An Oxidative Phenol Coupling Reaction Catalyzed by OxyB, a Cytochrome P450 from the Vancomycin‐Producing Microorganism

Zerbe, Katja; Woithe, Katharina; Li, Dong Bo; Vitali, Francesca; Bigler, Laurent; Robinson, John A.

doi:10.1002/anie.200461278

Cited by 117 publications

(91 citation statements)

References 18 publications

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“…The Oxy enzymes catalyze a critical step, probably the most distinctive feature in glycopeptide biosynthesis. Current evidence indicates that at least some of the Oxy enzymes act in concert with the NRPS while the growing peptide is still enzyme bound (3,36,46). In all glycopeptide clusters characterized to date, the oxy genes are linked and an oxyA ortholog is always the first gene, suggesting the existence of an operon in all cases.…”

Section: Discussionmentioning

confidence: 99%

Phosphate-Controlled Regulator for the Biosynthesis of the Dalbavancin Precursor A40926

et al. 2007

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The actinomycete Nonomuraea sp. strain ATCC 39727 produces the glycopeptide A40926, the precursor of the novel antibiotic dalbavancin. Previous studies have shown that phosphate limitation results in enhanced A40926 production. The A40926 biosynthetic gene (dbv) cluster, which consists of 37 genes, encodes two putative regulators, Dbv3 and Dbv4, as well as the response regulator (Dbv6) and the sensor-kinase (Dbv22) of a putative two-component system. Reverse transcription-PCR (RT-PCR) and real-time RT-PCR analysis revealed that the dbv14-dbv8 and the dbv30-dbv35 operons, as well as dbv4, were negatively influenced by phosphate. Dbv4 shows a putative helix-turn-helix DNA-binding motif and shares sequence similarity with StrR, the transcriptional activator of streptomycin biosynthesis in Streptomyces griseus. Dbv4 was expressed in Escherichia coli as an N-terminal His 6 -tagged protein. The purified protein bound the dbv14 and dbv30 upstream regions but not the region preceding dbv4. Bbr, a Dbv4 ortholog from the gene cluster for the synthesis of the glycopeptide balhimycin, also bound to the dbv14 and dbv30 upstream regions, while Dbv4 bound appropriate regions from the balhimycin cluster. Our results provide new insights into the regulation of glycopeptide antibiotics, indicating that the phosphate-controlled regulator Dbv4 governs two key steps in A40926 biosynthesis: the biosynthesis of the nonproteinogenic amino acid 3,5-dihydroxyphenylglycine and critical tailoring reactions on the heptapeptide backbone.

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Section: Discussionmentioning

confidence: 99%

Phosphate-Controlled Regulator for the Biosynthesis of the Dalbavancin Precursor A40926

et al. 2007

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“…Only recently has it become clear that whilst being shuffled along and remaining covalently anchored to the NRPS assembly line, the peptide intermediates may also be transformed by other enzymes acting in trans. This has been demonstrated in vitro for OxyB in vancomycin-like glycopeptides (Woithe et al, 2007;Zerbe et al, 2004), and may well also be true for the other cross-linking enzymes, and the halogenases which introduce Cl atoms into aromatic residues during glycopeptide biosynthesis.…”

Section: Figure-2mentioning

confidence: 70%

“…In contrast, several soluble microbial P450 enzymes have also been cloned and studied recently that catalyze phenol (and related) coupling reactions. These include a flaviolin oxidase (Zhao et al, 2005), a tetrahydroxynaphthalene oxidase (Funa et al, 2005), an indole-indole coupling enzyme (StaP) involved in staurosporine biosynthesis (Howard-Jones and Walsh, 2007;Makino et al, 2007), and last but not least, the enzyme OxyB, which catalyzes the first cross-linking step in the biosynthesis of vancomycin and related glycopeptides (Woithe et al, 2007;Zerbe et al, 2004).…”

Section: Figure-1mentioning

confidence: 99%

Chapter 19 In Vitro Studies of Phenol Coupling Enzymes Involved in Vancomycin Biosynthesis

Woithe

Geib

et al. 2009

Complex Enzymes in Microbial Natural Product Biosynthesis, Part A: Overview Articles and Peptides

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Oxidative phenol cross-linking reactions play a key role in the biosynthesis of glycopeptide antibiotics such as vancomycin. The vancomycin aglycone contains three cross-links between aromatic amino acid side-chains, which stabilize the folded backbone conformation required for binding to the target D-Ala-D-Ala dipeptide. At least the first cross-link is introduced into a peptide precursor whilst it is still bound as a thioester to a peptide carrier protein (PCP) domain (also called a thiolation domain) within the nonribosomal peptide synthetase. We described here methods for the solid-phase synthesis of peptides and their coupling to PCP domains, which may be useful for in vitro studies of cross-linking and related tailoring reactions during nonribosomal glycopeptide antibiotic biosynthesis.

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“…Alternatively, the possibility that phosphorylation occurs during peptide assembly cannot be ruled out, as other nonribosomal peptide-tailoring enzymes are suggested to modify peptidyl-thioester intermediates tethered to PCP domains. Indeed, there is evidence that oxidative coupling reactions between the phenolic side chains most likely occur on the peptidyl-thioester intermediates during the assembly of vancomycin and related glycopeptides (Zerbe et al, 2004;Bischoff et al, 2005).…”

Section: Discussionmentioning

confidence: 99%

An asparagine oxygenase (AsnO) and a 3-hydroxyasparaginyl phosphotransferase (HasP) are involved in the biosynthesis of calcium-dependent lipopeptide antibiotics

et al. 2007

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Nonribosomal peptides contain a wide range of unusual non-proteinogenic amino acid residues. As a result, these complex natural products are amongst the most structurally diverse secondary metabolites in nature, and possess a broad spectrum of biological activities. b-Hydroxylation of amino acid precursors or peptidyl residues and their subsequent processing by downstream tailoring enzymes are some of the most common themes in the biosynthetic diversification of these therapeutically important peptides. Identification and characterization of the biosynthetic intermediates and enzymes involved in these processes are thus pivotal in understanding nonribosomal peptide assembly and modification. To this end, the putative asparaginyl oxygenase-and 3-hydroxyasparaginyl phosphotransferase-encoding genes hasP and asnO were separately deleted from the calcium-dependent antibiotic (CDA) biosynthetic gene cluster of Streptomyces coelicolor. Whilst the parent strains produce a number of 3-hydroxyasparagine-and 3-phosphohydroxyasparagine-containing CDAs, the DhasP mutants produce exclusively non-phosphorylated CDAs. On the other hand, DasnO mutants produce several new Asn-containing CDAs not present in the wild-type, which retain calcium-dependent antimicrobial activity. This confirms that AsnO and HasP are required for the b-hydroxylation and phosphorylation of the Asn residue within CDA. INTRODUCTIONThe calcium-dependent antibiotics (CDAs) from Streptomyces coelicolor (Hojati et al., 2002;Kempter et al., 1997) belong to the group of structurally related acidic lipopetide antibiotics, which include A54145 (Fukuda et al., 1990; Miao et al., 2006), daptomycin (Baltz et al., 2005;Miao et al., 2005;Raja et al., 2003), friulimicins and amphomycins (Vértesy et al., 2000). All of these nonribosomally biosynthesized lipopeptides contain N-terminal fatty acid side chains, which is a trans-2,3-epoxyhexanoyl moiety in the case of CDA (Fig. 1), along with decapeptide lactone or lactam cores. Within the decapeptide cores are a number of conserved amino acids, including acidic residues responsible for co-ordination of calcium ions, which are essential for antimicrobial activity. Notably, daptomycin was recently approved for clinical use, becoming the first new structural class of natural antibiotic to reach the clinic in over 30 years (Baltz et al., 2005;Raja et al., 2003). As a result of this, there has been considerable interest in establishing the biosynthetic origins of the acidic lipopeptide group of antibiotics, with the specific aim of engineering new lipopeptide variants with improved therapeutic properties. To this end we have focused on engineering the biosynthesis of CDAs, using adenylation domain active site modifications to replace Asp In addition to D-HPG, CDA contains a number of other non-proteinogenic amino acids, including L-3-methylglutamic acid (3-MeGlu; at position 10), which is also present at the same relative position in the decapeptide cores of daptomycin and A54145 (Milne et al., 2006), and a Cterminal Z-dehydro...

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An Oxidative Phenol Coupling Reaction Catalyzed by OxyB, a Cytochrome P450 from the Vancomycin‐Producing Microorganism

Cited by 117 publications

References 18 publications

Phosphate-Controlled Regulator for the Biosynthesis of the Dalbavancin Precursor A40926

Phosphate-Controlled Regulator for the Biosynthesis of the Dalbavancin Precursor A40926

Chapter 19 In Vitro Studies of Phenol Coupling Enzymes Involved in Vancomycin Biosynthesis

An asparagine oxygenase (AsnO) and a 3-hydroxyasparaginyl phosphotransferase (HasP) are involved in the biosynthesis of calcium-dependent lipopeptide antibiotics

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