Biosynthesis of Argolaphos Illuminates the Unusual Biochemical Origins of Aminomethylphosphonate and N^ε-Hydroxyarginine Containing Natural Products

Abstract

Phosphonate natural products have a history of successful application in medicine and biotechnology due to their ability to inhibit essential cellular pathways. This has inspired efforts to discover phosphonate natural products by prioritizing microbial strains whose genomes encode uncharacterized biosynthetic gene clusters (BGCs). Thus, success in genome mining is dependent on establishing the fundamental principles underlying the biosynthesis of inhibitory chemical moieties to facilitate accurate prediction of BGCs and the bioactivities of their products. Here, we report the complete biosynthetic pathway for the argolaphos phosphonopeptides. We uncovered the biochemical origins of aminomethylphosphonate (AMPn) and Nε-hydroxyarginine, two noncanonical amino acids integral to the antimicrobial function of argolaphos. Critical to this pathway were dehydrogenase and transaminase enzymes dedicated to the conversion of hydroxymethylphosphonate to AMPn. The interconnected activities of both enzymes provided a solution to overcome unfavorable energetics, empower cofactor regeneration, and mediate intermediate toxicity during these transformations. Sequential ligation of l-arginine and l-valine was afforded by two GCN5-related N-acetyltransferases in a tRNA-dependent manner. AglA was revealed to be an unusual heme-dependent monooxygenase that hydroxylated the Nε position of AMPn-Arg. As the first biochemically characterized member of the YqcI/YcgG protein family, AglA enlightens the potential functions of this elusive group, which remains biochemically distinct from the well-established P450 monooxygenases. The widespread distribution of AMPn and YqcI/YcgG genes among actinobacterial genomes suggests their involvement in diverse metabolic pathways and cellular functions. Our findings illuminate new paradigms in natural product biosynthesis and realize a significant trove of AmPn and Nε-hydroxyarginine natural products that await discovery.

C-Diazeniumdiolate Graminine in the Siderophore Gramibactin Is Photoreactive and Originates from Arginine

“…In the BGC for Gbt (Figure ), the enzymes GrbE and GrbD have been identified through gene deletion studies to be essential for the formation of Gra . GrbE shares sequence homology to several reported arginine hydroxylases, including AglA/AlpD and Mhr24, which are N δ -hydroxylases, − and DcsA, which is a N ω -hydroxylase (Figure S1). GrbD shares sequence homology to only the cupin domain in SznF (Figure S1).…”

Tistrellabactins A and B Are Photoreactive C-Diazeniumdiolate Siderophores from the Marine-Derived Strain Tistrella mobilis KA081020-065

“…The biosynthetic genes encoding the enzymes that synthesize l -Gra have been identified in the biosynthetic gene cluster (BGC) for gramibactin as grbD and grbE through gene knockout studies with the strain P. graminis DSM 17151. 15 GrbE shares sequence homology to several Arg hydroxylases (DcsA, 23 Mhr24, 24 AglA 25 ), while GrbD shares sequence homology to the cupin domain of the N–N bond, forming enzyme SznF 7 /StzF 4 , which carries out the oxidative rearrangement of N δ -hydroxy- N ω -hydroxy- N ω -methyl- l -Arg. Targeted discovery of new C -diazeniumdiolate siderophores is achievable by searching microbial genomes containing a nonribosomal peptide synthetase (NRPS) adenylation (A) domain with the specificity code for Gra (DVHRTGLVAK) and FASTA sequences of grbD and grbE as queries.…”

Elucidation of chalkophomycin biosynthesis revealsN-hydroxypyrrole-forming enzymes

“…Recently, two members of this uncharacterized protein family, AglA and GntA, were shown to be heme-dependent guanidine N-oxygenases in argolaphos and guanitoxin biosynthesis, respectively (Figure 7A). 54,55 Additionally, DcsA, a related enzyme required for Dcycloserine biosynthesis was found to bind heme, although its activity could not be confirmed in vitro. 56 Although ChmN only shares 31% aa ID with AglA and 22% aa ID with GntA, we hypothesized that it could catalyze N-oxygenation of L-arginine, paralleling the transformation performed by SznF's HDO domain.…”