Sucrose synthase-1 from Arabidopsis thaliana (AtSus1) is a member of the retaining GT-4 glycosyltransferase subfamily (i.e. the product sucrose has the same alpha configuration as the sugar donor UDP-glucose) within the larger family of metal-independent GT-B glycosyltransferases. A typical GT-B fold comprises of two structurally similar Rossmann folds (GT-BN and GT-BC) connected by two hinge regions. We have determined the structure of AtSus1 in catalytically competent closed state with UDP-glucose or fructose and UDP bound at the active site. Active site analysis provides future evidence for SNi-like reaction mechanism and functions of conserved residues His438, Glu675, Lys585 and Arg580. Structural Comparison of AtSus1 with inverting and retaining GTs of known structures revealed two interesting features of GT-B glycosyltrasferase. First, three loop regions have distinct lengths and differing positions between the inverting and retaining GTs and play an important role in determining the direction of attack on the anomeric carbon of the sugar moiety by the sugar acceptor. Second, a simple rigid body motion of GT-BN domain relative to GT-BC domain may be sufficient for the movement of the loops to open or close the attacking route. This domain motion suggests the two hinge regions connecting the two individual Rossmann folds in a particular orientation will stabilize either retaining or inverting reaction outcome over the other. Further understanding of the hinge region will provide new insights for glycosyltransferase engineering and may open up a broader substrate spectrum for glycosyltransfer reactions.The work was supported in part by grants from the NIH (GM65501) and Michigan State University (REF03-016) to RMG.