Structure of the oligogalacturonate-specific KdgM porin.

The phytopathogenic Gram-negative bacterium Dickeya dadantii (Erwinia chrysanthemi) feeds on plant cell walls by secreting pectinases and utilizing the oligogalacturanate products. An outer membrane porin, KdgM, is indispensable for the uptake of these acidic oligosaccharides. Here, the crystal structure of KdgM determined to 1.9 Šresolution is presented. KdgM is folded into a regular 12-stranded antiparallel ß-barrel with a circular cross-section defining a transmembrane pore with a minimal radius of 3.1 Å. Most of the loops that would face the cell exterior in vivo are disordered, but nevertheless mediate contact between densely packed membrane-like layers in the crystal. The channel is lined by two tracks of arginine residues facing each other across the pore, a feature that is conserved within the KdgM family and is likely to facilitate the diffusion of acidic oligosaccharides.

Crystal structures of two human pyrophosphorylase isoforms in complexes with UDPGlc(Gal)NAc: role of the alternatively spliced insert in the enzyme oligomeric assembly and active site architecture.

The recently published human genome with its relatively modest number of genes has highlighted the importance of post-transcriptional and post-translational modifications, 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 resolution, respectively) and UDPGalNAc (at 2.2 and 2.3 A resolution, respectively). Comparison with known structures classifies AGX1 and AGX2 as two new members of the SpsA-GnT 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 significant modification of the active site architecture, a result suggesting that the two isoforms generated by alternative splicing may have distinct catalytic properties.

The crystal structures of Apo and complexed Saccharomyces cerevisiae GNA1 shed light on the catalytic mechanism of an amino-sugar N-acetyltransferase.

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-CoA-GlcNAc6P complexes and have refined them to 2.4, 1.3, and 1.8 A resolution, respectively. These structures not only reveal a stable, beta-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.

Crystal structure of Streptococcus pneumoniae N-acetylglucosamine-1-phosphate uridyltransferase bound to acetyl-coenzyme A reveals a novel active site architecture.

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 beta-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 A, respectively. The S. pneumoniae GlmU molecule is organized in two separate domains connected via a long alpha-helical linker and associates as a trimer, with the 50-A-long left-handed beta-helix (LbetaH) C-terminal domains packed against each other in a parallel fashion and the C-terminal region extended far away from the LbetaH core and exchanged with the beta-helix from a neighboring subunit in the trimer. AcCoA binding induces the formation of a long and narrow tunnel, enclosed between two adjacent LbetaH 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.

Characterisation and specificity of two single-chain Fv antibodies directed to the protein tyrosine kinase Syk.

In order to obtain single chain Fv fragments (scFv) specific for the protein tyrosine kinase Syk, we screened a human synthetic phage-display library. Two glutathione S-transferase (GST):Syk fusion proteins containing both SH2 domains of Syk were used to perform three rounds of selection of the library. Among the scFv fragments resulting from the third round of selection, the ones specific for the GST portion of the fusion proteins were eliminated by performing enzyme-linked immunosorbent assay tests on GST:Syk versus GST coated plates, and the monoclonal scFv fragments binding only to the GST:Syk coated plates with high affinities were further analysed. We report here the in vitro characterisation of G4G11 and G6G2 anti-Syk scFvs. G4G11 shows the best performance in immunoprecipitation and immunofluorescence experiments, and G6G2 is able to detect Syk in immunoprecipitation, immunofluorescence and on Western blots. Both scFvs are also able to detect the phosphorylated form of Syk, and neither of them binds to Zap-70, the other member of the Syk family of protein tyrosine kinases.