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

Martino L. Di Salvo, Rita Florio, Alessandro Paiardini, Mirella Vivoli, Simona D'Aguanno, Roberto Contestabile

Research output: Contribution to journalArticle

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.

Original languageEnglish
Pages (from-to)55-65
Number of pages11
JournalArchives of Biochemistry and Biophysics
Volume529
Issue number2
DOIs
Publication statusPublished - Jan 15 2013

Fingerprint

Alanine Racemase
Glycine Hydroxymethyltransferase
Biosynthesis
Cyclosporine
Enzymes
Cyclic Peptides
Pyridoxal Phosphate
Immunosuppressive Agents
Fungi
Alanine
Crystal structure

Keywords

  • Catalytic promiscuity
  • Cyclosporin
  • Fold type I alanine racemase
  • Nonribosomal peptide synthesis
  • Protein evolution
  • Pyridoxal 5′-phosphate

ASJC Scopus subject areas

  • Biochemistry
  • Biophysics
  • Molecular Biology

Cite this

Alanine racemase from Tolypocladium inflatum : A key PLP-dependent enzyme in cyclosporin biosynthesis and a model of catalytic promiscuity. / Di Salvo, Martino L.; Florio, Rita; Paiardini, Alessandro; Vivoli, Mirella; D'Aguanno, Simona; Contestabile, Roberto.

In: Archives of Biochemistry and Biophysics, Vol. 529, No. 2, 15.01.2013, p. 55-65.

Research output: Contribution to journalArticle

Di Salvo, Martino L. ; Florio, Rita ; Paiardini, Alessandro ; Vivoli, Mirella ; D'Aguanno, Simona ; Contestabile, Roberto. / Alanine racemase from Tolypocladium inflatum : A key PLP-dependent enzyme in cyclosporin biosynthesis and a model of catalytic promiscuity. In: Archives of Biochemistry and Biophysics. 2013 ; Vol. 529, No. 2. pp. 55-65.
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