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Titolo:
Coumarin formation in novobiocin biosynthesis: beta-hydroxylation of the aminoacyl enzyme tyrosyl-S-NovH by a cytochrome P450 NovI
Autore:
Chen, HW; Walsh, CT;
Indirizzi:
Harvard Univ, Sch Med, Dept Biol Chem & Mol Pharmacol, Boston, MA 02115 USA Harvard Univ Boston MA USA 02115 em & Mol Pharmacol, Boston, MA 02115 USA
Titolo Testata:
CHEMISTRY & BIOLOGY
fascicolo: 4, volume: 8, anno: 2001,
pagine: 301 - 312
SICI:
1074-5521(200104)8:4<301:CFINBB>2.0.ZU;2-L
Fonte:
ISI
Lingua:
ENG
Soggetto:
DNA GYRASE; ESCHERICHIA-COLI; PEPTIDE SYNTHETASES; ADENYLATION DOMAINS; GENE-CLUSTER; IDENTIFICATION; PROTEIN; PURIFICATION; SPECIFICITY; CLOROBIOCIN;
Keywords:
novobiocin; coumarin; beta-hydroxy-tyrosine; cytochrome P450 monooxygenase; non-ribosomal peptide synthetase;
Tipo documento:
Article
Natura:
Periodico
Settore Disciplinare:
Life Sciences
Citazioni:
33
Recensione:
Indirizzi per estratti:
Indirizzo: Walsh, CT Harvard Univ, Sch Med, Dept Biol Chem & Mol Pharmacol, 240 Longwood Ave, Boston, MA 02115 USA Harvard Univ 240 Longwood Ave Boston MA USA 02115 , MA 02115 USA
Citazione:
H.W. Chen e C.T. Walsh, "Coumarin formation in novobiocin biosynthesis: beta-hydroxylation of the aminoacyl enzyme tyrosyl-S-NovH by a cytochrome P450 NovI", CHEM BIOL, 8(4), 2001, pp. 301-312

Abstract

Background: Coumarin group antibiotics, such as novobiocin, coumermycin A(1) and clorobiocin, are potent inhibitors of DNA gyrase. These antibiotics have been isolated from various Streptomyces species and all possess a 3-amino-4-hydroxycoumarin moiety as their structural core. Prior labeling experiments on novobiocin established that the coumarin moiety was derived from L-tyrosine, probably via a beta -hydroxy-tyrosine (beta -OH-Tyr) intermediate. Recently the novobiocin gene cluster from Streptomyces spheroides was cloned 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, purified and characterized for tyrosine activation and oxygenation which are theinitial steps in coumarin formation. The 65 kDa NovH has two predicted domains, an adenylation (A) and a peptidyl carrier protein (PCP), reminiscent of non-ribosomal peptide synthetases. Purified NovH catalyzes L-tyrosyl-AMPFormation by its A domain, can be posttranslationally phosphopantetheinylated on the PCP domain, and accumulates the covalent L-tyrosyl-S-enzyme intermediate on the hole PCP domain. The second enzyme in the pathway, NovI, isa 45 kDa heme protein that functions as a cytochrome P450-type monooxygenase with specificity for the tyrosyl-S-NovH acyl enzyme. The product beta -OH-tyrosyl-S-NovH was detected by alkaline release and high performance liquid chromatography analysis of radioactive [H-3]beta -OH-Tyr and by mass spectrometry. Also detected was 4-OH-benzaldehyde, a retro aldol breakdown product of beta -OH-Tyr. The amino acid released was (3R,2S)-3-OH-Tyr by comparison with authentic standards. Conclusions: This work establishes that NovH and NovI are responsible for the formation of a beta -OH-Tyr intermediate that is covalently tethered toNovH in novobiocin biosynthesis. Comparable A-PCP/P450 pairs for amino acid beta -hydroxylation are found in various biosynthetic gene clusters, suchas ORF19/ORF20 in the chloroeremomycin cluster for tyrosine, CumC/CumD in the coumermycin A(1) cluster for tyrosine, and NikP1NikQ in the nikkomycin cluster for histidine. This phenomenon of covalent docking of the amino acid in a kinetically stable thioester linkage prior to chemical modification by downstream tailoring enzymes, could represent a common strategy for controlling the partitioning of the amino acid for incorporation into secondarymetabolites. (C) 2001 Elsevier Science Ltd. All rights reserved.

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Documento generato il 04/04/20 alle ore 11:59:56