L-TRYPTOPHAN 2',3'-OXIDASE FROM CHROMOBACTERIUM VIOLACEUM : SUBSTRATE SPECIFICITY AND MECHANISTIC IMPLICATIONS

Abstract L-Tryptophan 2′,3′-oxidase, an amino acid α,β-dehydrogenase isolated from Chromobacterium violaceum, catalyzes the formation of a double bond between the C and C carbons of various tryptophan derivatives provided that they possess: (i) a L-enantiomeric configuration, (ii) an α-carbonyl group, and (iii) an unsubstituted and unmodified indole nucleus. Kinetic parameters were evaluated for a series of tryptophan analogues, providing information on the contribution of each chemical group to substrate binding. The stereochemistry of the dehydro product was determined to be a Z-configuration from proton nuclear magnetic resonance assignments. No reaction can be observed in the presence of other aromatic β-substituted alanyl residues which behave neither as substrates nor as inhibitors and therefore do not compete against this reaction. The enzymatic synthesis of α,β-dehydrotryptophanyl peptides from 5 to 24 residues was successfully achieved without side product formation, irrespective of the position of the tryptophan residue in the amino acid sequence. A reactional mechanism involving a direct α,β-dehydrogenation of the tryptophan side chain is proposed.