Octanoylation of early intermediates of mycobacterial methylglucose lipopolysaccharides

Abstract

Mycobacteria synthesize unique intracellular methylglucose lipopolysaccharides (MGLP) proposed to modulate fatty acid metabolism. In addition to the partial esterification of glucose or methylglucose units with short-chain fatty acids, octanoate was invariably detected on the MGLP reducing end. We have identified a novel sugar octanoyltransferase (OctT) that efficiently transfers octanoate to glucosylglycerate (GG) and diglucosylglycerate (DGG), the earliest intermediates in MGLP biosynthesis. Enzymatic studies, synthetic chemistry, NMR spectroscopy and mass spectrometry approaches suggest that, in contrast to the prevailing consensus, octanoate is not esterified to the primary hydroxyl group of glycerate but instead to the C6 OH of the second glucose in DGG. These observations raise important new questions about the MGLP reducing end architecture and about subsequent biosynthetic steps. Functional characterization of this unique octanoyltransferase, whose gene has been proposed to be essential for M. tuberculosis growth, adds new insights into a vital mycobacterial pathway, which may inspire new drug discovery strategies.

Figure 1. M. tuberculosis H37Rv genomic clusters proposed to participate in MGLP biosynthesis.

Yellow and red, genes proposed to be essential for M. tuberculosis H37Rv growth by saturation transposon mutagenesis. Blue, genes with confirmed function but considered non-essential for growth. White, genes with unknown or putative function that lack experimental confirmation. MeTr, probable methyltransferase; GlcT; α(1→4)-glycosyltransferase; GpgS, glucosyl-3-phosphoglycerate synthase; GlgA, glycogen synthase; GlgC, glucose-1-phosphate adenylyltransferase; OctT, DGG-octanoyltransferase (Sequence data are shown in Figure S1); GpgP, glucosyl-3-phosphoglycerate phosphatase; TreS, trehalose synthase; Mak, maltokinase; GlgB, glycogen-branching enzyme; GlgE, maltosyltransferase.

Figure 2

(A) Synthesis of (2R)-2-O-[6-O-octanoyl-(α-d-glucopyranosyl-(1→6)-α-d-glucopyranosyl]-2,3-dihydroxypropanoic acid 1 (6-Oct-DGG). (B) Synthesis of (2R)-2-O-(α-d-glucopyranosyl-(1→6)-α-d-glucopyranosyl)-3-O-octanoyl-2,3-dihydroxypropanoic acid 2 (DGG-3-Oct).

Figure 3. ¹H-NMR spectra of natural Oct-DGG, chemical synthesized compounds 1 and 2 and DGG.

Peak assignment was supported by 2D correlation NMR experiments (COSY and HMQC).

Figure 4. Mass spectra of OctT reaction products.

DGG (blue spectra) and GG (red spectra) were incubated in the presence and absence of OctT from M. hassiacum with either Oct-CoA (a/c) or Hex-CoA (b/d). Unique peaks not present in control reactions are identified. Spectra were analyzed using the open-source mMass software package and all identified masses are the [M-H]^-1 ions.

Figure 5. MS/MS analysis of DGG and of its modified variants.

In order to validate the site of modification on the OctT reaction products MS/MS spectra were collected for the NMR-validated material (compounds 1 and 2; light blue), unmodified DGG (dark blue) and the C4, C6 and C8 OctT reaction products (red). A limited number of ions were observed which are unique to each modified reaction product and are shifted by 28 Da with the corresponding reduction in acyl-donor length (highlighted grey). Spectra were normalized and analyzed using the open-source mMass software. Parental ions are indicated in parenthesis and are the [M-H]⁻¹ ions.

Figure 6. OctT acyl-donor promiscuity.

DGG was incubated individually with acyl-CoAs including C2 (a), C3 (b), C4 (c), C6 (d), C8 (e), C10 (f), C12 (g) C14 (h) and C16 (i) in addition to succinyl-CoA (j) in the presence of OctT from M. hassiacum. Mass spectra from reactions where transferase activity was observed are colored red while spectra lacking product are colored blue. In all cases except for C14 the spectra from control reactions lacked the observed product peaks. A peak at the expected size of the C14 reaction product was observed in both control reactions and the C12 spectra. Its presence in those spectra and the lack of product in the C12 and C16 reactions makes it unlikely that this ion represents an authentic DGG-C14 reaction product. Spectra were normalized and analyzed using the open-source mMass software package and all identified masses are the [M-H]⁻¹ ions.

Figure 7. Early steps of the proposed pathway for MGLP biosynthesis.

Newly identified octanoyltransferase (OctT) is highlighted red. The dashed line indicates a hypothetical regulatory role of OctT. Experimentally validated functions are shaded blue. Open boxes represent unknown functions or those lacking biochemical confirmation. R groups (red) on the structure indicate acyl chains (acetate, propionate, isobutyrate or succinate) and methyl groups are in green. 3-PGA, d-3-phosphoglyceric acid. GpgS, glucosyl-3-phosphoglycerate synthase; GpgP, glucosyl-3-phosphoglycerate phosphatase; DggS, putative diglucosylglycerate synthase GgH, glucosylglycerate hydrolase (detected in rapidly-growing mycobacteria).