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 interaction of Citrobacter freundii methionine γ-lyase (MGL) and the mutant form in which Cys115 is replaced by Ala (MGL C115A) with the nonprotein amino acid (2R)-2-amino-3-[(S)-prop-2-enylsulfinyl]propanoic acid (alliin) was investigated. It was found that MGL catalyzes the β-elimination reaction of alliin to form 2-propenethiosulfinate (allicin), pyruvate and ammonia. The β-elimination reaction of alliin is followed by the inactivation and modification of SH groups of the wild-type and mutant enzymes. Three-dimensional structures of inactivated wild-type MGL (iMGL wild type) and a C115A mutant form (iMGL C115A) were determined at 1.85 and 1.45 Å resolution and allowed the identification of the SH groups that were oxidized by allicin. On this basis, the mechanism of the inactivation of MGL by alliin, a new suicide substrate of MGL, is proposed.

Tryptophan indole-lyase (Trpase) from Proteus vulgaris is a pyridoxal 5'-phosphate dependent enzyme that catalyzes the reversible hydrolytic cleavage of L-Trp to yield indole and ammonium pyruvate. Asp-133 and His-458 are strictly conserved in all sequences of Trpase, and they are located in the proposed substrate-binding region of Trpase. These residues were mutated to alanine to probe their role in substrate binding and catalysis. D133A mutant Trpase has no measurable activity with L-Trp as substrate, but still retains activity with S-(o-nitrophenyl)-L-cysteine, S-alkyl-L-cysteines, and beta-chloro-L-alanine. H458A mutant Trpase has 1.6% of wild-type Trpase activity with L-Trp, and high activity with S-(o-nitrophenyl)-L-cysteine, S-alkyl-L-cysteines, and beta-chloro-L-alanine. H458A mutant Trpase does not exhibit the pK(a) of 5.3 seen in the pH dependence of k(cat)/K(m) of L-Trp for wild-type Trpase. Both mutant enzymes are inhibited by L-Ala, L-Met, and L-Phe, with K(i) values similar to those of wild-type Trpase, but oxindolyl-L-alanine and beta-phenyl-DL-serine show much weaker binding to the mutant enzymes, suggesting that Asp-133 and His-458 are involved in the binding of these ligands. D133A and H458A mutant Trpase exhibit absorption and CD spectra in the presence of substrates and inhibitors that are similar to wild-type Trpase, with peaks at about 420 and 500 nm. The rate constants for formation of the 500 nm bands for the mutant enzymes are equal to or greater than those of wild-type Trpase, indicating that Asp-133 and His-458 do not play a role in the formation of quinonoid intermediates. In constrast to wild-type and H458A mutant Trpase, D133A mutant Trpase forms an intermediate from S-ethyl-L-Cys that absorbs at 345 nm, and is likely to be an alpha-aminoacrylate. Crystals of D133A and H458A mutant Trpase bind amino acids with similar affinity as the proteins in solution, except for L-Ala, which binds to D133A mutant Trpase crystals about 20-fold stronger than in solution. These results suggest that Asp-133 and His-458 play an important role in the elimination reaction of L-Trp. Asp-133 likely forms a hydrogen bond directly to the indole NH of the substrate, while His-458 probably is hydrogen bonded to Asp-133.

The problem of resistance to antibiotics requires the development of new classes of broad-spectrum antimicrobial drugs. The concept of pro-drugs allows researchers to look for new approaches to obtain effective drugs with improved pharmacokinetic and pharmacodynamic properties. Thiosulfinates, formed enzymatically from amino acid sulfoxides upon crushing cells of genus Allium plants, are known as antimicrobial compounds. The instability and high reactivity of thiosulfinates complicate their use as individual antimicrobial compounds. We propose a pharmacologically complementary pair: an amino acid sulfoxide pro-drug and vitamin B6 – dependent methionine γ-lyase, which metabolizes it in the patient’s body. The enzyme catalyzes the γ- and β-elimination reactions of sulfoxides, analogues of L-methionine and L-cysteine, which leads to the formation of thiosulfinates. In the present work, we cloned the enzyme gene from Clostridium sporogenes. Ionic and tautomeric forms of the internal aldimine were determined by lognormal deconvolution of the holoenzyme spectrum and the catalytic parameters of the recombinant enzyme in the γ- and β-elimination reactions of amino acids, and some sulfoxides of amino acids were obtained. For the first time, the possibility of usage of the enzyme for effective conversion of sulfoxides was established and the antimicrobial activity of thiosulfinates against Gram-negative and Gram-positive bacteria in situ was shown.