D-Arabinose 5-phosphate isomerase from Escherichia coli.

摘要

The outer membrane (OM) of Gram-negative bacteria is an asymmetric lipid bilayer composed predominantly of glycerophospholipids in the inner leaflet and the unique amphiphilic macromolecule lipopolysaccharide (LPS) in the outer leaflet. The OM is critical to numerous cellular functions, in addition to providing a measure of intrinsic resistance against the intracellular diffusion of toxic molecules and potential antimicrobials. The function of the OM is dependent upon the presence of LPS, with the minimal structure necessary to support growth of Escherichia coli under laboratory conditions having previously been recognized as two Kdo (3-deoxy D- manno-octulosonate) molecules attached to lipid A (Kdo2-lipid A). The first step in the Kdo biosynthetic pathway is the conversion of the pentose pathway intermediate D-ribulose 5-phosphate (Ru5P) to D-arabinose 5-phosphate (A5P). In this thesis, the identification of three independent A5P isomerase (API) enzymes (KdsD, GutQ, KpsF) from E. coli capable of catalyzing the reversible 2,1-keto/aldol isomerization of Ru5P to A5P is reported. All three API paralogues were cloned, and the biochemical properties of the recombinant APIs determined. Tentative roles for each API were assigned based on their biochemical properties, comparative genomics, regulation, and through chromosomal API deletion studies. During the process of investigating their respective roles within the cell, a DeltaAPI construct was made in E. coli K-12 MG1655. Consistent with previous reports that Kdo is necessary for viability, the construct (TCM15) was conditional for supplemental A5P in the growth medium. However, a strain (KPM22) was derived from TCM15 that no longer had the A5P auxotrophy, and was capable of division at 37 °C despite lacking Kdo. KPM22 was viable despite predominantly elaborating the endotoxically inactive LPS precursor molecule lipid IVA. These results challenge the established Kdo 2-lipid A viability theory, thus formally redefining the requisite LPS structure in E. coli.