The key step in the enzymatic reaction catalyzed by tyrosine phenol-lyase (TPL) is reversible cleavage of the Cβ–Cγ bond of l-tyrosine. Here, we present X-ray structures for two enzymatic states that form just before and after the cleavage of the carbon–carbon bond. As for most other pyridoxal 5′-phosphate-dependent enzymes, the first state, a quinonoid intermediate, is central for the catalysis. We captured this relatively unstable intermediate in the crystalline state by introducing substitutions Y71F or F448H in Citrobacter freundii TPL and briefly soaking crystals of the mutant enzymes with a substrate 3-fluoro-l-tyrosine followed by flash-cooling. The X-ray structures, determined at ∼2.0 Å resolution, reveal two quinonoid geometries: “relaxed” in the open and “tense” in the closed state of the active site. The “tense” state is characterized by changes in enzyme contacts made with the substrate’s phenolic moiety, which result in significantly strained conformation at Cβ and Cγ positions. We also captured, at 2.25 Å resolution, the X-ray structure for the state just after the substrate’s Cβ–Cγ bond cleavage by preparing the ternary complex between TPL, alanine quinonoid and pyridine N-oxide, which mimics the α-aminoacrylate intermediate with bound phenol. In this state, the enzyme–ligand contacts remain almost exactly the same as in the “tense” quinonoid, indicating that the strain induced by the closure of the active site facilitates elimination of phenol. Taken together, structural observations demonstrate that the enzyme serves not only to stabilize the transition state but also to destabilize the ground state.

The cyclometalated platinum complex [PtMe(bhq)(dppy)] (1), in which bhq = benzo{h}quinoline and dppy = 2-(diphenylphosphino)pyridine, was prepared by the reaction of [PtMe(SMe(2))(bhq)] with 1 equiv of dppy at room temperature. Complex 1 contains one free pyridyl unit and was readily characterized by multinuclear NMR spectroscopy and elemental microanalysis. The reaction of complex 1 with 1 equiv of [Ag(CH(3)CN)(4)]BF(4) gave the cyclometalated cluster complex [Pt(2)Me(2)(bhq)(2)(mu-dppy)(2)Ag(2)(mu-acetone)](BF(4))(2) (2) in 70% yield. The crystal structure of complex 2 was determined by X-ray crystallography, indicating a rare example of a butterfly cluster with a Pt(2)Ag(2) core in which the Ag atoms occupy the edge-sharing bond. In solution, the bridging acetone dissociates from the cluster complex 2, but as shown by NMR spectroscopy, the Pt(2)Ag(2) core is retained in solution and a dynamic equilibrium is suggested to be established between the planar and butterfly skeletal geometries.

Amino acid transformations catalyzed by a number of 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. In spite of 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 Å and 1.95 Å resolution, respectively, by cryocrystallography. 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, Lys257 and a water molecule. These two bases are connected by a hydrogen bonding system allowing internal transfer of the Cα proton.

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...

Synthetic peptides derived from ethylene-insensitive protein 2 (EIN2), a central regulator of ethylene signalling, were recently shown to delay fruit ripening by interrupting protein–protein interactions in the ethylene signalling pathway. Here, we show that the inhibitory peptide NOP-1 binds to the GAF domain of ETR1 – the prototype of the plant ethylene receptor family. Site-directed mutagenesis and computational studies reveal the peptide interaction site and a plausible molecular mechanism for the ripening inhibition.