The human ABO(H) blood group antigens are produced by specific glycosyltransferase enzymes. An N-acetylgalactosaminyltransferase (GTA) uses a UDP-GalNAc donor to convert the H-antigen acceptor to the A antigen, whereas a galactosyltransferase (GTB) uses a UDP-galactose donor to convert the H-antigen acceptor to the B antigen. GTA and GTB differ only in the identity of four critical amino acid residues. Crystal structures at 1.8-1.32 A resolution of the GTA and GTB enzymes both free and in complex with disaccharide H-antigen acceptor and UDP reveal the basis for donor and acceptor specificity and show that only two of the critical amino acid residues are positioned to contact donor or acceptor substrates. Given the need for stringent stereo- and regioselectivity in this biosynthesis, these structures further demonstrate that the ability of the two enzymes to distinguish between the A and B donors is largely determined by a single amino acid residue.

The crystal structure of the complex between the copper amine oxidase from Escherichia coli (ECAO) and a covalently bound inhibitor, 2-hydrazinopyridine, has been determined to a resolution of 2.0 A. The inhibitor covalently binds at the 5 position of the quinone ring of the cofactor, 2,4,5-trihydroxyphenylalaninequinone (TPQ). The inhibitor complex is analogous to the substrate Schiff base formed during the reaction with natural monoamine substrate. A proton is abstracted from a methylene group adjacent to the amine group by a catalytic base during the reaction. The inhibitor, however, has a nitrogen at this position, preventing proton abstraction and trapping the enzyme in a covalent complex. The electron density shows this nitrogen is hydrogen bonded to the side chain of Asp383, a totally conserved residue, identifying it as the probable catalytic base. The positioning of Asp383 is such that the pro-S proton of a substrate would be abstracted, consistent with the stereospecificity of the enzyme determined by 1H NMR spectroscopy. Site-directed mutagenesis and in vivo suppression have been used to substitute Asp383 for 12 other residues. The resulting proteins either lack or, in the case of glutamic acid, have very low enzyme activity consistent with an essential catalytic role for Asp383. The O4 position on the quinone ring is involved in a short hydrogen bond with the hydroxyl of conserved residue Tyr369. The distance between the oxygens is less than 2.5 A, consistent with a shared proton, and suggesting ionization at the O4 position of the quinone ring. The Tyr369 residue appears to play an important role in stabilizing the position of the quinone/inhibitor complex. The O2 position on the quinone ring is hydrogen bonded to the apical water ligand of the copper. The basal water ligand, which lies 2.0 A from the copper in the native structure, is at a distance of 3.0 A in the complex. In the native structure, the active site is completely buried, with no obvious route for entry of substrate. In the complex, the tip of the pyridine ring of the bound inhibitor is on the surface of the protein at the edge of the interface between domains 3 and 4, suggesting this as the entry point for the amine substrate.

Novel mutations in the RSW1 and KNOPF genes were identified in a large-scale screen for mutations that affect cell expansion in early Arabidopsis embryos. Embryos from both types of mutants were radially swollen with greatly reduced levels of crystalline cellulose, the principal structural component of the cell wall. Because RSW1 was previously shown to encode a catalytic subunit of cellulose synthase, the similar morphology of knf and rsw1-2 embryos suggests that the radially swollen phenotype of knf mutants is largely due to their cellulose deficiency. Map-based cloning of the KNF gene and enzyme assays of knf embryos demonstrated that KNF encodes α-glucosidase I, the enzyme that catalyzes the first step in N-linked glycan processing. The strongly reduced cellulose content of knf mutants indicates that N-linked glycans are required for cellulose biosynthesis. Because cellulose synthase catalytic subunits do not appear to be N glycosylated, the N-glycan requirement apparently resides in other component(s) of the cellulose synthase machinery. Remarkably, cellular processes other than extracellular matrix biosynthesis and the formation of protein storage vacuoles appear unaffected in knf embryos. Thus in Arabidopsis cells, like yeast, N-glycan trimming is apparently required for the function of only a small subset of N-glycoproteins.

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