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Octanoylation of early intermediates of mycobacterial methylglucose lipopolysaccharides.

Maranha A, Moynihan PJ, Miranda V, Correia Lourenço E, Nunes-Costa D, Fraga JS, José Barbosa Pereira P, Macedo-Ribeiro S, Ventura MR, Clarke AJ, Empadinhas N - Sci Rep (2015)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: CNC - Center for Neuroscience and Cell Biology, University of Coimbra, 3004-517 Coimbra, Portugal.

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.

No MeSH data available.


Related in: MedlinePlus

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 mutagenesis27. 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.
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f1: 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 mutagenesis27. 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.

Mentions: Although MGLP was identified in the 1960s19 and its chemical composition, structure and interactions with fatty acids have been thoroughly examined, its biosynthetic pathway remained largely unknown4. Glucosylglycerate (GG), the putative primer for MGLP synthesis, is now known to arise from the consecutive action of the enzymes glucosyl-3-phosphoglycerate synthase (GpgS) and glucosyl-3-phosphoglycerate phosphatase (GpgP), encoded in M. tuberculosis H37Rv by Rv1208 and Rv2419c, respectively20212223. A GG hydrolase was recently identified in rapidly-growing mycobacteria but its involvement in the regulation of MGLP biosynthesis has not been examined24. The third MGLP biosynthetic step was predicted to involve the transfer of an additional glucose to GG to yield DGG13. Given that bacterial genes involved in one pathway are often grouped into operons25, confirmation of the genetic implication of Rv3032 (α–(1→4)-glycosyltransferase) and Rv3030 (6–O–methyltransferase) in MGLP extension and methylation, respectively, linked Rv3031 (a putative glycoside hydrolase similar to α–(1→6)-branching enzymes) to DGG synthesis613 (Fig. 1). After assigning GpgP activity to Rv2419c we deemed it important to investigate the function of the neighboring gene considered essential for M. tuberculosis H37Rv growth232627. In addition to Rv2418c, we cloned the M. smegmatis ortholog and that from the thermophilic M. hassiacum, for protein stability constraints often curbing functional and structural studies28.


Octanoylation of early intermediates of mycobacterial methylglucose lipopolysaccharides.

Maranha A, Moynihan PJ, Miranda V, Correia Lourenço E, Nunes-Costa D, Fraga JS, José Barbosa Pereira P, Macedo-Ribeiro S, Ventura MR, Clarke AJ, Empadinhas N - Sci Rep (2015)

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 mutagenesis27. 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.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4555173&req=5

f1: 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 mutagenesis27. 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.
Mentions: Although MGLP was identified in the 1960s19 and its chemical composition, structure and interactions with fatty acids have been thoroughly examined, its biosynthetic pathway remained largely unknown4. Glucosylglycerate (GG), the putative primer for MGLP synthesis, is now known to arise from the consecutive action of the enzymes glucosyl-3-phosphoglycerate synthase (GpgS) and glucosyl-3-phosphoglycerate phosphatase (GpgP), encoded in M. tuberculosis H37Rv by Rv1208 and Rv2419c, respectively20212223. A GG hydrolase was recently identified in rapidly-growing mycobacteria but its involvement in the regulation of MGLP biosynthesis has not been examined24. The third MGLP biosynthetic step was predicted to involve the transfer of an additional glucose to GG to yield DGG13. Given that bacterial genes involved in one pathway are often grouped into operons25, confirmation of the genetic implication of Rv3032 (α–(1→4)-glycosyltransferase) and Rv3030 (6–O–methyltransferase) in MGLP extension and methylation, respectively, linked Rv3031 (a putative glycoside hydrolase similar to α–(1→6)-branching enzymes) to DGG synthesis613 (Fig. 1). After assigning GpgP activity to Rv2419c we deemed it important to investigate the function of the neighboring gene considered essential for M. tuberculosis H37Rv growth232627. In addition to Rv2418c, we cloned the M. smegmatis ortholog and that from the thermophilic M. hassiacum, for protein stability constraints often curbing functional and structural studies28.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: CNC - Center for Neuroscience and Cell Biology, University of Coimbra, 3004-517 Coimbra, Portugal.

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.

No MeSH data available.


Related in: MedlinePlus