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

L-Tryptophan 2',3'-oxidase, an amino acid alpha,beta-dehydrogenase isolated from Chromobacterium violaceum, catalyzes the formation of a double bond between the C-alpha and C-beta carbons of various tryptophan derivatives provided that they possess: (i) a L-enantiomeric configuration, (ii) an alpha-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 beta-substituted alanyl residues which behave neither as substrates nor as inhibitors and therefore do not compete against this reaction. The enzymatic synthesis of alpha,beta-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 alpha,beta-dehydrogenation of the tryptophan side chain is proposed.