Cloning, overexpression, and purification of novobiocic acid synthetase from Streptomyces spheroides NCIMB 11891.
Novobiocic acid synthetase, a key enzyme in the biosynthesis of the antibiotic novobiocin, was cloned from the novobiocin producer Streptomyces spheroides NCIMB 11891. The enzyme is encoded by the gene novL, which codes for a protein of 527 amino acids with a calculated mass of 56,885 Da. The protein was overexpressed as a His(6) fusion protein in Escherichia coli and purified to apparent homogeneity by affinity chromatography and gel chromatography. The purified enzyme catalyzed the formation of an amide bond between 3-dimethylallyl-4-hydroxybenzoic acid (ring A of novobiocin) and 3-amino-4,7-dihydroxy-8-methyl coumarin (ring B of novobiocin) in an ATP-dependent reaction. NovL shows homology to the superfamily of adenylate-forming enzymes, and indeed the formation of an acyl adenylate from ring A and ATP was demonstrated by an ATP-PP(i) exchange assay. The purified enzyme exhibited both activation and transferase activity, i.e. it catalyzed both the activation of ring A as acyl adenylate and the subsequent transfer of the acyl group to the amino group of ring B. It is active as a monomer as determined by gel filtration chromatography. The reaction was specific for ATP as nucleotide triphosphate and dependent on the presence of Mg(2+) or Mn(2+). Apparent K(m) values for ring A and ring B were determined as 19 and 131 micrometer respectively. Of several analogues of ring A, only 3-geranyl-4-hydroxybenzoate and to a lesser extent 3-methyl-4-aminobenzoate were accepted as substrates.
Steffensky
M
M
Eberhard-Karls-Universität Tübingen, Pharmazeutisches Institut, Pharmazeutische Biologie, 72076 Tübingen, Germany.
Li
S M
SM
Heide
L
L
D019732:Amide Synthases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D000595:Amino Acid Sequence; D001483:Base Sequence; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D004926:Escherichia coli; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009675:Novobiocin / Q000096:biosynthesis; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013302:Streptomyces / Q000201:enzymology* / Q000235:genetics*; D013379:Substrate Specificity
D011994:Recombinant Proteins; D009675:Novobiocin; D019732:Amide Synthases; C021884:novobiocic acid synthetase