Abstract
The biosynthesis of ansamycin antibiotics, like rifamycin B, involves formation of 3-amino-5-hydroxybenzoic acid (AHBA) by a novel variant of the shikimate pathway. AHBA then serves as the starter unit for the assembly of a polyketide which eventually links back to the amino group of AHBA to form the macrolactam ring. The terminal enzyme of AHBA formation, which catalyzes the aromatization of 5-deoxy-5-amino-3-dehydroshikimic acid, has been purified to homogeneity from Amycolatopsis mediterranei, the encoding gene has been cloned, sequenced, and overexpressed in Escherichia coli. The recombinant enzyme, a (His)6 fusion protein, as well as the native one, are dimers containing one molecule of pyridoxal phosphate per subunit. Mechanistic studies showed that the enzyme-bound pyridoxal phosphate forms a Schiff's base with the amino group of 5-deoxy-5-amino-3-dehydroshikimic acid and catalyzes both an alpha, beta-dehydration and a stereospecific 1,4-enolization of the substrate. Inactivation of the gene encoding AHBA synthase in the A. mediterranei genome results in loss of rifamycin formation; production of the antibiotic is restored when the mutant is supplemented with AHBA.
Publication types
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Comparative Study
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Research Support, U.S. Gov't, P.H.S.
MeSH terms
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Actinobacteria / enzymology*
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Actinobacteria / genetics
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Actinomycetales / enzymology*
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Actinomycetales / genetics
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Amino Acid Sequence
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Aminobenzoates / metabolism*
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Base Sequence
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Cloning, Molecular
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Cyclohexanecarboxylic Acids / pharmacology
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Escherichia coli / genetics
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Evolution, Molecular
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Hydro-Lyases / classification
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Hydro-Lyases / genetics
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Hydro-Lyases / isolation & purification
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Hydro-Lyases / metabolism*
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Hydroxybenzoates
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Molecular Sequence Data
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Phylogeny
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Pyridoxal Phosphate / metabolism
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Pyridoxamine / analogs & derivatives
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Pyridoxamine / metabolism
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Recombinant Proteins / isolation & purification
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Recombinant Proteins / metabolism
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Restriction Mapping
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Rifamycins / biosynthesis*
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Sequence Analysis, DNA
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Sequence Homology, Amino Acid
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Shikimic Acid / analogs & derivatives
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Shikimic Acid / metabolism
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Stereoisomerism
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Substrate Specificity
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Transaminases / classification
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Transaminases / genetics
Substances
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5-deoxy-5-amino-3-dehydroshikimic acid
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Aminobenzoates
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Cyclohexanecarboxylic Acids
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Hydroxybenzoates
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Recombinant Proteins
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Rifamycins
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Shikimic Acid
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gabaculine
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Pyridoxal Phosphate
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Pyridoxamine
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3-amino-5-hydroxybenzoic acid
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Transaminases
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3-amino-5-hydroxybenzoic acid synthase
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Hydro-Lyases
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pyridoxamine phosphate