Insights into the Mechanistic and Regulatory Properties of D-arabinose-5-phosphate

Abstract

Arabinose-5-phosphate isomerase (API) catalyzes the interconversion of D-ribulose-5-phosphate and D-arabinose-5-phosphate (A5P), which is the first step in the biosynthesis of 3-deoxy-D-manno-octulosonate (Kdo). Kdo, an important component of the highly conserved inner core of lipopolysaccharide (LPS), is biosynthesized and activated via four sequentially acting enzymes, API, 3-deoxy-D-manno-octulosonate-8-phosphate (Kdo8P) synthase, Kdo8P phosphatase, and cytidine-5’-monophosphate-Kdo synthetase. The Kdo produced from A5P serves an integral part of LPS as it bridges together lipid A, the membrane imbedded portion of LPS, and the inner oligosaccharide core of LPS. API isozymes can be split into two groups. Full-length APIs contain an N-terminal sugar isomerase (SIS) domain, where isomerization takes place, and tandem C-terminal cystathionine beta-synthase (CBS) domains, which are thought to be regulatory, while SIS-domain APIs are much shorter in primary amino acid sequence and only contain an SIS-domain. In an effort to identify unusual sources of A5P and explore how that A5P is utilized, this thesis examines several unique APIs from varying sources including Q723E8 from Bacteroides fragilis, CtAPI from Clostridium tetani, AtAPI from Arabidopsis thaliana, and c3406 from the uropathogenic Escherichia coli CFT037. Q723E8, from B. fragilis, was the first characterized SIS-domain API that does not also contain a full-length API within the host genome. Q723E8 was also the first API shown to be inhibited by CMP-Kdo, the end product of the Kdo biosynthetic pathway. CtAPI from C. tetani, a SIS-domain API, is the first API described and characterized in a Gram-positive bacterium and is speculated to play a role in regulation of an operon involved in ribose transport and metabolism. AtAPI from A. thaliana, which is a full-length API, is required for Kdo biosynthesis for incorporation of Kdo into Rhamnogalacturonan-II, an important part of plant cell walls. Kdo produced from the Kdo biosynthetic pathway in A. thaliana may also be incorporated onto a lipid A-like molecule. Finally, c3406 from E. coli CFT073 was crystallized in complex with D-arabinose-5-phosphate, key active site mutants were made and compared to active site mutants of E. coli KdsD. A mechanism was proposed for the isomerization of D-ribulose-5-phosphate and D-arabinose-5-phosphate. These studies demonstrate that APIs are not only found in Gram-negative bacteria, but also in Gram-positive bacteria and some plants. APIs in these unusual sources have sparked speculation that A5P may be used for more than lipopolysaccharide biosynthesis. This thesis provides unique insight into the first proposed API mechanism, AtAPI’s role in RGII biosynthesis and the possibility of biosynthesis of a lipid A-like molecule in Arabidopsis thaliana, regulation of operons by A5P, regulation of APIs and the Kdo biosynthetic pathway via CMP-Kdo, and the functional assignment of active site residues within this enzyme.