The Isopropylstilbene Precursor Cinnamic Acid Inhibits Anthraquinone Pigment Production by Targeting AntI

Abstract

Photorhabdus strains, Gram-negative bacteria pathogenic to insect larvae, produce two signature compounds: the multifunctional isopropylstilbene (IPS), known for its antibiotic, insecticidal, and immunosuppressive activities, and orange-to-red pigmented anthraquinones (AQs), which attenuate oxidative stress. Here, we demonstrate an inverse correlation between the production of AQs and cinnamic acid (CA), the primary precursor for IPS formation in the model strain P. laumondii TTO1. Metabolic and proteomic analyses following CA treatment show that CA inhibits AntI, a key enzyme in the final step of AQ-256 biosynthesis. The crystal structure of AntI in complex with CA reveals that cinnamic acid functions as a competitive inhibitor by inducing specific structural rearrangements in the lyase, resulting in noncovalent, reversible inhibition. These findings provide atomic insights into the intricate regulatory control of pigment biosynthesis and the production of bioactive compounds.

1

Biosynthesis pathway of (a) anthraquinone (AQ) and its shunt products (SP1–5), and (b) isopropylstilbene (IPS).

2

Extracted ion chromatograms (EICs) of AQs and AQ shunt products (SPs) from wild-type TTO1 in different culture conditions after 2 days. Asterisk means peaks of AQ-270a were 5-fold decreased in order to fit it into the chromatogram.

3

HR-LCMS analysis of the production of AQs and AQ shunt products SP1-SP5 in the absence/presence of fed CA after 2 days. Error bars indicate mean ± s.d. for four replicate samples. Statistical analysis was performed with the Bonferroni’s multiple comparisons test. Asterisk show significant difference between CA-fed and non CA-fed samples (ns = not significant, * = P ≤ 0.033, **= P ≤ 0.002, *** = P ≤ 0.001.). Figure S4 shows the same analysis after 1 day.

4

Volcano plot of the comparative proteomic analysis for TTO1, ΔstlA, and ΔstlA ΔMTs in the presence and absence of CA. Red dots indicate the proteins involved in AQ biosynthesis, AntI is highlighted with black circle. Dashed lines show cutoff values q = 0.05, and FC (fold change) = 2.

5

Binding mode of CA to AntI. a) Ribbon representation of dimeric AntI in complex with the inhibitor CA (carbon atoms in green; PDB ID 9GLF). b) The superposition of AntI^apo (gray, PDB ID 6HXA, open state) with AntI:CA complex structure (closed state) shows the inhibitor bound in a central cavity (region II). The loop connecting residues Pro281-Arg283 is rotated by 90° from black (AntI^apo) to gold (AntI:CA). c) Sliced surface representations of the closed (ligand bound) and open states of AntI with electrostatic potentials. d) Atomic view of the active site in the CA-bound complex (left panel) and ligand-free open state structure (right panel). Yellow sticks represent the aligned catalytic triad, while surrounding residues are shown in gray. Water molecules are depicted as red spheres, with H-bonds indicated by dashed lines and π-stacking interactions by semicircles. CA is shown in green. The Fo-Fc electron density map for CA and the active site residues is displayed as blue mesh and contoured at 3.0 σ.