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. (C) 2012 Elsevier Inc. All rights reserved.

Alanine racemase from Tolypocladium inflatum: A key PLP-dependent enzyme in cyclosporin biosynthesis and a model of catalytic promiscuity / DI SALVO, Martino Luigi; Florio, Rita; Paiardini, Alessandro; Vivoli, Mirella; Simona, D'Aguanno; Contestabile, Roberto. - In: ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS. - ISSN 0003-9861. - STAMPA. - 529:2(2013), pp. 55-65. [10.1016/j.abb.2012.11.011]

Alanine racemase from Tolypocladium inflatum: A key PLP-dependent enzyme in cyclosporin biosynthesis and a model of catalytic promiscuity

DI SALVO, Martino Luigi;FLORIO, Rita;PAIARDINI, ALESSANDRO;VIVOLI, MIRELLA;CONTESTABILE, Roberto
2013

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. (C) 2012 Elsevier Inc. All rights reserved.
catalytic promiscuity; cyclosporin; fold type i alanine racemase; nonribosomal peptide synthesis; protein evolution; protein evolution.; pyridoxal 5 '-phosphate; pyridoxal 5'-phosphate; pyridoxal 5′-phosphate
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Alanine racemase from Tolypocladium inflatum: A key PLP-dependent enzyme in cyclosporin biosynthesis and a model of catalytic promiscuity / DI SALVO, Martino Luigi; Florio, Rita; Paiardini, Alessandro; Vivoli, Mirella; Simona, D'Aguanno; Contestabile, Roberto. - In: ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS. - ISSN 0003-9861. - STAMPA. - 529:2(2013), pp. 55-65. [10.1016/j.abb.2012.11.011]
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11573/501940
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