Enzymatic Cβ-H Functionalization of l-Arg and l-Leu in Nonribosomally Derived Peptidyl Natural Products: A Tale of Two Oxidoreductases

Abstract

Muraymycins are peptidyl nucleoside antibiotics that contain two C_β-modified amino acids, (2S,3S)-capreomycidine and (2S,3S)-β-OH-Leu. The former is also a component of chymostatins, which are aldehyde-containing peptidic protease inhibitors that─like muraymycin─are derived from nonribosomal peptide synthetases (NRPSs). Using feeding experiments and in vitro characterization of 12 recombinant proteins, the biosynthetic mechanism for both nonproteinogenic amino acids is now defined. The formation of (2S,3S)-capreomycidine is shown to involve an FAD-dependent dehydrogenase:cyclase that requires an NRPS-bound pathway intermediate as a substrate. This cryptic dehydrogenation strategy is both temporally and mechanistically distinct in comparison to the biosynthesis of other capreomycidine diastereomers, which has previously been shown to proceed by C_β-hydroxylation of free l-Arg catalyzed by a member of the nonheme Fe²⁺- and α-ketoglutarate (αKG)-dependent dioxygenase family and (eventually) a dehydration-mediated cyclization process catalyzed by a distinct enzyme(s). Contrary to our initial expectation, the sole nonheme Fe²⁺- and αKG-dependent dioxygenase candidate Mur15 encoded within the muraymycin gene cluster is instead demonstrated to catalyze specific C_β hydroxylation of the Leu residue to generate (2S,3S)-β-OH-Leu that is found in most muraymycin congeners. Importantly, and in contrast to known l-Arg-C_β-hydroxylases, the Mur15-catalyzed reaction occurs after the NRPS-mediated assembly of the peptide scaffold. This late-stage functionalization affords the opportunity to exploit Mur15 as a biocatalyst, proof of concept of which is provided.

Figure 1.

Structures of representative 1-containing and related natural products. (a) The structure of the (2S,3S)-1-containing muraymycins and differences between the series. (b) The structure of representative chymostatins that contain (2S,3S)-1 and representative analogues that contain unmodified l-Arg instead of (2S,3S)-1. (c) The mostly established (viomycin) and proposed (faulknamycin) biosynthetic pathways leading to the respective diastereomers, (2S,3R)-1 and (2R,3S)-1. Whether amide bond formation with the upstream amino acid occurs prior to (R = H) or after (R = Val) conjugate addition during faulknamycin biosynthesis is unknown.

Figure 2.

MS analysis of (2S,3S)-1-containing natural products following isotope enrichment with Arg isotopologues. (a) HR-MS of 3 following feeding of the producing strain with l-[D₇,¹⁵N₄]Arg or with l-[D₇]Arg. The expected (M + H)⁺ parent ions are m/z = 930.4533 for 3; m/z = 939.4728 for [D₅,¹⁵N₄]3; m/z = 940.4698 for [D₅,¹⁵N₅]3, and m/z = 935.4846 for [D₅]3. (b) (+)-HR-ESI-MS/MS of natural abundance 3 (top) and [D₅]3 following feeding with l-[D₇]Arg. The expected (M + H)⁺ fragment ions are m/z = 98.0718 for 3 and m/z = 103.1027 for [D₅]3. (c) (+)-HR-ESI-MS of 6 and 7 following feeding with l-[D₇]Arg. The expected (M + H)⁺ parent ions are m/z = 608.3197 for 6; m/z = 613.3510 for [D₅]6; m/z = 594.3040 for 7; and m/z = 599.3354 for [D₅]7. (d) (+)-HR-ESI-MS/MS of natural abundance 7 (top) and [D₅]7 following feeding with l-[D₇]Arg.

Figure 3.

Muraymycin NRPS A domain specificity. Relative activity of (a) Mur12, (b) Mur21, and (c) Mur27 with representative amino acids. Counts were processed by subtracting the background prior to calculating the relative activity. The proposed reaction catalyzed by the respective NRPS is shown.

Figure 4.

Characterization of Mur15. (a) The Mur15-catalyzed reaction. (b) HPLC traces of reactions with 3 and (i) the exclusion of Mur15, (ii) all components, and (iii) all components co-injected with authentic 8. (c) Single-substrate kinetic analysis with variable 3. (d) Structure of the synthetic deaminoribose-3 analogue and HPLC traces of Mur15-catalyzed reactions with (i) the exclusion of Mur15 and (ii) all components.

Figure 5.

Characterization of AnpI. (a) Reactions catalyzed by the indicated NRPS and AnpI, and hydrolysis of the product to generate 11 and 12, respectively. (b) HPLC traces detecting 11 from the indicated enzymes using natural abundance l-Phe, l-Arg, and NaHCO₃ (bottom two traces) or substituting the indicated isotopically labelled substrate for the natural abundance counterpart. Data were collected using the indicated mass with m/z expansion set to ± 100 ppm. The limit of detection for 11 was estimated to be 200 fmol based on a signal-to-noise ratio ≥ 3 for the EIC. This value corresponds to a concentration of 20 nM (10 mL injection) from a 50 mL reaction. (c) HPLC traces detecting 12 from the indicated enzymes using natural abundance l-Phe, l-Arg, and NaHCO₃ (bottom two traces) or substituting the indicated isotopically labelled substrate for the natural abundance counterpart. Data were collected using the indicated mass with m/z expansion set to ± 100 ppm. The limit of detection for 12 was estimated to be 200 fmol based on a signal-to-noise ratio ≥ 3 for the EIC. This value corresponds to a concentration of 20 nM (10 mL injection) from a 50 mL reaction.

Scheme 1.

Biosynthesis of the peptide component of muraymycins and chymostatins and the two oxidative transformations characterized in this study.