Cyclic dipeptide oxidase from Streptomyces noursei - Isolation, purification and partial characterization of a novel, amino acyl alpha,beta-dehydrogenase

Cyclic dipeptide oxidase is a novel enzyme that specifically catalyzes the formation of alpha,beta -dehydro-Phe (Delta Phe) and alpha,beta -dehydro-Le u (Delta Leu) residues during the biosynthesis of albonoursin, cyclo(Delta Phe-Delta Leu), an antibiotic produced by Streptomyces noursei. It was puri fied 600-fold with a 30% overall recovery, and consists of the association of a single type of subunit with a relative molecular mass of 21 066 result ing in a large homopolymer of relative molecular mass over 2 000 000. The e nzyme exhibits a typical flavoprotein spectrum with maxima at 343.5 and 447 .5 nm, the flavin prosthetic group being covalently bound to the protein. T he catalytic reaction of the natural substrate cyclo(L-Phe-L-Leu) occurs in a two-step sequential reaction leading first to cyclo(alpha,beta -dehydro- Phe-L-Leu) and finally to albonoursin. Kinetic parameters for the first ste p were determined (K-m = 53 mum; k = 0.69 s(-1)). The enzyme was shown to c atalyze the conversion of a variety of cyclo(dipeptides) and can be reoxidi zed at the expense of molecular oxygen by producing H2O2. This reaction mec hanism, which differs from those already described for the formation of alp ha,beta -dehydro-amino acids, might consist of the transient formation of a n intermediate imine followed by its rearrangement into an alpha,beta -dehy dro-residue.