Abstract
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.
Highlights
Amino acid oxidases and dehydrogenases are widely distributed in living cells where they play a key role in the metabolic fate of amino acids, generating ␣-keto acids by oxidative deamination or dehydration reactions (1–3)
We recently isolated a novel enzyme from Chromobacterium violaceum (ATCC 12472), and showed it to convert N-acetyl-L-tryptophanamide (NATA)1 into N-acetyl␣,-dehydrotryptophanamide (⌬NATA) (4)
The data obtained suggest that L-tryptophan 2Ј,3Ј-oxidase proceeds to a direct ␣,-dehydrogenation of the tryptophan side chain via a mechanism closely related to that proposed for fatty acyl-CoA dehydrogenases (9), in sharp contrast with the complex pH-dependent dichotomous mechanism postulated for the Pseudomonas tryptophan side chain oxidase (EC 1.13.99.3) (10, 11)
Summary
Amino acid oxidases and dehydrogenases are widely distributed in living cells where they play a key role in the metabolic fate of amino acids, generating ␣-keto acids by oxidative deamination or dehydration reactions (1–3). In this context, we recently isolated a novel enzyme from Chromobacterium violaceum (ATCC 12472), and showed it to convert N-acetyl-L-tryptophanamide (NATA) into N-acetyl␣,-dehydrotryptophanamide (⌬NATA) (4). L-Tryptophan 2Ј,3Ј-oxidase is a novel hemoprotein exhibiting a number of interesting features (4) It seems to directly catalyze the conversion of NATA into ⌬NATA and in a stoichiometric manner the reduction of molecular O2 to generate H2O2. This finding offers new enzymatic issues as to the modification and labeling of this peculiar side chain in proteins
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