As an extension of previous study (de Vries et al., 1995, J. Biol. Chem., 270, 8712-8722) the acceptor specificity of recombinant FucT VI, expressed in insect cells as soluble enzyme, and purified from the growth medium by affinity chromatography, was analyzed toward a broad panel of oligosaccharide and glycoprotein substrates. It was found that FucT VI effectively utilizes any type-2-chain based structure (Gal beta 1-->4GlcNAc-R). Neutral as well as sialylated structures are fucosylated with high efficiency. To identify polar groups on acceptors that function in enzyme binding, deoxygenated substrate analogs were tested as acceptors. FucT VI had an absolute requirement for a hydroxyl at C-6 of galactose in addition to the accepting hydroxyl at C-3. Thus, FucT VI, although different from FucT III, IV, and V in acceptor properties, seems to bind the acceptor in a similar way.

Poster Sessions C787 not resemble any known structure. SsAPRTase is to our knowledge the fi rst archaean APRTase to be structurally characterized.We will present dimeric P6 1 structures of "apo" SsAPRTase (with PO 4 3-) together with the complexes SsAPRTase:AMP (product) and SsAPRTase:ADP (inhibitor) based on ESRF (Grenoble) synchrotron data to about 2.4 Å resolution. The current work concentrates on obtaining substrate complexes of SsAPRTase. Glycosyltransferases (GTs) are carbohydrate-active enzymes with essential roles in numerous fundamental biological processes such as cellular adhesion, cell signaling, carcinogenesis and cell wall biosynthesis in human pathogens. These enzymes therefore underpin human health and disease and thus inhibitors of GTs are highly sought after as small molecular tools for chemical biology and as lead compounds for drug discovery. Previously, reported ground-state GT donor or acceptor analogues often possess only limited inhibitory potency and the design is complicated by the complex reaction mechanism.Recently, new and potent GT inhibitors were developed by structural modifi cation of the UDP-galactose donor at position 5 of the uracil base (fi gure 1) [1]. Initially, we solved the crystal structure of a representative GT with the most potent UDP-sugar derivative bound. The complex structure reveals that the derivative binds in the active site in a similar manner as the natural donor, but almost completely abolishes sugar transfer by locking the target enzyme in a catalytically inactive conformation [2]. This unique mode of inhibition for GTs seems to be generally applicable to other enzymes in this family. Interestingly, new structures of other similar UDP-sugar derived inhibitors bound to the active site including binding of acceptor reveal striking adaptive dynamics of the enzyme and provide an explanation for its ability to remain slightly active. In addition, the new structures provide an explanation for the dependence of inhibitory potency on the subsituent structure. Finally, by modifying a UDP-GalNAc we have for the fi rst time obtained a structure with an intact UDP-GalNAc in the binding site of human blood group GTs. This structure reveals important aspects of the specifi city of the enzymes responsible for creating blood type A and B. These results demonstrate the dynamics of the GTs and provide a template for the development of a new class of allosteric GT inhibitors.