NovJ/NovK Catalyze Benzylic Oxidation of a â-Hydroxyl Tyrosyl-S-pantetheinyl Enzyme during Aminocoumarin Ring Formation in Novobiocin Biosynthesis†

ABSTRACT: The bicyclic coumarin ring in the aminocoumarin natural product antibiotics that target bacterial DNA gyrase is assembled from tyrosine by nonribosomal peptide synthetase logic. Tyrosine has previously been shown to be activated and installed as a phosphopantetheinyl thioester on the thiolation domain of NovH and then hydroxylated on the benzylic carbon by the heme protein NovI, generating â-OH-Tyr-S-NovH. This aminoacyl-S-protein is the substrate for the next two orfs, Streptomyces sphaeroides NovJ and NovK, that have now been expressed in and purified from Escherichia coli as a J2K2 heterotetramer. NovJ/NovK use NADP as an electron acceptor to oxidize the â-OH of the tyrosyl moiety to yield the tethered â-ketotyrosyl-S-NovH. The enol tautomer is the form that predominates in the subsequently cyclized aminocoumarin scaffold. The labile â-ketotyrosyl thioester moiety was identified by hydrolytic release from NovH, analysis by mass spectroscopy, and comparison with a synthetic sample. We also have identified a residue in NovJ that when mutated results in a 50-fold reduction in catalytic activity. The antibiotics novobiocin, clorobiocin, and coumermycin A1 target bacterial DNA gyrase with KDs of 10-8 to 10-10 M (1). These natural products have in common a 7-hydroxy-2-aminocoumarin core. During biosynthesis, the phenoli