and TIL are catalytic as dimers, which are stabilized by the binding of two potassium cations, as such, the active site of each monomer is closed off from solvent by dimerization (31,(37)(38)(39). These dimers form higher-order tetramers in solution and crystal structures.…”

Crystal Structure of Citrobacter freundii Asp214Ala Tyrosine Phenollyase Reveals that Asp214 is Critical for Maintaining a Strain in the Internal Aldimine

“…Significantly, in the D214A TPL the torsion angle C3-C4-C4′-Nζ of the internal aldimine is reduced to 4.2° (subunit A) or 1.3° (subunit B), in comparison with ≈21° angle found in the structure of the wild-type holoenzyme. 13 The most prominent changes in the active site of the D214A TPL are in the conformations of the side chains of Glu103 (χ 2 is changed by ≈120°) and Asn185 (χ 1 is changed by ≈111°). The side chain of Phe123 is stacked with the pyridine ring of PLP at an angle of 4° and 7° in the A and B active sites, respectively, which is a somewhat smaller angle than in the wild-type holoenzyme (12°).…”

“…The data were processed using DENZO and SCALEPACK. 14 Since the unit-cell parameters for D214A TPL crystal were only slightly different than for the native TPL holoenzyme, 13 it was possible to refine the D214A TPL structure in REFMAC 15 using the wildtype TPL holoenzyme structure (PDB ID: 2EZ1), modified by omitting PLP and solvent atoms, as a starting model. During the refinement, the small (residues 19-44, 346-404, 434-456) and the large domain (residues 2-13, 45-345, 405-422) of each subunit were used as separate TLS 16 groups.…”

“…The largest difference of 1.06 Å is for the Cα atoms of Asn390 which is situated in a flexible loop. Differences between the structure of the D214A TPL and the wild-type TPL 13 are more pronounced with the Cα atom RMSD of 0.44 Å. The largest difference is in the conformation of the small domain (residues 19-44, 346-404, and 434-456; RMSD of 0.77 Å for subunit A, and 0.44 Å for subunit B) and a part of the large domain comprising residues 108-211 (RMSD of 0.60 Å and 0.49 Å for subunits A and B, respectively).…”