Biochemical characterization of a process that occurs at the membrane-cytosol interface is inherently challenging due to the bimodal nature of the reactants and products involved. A representative process potentially amenable to biochemical analysis resides in the biosynthesis of bacillosamine from the gram-negative enteropathogen Campylobacter jejuni. In C.jejuni bacillosamine is the first carbohydrate of a heptameric, prenylpyrophosphate-linked glycan that is assembled in the cytosol, transported across the membrane, and N-linked to nascent proteins in the periplasmic space. The locus that codes for enzymes involved in bacillosamine biosynthesis has been cloned and the heptameric glycan identified. However, the roles of the plasma membrane, transmembrane domains of the enzymes, and the membrane bound bactoprenolpyrophosphate molecule during glycan biosynthesis remains unclear. Outlined in this proposal are approaches designed to detail the biological mechanism of lipid anchored bacillosamine biosynthesis in model membranes. Full-length or separable domain constructs for each enzyme in the pathway will be heterologously expressed, purified, and respective enzymatic activity characterized in cell-free systems. Purified material will then be reconstituted in model membranes, and biochemical reactions characterized using lipid anchored substrate. The assays will also be used to identify the roles of the membrane, protein transmembrane domains, and the membrane bound prenylpyrophosphate molecule in bacillosamine biosynthesis. ? ?

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Postdoctoral Individual National Research Service Award (F32)
Project #
5F32GM075415-02
Application #
7172316
Study Section
Special Emphasis Panel (ZRG1-F13 (20))
Program Officer
Marino, Pamela
Project Start
2005-09-01
Project End
2008-08-31
Budget Start
2006-09-01
Budget End
2007-08-31
Support Year
2
Fiscal Year
2006
Total Cost
$45,976
Indirect Cost
Name
Massachusetts Institute of Technology
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
001425594
City
Cambridge
State
MA
Country
United States
Zip Code
02139
Olivier, Nelson B; Imperiali, Barbara (2008) Crystal structure and catalytic mechanism of PglD from Campylobacter jejuni. J Biol Chem 283:27937-46
Olivier, Nelson B; Chen, Mark M; Behr, Jonathan R et al. (2006) In vitro biosynthesis of UDP-N,N'-diacetylbacillosamine by enzymes of the Campylobacter jejuni general protein glycosylation system. Biochemistry 45:13659-69