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 A1 and clorobiocin, are potent inhibitors of DNA gyrase. These antibiotics have been isolated from various Streptomyces species and all possess a 3-amino-4-hydroxy-coumarin 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 and 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 the initial 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-AMP formation by its A domain, can be posttranslationally phosphopantetheinylated on the PCP domain, and accumulates the covalent L-tyrosyl-S-enzyme intermediate on the holo PCP domain. The second enzyme in the pathway, NovI, is a 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 [3H]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 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 the coumermycin A1 cluster for tyrosine, and NikP1/NikQ 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 secondary metabolites.
Chen
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Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, 240 Longwood Avenue, Boston, MA 02115, USA.
Walsh
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D000215:Acylation; D000255:Adenosine Triphosphate / Q000378:metabolism; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D003374:Coumarins / Q000737:chemistry / Q000378:metabolism*; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry / Q000378:metabolism*; D005798:Genes, Bacterial / Q000235:genetics; D006900:Hydroxylation; D009675:Novobiocin / Q000096:biosynthesis* / Q000737:chemistry / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D013237:Stereoisomerism; D013302:Streptomyces / Q000201:enzymology / Q000235:genetics / Q000378:metabolism*
D001426:Bacterial Proteins; D003374:Coumarins; D011994:Recombinant Proteins; D009675:Novobiocin; D000255:Adenosine Triphosphate; D003577:Cytochrome P-450 Enzyme System; C030123:coumarin