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

Tyrosine phenol-lyase (TPL) is a pyridoxal-5′-phosphate (PLP) dependent enzyme which catalyzes β-elimination of L-tyrosine. In the holoenzyme the protonated pyridinium N1 atom of the PLP cofactor is hydrogen-bonded to the side chain of Asp214. Here we report the X-ray structure of C. freundii D214A TPL determined at 1.9 Å resolution. Comparison with the structure of the wild-type TPL shows that the D214A replacement induced significant conformational reorganization in the active site leading to its partial closure. Significantly, in D214A TPL the strain in the internal aldimine is completely released and the pyridine N1 atom of PLP is deprotonated. These observations explain the considerably reduced activity of the D214A TPL towards its substrates [T. V. Demidkina et al., Biochim. Biophys. Acta, Proteins Proteomics 1764 (2006) 1268–1276]. The reported structure reveals that Asp214 is critical for maintaining the strain in the internal aldimine. We argue that this strain is used by the enzyme to accelerate the transaldimination reaction, the first step in the enzymatic catalysis.(doi: 10.5562/cca1915

Crystals of tryptophan indole-lyase and tyrosine phenol-lyase form stable quinonoid complexes.

The binding of substrates and inhibitors to wild-type Proteus vulgaris tryptophan indole-lyase and to wild type and Y71F Citrobacter freundii tyrosine phenol-lyase was investigated in the crystalline state by polarized absorption microspectrophotometry. Oxindolyl-lalanine binds to tryptophan indole-lyase crystals to accumulate predominantly a stable quinonoid intermediate absorbing at 502 nm with a dissociation constant of 35 microm, approximately 10-fold higher than that in solution. l-Trp or l-Ser react with tryptophan indole-lyase crystals to give, as in solution, a mixture of external aldimine and quinonoid intermediates and gem-diamine and external aldimine intermediates, respectively. Different from previous solution studies (Phillips, R. S., Sundararju, B.,Faleev, N. G. (2000) J. Am. Chem. Soc. 122, 1008-1114), the reaction of benzimidazole and l-Trp or l-Ser with tryptophan indole-lyase crystals does not result in the formation of an alpha-aminoacrylate intermediate, suggesting that the crystal lattice might prevent a ligand-induced conformational change associated with this catalytic step. Wild-type tyrosine phenol-lyase crystals bind l-Met and l-Phe to form mixtures of external aldimine and quinonoid intermediates as in solution. A stable quinonoid intermediate with lambda(max) at 502 nm is accumulated in the reaction of crystals of Y71F tyrosine phenol-lyase, an inactive mutant, with 3-F-l-Tyr with a dissociation constant of 1 mm, approximately 10-fold higher than that in solution. The stability exhibited by the quinonoid intermediates formed both by wild-type tryptophan indole-lyase and by wild type and Y71F tyrosine phenol-lyase crystals demonstrates that they are suitable for structural determination by x-ray crystallography, thus allowing the elucidation of a key species of pyridoxal 5'-phosphate-dependent enzyme catalysis