Structure of a putative terminal amidation domain in natural product biosynthesis

Abstract

Bacteria are rich sources of pharmaceutically valuable natural products, many crafted by modular polyketide synthases (PKS) and non-ribosomal peptide synthetases (NRPS). PKS and NRPS systems typically contain a thioesterase (TE) to offload a linear or cyclized product from a carrier protein, but alternative chemistry is needed for products with a terminal amide. Several pathways with amidated products also possess an uncharacterized 400-amino acid terminal domain. We present the characterization and structure of this putative terminal amidation domain (TAD). TAD binds NAD with the nicotinamide near an invariant cysteine that is also accessible to an intermediate on a carrier protein, indicating a catalytic role. The TAD structure resembles cyanobacterial acyl-ACP reductase (AAR), which binds NADPH near an analogous catalytic cysteine. Bioinformatic analysis reveals that TADs are broadly distributed across bacterial phyla and often occur at the end of terminal NRPS modules, suggesting many amidated products may yet be discovered.

Keywords: biosynthesis; crosslinking; crystallography; genome mining; mass spectrometry; natural products.

Figure 1.

Terminal amide natural products. A) Final modules of carmabin A^, (genome accession number: GCA_001942475.1), vatiamide E (GCA_010671925.1), and hectoramide B (GCA_001854205.2) biosynthesis. Each TAD may release terminally amidated products. Blue atoms and bonds indicate the monomer installed by the terminal module, and red atoms indicate the offloaded amide. C: condensation; A: adenylation; PCP: peptidyl carrier protein; MT: methyltransferase. B) Chemical structures of “orphan” C-terminally amidated molecules of interest that have no defined BGC.

Figure 2.

CarI TAD homologs identified in diverse bacteria. A) Sequence similarity network (SSN)^, for proteins related to CarI TAD. Lines connect sequence nodes with high identity (approx. 65%). Clusters defined in this manner are separately colored, with cyanobacterial sequences highlighted in yellow. B) Genome neighborhoods for representative examples of the five largest clusters. Many TAD homologs are at the C-terminus of an NRPS module, but those in Cluster 4 are discrete proteins. UniProt IDs are displayed for each TAD homolog; the CarI TAD represents cluster 2.

Figure 3.

Crystal structure of the CarI TAD. A) TAD monomer with cyan NTD and magenta CTD. NAD is bound in a cleft between the two lobes, near putative catalytic Cys2238. B) Details NAD binding site. Cys2238 is near the hydride-reactive nicotinamide C4 atom. Black dashed lines indicate hydrogen bonds. C) Domain schematic for the TAD region of CarI. The PCP is separated from the TAD by a 26-residue linker. The CTD is interrupted by a 32-residue excursion into the NTD.

Figure 4.

Interaction of ligands with the CarI TAD. A) Thermal shift assay (TSA) showing the impact of potential ligands on CarI TAD stability. Notably, NAD(H), but not NADP(H), stabilizes the TAD and increases its melting temperature (T_m) substantially. Ribbons indicate the standard error of four measurements. B,C) Fluorescence spectra with an excitation wavelength of 280 nm. Increasing concentrations (from 0.18 uM in light blue to 3 mM in dark blue) of NAD (B) or NADH (C) decrease fluorescence emission at 340 nm. D,E) Binding curves for the interaction of CarI TAD and NAD (440 ± 50 μM) (D) and NADH (85 ± 7 μM) (E). Error bars represent standard error of three replicates.

Figure 5.

Comparison of the CarI TAD and S. elongatus PCC 7942 AAR (green, PDB 6JZY) structures. A) Superposition of N-terminal domains; RMSD = 3.2 Å for 79 Cα atoms. These domains have only 9% sequence identity. B) Superposition of C-terminal domains; RMSD = 1.6 Å for 120 Cα atoms. The NAD⁺ (TAD, yellow) and NADPH (AAR, salmon) cofactors are similarly bound. The TAD and AAR CTDs are 19% identical. Compared to the NTDs, this higher identity is reflected in the greater structural similarity (3.2 Å vs. 1.6 Å RMSD). C) Schematic of TAD and AAR sequences. Regions of AAR that do not align structurally with TAD are indicated by dashed lines. AAR residues between 270 and 290 do not form an excursion into the NTD. The analogous conserved TAD Cys2238 and AAR Cys294 are indicated as well as the conserved GxGxxG “P loop” for cofactor phosphate binding. The conserved TAD Asp2102 and Asp2170 are not aspartates in AAR.

Figure 6.

Crosslinking of the PCP Ppant and TAD Cys2238. A) SDS-PAGE gel of CarI PCP, TAD, PCP-TAD didomain, or PCP and TAD proteins incubated with and without BMOE. B) Cys2238 central location in the cleft between TAD NTD (cyan) and CTD (magenta). This residue is adjacent to a pocket that may accommodate the waiting substrate and the tunnel (arrow) where NAD(H) binds. C) Deconvoluted mass spectrum of intact CarI PCP and TAD proteins following incubation with (blue) and without (red) BMOE. Cysteine, added to quench the crosslinking reaction, was detected as a cap to several BMOE modification sites. See Figure S9 for details. D) Detection of a crosslinked peptide consisting of TAD residues 2234–2240 and Ppant-PCP 1830–1841 linked by BMOE. The MS/MS spectrum includes a Ppant ejection fragment linked to the TAD peptide containing Cys2238. The first isotopic peaks for important ions are highlighted. See Table S3 for details. E) Chemical structure of the crosslink between chymotryptic fragments of TAD Cys2238 and the Ppant-PCP thiol.

Figure 7.

PCP-TAD crosslinking ability with mutagenized TAD proteins. A) SDS-PAGE gel of crosslinked PCP and mutagenized TADs. Both Cys2238 and Cys2258 participate in PCP crosslinking, but the effect from Cys2238 is dominant. B) Cartoon representation of the CarI TAD with both cysteines highlighted. Cys2258 is on the periphery of the protein and is an off-target crosslinking hit. C) Deconvoluted mass spectrum of PCP-TAD crosslinking experiments with (blue) and without (red) BMOE. Mutagenesis of Cys2238 results in substantial decrease of PCP-TAD crosslink formation. Datasets are scaled to the highest peak of each spectrum. PCP-TAD crosslinking ability with mutagenized TAD proteins. See Figure S9 for details.

Figure 8.

Competing Ppant and NAD interaction with TAD. A) BMOE crosslinking of CarI PCP and CarI TAD C2258S. NADH prevented PCP-Ppant access to Cys2238. B) Co-crystal structure of Ppant mimic dPCoA with the CarI TAD. The Ppant formed a disulfide with Cys2238 (Polder density contoured at 3σ). C) Detail at the ligand site for superposition of TAD:NAD (cyan, dark magenta) and TAD:dPCoA (light cyan, light pink) complexes (RMSD = 0.2 Å for the full subunits).

Figure 9.

Proposed reaction schemes for TAD activity. After transthioesterification of the substrate to the TAD Cys2238, a terminal amide may be formed by ammonia attack on the peptidyl-S-enzyme thioester. Ammonia may be generated by (1) NAD⁺-dependent oxidation of an amino acid, (2) salvaged from the NAD amide, (3) produced by an unknown hydrolytic enzyme, or (4) supplied exogenously. (R₁: side chain of the C-terminal amino acid of the natural product intermediate, R₂: remainder of the intermediate, wavy line: Ppant).