Chemoenzymatic synthesis of polyprenyl phosphates

Hartley, Meredith D.; Larkin, Angelyn; Imperiali, Barbara

doi:10.1016/j.bmc.2008.03.025

Cited by 22 publications

(31 citation statements)

References 28 publications

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“…Undecaprenol was extracted from the leaves of the staghorn sumac (Rhus typhina) and phosphorylated using chemical or enzymatic methods (SI Text). Und-[ 33 P]P was prepared by using Streptococcus mutans kinase and [γ 33 P] ATP (46). UDP-diNAcBac was prepared using a chemoenzymatic approach exploiting PglF, PglE, and PglD of the pgl pathway (47).…”

Section: Methodsmentioning

confidence: 99%

Lipid bilayer nanodisc platform for investigating polyprenol-dependent enzyme interactions and activities

Hartley

Schneggenburger

Imperiali

2013

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

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Membrane-bound polyprenol-dependent pathways are important for the assembly of essential glycoconjugates in all domains of life. However, despite their prevalence, the functional significance of the extended linear polyprenyl groups in the interactions of the glycan substrates, the biosynthetic enzymes that act upon them, and the membrane bilayer in which they are embedded remains a mystery. These interactions are investigated simultaneously and uniquely through application of the nanodisc membrane technology. The Campylobacter jejuni N-linked glycosylation pathway has been chosen as a model pathway in which all of the enzymes and substrates are biochemically accessible. We present the functional reconstitution of two enzymes responsible for the early membrane-committed steps in glycan assembly. Protein stoichiometry analysis, fluorescence-based approaches, and biochemical activity assays are used to demonstrate the colocalization of the two enzymes in nanodiscs. Isotopic labeling of the substrates reveals that undecaprenyl-phosphate is coincorporated into discs with the two enzymes, and furthermore, that both enzymes are functionally reconstituted and can sequentially convert the coembedded undecaprenyl-phosphate into undecaprenyl-diphosphate-linked disaccharide. These studies provide a proof-of-concept demonstrating that the nanodisc model membrane system represents a promising experimental platform for analyzing the multifaceted interactions among the enzymes involved in polyprenol-dependent glycan assembly pathways, the membrane-associated substrates, and the lipid bilayer. The stage is now set for exploration of the roles of the conserved polyprenols in promoting protein-protein interactions among pathway enzymes and processing of substrates through sequential steps in membrane-associated glycan assembly.

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Section: Methodsmentioning

confidence: 99%

Lipid bilayer nanodisc platform for investigating polyprenol-dependent enzyme interactions and activities

Hartley

Schneggenburger

Imperiali

2013

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

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“…Interestingly, unlike Gram-negative bacteria which possess undetectable levels of free undecaprenol, Gram-positive bacteria maintain a significant level of free undecaprenol, but their biosynthetic pathway remains to be determined (Van Horn & Sanders, 2012). Similar to eukaryotes which encode a DK, Gram-positive bacteria encode an undecaprenol kinase (dgkA) (Hartley et al, 2008;Jerga et al, 2007), indicating that the evolutionarily older Gram-positive bacteria can yield undecaprenyl monophosphate both through phosphorylation and more universally in all eubacteria through phosphatase activity. Fig.…”

Section: Biosynthesis Of the Eubacterial Undecaprenyl Lipid Carriersmentioning

confidence: 99%

Conservation of the PTEN catalytic motif in the bacterial undecaprenyl pyrophosphate phosphatase, BacA/UppP

Bickford

Nick

2013

Microbiology

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Isoprenoid lipid carriers are essential in protein glycosylation and bacterial cell envelope biosynthesis. The enzymes involved in their metabolism (synthases, kinases and phosphatases) are therefore critical to cell viability. In this review, we focus on two broad groups of isoprenoid pyrophosphate phosphatases. One group, containing phosphatidic acid phosphatase motifs, includes the eukaryotic dolichyl pyrophosphate phosphatases and proposed recycling bacterial undecaprenol pyrophosphate phosphatases, PgpB, YbjB and YeiU/LpxT. The second group comprises the bacterial undecaprenol pyrophosphate phosphatase, BacA/UppP, responsible for initial formation of undecaprenyl phosphate, which we predict contains a tyrosine phosphate phosphatase motif resembling that of the tumour suppressor, phosphatase and tensin homologue (PTEN). Based on protein sequence alignments across species and 2D structure predictions, we propose catalytic and lipid recognition motifs unique to BacA/UppP enzymes. The verification of our proposed active-site residues would provide new strategies for the development of substratespecific inhibitors which mimic both the lipid and pyrophosphate moieties, leading to the development of novel antimicrobial agents.

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“…First, purified Dol 14 (10 mg) was incubated at 258C for 10 min with (15 mg) purified recombinant polyprenyl-dolichol kinase cloned from Streptococcus mutans DGK (Hartley et al, 2008) in a reaction mixture (100 mL) containing 50 mM Tris-HCl, pH 7.6, 2 mM [g-32 P]ATP (10 mCi/mmol; Hartmann Analytic), 1 mM DTT, 5 mM MgCl 2 , and 0.1% Tween 80. Upon completion of this step, 50 mL of liposomes containing purified DPMS protein components (equivalent to 20 mg) as specified in the text were added to the reaction mixture supplemented with 2 mM GDP-Man, and the reaction was allowed to proceed for 30 min.…”

Section: Purification Of Dolichols and Assay Of Dpms Activitymentioning

confidence: 99%

DOLICHOL PHOSPHATE MANNOSE SYNTHASE1 Mediates the Biogenesis of Isoprenyl-Linked Glycans and Influences Development, Stress Response, and Ammonium Hypersensitivity in Arabidopsis

et al. 2011

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The most abundant posttranslational modification in nature is the attachment of preassembled high-mannose-type glycans, which determines the fate and localization of the modified protein and modulates the biological functions of glycosylphosphatidylinositol-anchored and N-glycosylated proteins. In eukaryotes, all mannose residues attached to glycoproteins from the luminal side of the endoplasmic reticulum (ER) derive from the polyprenyl monosaccharide carrier, dolichol P-mannose (Dol-P-Man), which is flipped across the ER membrane to the lumen. We show that in plants, Dol-P-Man is synthesized when Dol-P-Man synthase1 (DPMS1), the catalytic core, interacts with two binding proteins, DPMS2 and DPMS3, that may serve as membrane anchors for DPMS1 or provide catalytic assistance. This configuration is reminiscent of that observed in mammals but is distinct from the single DPMS protein catalyzing Dol-P-Man biosynthesis in bakers' yeast and protozoan parasites. Overexpression of DPMS1 in Arabidopsis thaliana results in disorganized stem morphology and vascular bundle arrangements, wrinkled seed coat, and constitutive ER stress response. Loss-of-function mutations and RNA interference-mediated reduction of DPMS1 expression in Arabidopsis also caused a wrinkled seed coat phenotype and most remarkably enhanced hypersensitivity to ammonium that was manifested by extensive chlorosis and a strong reduction of root growth. Collectively, these data reveal a previously unsuspected role of the prenyl-linked carrier pathway for plant development and physiology that may help integrate several aspects of candidate susceptibility genes to ammonium stress.

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Chemoenzymatic synthesis of polyprenyl phosphates

Cited by 22 publications

References 28 publications

Lipid bilayer nanodisc platform for investigating polyprenol-dependent enzyme interactions and activities

Lipid bilayer nanodisc platform for investigating polyprenol-dependent enzyme interactions and activities

Conservation of the PTEN catalytic motif in the bacterial undecaprenyl pyrophosphate phosphatase, BacA/UppP

DOLICHOL PHOSPHATE MANNOSE SYNTHASE1 Mediates the Biogenesis of Isoprenyl-Linked Glycans and Influences Development, Stress Response, and Ammonium Hypersensitivity in Arabidopsis

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