An Enzymatic Pathway for the Biosynthesis of the Formylhydroxyornithine Required for Rhodochelin Iron Coordination

Rhodochelin, a mixed catecholate–hydroxamate type siderophore isolated from <i>Rhodococcus jostii</i> RHA1, holds two l-δ-<i>N</i>-formyl-δ-<i>N</i>-hydroxyornithine (l-fhOrn) moieties essential for proper iron coordination. Previously, bioinformatic and genetic analysis proposed <i>rmo</i> and <i>rft</i> as the genes required for the tailoring of the l-ornithine (l-Orn) precursor [Bosello, M. (2011) <i>J. Am. Chem. Soc.</i> <i>133</i>, 4587–4595]. In order to investigate if both Rmo and Rft constitute a pathway for l-fhOrn biosynthesis, the enzymes were heterologously produced and assayed <i>in vitro</i>. In the presence of molecular oxygen, NADPH and FAD, Rmo monooxygenase was able to convert l-Orn into l-δ-<i>N</i>-hydroxyornithine (l-hOrn). As confirmed in a coupled reaction assay, this hydroxylated intermediate serves as a substrate for the subsequent <i>N</i>¹⁰-formyl-tetrahydrofolate-dependent (<i>N</i>¹⁰-fH₄F) Rtf-catalyzed formylation reaction, establishing a route for the l-fhOrn biosynthesis, prior to its incorporation by the NRPS assembly line. It is of particular interest that a major improvement to this study has been reached with the use of an alternative approach to the chemoenzymatic FolD-dependent <i>N</i>¹⁰-fH₄F conversion, also rescuing the previously inactive CchA, the Rft-homologue in coelichelin assembly line [Buchenau, B. (2004) <i>Arch. Microbiol.</i> <i>182</i>, 313–325; Pohlmann, V. (2008) <i>Org. Biomol. Chem.</i> <i>6</i>, 1843–1848]

Structural Characterization of the Heterobactin Siderophores from <i>Rhodococcus erythropolis</i> PR4 and Elucidation of Their Biosynthetic Machinery

In this study, the isolation, the structural characterization, and the elucidation of the biosynthetic origin of heterobactins, catecholate-hydroxamate mixed-type siderophores from <i>Rhodococcus erythropolis</i> PR4, are reported. The structure elucidation of heterobactin A was accomplished via MS^<i>n</i> analysis and NMR spectroscopy and revealed the noteworthy presence of a peptide bond between the guanidine group of an arginine residue and a 2,3-dihydroxybenzoate moiety. The two heterobactin S1 and S2 variants are derivatives of heterobactin A that have sulfonation modifications on the aromatic rings. The bioinformatic analysis of the <i>R. erythropolis</i> PR4 genome and the subsequent genetic and biochemical characterization of the putative biosynthetic machinery identified the gene cluster responsible for the biosynthesis of the heterobactins. Interestingly, the HtbG NRPS presents an unprecedented C-PCP-A domain organization within the second module of the synthetase that may help the correct elongation of the peptide intermediate. Finally, the present work revises the structure of heterobactin A that was described by Carrano et al. in 2001

List of primers used in the study.

<p>F corresponds to forward primer and R to reverse primer. Restriction sites are underlined.</p

Human sera specifically recognize SAP.

<p>(A) Immuno-reactivity of human sera towards recombinant SAP(H+L) and SAP(L). The data were obtained by ELISA and represent the mean±SD of 4 human sera. (B) Results of competitive-inhibition ELISA demonstrating antigenic specificity of human antibodies reacting with plates coated with recombinant SAP. Percent inhibition of binding of human serum by each inhibiting antigen was determined by comparison of absorbance at 492 nm in the presence and absence of inhibitor. White circle labels indicate the mean±SD of the % inhibition by SAP of 4 human sera. Black square labels indicate % inhibition by an unrelated GBS protein (AP-1).</p

Exposure of SAP on bacterial surface in the presence of different carbohydrates.

*<p>Numbers indicate the delta mean of fluorescence relative to bacteria incubated with a SAP immune serum versus bacteria incubated with a pre-immune serum.</p

The capacity of GBS to grown in pullulan and glycogen depends on SAP expression.

<p>The graphs represent the growth curves relative to GBS COH1 wild type strain and COH1Δsap mutant strain grown in complex medium alone or with the addition of glucose (A), maltose (B), pullulan (C) and glycogen (D). White circles indicate the COH1 wild type strain incubated in the presence of sugars, while white squares the same strain incubated in complex medium alone. Black circles represent the COH1Δsap strain grown in complex medium supplemented with sugars, while black squares are relative to the same strain grown in complex medium alone. A typical experiment, out of 4 performed giving identical results, is shown. (E–F) Comparison of CFU/ml recovered after growing GBS COH1 wild type and COH1Δsap for 3 h in the presence of pullulan (E) or glycogen (F). The data are the mean of 3 independent experiments ± SD. The asterisk indicates a significant difference between values (p<0,01).</p

In vivo SAP protein expression is modulated by the presence of α-glucans.

<p>(A) RT-PCR and WB analysis of SAP expression in COH1 wt strain and COH1Δsap strain grown in the presence of different sugars. Peptidoglycan-associated protein fraction was separated by 10% (w/v) SDS-PAGE. Blots were overlaid with a mouse anti-SAP polyclonal antibody and stained with HRP-conjugated antibody. (B) Immunogold electron microscopy and confocal imaging of SAP expression in COH1 wild type strain and COH1Δsap strain grown as in (A). For IEM, fixed bacteria were incubated with an anti-SAP serum and then labeled with secondary antibody conjugated to 10-nm gold particles. Scale bars 200 nm. In confocal imaging experiments, bacteria were stained with mouse polyclonal anti-capsular type III antibodies (red) and the SAP protein with rabbit polyclonal anti-SAP antibodies (green). Magnification, ×100.</p

Expression of SAP recombinant protein.

<p>(A) SDS-PAGE of the mixture of the high and low molecular weight forms of SAP (left lane) as obtained after affinity chromatography and SAP(L) as obtained after anionic exchange chromatography (right lane). (B) Schematic representation of the recombinant form of SAP. Due to the presence of an alternative translation site the protein is expressed in two forms: SAP(H), the full-length form of the enzyme; SAP(L), the truncated form without the CBMs.</p

Anti-SAP antibodies block SAP and PulA enzymatic activity.

<p>(A) COH1 strain was grown in pullulan and assayed for the capacity to degrade pullulan in the presence of different anti-sera. The effect of specific anti-sera was tested in a dose range between 0.5 and 2%. White circles indicate the effect of a mouse anti-PBS serum; black circles indicate the effect of an antiserum from an unrelated surface-associated protein; black squares indicate the effect of a mouse anti-SAP serum; white triangles indicate the effect of a mouse anti-SAP serum absorbed to a CNBR resin coated with SAP. (B) as in (A) except for testing the inhibitory activity of an anti-SAP serum on GAS SF370 strain.</p

Analysis of SAP(H+L) enzymatic activity on pullulan and glycogen.

<p>(A) NMR spectra indicate the generation of maltotriose units after the addition of SAP(H+L) to the reaction mixture containing pullulan. Pullulan NMR spectra were recorded on the native polysaccharide (−SAP) and after the addition of the recombinant enzyme (+SAP). NMR experiments were recorded at 25°C on Bruker Avance 600 MHz spectrometer and using 5-mm probe (Bruker). For details see the Experimental Procedures section. (B) SEC-HPLC analysis indicates that SAP(H+L) is active on glycogen. Two chromatograms were recorded at 214nm, one on the native glycogen polysaccharide (black line) and the other 1 h later the addition of SAP(H+L) (blue line). A gel filtration analytical column with a fractionation range of Mw PEG/PEO 2×10³–3×10⁵ Da was used. For details see the Experimental Procedures section.</p