The recently published human genome with its relatively modest number of genes has highlighted the importance of post-transcriptional and post-translational modi®cations, such as alternative splicing or glycosylation, in generating the complexities of human biology. The human UDP-N-acetylglucosamine (UDPGlcNAc) pyrophosphorylases AGX1 and AGX2, which differ in sequence by an alternatively spliced 17 residue peptide, are key enzymes synthesizing UDPGlcNAc, an essential precursor for protein glycosylation. To better understand the catalytic mechanism of these enzymes and the role of the alternatively spliced segment, we have solved the crystal structures of AGX1 and AGX2 in complexes with UDPGlcNAc (at 1.9 and 2.4 A Ê resolution, respectively) and UDPGalNAc (at 2.2 and 2.3 A Ê resolution, respectively). Comparison with known structures classi®es AGX1 and AGX2 as two new members of the SpsAGnT I Core superfamily and, together with mutagenesis analysis, helps identify residues critical for catalysis. Most importantly, our combined structural and biochemical data provide evidence for a change in the oligomeric assembly accompanied by a signi®cant modi®cation of the active site architecture, a result suggesting that the two isoforms generated by alternative splicing may have distinct catalytic properties. Keywords: alternative splicing/crystallography/human enzyme/oligomeric assembly/UDPGlcNAc pyrophosphorylase Introduction Carbohydrates, N-or O-linked to proteins via posttranslational modi®cations, play diverse and crucial biological roles in eukaryotes. When present on cell surface glycoproteins, they are instrumental in cell±cell/matrix interactions, immune reactions and tumour development (Hakomori, 1991;Rudd et al., 2001). They are important for the transport, biological activity and clearance from the circulation of secreted glycoproteins and modify the structure of cytosolic and nuclear proteins with implications in transcription, translation, neuronal pathology and other biological processes (Hart et al., 1989;Dennis et al., 1999;Wells et al., 2001).UDP-N-acetylglucosamine (UDPGlcNAc), the activated form of GlcNAc, is a key precursor of these Nand O-linked glycosylations (Hart et al., 1989;Herscovics and Orlean, 1993). It is also essential for the synthesis of chitin (a major component of the fungal cell wall) (Cabib et al., 1982) and of the glycosylphosphatidylinositol (GPI) linker which anchors a variety of cell surface proteins to the plasma membrane (Udenfriend and Kodukula, 1995). In bacteria, UDPGlcNAc represents an essential precursor for both peptidoglycan and lipopolysaccharide biosynthesis (Raetz, 1996).UDPGlcNAc is synthesized in the cell cytoplasm by an enzyme which catalyses the following reversible reaction (in the presence of Mg 2+ /Mn 2+ ):This enzyme is named UDPGlcNAc pyrophosphorylase (UAP) or GlcNAc1P uridyltransferase, depending on the direction of the reaction considered. In eukaryotes, initial characterization of UAPs was undertaken on partially puri®ed enzymes from yeast, Neurospora cra...

␥-Aminobutyric acid aminotransferase (GABA-AT) is a pyridoxal 5-phosphate-dependent enzyme responsible for the degradation of the inhibitory neurotransmitter GABA. GABA-AT is a validated target for antiepilepsy drugs because its selective inhibition raises GABA concentrations in brain. The antiepilepsy drug, ␥-vinyl-GABA (vigabatrin) has been investigated in the past by various biochemical methods and resulted in several proposals for its mechanisms of inactivation. In this study we solved and compared the crystal structures of pig liver GABA-AT in its native form (to 2.3-Å resolution) and in complex with vigabatrin as well as with the close analogue ␥-ethynyl-GABA (to 2.3 and 2.8 Å, respectively). Both inactivators form a covalent ternary adduct with the active site Lys-329 and the pyridoxal 5-phosphate (PLP) cofactor. The crystal structures provide direct support for specific inactivation mechanisms proposed earlier on the basis of radiolabeling experiments. The reactivity of GABA-AT crystals with the two GABA analogues was also investigated by polarized absorption microspectrophotometry. The spectral data are discussed in relation to the proposed mechanism. Intriguingly, all three structures revealed a [2Fe-2S] cluster of yet unknown function at the center of the dimeric molecule in the vicinity of the PLP cofactors.

The bifunctional bacterial enzyme N-acetyl-glucosamine-1-phosphate uridyltransferase (GlmU) catalyzes the two-step formation of UDP-GlcNAc, a fundamental precursor in bacterial cell wall biosynthesis. With the emergence of new resistance mechanisms against ␤-lactam and glycopeptide antibiotics, the biosynthetic pathway of UDP-GlcNAc represents an attractive target for drug design of new antibacterial agents. The crystal structures of Streptococcus pneumoniae GlmU in unbound form, in complex with acetyl-coenzyme A (AcCoA) and in complex with both AcCoA and the end product UDP-GlcNAc, have been determined and refined to 2.3, 2.5, and 1.75 Å, respectively. The S. pneumoniae GlmU molecule is organized in two separate domains connected via a long ␣-helical linker and associates as a trimer, with the 50-Å-long left-handed ␤-helix (L␤H) Cterminal domains packed against each other in a parallel fashion and the C-terminal region extended far away from the L␤H core and exchanged with the ␤-helix from a neighboring subunit in the trimer. AcCoA binding induces the formation of a long and narrow tunnel, enclosed between two adjacent L␤H domains and the interchanged C-terminal region of the third subunit, giving rise to an original active site architecture at the junction of three subunits.

The yeast enzymes involved in UDP-GlcNAc biosynthesis are potential targets for antifungal agents. GNA1, a novel member of the Gcn5-related N-acetyltransferase (GNAT) superfamily, participates in UDP-GlcNAc biosynthesis by catalyzing the formation of GlcNAc6P from AcCoA and GlcN6P. We have solved three crystal structures corresponding to the apo Saccharomyces cerevisiae GNA1, the GNA1-AcCoA, and the GNA1-CoAGlcNAc6P complexes and have refined them to 2.4, 1.3, and 1.8 Å resolution, respectively. These structures not only reveal a stable, ␤-intertwined, dimeric assembly with the GlcNAc6P binding site located at the dimer interface but also shed light on the catalytic machinery of GNA1 at an atomic level. Hence, they broaden our understanding of structural features required for GNAT activity, provide structural details for related aminoglycoside N-acetyltransferases, and highlight the adaptability of the GNAT superfamily members to acquire various specificities.