MoeH5: a natural glycorandomizer from the moenomycin biosynthetic pathway

Abstract

The biosynthesis of the phosphoglycolipid antibiotic moenomycin A attracts the attention of researchers hoping to develop new moenomycin-based antibiotics against multidrug resistant Gram-positive infections. There is detailed understanding of most steps of this biosynthetic pathway in Streptomyces ghanaensis (ATCC14672), except for the ultimate stage, where a single pentasaccharide intermediate is converted into a set of unusually modified final products. Here we report that only one gene, moeH5, encoding a homologue of the glutamine amidotransferase (GAT) enzyme superfamily, is responsible for the observed diversity of terminally decorated moenomycins. Genetic and biochemical evidence support the idea that MoeH5 is a novel member of the GAT superfamily, whose homologues are involved in the synthesis of various secondary metabolites as well as K and O antigens of bacterial lipopolysaccharide. Our results provide insights into the mechanism of MoeH5 and its counterparts, and give us a new tool for the diversification of phosphoglycolipid antibiotics.

Fig. 1

Structures of the moenomycins mentioned in this work.

Fig. 2

Genes and constructs described in this work. A. Genetic organization of moe clusters 1 and 2. The fragment of moe cluster 1 between moeK5 and moeR5, not relevant to this study, is not shown on the figure. Amide synthase gene moeB4 and GAT superfamily genes moeF5 and moeH5 are shown as grey arrows. Cosmids and plasmids used to express segments of the moe clusters are shown below. Genes moeH5 and moeB4 were replaced in moeno38-6 and pOOB64bd, respectively, with the kanamycin resistance cassette neo (marked as grey rectangle). B. Relative localization of moe clusters 1 and 2 in the S. ghanaensis chromosome (according to available WGS data at www.broadinstitute.org).

Fig. 3

Moenomycin production profiles of moeB4- and moeH5-deficient S. ghanaensis mutants. Note the four distinct x-axes. Extracted ion chromatograms showing the presence of moenomycins in the methanol extracts from S. ghanaensis strains dB4 (ΔmoeB4), dH5 (ΔmoeH5) and complemented dH5 (ΔmoeH5+pOOB47h). The moeGT1-deficient moenomycin nonproducer mutant (ΔmoeGT1; Ostash et al., 2007) was used as a negative control. Compounds are shown as graphical symbols, where the rectangle indicates NoA; masses (in Da) are given to the right. Red arrows indicate peaks corresponding to NoA (1485 Da), that overlap with the 1485.6 isotope of NoB.

Fig. 4

Unrooted maximum-likelihood tree showing the relationships within a set of selected MoeH5 homologues found in primary and secondary metabolic pathways. Complete amino acid (aa) sequences of the proteins were used for analysis. Double black triangles mark the proteins having truncated Ntn domains (see Fig. S6, ESM), while single triangles mark those with complete Ntn domains that lack the invariable Cys₁ residue. The tree was built with the help of phylogeny.fr v.2 server (Dereeper et al., 2008) using default parameters. Confidence indices aLRT (Anisimova and Gascuel 2006) are shown on the nodes; the branches having aLRT values below stringent threshold level (0.75) were collapsed. The scale bar represents 2.0 aa substitution per aa position. Source data are given in supplementary table S1.

Fig. 5

Purification of proteins MoeH5 and MoeF5. A. SDS-PAGE of protein fraction obtained after IMAC of lysate from S. lividans strain TK24 pOOB83e⁺. Lanes: 1: soluble fraction; 2–4: fractions eluted with 200 mM imidazole; 5–6: 250 mM imidazole. The 56 kDa band corresponds to rMoeH5 as judged by Western blot analysis (not shown). The lower band is a MoeH5 degradation product. Lane 7: marker (Precision Plus protein ladder, BioRad) B. SDS-PAGE of concentrated MoeH5 (lane 1) and MoeF5 (lane 2). Lane 3: Long Range protein ladder (Sigma).

Fig. 6

In vitro assay of rMoeH5 activity. NoA, ATP and rMoeH5 were present in each reaction mixture. Conversion of NoA into respective products was measured using 10 mM ATP, 10 mM amide donor substrate, 25 μM NoA and ~40 nM enzyme. Conversion was between 2 and 20% for different donor substrates. Reactions were run for 4 h, quenched and immediately analyzed by MS and MS-MS. Extracted ion chromatograms (representative result of at least four independent experiments) showing production of moenomycins by MoeH5 in the presence of D,L-glutamine (A), D,L-glutamine and MoeF5 (B), NH₄Cl (C), NH₄Cl and MoeF5 (D), glycine (E), D,L-serine (F), A ring (G) and cyclopentylamine CPA (H). Masses of the compounds are given in Table 1. Relevant control reactions are given in SI, Fig. S9; MS-MS data are given in Figs. S10–S12.

Fig. 7

Summary of the final stage of moenomycin biosynthesis in S. ghanaensis.