RESUMEN

L-DOPA-2,3-dioxygenase from Streptomyces lincolnensis is a single-domain type I extradiol dioxygenase of the vicinal oxygen chelate superfamily and catalyzes the second step in the metabolism of tyrosine to the propylhygric acid moiety of the antibiotic, lincomycin. S. lincolnensis L-DOPA-2,3-dioxygenase was overexpressed, purified and reconstituted with Fe(II). The activity of L-DOPA-2,3-dioxygenase was kinetically characterized with L-DOPA (K(M)=38 microM, k(cat)=4.2 min(-1)) and additional catecholic substrates including dopamine, 3,4-dihydroxyhydrocinnamic acid, catechol and D-DOPA. 3,4-Dihydroxyphenylacetic acid was characterized as a competitive inhibitor of the enzyme (K(i) =2.2 mM). Site-directed mutagenesis and its effects on enzymatic activity were used to identify His14 and His70 as iron ligands.

Asunto(s)

Proteínas Bacterianas/química , Oxigenasas/química , Streptomyces/enzimología , Ácido 3,4-Dihidroxifenilacético/química , Proteínas Bacterianas/antagonistas & inhibidores , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Catecoles/química , Catecoles/metabolismo , Inhibidores Enzimáticos/química , Hierro/química , Hierro/metabolismo , Cinética , Ligandos , Lincomicina/biosíntesis , Lincomicina/química , Mutagénesis Sitio-Dirigida/métodos , Oxigenasas/antagonistas & inhibidores , Oxigenasas/genética , Oxigenasas/metabolismo , Estructura Terciaria de Proteína/fisiología , Proteínas Recombinantes/antagonistas & inhibidores , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Streptomyces/genética , Tirosina/química , Tirosina/genética , Tirosina/metabolismo