title: An engineered folded PLP-bound monomer of Treponema denticola cystalysin reveals the effect of the dimeric structure on the catalytic properties of the enzyme.
abstract: Cystalysin, a dimeric pyridoxal 5'-phosphate (PLP)-dependent lyase, is a virulence factor of the human oral pathogen Treponema denticola. Guided by bioinformatic analysis, two interfacial residues (Leu57 and Leu62) and an active site residue (Tyr64*), hydrogen-bonded with the PLP phosphate group of the neighboring subunit, have been mutated. The wild-type and the L57A, L62A, Y64*A, L57A/L62A, L57A/Y64*A, L57A/L62A/Y64*A mutants, all having a C-terminal histidine tag, have been constructed, expressed, and purified. The impact of these mutations on the dimeric state of cystalysin in the apo- and holo-form has been analyzed by size-exclusion chromatography. The results demonstrate that (i) Leu57 is more critical than Leu62 for apodimer formation, (ii) Tyr64*, more than Leu62, interferes with dimerization of holocystalysin without affecting that of apoenzyme, (iii) while each single mutation is inadequate in significantly altering the extent of monomerization of both apo- and holo-cystalysin, their combination leads to species which remain in a folded monomeric state at a reasonably high concentration in both the apo- and holo-forms. Although L57A/L62A or L57A/Y64*A, even to a different extent, are stimulated to dimer formation in the presence of either unproductive or productive ligands, L57A/L62A/Y64*A remains prevalently monomer at a concentration up to 50 microM. Kinetic analyses show that in this monomeric species the alpha,beta-eliminase, alanine racemase, and D-alanine half-transaminase activities are almost abolished, while the L-alanine half-transaminase activity is slightly enhanced when compared with that of wild-type. The structural basis of the stereospecific transaminase activity displayed by the engineered folded PLP-bound monomer has been analyzed and discussed.
authors.affiliation: Dipartimento di Scienze Morfologico-Biomediche, Sezione di Chimica Biologica, Facoltà di Medicina e Chirurgia, Università degli Studi di Verona, Strada Le Grazie 8, 37134 Verona, Italy.
authors.forename: Riccardo
authors.initials: R
authors.lastname: Montioli
authors.forename: Barbara
authors.initials: B
authors.lastname: Cellini
authors.forename: Mariarita
authors.initials: M
authors.lastname: Bertoldi
authors.forename: Alessandro
authors.initials: A
authors.lastname: Paiardini
authors.forename: Carla Borri
authors.initials: CB
authors.lastname: Voltattorni
mesh_terms: D000818:Animals; D001051:Apoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D020134:Catalytic Domain; D003542:Cystathionine gamma-Lyase / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D004926:Escherichia coli; D020035:Holoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D007930:Leucine / Q000235:genetics / Q000378:metabolism; D008190:Lyases / Q000378:metabolism; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D020836:Protein Structure, Quaternary; D011732:Pyridoxal Phosphate / Q000737:chemistry / Q000378:metabolism; D013329:Structure-Activity Relationship; D048373:Treponema denticola / Q000201:enzymology* / Q000235:genetics; D014443:Tyrosine / Q000235:genetics / Q000378:metabolism
chemical_list: D001051:Apoenzymes; D001426:Bacterial Proteins; D020035:Holoenzymes; D014443:Tyrosine; D011732:Pyridoxal Phosphate; D008190:Lyases; C400145:cystalysin; D003542:Cystathionine gamma-Lyase; D007930:Leucine