title: Alanine racemase from Tolypocladium inflatum: a key PLP-dependent enzyme in cyclosporin biosynthesis and a model of catalytic promiscuity.
abstract: Cyclosporin A, a cyclic peptide produced by the fungus Tolypocladium inflatum, is a widely employed immunosuppressant drug. Its biosynthesis is strictly dependent on the action of the pyridoxal 5'-phosphate-dependent enzyme alanine racemase, which produces the d-alanine incorporated in the cyclic peptide. This enzyme has a different fold with respect to bacterial alanine racemases. The interest elicited by T. inflatum alanine racemase not only relies on its biotechnological relevance, but also on its evolutionary and structural similarity to the promiscuous enzymes serine hydroxymethyltransferase and threonine aldolase. The three enzymes represent a model of divergent evolution from an ancestral enzyme that was able to catalyse all the reactions of the modern enzymes. A protocol to express and purify with high yield recombinant T. inflatum alanine racemase was developed. The catalytic properties of the enzyme were characterized. Similarly to serine hydroxymethyltransferase and threonine aldolase, T. inflatum alanine racemase was able to catalyse retroaldol cleavage and transamination reactions. This observation corroborates the hypothesis of the common evolutionary origin of these enzymes. A three-dimensional model of T. inflatum alanine racemase was constructed on the basis of threonine aldolase crystal structure. The model helped rationalise the experimental data and explain the catalytic properties of the enzymes.
authors.affiliation: Dipartimento di Scienze Biochimiche A. Rossi Fanelli, Sapienza Università di Roma, Piazzale Aldo Moro 5, 00185 Roma, Italy. martino.disalvo@uniroma1.it
authors.forename: Martino L
authors.initials: ML
authors.lastname: di Salvo
authors.forename: Rita
authors.initials: R
authors.lastname: Florio
authors.forename: Alessandro
authors.initials: A
authors.lastname: Paiardini
authors.forename: Mirella
authors.initials: M
authors.lastname: Vivoli
authors.forename: Simona
authors.initials: S
authors.lastname: D'Aguanno
authors.forename: Roberto
authors.initials: R
authors.lastname: Contestabile
mesh_terms: D000411:Alanine Racemase / Q000737:chemistry* / Q000378:metabolism* / Q000648:ultrastructure; D000595:Amino Acid Sequence; D001203:Ascomycota / Q000201:enzymology*; D002384:Catalysis; D003198:Computer Simulation; D016572:Cyclosporine / Q000378:metabolism*; D004789:Enzyme Activation; D008956:Models, Chemical; D008958:Models, Molecular; D008969:Molecular Sequence Data
chemical_list: D016572:Cyclosporine; D000411:Alanine Racemase