Biosynthesis of fluopsin C, a copper-containing antibiotic from Pseudomonas aeruginosa

Abstract

Metal-binding natural products contribute to metal acquisition and bacterial virulence, but their roles in metal stress response are underexplored. We show that a five-enzyme pathway in Pseudomonas aeruginosa synthesizes a small-molecule copper complex, fluopsin C, in response to elevated copper concentrations. Fluopsin C is a broad-spectrum antibiotic that contains a copper ion chelated by two minimal thiohydroxamates. Biosynthesis of the thiohydroxamate begins with cysteine and requires two lyases, two iron-dependent enzymes, and a methyltransferase. The iron-dependent enzymes remove the carboxyl group and the α carbon from cysteine through decarboxylation, N-hydroxylation, and methylene excision. Conservation of the pathway in P. aeruginosa and other bacteria suggests a common role for fluopsin C in the copper stress response, which involves fusing copper into an antibiotic against other microbes.

Fig. 1.. Identification of the fluopsin C gene cluster (flc).

(A) The flc cluster (PA3515–PA3519) in P. aeruginosa PAO1 is located in a copper island regulated by CueR. PA3521–PA3523 encodes an efflux pump (MexPQ-OpmE). (B) Genes flcA to flcE in PAO1 are essential for fluopsin C production in CuSO₄-containing media. Extracted ion chromatograms (EICs) of fluopsin C (1) (m/z 152.9304 [M–C₂H₄NOS]⁺) are shown. (C) Structure of fluopsin C. (D) X-band room temperature EPR spectra of fluopsin C in toluene show the cis isomer (g_iso = 2.0900, A_iso(⁶³Cu) = 259 MHz) and trans isomer (g_iso = 2.0808, A_iso(⁶³Cu) = 247 MHz) with approximately equal concentrations. g_iso, isotropic g value; A_iso, isotropic A value; Exp., experimental; Sim., simulated. (E) X-band frozen-solution EPR spectra of fluopsin C in toluene at 15 K show different electronic structures for the cis isomer (g = [2.151, 2.064, 2.064], A(⁶³Cu) = [580, 173, 173] MHz) and trans isomer (g = [2.166, 2.036, 2.036], A(⁶³Cu) = [583, 108, 108] MHz) in ~1:6 ratio. (F) Q-band electron spin-echo–detected field-swept EPR spectrum confirms 1:6 ratio of cis:trans. (G) Q-band variable mixing time (VMT) Mims-ENDOR spectra of [¹⁵N,¹³C₂]-fluopsin C in toluene at the magnetic field position (1198.0 mT) with contribution from only the trans isomer. tmix, mixing time.

Fig. 2.. FlcB catalyzes S-succinylation of cysteine, and FlcE performs oxidative decarboxylation.

(A) Feeding studies in P. aeruginosa PAO1 using [¹⁵N,¹³C₅] l-methionine or [¹⁵N,¹³C₃] l-cysteine. Mass spectra of fluopsin C with a single ligand are shown. Positions of isotopic labeling are inferred. (B) EICs of 2 (m/z 238.0380 [M+H]⁺) from the FlcB reaction and controls. A small amount of racemic 2 forms nonenzymatically. (C) FlcE converts 2 to 3 in an Fe(II)-dependent manner. EICs of 3 (m/z 230.0094 [M+Na]⁺) are shown. (D) NMR spectrum of 3 in deuterated dimethylsulfoxide supports an oxime structure that exists in two diastereomers. HMBC, heteronuclear multiple bond correlation; ppm, parts per million.

Fig. 3.. FlcD excises a methylene carbon from 3 as formate.

(A) FlcD-catalyzed formation of 4 (m/z 194.0118 [M+H]⁺) depends on Fe(II). (B) Mass spectra show that the 3-¹³C atom of [3-¹³C] l-cysteine is retained in 4 in a one-pot reaction containing FlcB, FlcE, and FlcD. (C) NMR analysis of the FlcD reaction in D₂O reveals the production of formate and 4 in a 1:1 stoichiometry.

Fig. 4.. FlcC and FlcA catalyze the final steps of fluopsin C biosynthesis.

(A) NMR time-course study of the lyase FlcC. (B) FlcA and FlcC catalyze tandem transformations of 4 to fluopsin C, with addition of CuSO₄ at the end of the reaction. Combined EICs of 1 (m/z 152.9304 [M–C₂H₄NOS]⁺ and 265.9215 [M+Na]⁺) are shown. (C) Complete fluopsin C biosynthetic pathway. SAH, S-adenosyl-l-homocysteine.