ABSTRACT
Cystobactamids are myxobacteria-derived topoisomerase inhibitors with potent anti-Gram-negative activity. They are formed by a non-ribosomal peptide synthetase (NRPS) and consist of tailored para-aminobenzoic acids, connected by a unique α-methoxy-L-isoasparagine or a ß-methoxy-L-asparagine linker moiety. We describe the heterologous expression of the cystobactamid biosynthetic gene cluster (BGC) in Myxococcus xanthus. Targeted gene deletions produce several unnatural cystobactamids. Using in vitro experiments, we reconstitute the key biosynthetic steps of linker formation and shuttling via CysB to the NRPS. The biosynthetic logic involves a previously uncharacterized bifunctional domain found in the stand-alone NRPS module CysH, albicidin biosynthesis and numerous BGCs of unknown natural products. This domain performs either an aminomutase (AM) or an amide dehydratase (DH) type of reaction, depending on the activity of CysJ which hydroxylates CysH-bound L-asparagine. Furthermore, CysQ O-methylates hydroxyl-L-(iso)asparagine only in the presence of the AMDH domain. Taken together, these findings provide direct evidence for unique steps in cystobactamid biosynthesis.
Subject(s)
Amides/metabolism , Anti-Bacterial Agents/biosynthesis , Amides/chemistry , Asparagine/metabolism , Biosynthetic Pathways , Hydroxylation , Models, Biological , Molecular Weight , Myxococcus xanthus/metabolism , Substrate SpecificityABSTRACT
B12-dependent radical SAM enzymes that can perform methylations on sp3 carbon centers are important for functional diversity and regulation of biological activity in several nonribosomal peptides. Detailed studies on these enzymes are hindered by the complexity of the substrates and low levels of expression of active enzymes. CysS can catalyze iterative methylations of a methoxybenzene moiety during the biosynthesis of the cystobactamids. Here, we describe the overexpression, purification, substrate identification, and mechanism of this enzyme.
Subject(s)
Bacterial Proteins/metabolism , Enzyme Assays/methods , Methyltransferases/metabolism , Nitro Compounds/metabolism , Anisoles/metabolism , Bacterial Proteins/isolation & purification , Biocatalysis , Biosynthetic Pathways , Esters/metabolism , Free Radicals/metabolism , Methylation , Methyltransferases/isolation & purification , Myxococcales , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , S-Adenosylmethionine/metabolism , Vitamin B 12/metabolismABSTRACT
The biosynthesis of branched alkoxy groups, such as the unique t-butyl group found in a variety of natural products, is still poorly understood. Recently, cystobactamids were isolated and identified from Cystobacter sp as novel antibacterials. These metabolites contain an isopropyl group proposed to be formed using CysS, a cobalamin-dependent radical S-adenosylmethionine (SAM) methyltransferase. Here, we reconstitute the CysS-catalyzed reaction, on p-aminobenzoate thioester substrates, and demonstrate that it not only catalyzes sequential methylations of a methyl group to form ethyl and isopropyl groups but remarkably also sec-butyl and t-butyl groups. To our knowledge, this is the first in vitro reconstitution of a cobalamin-dependent radical SAM enzyme catalyzing the conversion of a methyl group to a t-butyl group.
