Abstract
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.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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3,4-Dihydroxyphenylacetic Acid / chemistry
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Bacterial Proteins / antagonists & inhibitors
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Bacterial Proteins / chemistry*
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Bacterial Proteins / genetics
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Bacterial Proteins / metabolism
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Catechols / chemistry
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Catechols / metabolism
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Enzyme Inhibitors / chemistry
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Iron / chemistry
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Iron / metabolism
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Kinetics
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Ligands
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Lincomycin / biosynthesis
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Lincomycin / chemistry
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Mutagenesis, Site-Directed / methods
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Oxygenases / antagonists & inhibitors
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Oxygenases / chemistry*
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Oxygenases / genetics
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Oxygenases / metabolism
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Protein Structure, Tertiary / physiology
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Recombinant Proteins / antagonists & inhibitors
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Recombinant Proteins / chemistry
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Recombinant Proteins / genetics
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Recombinant Proteins / metabolism
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Streptomyces / enzymology*
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Streptomyces / genetics
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Tyrosine / chemistry
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Tyrosine / genetics
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Tyrosine / metabolism
Substances
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Bacterial Proteins
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Catechols
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Enzyme Inhibitors
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Ligands
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Recombinant Proteins
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3,4-Dihydroxyphenylacetic Acid
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Tyrosine
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Lincomycin
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Iron
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Oxygenases
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extradiol dioxygenase