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-Leu (Delta Leu) residues during the biosynthesis of albonoursin, cyclo(Delta Phe-Delta Leu), an antibiotic produced by Streptomyces noursei. It was purified 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 resulting in a large homopolymer of relative molecular mass over 2,000,000. The enzyme exhibits a typical flavoprotein spectrum with maxima at 343.5 and 447.5 nm, the flavin prosthetic group being covalently bound to the protein. The 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 step were determined (K(m) = 53 microM; k = 0.69 s(-1)). The enzyme was shown to catalyze the conversion of a variety of cyclo(dipeptides) and can be reoxidized at the expense of molecular oxygen by producing H(2)O(2). This reaction mechanism, which differs from those already described for the formation of alpha,beta-dehydro-amino acids, might consist of the transient formation of an intermediate imine followed by its rearrangement into an alpha,beta-dehydro-residue.
Gondry
M
M
CEA/Saclay, Département d'Ingénierie et d'Etudes des Protéines, Gif-sur-Yvette, France. muriel.gondry@cea.fr
Lautru
S
S
Fusai
G
G
Meunier
G
G
Ménez
A
A
Genet
R
R
D002384:Catalysis; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D007525:Isoelectric Focusing; D007700:Kinetics; D013058:Mass Spectrometry; D008970:Molecular Weight; D010088:Oxidoreductases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D011487:Protein Conformation; D013302:Streptomyces / Q000201:enzymology*; D013379:Substrate Specificity
D010088:Oxidoreductases; C424906:cyclic dipeptide oxidase