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Comparative Study
. 2017 Feb 17;292(7):2944-2955.
doi: 10.1074/jbc.M116.759340. Epub 2016 Dec 30.

Comparing Galactan Biosynthesis in Mycobacterium tuberculosis and Corynebacterium diphtheriae

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Free PMC article
Comparative Study

Comparing Galactan Biosynthesis in Mycobacterium tuberculosis and Corynebacterium diphtheriae

Darryl A Wesener et al. J Biol Chem. .
Free PMC article

Abstract

The suborder Corynebacterineae encompasses species like Corynebacterium glutamicum, which has been harnessed for industrial production of amino acids, as well as Corynebacterium diphtheriae and Mycobacterium tuberculosis, which cause devastating human diseases. A distinctive component of the Corynebacterineae cell envelope is the mycolyl-arabinogalactan (mAG) complex. The mAG is composed of lipid mycolic acids, and arabinofuranose (Araf) and galactofuranose (Galf) carbohydrate residues. Elucidating microbe-specific differences in mAG composition could advance biotechnological applications and lead to new antimicrobial targets. To this end, we compare and contrast galactan biosynthesis in C. diphtheriae and M. tuberculosis In each species, the galactan is constructed from uridine 5'-diphosphate-α-d-galactofuranose (UDP-Galf), which is generated by the enzyme UDP-galactopyranose mutase (UGM or Glf). UGM and the galactan are essential in M. tuberculosis, but their importance in Corynebacterium species was not known. We show that small molecule inhibitors of UGM impede C. glutamicum growth, suggesting that the galactan is critical in corynebacteria. Previous cell wall analysis data suggest the galactan polymer is longer in mycobacterial species than corynebacterial species. To explore the source of galactan length variation, a C. diphtheriae ortholog of the M. tuberculosis carbohydrate polymerase responsible for the bulk of galactan polymerization, GlfT2, was produced, and its catalytic activity was evaluated. The C. diphtheriae GlfT2 gave rise to shorter polysaccharides than those obtained with the M. tuberculosis GlfT2. These data suggest that GlfT2 alone can influence galactan length. Our results provide tools, both small molecule and genetic, for probing and perturbing the assembly of the Corynebacterineae cell envelope.

Keywords: cell wall; enzyme catalysis; galactofuranose; glycobiology; glycosyltransferase; mycobacteria.

Conflict of interest statement

The authors declare that they have no conflicts of interest with the contents of this article

Figures

FIGURE 1.
FIGURE 1.
Comparative models of the structure of the mAG complex. Schematic comparison of the mAG complex from M. tuberculosis and C. diphtheriae cell walls.
FIGURE 2.
FIGURE 2.
Proposed enzymatic reactions in galactan biosynthesis in C. diphtheriae.
FIGURE 3.
FIGURE 3.
Chemical inhibition of C. glutamicum growth. A, chemical structure of 2-aminothiazole small molecule UGM inhibitors employed in this study. B, in vitro inhibition of C. diphtheriae UGM activity by the small molecules (15 μm) shown in A. Compound 3 has the same chemotype as compounds 1 and 2 yet fails to inhibit C. diphtheriae UGM. Data are shown as the mean ± S.D. (n = 3 technical replicates). C, inhibition of C. glutamicum growth by UGM inhibitor 1. The antibiotic kanamycin was used as a known inhibitor of cell growth. DMSO treatment was used as a vehicle control, and samples treated with compound 3 were tested as a chemotype control. The concentration of a 10-μl solution of 1 added to each well is included below the image. The result is representative of three biological replicates.
FIGURE 4.
FIGURE 4.
Assessing the polymerase activity of C. diphtheriae GlfT2. A, chemical structures of the synthetic acceptors used in this study. B, left, polymerase activity of C. diphtheriae GlfT2 with synthetic acceptor 4a. Right, activity of C. diphtheriae GlfT2 using a β(1–5) linked disaccharide acceptor substrate, compound 5. Polymer lengths from C. diphtheriae GlfT2 are similar whether the initial disaccharide is β(1–5)- or β(1–6)-linked. C, comparison of the in vitro polymerase activity of C. diphtheriae (C. dip) GlfT2 and M. tuberculosis (M. tb) GlfT2. Spectra from the reaction of 4a with either M. tuberculosis GlfT2 or C. diphtheriae GlfT2 (Fig. 4B, left) were overlaid. Spectra were attained from reactions conducted under identical conditions. Product mixtures were analyzed sequentially using MALDI mass spectrometry. From the raw data spectrum, the maximum intensity of each peak is divided by the total number of laser shots used to obtain that spectrum. Results are representative of at least two replicate reactions.
FIGURE 5.
FIGURE 5.
Polysaccharide product profiles attained using C. diphtheriae GlfT2. In vitro polymerase activity of C. diphtheriae GlfT2 using different acceptors. A, MALDI mass spectrum obtained from analysis of the reaction of C. diphtheriae GlfT2 with 4b, a compound with a shorter lipid substituent than 4a. B, MALDI mass spectrum obtained from analysis of the reaction of C. diphtheriae GlfT2 with 4c, a compound with a longer lipid substituent than 4a. C, MALDI mass spectrum resulting from analysis of the reaction of GlfT2 with a tetrasaccharide acceptor generated chemoenzymatically from compound 4a. Results are representative of at least two replicate reactions.
FIGURE 6.
FIGURE 6.
Genetic modulation of C. diphtheriae GlfT2 polymerase length control. A, proposed active site structure of C. diphtheriae GlfT2 based on mutagenesis results and bioinformatic analysis. B, a single Asp→Glu mutation in the glycosyl donor binding site of C. diphtheriae GlfT2 yields polymers roughly half the length of wild-type polymer products when using compound 4a as the acceptor. The tetrasaccharide used is derived from elongation of 4a by two Galf residues. The result is representative of two replicate reactions.

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