Biochemical characterization of L-DOPA 2,3-dioxygenase, a single-domain type I extradiol dioxygenase from lincomycin biosynthesis.
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.
Colabroy
Keri L
KL
Department of Chemistry, Muhlenberg College, Allentown, PA 18104, USA.
Hackett
William T
WT
Markham
Andrew J
AJ
Rosenberg
Jennifer
J
Cohen
David E
DE
Jacobson
Ariel
A
D015102:3,4-Dihydroxyphenylacetic Acid / Q000737:chemistry; D001426:Bacterial Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002396:Catechols / Q000737:chemistry / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000737:chemistry; D007501:Iron / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D008024:Ligands; D008034:Lincomycin / Q000096:biosynthesis / Q000737:chemistry; D016297:Mutagenesis, Site-Directed / Q000379:methods; D010105:Oxygenases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D017434:Protein Structure, Tertiary / Q000502:physiology; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013302:Streptomyces / Q000201:enzymology* / Q000235:genetics; D014443:Tyrosine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D001426:Bacterial Proteins; D002396:Catechols; D004791:Enzyme Inhibitors; D008024:Ligands; D011994:Recombinant Proteins; D015102:3,4-Dihydroxyphenylacetic Acid; D014443:Tyrosine; D008034:Lincomycin; D007501:Iron; D010105:Oxygenases; C439587:extradiol dioxygenase