Article

Polyisoprenol Specificity in the Campylobacter jejuni N-Linked Glycosylation Pathway †

Department of Chemistry, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, Massachusetts 02139, USA.
Biochemistry (Impact Factor: 3.02). 01/2008; 46(50):14342-8. DOI: 10.1021/bi701956x
Source: PubMed

ABSTRACT

Campylobacter jejuni contains a general N-linked glycosylation pathway in which a heptasaccharide is sequentially assembled onto a polyisoprenyl diphosphate carrier and subsequently transferred to the asparagine side chain of an acceptor protein. The enzymes in the pathway function at a membrane interface and have in common amphiphilic membrane-bound polyisoprenyl-linked substrates. Herein, we examine the potential role of the polyisoprene component of the substrates by investigating the relative substrate efficiencies of polyisoprene-modified analogues in individual steps of the pathway. Chemically defined substrates for PglC, PglJ, and PglB are prepared via semisynthetic approaches. The substrates included polyisoprenols of varying length, double bond geometry, and degree of saturation for probing the role of the hydrophobic polyisoprene in substrate specificity. Kinetic analysis reveals that all three enzymes exhibit distinct preferences for the polyisoprenyl carrier whereby cis-double bond geometry and alpha-unsaturation of the native substrate are important features, while the precise polyisoprene length may be less critical. These findings suggest that the polyisoprenyl carrier plays a specific role in the function of these enzymes beyond a purely physical role as a membrane anchor. These studies underscore the potential of the C. jejuni N-linked glycosylation pathway as a system for investigating the biochemical and biophysical roles of polyisoprenyl carriers common to prokaryotic and eukaryotic glycosylation.

Full-text preview

Available from: ncbi.nlm.nih.gov
  • Source
    • "A striking observation in our study was the strong specificity of WbaP CT for Und-P. Kinetic analysis of purified PglC with PI phosphates varying in length, double-bond geometry and degree of saturation has also revealed a preference for the native substrate (Chen et al. 2007). An interaction of the enzyme with the Und-P (C55-P) acceptor presumably occurs in the inner membrane via regions of the protein within or at the boundaries of TM helices. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Two families of membrane enzymes catalyze the initiation of the synthesis of O-antigen lipopolysaccharide. The Salmonella enterica Typhimurium WbaP is a prototypic member of one of these families. We report here the purification and biochemical characterization of the WbaP C-terminal (WbaPCT) domain harboring one putative transmembrane helix and a large cytoplasmic tail. An N-terminal thioredoxin fusion greatly improved solubility and stability of WbaPCT allowing us to obtain highly purified protein. We demonstrate that WbaPCT is sufficient to catalyze the in vitro transfer of galactose (Gal)-1-phosphate from uridine monophosphate (UDP)-Gal to the lipid carrier undecaprenyl monophosphate (Und-P). We optimized the in vitro assay to determine steady-state kinetic parameters with the substrates UDP-Gal and Und-P. Using various purified polyisoprenyl phosphates of increasing length and variable saturation of the isoprene units, we also demonstrate that the purified enzyme functions highly efficiently with Und-P, suggesting that the WbaPCT domain contains all the essential motifs to catalyze the synthesis of the Und-P-P-Gal molecule that primes the biosynthesis of bacterial surface glycans.
    Preview · Article · Aug 2011 · Glycobiology
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Abstract N-glycosylation is a metabolic process found inall three domains,of life. It is the attachment of a polysaccharide glycan to asparagine (Asn) residues within the amino acid motif,
    Preview · Article ·
  • [Show abstract] [Hide abstract]
    ABSTRACT: Theoretically and experimentally, it has been shown that the strained heterostructures grown on polar substrates, which an oriented on axes other than the [100], have piezoelectric fields due to off-diagonal components of strain, and the internal electric fields result in a redshift of the excitonic transitions within the strained heterostructures. Based on this, several groups have demonstrated a tunable blueshift of the absorption edge by modulating the internal fields with an external reverse bias in strained piezoelectric quantum well (QW) diodes. The experimental determination of partial screening of the piezoelectric fields under forward biased current injection has been also demonstrated. In this paper, we report theoretical study of this screening in strained InGaAsP based QW grown on (111) substrate and its gain spectrum under current injection
    No preview · Conference Paper · Dec 1997
Show more