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

Abstract

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...

A family of diiron monooxygenases catalyzing amino acid beta-hydroxylation in antibiotic biosynthesis

“…One commonly encountered modification is the formation of β-hydroxy amino acids. The resulting hydroxylated amino acid is more hydrophilic than its precursor and often participates in secondary reactions (2), including further oxidation (3), glycosylation (4,5), methylation (6), phosphorylation (7), and macrolactonization during antibiotic heterocyclization (8). Many of the nonribosomal peptides that are modified in this fashion are mainstays of the pharmaceutical industry for use as chemotherapeutics and antimicrobial agents, such as the widely used antitumor drug bleomycin and the teicoplanin group of glycopeptide antibiotics (5).…”

“…Amino acid β-hydroxylation in natural product biosynthetic pathways has been shown to be catalyzed by either nonheme iron-containing alpha-ketoglutarate (αKG)-dependent dioxygenase enzymes (7,9) or cytochrome P450 monooxygenases (10). The high C-H bond strength at the β-carbon of the amino acid (∼85 kcal∕mol) necessitates the formation of a potent oxidant, which is accommodated by the formation of activated and highly reactive metal-oxygen adducts by these enzymes, Fe IV -oxo (11) and Fe IV -oxo heme π-cation radical (reviewed in refs.…”

Biosynthesis and biosynthetic engineering of calcium-dependent lipopeptide antibiotics

“…Downsteam of asnO is another gene that encodes a protein similar to a number of ATP-dependent phosphotransferases, which was suggested to phosphorylate the CDA 3-OHAsn residue. As predicted, deletion of the AsnO encoding gene results in a mutant strain (∆asnO), which produces new CDA variants possessing exclusively Asn residues at position 9 (CDA5b, 5a, 6a) [9]. Similarly, deletion of the putative phosphotransferase encoding gene resulted in a mutant (∆hasP) which, when grown under conditions that favor phosphorylation of CDA, produces exclusively non-phosphorylated CDAs.…”

“…Similarly, deletion of the putative phosphotransferase encoding gene resulted in a mutant (∆hasP) which, when grown under conditions that favor phosphorylation of CDA, produces exclusively non-phosphorylated CDAs. In addition, Strieker et al overproduced AsnO and have shown that the enzyme is a nonhaem Fe(II)/2-oxoglutarate-dependent oxygenase that β-hydroxylates free Asn [10], as we had earlier proposed [9]. Currently, it is suggested that hydroxylation of Asn occurs prior to activation and incorporation into the CDA lipopeptide core and that phosphorylation is most likely to occur during or after lipopeptide assembly (Fig.…”

“…The first, asnO, encodes a protein similar to the Fe(II)/2-oxoglutarate-dependent oxygenase clavaminate synthase (CAS) which catalyzes β-hydroxylation of an Arg derivative during the biosynthesis of clavulanic acid. On this basis, we suggested that AsnO is an asparagine oxygenase that β-hydroxylates the Asn residue of CDA [9]. Downsteam of asnO is another gene that encodes a protein similar to a number of ATP-dependent phosphotransferases, which was suggested to phosphorylate the CDA 3-OHAsn residue.…”