Discovery of a metagenome-derived enzyme that produces branched-chain acyl-(acyl-carrier-protein)s from branched-chain α-keto acids.

Clones that confer the ability to produce N-acyl amino acids onto model cultured bacterial hosts are frequently identified in activity-based screens of soil DNA libraries. The N-acyl amino acids encoded by these clones are synthesized from acyl-(acylcarrier-protein)s (acyl-ACPs) and amino acids by a diverse group of bacterial enzymes referred to as N-acyl amino acid synthases (NASs). 2] The acyl-ACP substrates of NASs are common intermediates in the de novo synthesis of fatty acids by the type-II fatty acid synthase system (FAS II). In addition to N-acyl amino acids, FAS II-derived acyl-ACPs also serve as acyl donors in the production of N-acyl homoserine lactone autoinducers, the cofactor lipoic acid, and several lipopeptide antibiotics (Scheme 1A, compounds 1–4). During a recent screen of soil metagenomic libraries for clones exhibiting antimicrobial activity, we uncovered an NAS-containing clone (EC5) that confers the ability to produce long-chain N-acyl-phenylalanines and N-acyl-tryptophans onto multiple Gram-negative host species. The NAS-encoding gene found on clone EC5, nasA, is the first gene in a predicted two-gene operon, nasAB (GenBank accession no. GQ869383). The second gene in this operon, nasB, encodes a putative 1118-residue protein that contains six well-characterized Pfam homology domains (Scheme 1B). Based on generic functional predictions for these domains, we hypothesized that this enzyme was likely to be involved in the formation of ACP-linked fatty acids similar to those produced by bacterial FAS II systems. Here we report results from a series of heterologous-expression experiments in Burkholderia graminis C4D1M that show that NasB functions to provide branched-chain acyl-ACPs for NasA. Many bacteria, particularly Gram-negative species, do not naturally produce branched-chain fatty acids. NasB and its homologues are likely used by bacteria with straight-chain specific FAS II systems to generate branched-chain acyl-ACP substrates and should be useful as tools for synthesizing these substrates in model heterologous expression systems in which they are not natively produced (e.g. , E. coli). The N-terminal region of NasB contains the same set of domains as are found in the E1a and E1b subunits of 2-oxo acid dehydrogenase complexes (e.g. , the pyruvate dehydrogenase complex, which converts pyruvate to acetyl-CoA). Following these domains is an E2-like (E2*) lipoyl attachment site. Although lipoyl attachment sites are found in the E2 subunits of 2-oxo acid dehydrogenase complexes, NasB lacks the catalytic lipoamide acyltransferase domain found at the C terminus of canonical E2 subunit proteins (Pfam PF00198; exchanges the acyl-lipoate thioester with CoA, yielding a soluble acyl-CoA). Instead, the C-terminal portion of NasB contains both of the protein domains that are found in the E. coli FabH protein, a bketoacyl-ACP synthase III (KASIII)-type enzyme that performs the initial condensation reaction of FAS II (acetyl-CoA and malonyl-ACP, forming acetoacetyl-ACP). To test our original hypothesis that NasB provides substrates for the biosynthesis of N-acyl amino acids, the nasA gene was subcloned alone and in combination with nasB (i.e. , the nasAB operon) into the broad host range vector pJWC1, and these constructs were then electroporated into B. graminis for heterologous expression studies. Ethyl acetate extracts from cultures of B. graminis were examined by HPLC-MS for differences Scheme 1. A) Incorporation of FAS II intermediates (acyl-ACPs) into secondary metabolites and cofactors : N-dodecanoyl-phenylalanine (1), N-octanoyl homoserine lactone (2), enzyme-bound lipoic acid (3), and calcium-dependent antibiotic (CDA) (4). B) The domain architecture of NasB includes all three Pfam homology domains found in the E1a/E1b subunits of 2-oxo acid dehydrogenase complexes, as well as both of the Pfam homology domains found in the E. coli FabH protein (b-ketoacyl-ACP synthase III). Separating these two sets of domains is an E2-like (E2*) lipoyl-attachment site.