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Alanine racemase from Tolypocladium inflatum: a key PLP-dependent enzyme in cyclosporin biosynthesis and a model of catalytic promiscuity.
Arch Biochem Biophys. 2013 Jan 15; 529(2):55-65.AB

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+Show Affiliations

Dipartimento di Scienze Biochimiche A. Rossi Fanelli, Sapienza Università di Roma, Piazzale Aldo Moro 5, 00185 Roma, Italy. martino.disalvo@uniroma1.itNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

23219598

Citation

di Salvo, Martino L., et al. "Alanine Racemase From Tolypocladium Inflatum: a Key PLP-dependent Enzyme in Cyclosporin Biosynthesis and a Model of Catalytic Promiscuity." Archives of Biochemistry and Biophysics, vol. 529, no. 2, 2013, pp. 55-65.
di Salvo ML, Florio R, Paiardini A, et al. Alanine racemase from Tolypocladium inflatum: a key PLP-dependent enzyme in cyclosporin biosynthesis and a model of catalytic promiscuity. Arch Biochem Biophys. 2013;529(2):55-65.
di Salvo, M. L., Florio, R., Paiardini, A., Vivoli, M., D'Aguanno, S., & Contestabile, R. (2013). Alanine racemase from Tolypocladium inflatum: a key PLP-dependent enzyme in cyclosporin biosynthesis and a model of catalytic promiscuity. Archives of Biochemistry and Biophysics, 529(2), 55-65. https://doi.org/10.1016/j.abb.2012.11.011
di Salvo ML, et al. Alanine Racemase From Tolypocladium Inflatum: a Key PLP-dependent Enzyme in Cyclosporin Biosynthesis and a Model of Catalytic Promiscuity. Arch Biochem Biophys. 2013 Jan 15;529(2):55-65. PubMed PMID: 23219598.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Alanine racemase from Tolypocladium inflatum: a key PLP-dependent enzyme in cyclosporin biosynthesis and a model of catalytic promiscuity. AU - di Salvo,Martino L, AU - Florio,Rita, AU - Paiardini,Alessandro, AU - Vivoli,Mirella, AU - D'Aguanno,Simona, AU - Contestabile,Roberto, Y1 - 2012/12/03/ PY - 2012/10/23/received PY - 2012/11/22/revised PY - 2012/11/28/accepted PY - 2012/12/11/entrez PY - 2012/12/12/pubmed PY - 2013/3/21/medline SP - 55 EP - 65 JF - Archives of biochemistry and biophysics JO - Arch Biochem Biophys VL - 529 IS - 2 N2 - 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. SN - 1096-0384 UR - https://www.unboundmedicine.com/medline/citation/23219598/Alanine_racemase_from_Tolypocladium_inflatum:_a_key_PLP_dependent_enzyme_in_cyclosporin_biosynthesis_and_a_model_of_catalytic_promiscuity_ L2 - https://linkinghub.elsevier.com/retrieve/pii/S0003-9861(12)00404-3 DB - PRIME DP - Unbound Medicine ER -