Structure ofEscherichia colitryptophanase

Abstract

Pyridoxal 5'-phosphate (PLP) dependent tryptophanase has been isolated from Escherichia coli and its crystal structure has been determined. The structure shares the same fold with and has similar quaternary structure to Proteus vulgaris tryptophanase and tyrosine-phenol lyase, but is found in a closed conformation when compared with these two enzymes. The tryptophanase structure, solved in its apo form, does not have covalent PLP bound in the active site, but two sulfate ions. The sulfate ions occupy the phosphoryl-binding site of PLP and the binding site of the alpha-carboxyl of the natural substrate tryptophan. One of the sulfate ions makes extensive interactions with both the transferase and PLP-binding domains of the protein and appears to be responsible for holding the enzyme in its closed conformation. Based on the sulfate density and the structure of the P. vulgaris enzyme, PLP and the substrate tryptophan were modeled into the active site. The resulting model is consistent with the roles of Arg419 in orienting the substrate to PLP and acidifying the alpha-proton of the substrate for beta-elimination, Lys269 in the formation and decomposition of the PLP quinonoid intermediate, Arg230 in orienting the substrate-PLP intermediates in the optimal conformation for catalysis, and His463 and Tyr74 in determining substrate specificity and suggests that the closed conformation observed in the structure could be induced by substrate binding and that significant conformational changes occur during catalysis. A catalytic mechanism for tryptophanase is proposed. Since E. coli tryptophanase has resisted forming diffraction-quality crystals for many years, the molecular surface of tryptophanase has been analyzed in various crystal forms and it was rationalized that strong crystal contacts occur on the flat surface of the protein and that the size of crystal contact surface seems to correlate with the diffraction quality of the crystal.

Binding of Silver Nanoparticles to Bacterial Proteins Depends on Surface Modifications and Inhibits Enzymatic Activity

“…Reproducibly high-binding TNase fragments (defined as peaks at least 10% the height of the base peak) from at least two experiments are shown in Table 1. High-binding TNase fragments specific only to one type of AgNP reinforce the prior observation that In addition, one high-binding protein fragment (NIF-GYQYTIPTHQGR) contains an amino acid (Arg103) that is part of the enzyme's active site (29). Although the amino acid Arg103 is buried within the molecule, the bulk of the peptide containing Arg103 and binding to AgNPs is located on the protein surface.…”

Identification and molecular characterization of tryptophanase encoded by tnaA in Porphyromonas gingivalis

“…X-ray crystal structural analyses demonstrated that the tryptophanases from E. coli and Pr. vulgaris contain K + -binding sites close to the active site, and that K + ions interact with the O e atom of Glu-72 in one subunit and the backbone carbonyl O atoms of Gly-55 and Pro-275 (Isupov et al, 1998;Ku et al, 2006). P. gingivalis TnaA contained Glu-72 and Gly-55, even though Pro-275 was replaced by Asn.…”

New tryptophanase inhibitors: Towards prevention of bacterial biofilm formation

“…The structure of E. coli apo enzyme was solved in two crystal forms [26,27] and the structure of a highly homologous Tnase from P. vulgaris was solved in the holo form [28]. 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.…”