Enzymatic tailoring of ornithine in the biosynthesis of the Rhizobium cyclic trihydroxamate siderophore vicibactin

Abstract

To acquire iron, the N(2)-fixing, symbiotic bacterium Rhizobium sp. produce the cyclic trihydroxamate siderophore vicibactin, containing a 30-membered trilactone scaffold. Herein we report the overproduction and purification of the six proteins VbsACGOLS in the bacterial host Escherichia coli and the reconstitution of the biosynthesis of vicibactin from primary metabolites. The flavoprotein VbsO acts as a pathway-initiating l-ornithine N(5)-hydroxylase, followed by VbsA, which transfers (R)-3-hydroxybutyryl- from the CoA thioester to N(5)-hydroxyornithine to yield N(5)-((R)-3-hydroxybutyryl)-N(5)-hydroxy-l-ornithine. VbsL is a PLP-dependent epimerase acting at C(2) of the 10 atom monomer unit. VbsS, a nonribosomal peptide synthetase free-standing module, then activates N(5)-((R)-3-hydroxybutyryl)-N(5)-hydroxy-d-ornithine as the AMP anhydride on the way to cyclotrimerization to the vicibactin scaffold. The last step, tris-acetylation of the C(2) amino group of desacetyl-d-vicibactin to the mature siderophore vicibactin, is catalyzed distributively by VbsC, using three molecules of acetyl-CoA.

Figure 1

A. Structures of bacterial (vicibactin and desferrioxamine E) and fungal (fusarinines) trimeric hydroxamate siderophores. B. X-ray structure of the neurosporin-Fe complex. Neurosporin, isolated from the fungus, Neurospora, is identical to vicibactin.

Figure 2

Vicibactin gene cluster and annotated protein functions. The multidomain NRPS, VbsS, is thought to be the central catalyst responsible for trimerization of the functionalized ornithine monomer. The N-terminal condensation domain (marked C*) of VbsS lacks required conserved residues in the active site and is thought to be inactive. (A = adenylation domain, T = thiolation domain, TE = thioesterase domain).

Figure 3

Characterization of VbsO as an L-ornithine N⁵-hydroxylase. (A) Schematic of the VbsO-catalyzed conversion of L-Orn to N⁵-hydroxy-L-ornithine (L-3) and proposed structure of the Fmoc-derivatized product. ESI-HRMS m/z data of the purified product resulting from incubation of VbsO with L-Orn and subsequent Fmoc-derivatization is consistent with bisFmoc-N⁵-hydroxyornithine (bisFmoc-3). (B) HPLC traces (263 nm) showing the Fmoc-derivatized products resulting from incubation of L- or D-Orn (500 μM) with VbsO (5 μM) in the presence of FAD (50 μM), NADPH (2 mM) and pH 8.0 Tris (50 mM). Peaks corresponding to bisFmoc-Orn appear at t_R 21.9 min and bisFmoc-L-3 at t_R 20.8 min. (C) Plot of initial velocity versus L-Orn concentration, with the best fit curve to the Michaelis-Menten equation. Error bars are drawn at one standard deviation.

Figure 4

Characterization of VbsA as a CoA-dependent N⁵-hydroxyornithine N-((R)-3-hydroxybutryl)- transferase. (A) Schematic of the VbsA-catalyzed transfer of (R)-3-hydroxybutyryl- from the CoA thioester (11) to N⁵ of N⁵-hydroxy-L- or D-ornithine (L- or D-3). ESI-HRMS m/z data of the purified products are consistent with N⁵-((R)-3-hydroxybutyryl)-N⁵-hydroxy-L- and D-ornithine (9) (B) HPLC traces (220 nm) showing products resulting from the incubation of 11 (1 mM) and L- or D-3 (1.2 mM) with and without VbsA (1 μM) in pH 7.5 Tris (50 mM). Peaks corresponding to L-9 appear at t_R 6.6 min, D-9 at t_R 6.8 min, CoASH at t_R 8.5 min and 11 at t_R 9.4 min. (C) Kinetic traces for the VbsA-catalyzed acyltransfer from 11 to L- or D-3, with the best-fit curve to the Michaelis-Menten equation. Error bars are drawn at one standard deviation.

Figure 5

Characterization of VbsL as a PLP-dependent epimerase. (A) Schematic of the VbsL-catalyzed racemization of N⁵-((R)-3-hydroxybutyryl)-N⁵-hydroxy-L- or D-ornithine (L- or D-9) and proposed structure of the Fmoc-derivatized products (bisFmoc-L- and D-9). (B) Chiral HPLC traces (263 nm) showing racemization of L-9 (1 mM) resulting from incubation with VbsL (500 nM), PLP (50 μM) and pH 7.75 HEPES (50 mM) followed by derivatization with Fmoc-Cl. (C) Chiral HPLC traces (263 nm) showing racemization of D-9 (1 mM) resulting from incubation with VbsL (500 nM), PLP (50 μM) and pH 7.75 HEPES (50 mM) followed by derivatization with Fmoc-Cl. Peaks corresponding to bisFmoc-D-9 appear at t_R 5.4 min and bisFmoc-L-9 at t_R 7.6 min. Peak at t_R 6.4 min is an artifact of Fmoc-Cl decomposition in aqueous solution.

Figure 6

Relative activities of the VbsS adenylation (A) domain with L- and D-Orn, L- and D-3 and L- and D-9 as assessed by ATP-[³²P]PPi exchange assays.

Figure 7

Characterization of VbsC as a CoA-dependent desacetyl-D-vicibactin N²-acetyltransferase. (A) Schematic of the VbsC-catalyzed, tri-acetylation of desacetyl-D-vicibactin (D-14) with 3 equivalents of acetyl-CoA and ESI-HRMS m/z data of purified products. (B) HPLC traces (220 nm) showing the time-dependent appearance of mono-, di- and tri-acetylated products resulting from the incubation of D-14 (1 mM) and acetyl-CoA (4 mM) with VbsC (10 μM) in pH 7.75 HEPES (50 mM). Peaks corresponding to CoASH appear at t_R 8.1 min, Acetyl-CoA at t_R 9.1 min, D-14 at t_R 10.2 min, monoacetylated D-15 at t_R 11.0 min, diacetylated D-16 at t_R 11.9 min and VB at t_R 12.8 min.

Figure 8

Logic of vicibactin biosynthesis involves complete functionalization of the Orn-based monomer prior to VbsS catalyzed desacetylvicibactin assembly. Selection for the C²-D hydroxamate (D-9) monomer is maintained by the adenylation (A) domain of VbsS. Tailoring catalyzed by VbsC leads to the fully-functionalized vicibactin siderophore scaffold. (NOH = amine N-hydroxylase, AT = acyltransferase, E = epimerase)

Scheme 1

Synthesis of hydroxylamines L- and D-3. Yields shown in parentheses.

Scheme 2

Synthesis of hydroxamates D- and L-9. Yields shown in parentheses.