Coumarin formation in novobiocin biosynthesis: beta-hydroxylation of the aminoacyl enzyme tyrosyl-S-NovH by a cytochrome P450 NovI

Background: Coumarin group antibiotics, such as novobiocin, coumermycin A(1 ) and clorobiocin, are potent inhibitors of DNA gyrase. These antibiotics h ave been isolated from various Streptomyces species and all possess a 3-ami no-4-hydroxycoumarin moiety as their structural core. Prior labeling experi ments on novobiocin established that the coumarin moiety was derived from L -tyrosine, probably via a beta -hydroxy-tyrosine (beta -OH-Tyr) intermediat e. Recently the novobiocin gene cluster from Streptomyces spheroides was cl oned and sequenced acid allows analysis of the biosynthesis of the coumarin at the biochemical level using overexpressed and purified proteins. Results: Two open reading frames (ORFs), NovH and NovI, from the novobiocin producer S. spheroides have been overexpressed in Escherichia coli, purifi ed and characterized for tyrosine activation and oxygenation which are the initial steps in coumarin formation. The 65 kDa NovH has two predicted doma ins, an adenylation (A) and a peptidyl carrier protein (PCP), reminiscent o f non-ribosomal peptide synthetases. Purified NovH catalyzes L-tyrosyl-AMP Formation by its A domain, can be posttranslationally phosphopantetheinylat ed on the PCP domain, and accumulates the covalent L-tyrosyl-S-enzyme inter mediate on the hole PCP domain. The second enzyme in the pathway, NovI, is a 45 kDa heme protein that functions as a cytochrome P450-type monooxygenas e with specificity for the tyrosyl-S-NovH acyl enzyme. The product beta -OH -tyrosyl-S-NovH was detected by alkaline release and high performance liqui d chromatography analysis of radioactive [H-3]beta -OH-Tyr and by mass spec trometry. Also detected was 4-OH-benzaldehyde, a retro aldol breakdown prod uct of beta -OH-Tyr. The amino acid released was (3R,2S)-3-OH-Tyr by compar ison with authentic standards. Conclusions: This work establishes that NovH and NovI are responsible for t he formation of a beta -OH-Tyr intermediate that is covalently tethered to NovH in novobiocin biosynthesis. Comparable A-PCP/P450 pairs for amino acid beta -hydroxylation are found in various biosynthetic gene clusters, such as ORF19/ORF20 in the chloroeremomycin cluster for tyrosine, CumC/CumD in t he coumermycin A(1) cluster for tyrosine, and NikP1NikQ in the nikkomycin c luster for histidine. This phenomenon of covalent docking of the amino acid in a kinetically stable thioester linkage prior to chemical modification b y downstream tailoring enzymes, could represent a common strategy for contr olling the partitioning of the amino acid for incorporation into secondary metabolites. (C) 2001 Elsevier Science Ltd. All rights reserved.