Tetrameric Structure of the GlfT2 Galactofuranosyltransferase Reveals a Scaffold for the Assembly of Mycobacterial Arabinogalactan

Wheatley, R.W.; Zheng, Ruixiang Blake; Richards, Michele R.; Lowary, Todd L.; Ng, Kenneth

doi:10.1074/jbc.m112.347484

Cited by 56 publications

(91 citation statements)

References 53 publications

(75 reference statements)

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“…We confirmed the PMf association of six proteins by density gradient fractionation and performed further analyses on GlfT2, Ppm1, and PyrD. GlfT2 does not contain predicted TM domains, but rather, its crystal structure implies peripheral membrane association (40). Extending previous studies showing that GlfT2 cofractionates with both membrane and CW (24), our data demonstrated stable and specific binding of GlfT2 to the PMf membrane.…”

Section: Discussionsupporting

confidence: 83%

Spatially distinct and metabolically active membrane domain in mycobacteria

Hayashi

Luo

Mayfield

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

218

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Protected from host immune attack and antibiotic penetration by their unique cell envelope, mycobacterial pathogens cause devastating human diseases such as tuberculosis. Seamless coordination of cell growth with cell envelope elongation at the pole maintains this barrier. Unraveling this spatiotemporal regulation is a potential strategy for controlling mycobacterial infections. Our biochemical analysis previously revealed two functionally distinct membrane fractions in Mycobacterium smegmatis cell lysates: plasma membrane tightly associated with the cell wall (PM-CW) and a distinct fraction of pure membrane free of cell wall components (PMf). To provide further insight into the functions of these membrane fractions, we took the approach of comparative proteomics and identified more than 300 proteins specifically associated with the PMf, including essential enzymes involved in cell envelope synthesis such as a mannosyltransferase, Ppm1, and a galactosyltransferase, GlfT2. Furthermore, comparative lipidomics revealed the distinct lipid composition of the PMf, with specific association of key cell envelope biosynthetic precursors. Live-imaging fluorescence microscopy visualized the PMf as patches of membrane spatially distinct from the PM-CW and notably enriched in the pole of the growing cells. Taken together, our study provides the basis for assigning the PMf as a spatiotemporally distinct and metabolically active membrane domain involved in cell envelope biogenesis.

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Section: Discussionsupporting

confidence: 83%

Spatially distinct and metabolically active membrane domain in mycobacteria

Hayashi

Luo

Mayfield

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

218

View full text Add to dashboard Cite

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“…Apart from being a bifunctional GalT, GlfT2 displays additional interesting features: By using a substrate tethering mechanism, it is able to have intrinsic control of the chain length of the galactan product (May et al 2009). Further structural analysis of GlfT2 using X-ray crystallography and NMR has shed further light on how this unique enzyme is tetrameric and is able to control chain length processivity and bifunctionality using a single active site (Szczepina et al 2009;Wheatley et al 2012). …”

Section: Galactan Biosynthesismentioning

confidence: 99%

The Mycobacterial Cell Wall—Peptidoglycan and Arabinogalactan

Alderwick

Harrison

Lloyd

et al. 2015

Cold Spring Harb Perspect Med

190

156

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The mycobacterial bacillus is encompassed by a remarkably elaborate cell wall structure. The mycolyl-arabinogalactan-peptidoglycan (mAGP) complex is essential for the viability of Mycobacterium tuberculosis and maintains a robust basal structure supporting the upper "myco-membrane." M. tuberculosis peptidoglycan, although appearing to be unexceptional at first glance, contains a number of unique molecular subtleties that become particularly important as the TB-bacilli enters into nonreplicative growth during dormancy. Arabinogalactan, a highly branched polysaccharide, serves to connect peptidoglycan with the outer mycolic acid layer, and a variety of unique glycolsyltransferases are used for its assembly. In this review, we shall explore the microbial chemistry of this unique heteropolysacchride, examine the molecular genetics that underpins its fabrication, and discuss how the essential biosynthetic process might be exploited for the development of future anti-TB chemotherapies. THE MYCOBACTERIAL CELL WALL-PEPTIDOGLYCAN AND ARABINOGALACTAN

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“…Of all the structure templates surveyed, the bestthreaded positional or conformational fit was with UDP-GALACTOFURANOSYL TRANSFERASE2 (GalfT2; PDB code 4FIX; Wheatley et al, 2012). The sequence-structure comparison threaded with FUGUE (Shi et al, 2001) gave an identity of 14% between RGP2 and 4FIX (Supplemental Table S2).…”

Section: Protein Modeling Indicates That the Mur5 Mutation Impacts Prmentioning

confidence: 99%

“…Improvements in sequencing technologies have reduced the years required to complete the reference genome of Arabidopsis (Arabidopsis Genome Initiative, 2000) to only days needed to assemble the genomes of hundreds of Arabidopsis ecotypes from extensive numbers of short-read sequences (Schneeberger . RGP2 threaded to GalfT2 (PDB code 4FIX_A; Wheatley et al, 2012). The model preserves the classical nucleotide-binding fold and places Cys-252 and Cys-257 on the face distal to the catalytic residues.…”

Section: High-throughput Sequencing Accelerates Mutant Identificationmentioning

confidence: 99%

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The Cell Wall Arabinose-Deficient Arabidopsis thaliana Mutant murus5 Encodes a Defective Allele of REVERSIBLY GLYCOSYLATED POLYPEPTIDE2

et al. 2016

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Traditional marker-based mapping and next-generation sequencing was used to determine that the Arabidopsis (Arabidopsis thaliana) low cell wall arabinose mutant murus5 (mur5) encodes a defective allele of REVERSIBLY GLYCOSYLATED POLYPEPTIDE2 (RGP2). Marker analysis of 13 F2 confirmed mutant progeny from a recombinant mapping population gave a rough map position on the upper arm of chromosome 5, and deep sequencing of DNA from these 13 lines gave five candidate genes with G→A (C→T) transitions predicted to result in amino acid changes. Of these five, only insertional mutant alleles of RGP2, a gene that encodes a UDP-arabinose mutase that interconverts UDP-arabinopyranose and UDP-arabinofuranose, exhibited the low cell wall arabinose phenotype. The identities of mur5 and two SALK insertional alleles were confirmed by allelism tests and overexpression of wild-type RGP2 complementary DNA placed under the control of the 35S promoter in the three alleles. The mur5 mutation results in the conversion of cysteine-257 to tyrosine-257 within a conserved hydrophobic cluster predicted to be distal to the active site and essential for protein stability and possible heterodimerization with other isoforms of RGP.

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Tetrameric Structure of the GlfT2 Galactofuranosyltransferase Reveals a Scaffold for the Assembly of Mycobacterial Arabinogalactan

Cited by 56 publications

References 53 publications

Spatially distinct and metabolically active membrane domain in mycobacteria

Spatially distinct and metabolically active membrane domain in mycobacteria

The Mycobacterial Cell Wall—Peptidoglycan and Arabinogalactan

The Cell Wall Arabinose-Deficient Arabidopsis thaliana Mutant murus5 Encodes a Defective Allele of REVERSIBLY GLYCOSYLATED POLYPEPTIDE2

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