| Deoxy sugars are vital constituents of many biological molecules and play an important role in these molecules' activities and/or functions. The biosynthetic pathways for this class of carbohydrate are complex, containing many enzymatic steps. Examination of 3,6-dideoxyhexose biosynthesis reveals that many of these steps require nicotinamide adenine dinucleotide (NAD(P) + or NAD(P)H as a cofactor. This thesis highlights the versatility of NAD(P), including its attribute of stereospecificity with regard to theories of its origin and trends in enzymes and the various mechanisms used by enzymes to accomplish epimerization and ring rearrangement of sugars. However, the main focus is on those enzymes in the 3,6-dideoxyhexose biosynthetic pathway that include NAD(P)+/NAD(P)H chemistry in their catalysis. CDP-D-glucose 4,6-dehydratase (Eod) is responsible for effecting deoxygenation at C-6 via a reversible NAD-dependent oxidation process. Determination of the stereospecificity of hydride transfer to and from NAD for Eod was made possible by the fortuitous discovery that apo-Eod reconstituted with NADH can reduce the C-4 keto moiety of its own product, 3. In a subsequent step in the 3,6-dideoxyhexose pathway, the common intermediate CDP-3,6-dideoxy-D- glycero-D-glycero-4-hexulose ( 4) is stereospecifically reduced to generate CDP-paratose. The reductase responsible for this transformation, CDP-paratose synthase, was cloned, overexpressed, and thoroughly characterized. The kinetic mechanism and the binding constant of NADPH were also determined. Next in this branch of the pathway is the C-2 epimerization of CDP-paratose by CDP-tyvelose 2-epimerase to provide CDP-tyvelose. This enzyme was also cloned, overexpressed and characterized. Several mechanistic studies were performed on CDP-tyvelose 2-epimerase in an attempt to discern the chemistry used to accomplish epimerization at C-2. Colitose is a 3,6-dideoxyhexose produced via a separate biosynthetic pathway. Sequencing of the colitose gene cluster identified by Dr. Zhihong Guo was continued, and the functions of three additional genes were proposed. The Eod analog of this pathway, colB, was cloned, overexpressed and characterized. In the tylosin biosynthetic pathway, the formation of the branched-chain sugar, mycarose, was examined. As a result of a collaboration with Dr. Huawei Chen, the function of the TylC3 methyltrawferase was confirmed and kinetic studies were performed. |
