Biosynthesis and structure-activity relationships of the lipid a family of glycolipids.

Lipopolysaccharide (LPS), a glycolipid found in the outer membrane of Gram-negative bacteria, is a potent elicitor of innate immune responses in mammals. A typical LPS molecule is composed of three different structural domains: a polysaccharide called the O-antigen, a core oligosaccharide, and Lipid A. Lipid A is the amphipathic glycolipid moiety of LPS. It stimulates the immune system by tightly binding to Toll-like receptor 4. More recently, Lipid A has also been shown to activate intracellular caspase-4 and caspase-5. An impressive diversity is observed in Lipid A structures from different Gram-negative bacteria, and it is well established that subtle changes in chemical structure can result in dramatically different immune activities. For example, Lipid A from Escherichia coli is highly toxic to humans, whereas a biosynthetic precursor called Lipid IVA blocks this toxic activity, and monophosphoryl Lipid A from Salmonella minnesota is a vaccine adjuvant. Thus, an understanding of structure-activity relationships in this glycolipid family could be used to design useful immunomodulatory agents. Here we review the biosynthesis, modification, and structure-activity relationships of Lipid A.

Partial In Vitro Reconstitution of an Orphan Polyketide Synthase Associated with Clinical Cases of Nocardiosis.

Although a few well-characterized polyketide synthases (PKSs) have been functionally reconstituted in vitro from purified protein components, the use of this strategy to decode "orphan" assembly line PKSs has not been described. To begin investigating a PKS found only in Nocardia strains associated with clinical cases of nocardiosis, we reconstituted in vitro its five terminal catalytic modules. In the presence of octanoyl-CoA, malonyl-CoA, NADPH, and S-adenosyl methionine, this pentamodular PKS system yielded unprecedented octaketide and heptaketide products whose structures were partially elucidated using mass spectrometry and NMR spectroscopy. The PKS has several notable features, including a "split, stuttering" module and a terminal reductive release mechanism. Our findings pave the way for further analysis of this unusual biosynthetic gene cluster whose natural product may enhance the infectivity of its producer strains in human hosts.

Metabolic flux between unsaturated and saturated fatty acids is controlled by the FabA:FabB ratio in the fully reconstituted fatty acid biosynthetic pathway of Escherichia coli.

The entire fatty acid biosynthetic pathway of Escherichia coli, starting from the acetyl-CoA carboxylase, has been reconstituted in vitro from 14 purified protein components. Radiotracer analysis verified stoichiometric conversion of acetyl-CoA and NAD(P)H to the free fatty acid product, allowing implementation of a facile spectrophotometric assay for kinetic analysis of this multienzyme system. At steady state, a maximal turnover rate of 0.5 s(-1) was achieved. Under optimal turnover conditions, the predominant products were C16 and C18 saturated as well as monounsaturated fatty acids. The reconstituted system allowed us to quantitatively interrogate the factors that influence metabolic flux toward unsaturated versus saturated fatty acids. In particular, the concentrations of the dehydratase FabA and the ß-ketoacyl synthase FabB were found to be crucial for controlling this property. Via changes in these variables, the percentage of unsaturated fatty acid produced could be adjusted between 10 and 50% without significantly affecting the maximal turnover rate of the pathway. Our reconstituted system provides a powerful tool for understanding and engineering rate-limiting and regulatory steps in this complex and practically significant metabolic pathway.

Competition-based transfer of carbohydrate expression information from a cell-adhered surface to a secondary surface.

An information transfer strategy was developed for the visualization of carbohydrate expression by the competition of a primary cell-adhered solid surface with a carbohydrate assembled surface as an artificial secondary surface for one species. The strategy could be effectively utilized for in situ monitoring of dynamic carbohydrate expression on an adhesive cell surface.

Noncovalent functionalization of carbon nanotubes with lectin for label-free dynamic monitoring of cell-surface glycan expression.

A kind of concanavalin A functionalized multiwalled carbon nanotube (ConA-MWCNT) was constructed by noncovalent assembly of ConA on carboxylated MWCNT with poly(diallyldimethylammonium) as a linker. The novel nanomaterial was characterized with scanning electron microscopy and atomic force microscopy. It incorporated both the specific recognition ability of lectin for cell-surface mannosyl groups and the unique electronic and mechanical properties of MWCNT. An electrochemical label-free method for cytosensing was proposed by constructing a ConA-MWCNT interface on a glassy carbon electrode, which showed a linear response to K562 cells ranging from 1 × 10(4) to 1 × 10(7) cellsmL(-1). The ConA-MWCNT interface could be further used for monitoring of dynamic variation of glycan expression on K562 cells in response to drugs. A facile and high-throughput optical method for the analysis of dynamic glycan expression on living cells was also developed by constructing an array of ConA-MWCNT spots on a glass slide. This method showed acceptable rapidity and low cost. The noncovalent functionalization of MWCNTs with lectins could be potentially applied in cell biological studies based on cell-surface glycan expression.