Sharon M. Shepherd

Structural insights into mechanism and specificity of O ‐GlcNAc transferase

Post‐translational modification of protein serines/threonines with N‐acetylglucosamine (O‐GlcNAc) is dynamic, inducible and abundant, regulating many cellular processes by interfering with protein phosphorylation. O‐GlcNAcylation is regulated by O‐GlcNAc transferase (OGT) and O‐GlcNAcase, both encoded by single, essential, genes in metazoan genomes. It is not understood how OGT recognises its sugar nucleotide donor and performs O‐GlcNAc transfer onto proteins/peptides, and how the enzyme recognises specific cellular protein substrates. Here, we show, by X‐ray crystallography and mutagenesis, that OGT adopts the (metal‐independent) GT‐B fold and binds a UDP‐GlcNAc analogue at the bottom of a highly conserved putative peptide‐binding groove, covered by a mobile loop. Strikingly, the tetratricopeptide repeats (TPRs) tightly interact with the active site to form a continuous 120 Å putative interaction surface, whereas the previously predicted phosphatidylinositide‐binding site locates to the opposite end of the catalytic domain. On the basis of the structure, we identify truncation/point mutants of the TPRs that have differential effects on activity towards proteins/peptides, giving first insights into how OGT may recognise its substrates.

Molecular Mechanisms of Yeast Cell Wall Glucan Remodeling

Yeast cell wall remodeling is controlled by the equilibrium between glycoside hydrolases, glycosyltransferases, and transglycosylases. Family 72 glycoside hydrolases (GH72) are ubiquitous in fungal organisms and are known to possess significant transglycosylase activity, producing elongated β(1–3) glucan chains. However, the molecular mechanisms that control the balance between hydrolysis and transglycosylation in these enzymes are not understood. Here we present the first crystal structure of a glucan transglycosylase, Saccharomyces cerevisiae Gas2 (ScGas2), revealing a multidomain fold, with a (βα)8 catalytic core and a separate glucan binding domain with an elongated, conserved glucan binding groove. Structures of ScGas2 complexes with different β-glucan substrate/product oligosaccharides provide “snapshots” of substrate binding and hydrolysis/transglycosylation giving the first insights into the mechanisms these enzymes employ to drive β(1–3) glucan elongation. Together with mutagenesis and analysis of reaction products, the structures suggest a “base occlusion” mechanism through which these enzymes protect the covalent protein-enzyme intermediate from a water nucleophile, thus controlling the balance between hydrolysis and transglycosylation and driving the elongation of β(1–3) glucan chains in the yeast cell wall.

Crystal structures of penicillin-binding protein 3 from Pseudomonas aeruginosa: comparison of native and antibiotic-bound forms

We report the first crystal structures of a penicillin-binding protein (PBP), PBP3, from Pseudomonas aeruginosa in native form and covalently linked to two important β-lactam antibiotics, carbenicillin and ceftazidime. Overall, the structures of apo and acyl complexes are very similar; however, variations in the orientation of the amino-terminal membrane-proximal domain relative to that of the carboxy-terminal transpeptidase domain indicate interdomain flexibility. Binding of either carbenicillin or ceftazidime to purified PBP3 increases the thermostability of the enzyme significantly and is associated with local conformational changes, which lead to a narrowing of the substrate-binding cleft. The orientations of the two β-lactams in the active site and the key interactions formed between the ligands and PBP3 are similar despite differences in the two drugs, indicating a degree of flexibility in the binding site. The conserved binding mode of β-lactam-based inhibitors appears to extend to other PBPs, as suggested by a comparison of the PBP3/ceftazidime complex and the Escherichia coli PBP1b/ceftoxamine complex. Since P. aeruginosa is an important human pathogen, the structural data reveal the mode of action of the frontline antibiotic ceftazidime at the molecular level. Improved drugs to combat infections by P. aeruginosa and related Gram-negative bacteria are sought and our study provides templates to assist that process and allows us to discuss new ways of inhibiting PBPs.

The structure of Serratia marcescens Lip, a membrane-bound component of the type VI secretion system

The high-resolution crystal structure of S. marcescens Lip reveals a new member of the transthyretin family of proteins. Lip, a core component of the type VI secretion apparatus, is localized to the outer membrane and is positioned to interact with other proteins forming this complex system.

Structural and Kinetic Differences between Human and Aspergillus Fumigatus D-Glucosamine-6- Phosphate N-Acetyltransferase.

Aspergillus fumigatus is the causative agent of aspergillosis, a frequently invasive colonization of the lungs of immunocompromised patients. GNA1 (D-glucosamine-6-phosphate N-acetyltransferase) catalyses the acetylation of GlcN-6P (glucosamine-6-phosphate) to GlcNAc-6P (N-acetylglucosamine-6-phosphate), a key intermediate in the UDP-GlcNAc biosynthetic pathway. Gene disruption of gna1 in yeast and Candida albicans has provided genetic validation of the enzyme as a potential target. An understanding of potential active site differences between the human and A. fumigatus enzymes is required to enable further work aimed at identifying selective inhibitors for the fungal enzyme. In the present study, we describe crystal structures of both human and A. fumigatus GNA1, as well as their kinetic characterization. The structures show significant differences in the sugar-binding site with, in particular, several non-conservative substitutions near the phosphate-binding pocket. Mutagenesis targeting these differences revealed drastic effects on steady-state kinetics, suggesting that the differences could be exploitable with small-molecule inhibitors.

Pseudomonas aeruginosa 4-Amino-4-Deoxychorismate Lyase: Spatial Conservation of an Active Site Tyrosine and Classification of Two Types of Enzyme

4-Amino-4-deoxychorismate lyase (PabC) catalyzes the formation of 4-aminobenzoate, and release of pyruvate, during folate biosynthesis. This is an essential activity for the growth of Gram-negative bacteria, including important pathogens such as Pseudomonas aeruginosa. A high-resolution (1.75 Å) crystal structure of PabC from P. aeruginosa has been determined, and sequence-structure comparisons with orthologous structures are reported. Residues around the pyridoxal 5′-phosphate cofactor are highly conserved adding support to aspects of a mechanism generic for enzymes carrying that cofactor. However, we suggest that PabC can be classified into two groups depending upon whether an active site and structurally conserved tyrosine is provided from the polypeptide that mainly forms an active site or from the partner subunit in the dimeric assembly. We considered that the conserved tyrosine might indicate a direct role in catalysis: that of providing a proton to reduce the olefin moiety of substrate as pyruvate is released. A threonine had previously been suggested to fulfill such a role prior to our observation of the structurally conserved tyrosine. We have been unable to elucidate an experimentally determined structure of PabC in complex with ligands to inform on mechanism and substrate specificity. Therefore we constructed a computational model of the catalytic intermediate docked into the enzyme active site. The model suggests that the conserved tyrosine helps to create a hydrophobic wall on one side of the active site that provides important interactions to bind the catalytic intermediate. However, this residue does not appear to participate in interactions with the C atom that undergoes an sp 2 to sp 3 conversion as pyruvate is produced. The model and our comparisons rather support the hypothesis that an active site threonine hydroxyl contributes a proton used in the reduction of the substrate methylene to pyruvate methyl in the final stage of the mechanism.

Glucose‐6‐phosphate as a probe for the glucosamine‐6‐phosphate N‐acetyltransferase Michaelis complex

Glucosamine‐6‐phosphate N‐acetyltransferase (GNA1) catalyses the N‐acetylation of d‐glucosamine‐6‐phosphate (GlcN‐6P), using acetyl‐CoA as an acetyl donor. The product GlcNAc‐6P is an intermediate in the biosynthesis UDP‐GlcNAc. GNA1 is part of the GCN5‐related acetyl transferase family (GNATs), which employ a wide range of acceptor substrates. GNA1 has been genetically validated as an antifungal drug target. Detailed knowledge of the Michaelis complex and trajectory towards the transition state would facilitate rational design of inhibitors of GNA1 and other GNAT enzymes. Using the pseudo‐substrate glucose‐6‐phosphate (Glc‐6P) as a probe with GNA1 crystals, we have trapped the first GNAT (pseudo‐)Michaelis complex, providing direct evidence for the nucleophilic attack of the substrate amine, and giving insight into the protonation of the thiolate leaving group.

Structure of Pseudomonas aeruginosa inosine 5′-monophosphate dehydrogenase

The crystal structure of inosine 5′-monophosphate dehydrogenase from P. aeruginosa has been determined to 2.25 Å resolution.