Lipid A, biosynthesis

F. Whole-Cell Screening of Enzyme Inhibitors Lipid A Biosynthesis... 

HR Onishi, BA Pelak, LS Gerckens, LL Silver, FM Kahan, MH Chen, AA Patchett, SM Galloway, SA Hyland, MS Anderson, CRH Raetz. Antibacterial agents that inhibit lipid A biosynthesis. Science 274 980-982, 1996. 

MH Chen, MG Steiner, SE de Laszlo, AA Patchett, MS Anderson, SA Hyland, HR Onishi, LL Silver, CRH Raetz. Carbohydroxamido-oxazolidines antibacterial agents that target lipid A biosynthesis. Bioorg Med Chem Lett 9 313-318, 1999. 

K Young, LL Silver, D Bramhill, P Cameron, SS Eveland, CR Raetz, SA Hyland, MS Anderson. The envA permeability/cell division gene of Escherichia coli encodes the second enzyme of lipid A biosynthesis. UDP-3-0-(R-3-hydroxymyristoyl)-N-acetylglucosamine deacetylase. J Biol Chem 270 30384-30391, 1995. 

S Mohan, TM Kelly, SS Eveland, CR Raetz, MS Anderson. An Escherichia coli gene (FabZ) encoding (3R)-hydroxymyristoyl acyl carrier protein dehydrase. Relation to fabA and suppression of mutations in lipid A biosynthesis. J Biol Chem 269 32896-32903, 1994. 

Fig. 1.1 Structure and biosynthetic pathway of Kdo2-lipid A in E. coli. Each reaction is catalyzed by a single enzyme. The names of the enzyme and substrate are highlit. The carbon position and the carbon number of fatty acid chains in lipid A are labeled. The genes encoding the enzymes of Kdo2-lipid A biosynthesis are present in single copy and highly conserved among bacteria (Raetz et al., 2007 Raetz and Whitfield, 2002)...

Babinski, K.J., Ribeiro, A.A., Raetz, C.R.H. The Escherichia coli gene encoding the UDP-2,3-diacylglucosamine pyrophosphatase of lipid A biosynthesis. J Biol Chem 277 (2002b)... 

Battling, C.M., Raetz, C.R. Crystal structure and acyl chain selectivity of Escherichia coli LpxD, the JV-acyltransferase of lipid a biosynthesis. Biochemistry 48 (2009) 8672-8683. 

Buetow, L., Smith, T.K., Dawson, A., Fyffe, S., Hunter, W.N. Structure and reactivity of LpxD, the IV-acyltransferase of lipid A biosynthesis. Proc Natl Acad Sci USA 104 (2007) 4321 1326. 

Steeghs, L., Jennings, M.P., Poolman, J.T., van der Ley, P. Isolation and characterization of the Neisseria meningitidis IpxD-fabZ-lpxA gene cluster involved in lipid A biosynthesis. Gene 190 (1997) 263-270. 

Figure 2. The constitutive pathway for lipid A biosynthesis in E. coli. The genes encoding the proteins which catalyze each reaction are shown. Nine enzymes are required for the biosynthesis of Kdo2-iipid A, also known as Re endotoxin.

After disaccharide formation, the lipid A 4 kinase, catalyzes the transfer of the y-phosphate from ATP to the 4 position of DS-l-P to form tetraacyldisaccharide l,4 -ftw-phosphate (also known as lipid IVa) (Figure 2) [34], Incorporation of the 4 phosphate is required for subsequent reactions in lipid A biosynthesis [35], Also, lipid A-like molecules are maximally active as activators of signal transduction in mammalian cells when both the 1 and 4 phosphates are present [13, 36, 37],... 

The 4 kinase activity was first identified in 1987 [34], It has since proven useful in the preparation of (4 - P)-lipid IVa, and (4 - P)-Kdo2-lipid IVa for the study of late reactions in lipid A biosynthesis (see below) [34, 38]. Such probes are also useful for the detection of lipid A binding proteins in mammalian cells [37, 39]. 

The Kdo transferase from E coH has been cloned, overexpressed and purified [35]. The purified protein displays at least 1000-fold preference for substrates which contain a 4 phosphate group, indicating the importance of the 4 kinase in lipid A biosynthesis [35]. 

The gene encoding the palmitoyl transferase has recently been identified in Salmonella as the PhoPQ-activated gene pagP [51, 54, 55]. Overexpression of the E. coli homolog, crcA (now refered to as IpxY), leads to massive overproduction of palmitoyl transferase activity [51]. Analysis of inner and outer membrane fractions for palmitoyl transferase activity shows that LpxY (Figure 3) is located in the outer membrane [51], This is in contrast to the inner membrane localization of all other enzymes involved in lipid A biosynthesis. 

Figure 3. Other regulated acylation reactions involved in E. coli lipid A biosynthesis under special conditions. The reactions catalyzed by LpxP (formerly known as Ddg) [59] and LpxY (also known as PagP or CrcA) [51] are shown. LpxP is a cold induced protein which incorporates an unsaturated 16 carbon fatty acyl chain in place of the laurate normally incorporated by HtrB. LpxY, the expression of which is activated by the PhoP/PhoQ system [51], is an outer membrane protein that incorporates palmitate to make hepta-acylated lipid A. LpxY is capable of acylating lipid X, lipid IVa, and lipid A, as well as Kdo2-lipid IVa (as shown). The physiological substrate is probably lipid A, given that LpxY is an outer membrane protein.

Sorensen, P. G., Lutkenhaus, J., Young, K., Eveland, S. S., Anderson, M. S., and Raetz, C. R. H. (1996). Regulation of UDP-3-0-[R-3-hydroxymyristoyl]-JV-acetyl-glucosamine deacetylase in Escherichia coli. The second enzymatic step of lipid A biosynthesis. J. Biol. Chem. 271, 25898-25905. 

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