Potentiometric analysis of UDP-galactopyranose mutase: stabilization of the flavosemiquinone by substrate

UDP-galactopyranose mutase is a flavoprotein which catalyses the interconversion of UDP-galactopyranose and UDP-galactofuranose. The enzyme is of interest because it provides the activated biosynthetic precursor of galactofuranose, a key cell wall component of many bacterial pathogens. The reaction mechanism of this mutase is intriguing because the anomeric oxygen forms a glycosidic bond, which means that the reaction must proceed by a novel mechanism involving ring breakage and closure. The structure of the enzyme is known, but the mechanism, although speculated on, is not resolved. The overall reaction is electrically neutral but a crypto-redox reaction is suggested by the requirement that the flavin must adopt the reduced form for activity. Herein we report a thermodynamic analysis of the enzyme's flavin cofactor with the objective of defining the system and setting parameters for possible reaction schemes. The analysis shows that the neutral semiquinone (FADH(.)) is stabilized in the presence of substrate and the fully reduced flavin is the anionic FADH(-) rather than the neutral FADH(2). The anionic FADH(-) has the potential to act as a rapid 1-electron donor/acceptor without being slowed by a coupled proton transfer and is therefore an ideal crypto-redox cofactor.</p

Mechanism of the Class I KDPG aldolase

In vivo, 2-keto-3-deoxy-6-pliosphogluconate (KDPG) aldolase catalyzes the reversible, stereospecific retro-aldol cleavage of KDPG to pyruvate and D-glyceraldehyde-3-phosphate. The enzyme is a lysine-dependent (Class 1) aldolase that functions through the intermediacy of a Schiff base. Here, we propose a mechanism for this enzyme based on crystallographic studies of wild-type and mutant aldolases. The three dimensional Structure of KDPG aldolase from the thermophile Thermotoga maritima was determined to 1.9 angstrom. The Structure is the standard alpha/beta barrel observed for all Class I aldolases. At the active site Lys we observe clear density for a pyruvate Schiff base. Density for a sulfate ion bound in a conserved Cluster of residues close to the Schiff base is also observed. We have also determined the structure of a mutant of Escherichia coli KDPG aldolase in which the proposed general acid/base catalyst has been removed (E45N). One Subunit of the trimer contains density suggesting a trapped pyruvate carbinolamine intermediate. All three Subunits contain a phosphate ion bound in a location effectively identical to that of the Sulfate ion bound in the T maritima enzyme. The sulfate and phosphate ions experimentally locate the putative phosphate binding site of the aldolase and, together with the position of the bound pyruvate, facilitate construction of a model for the full-length KDPG substrate complex. The model requires only minimal positional adjustments of the experimentally determined covalent intermediate and bound anion to accommodate full-length substrate. The model identifies the key catalytic residues of the protein and Suggests important roles for two observable water molecules. The first water molecule remains bound to the enzyme during the entire catalytic cycle, shuttling protons between the catalytic glutamate and the substrate. The second water molecule arises from dehydration of the carbinolamine and serves as the nucleophilic water during hydrolysis of the enzyme-product Schiff base. The second water Molecule may also mediate the base-catalyzed enolization required to form the carbon nucleophile, again bridging to the catalytic glutamate. Many aspects of this mechanism are observed in other Class I aldolases and suggest a mechanistically and, perhaps, evolutionarily related family of aldolases distinct from the N-acetyineuraminate lyase (NAL) family. (c) 2005 Elsevier Ltd. All rights reserved.</p

Crystal structures of <em>Mycobacteria tuberculosis</em> and <em>Klebsiella pneuoniae</em> UDP-galactopyranose mutase in the oxidised state and <em>Klebsiella pneumoniae</em> UDP-galactopyranose mutase in the (active) reduced state.

Uridine diphosphogalactofuranose (UDP-Galf) is the precursor of the D-galactofuranose sugar found in bacterial and parasitic cell walls, including those of many pathogens. UDP-Galf is made from UDP-galactopyranose by the enzyme UDP-galactopyranose mutase. The enzyme requires the reduced FADH(−) co-factor for activity. The structure of the Mycobacterium tuberculosis mutase with FAD has been determined to 2.25Å. The structures of Klebsiella pneumoniae mutase with FAD and with FADH(−) bound have been determined to 2.2Å and 2.35Å resolutions respectively. This is the first report of the FADH(−) containing structure. Two flavin dependent mechanisms for the enzyme have been proposed, one which involves a covalent adduct being formed at the flavin and the other based on electron transfer. Using our structural data, we have examined the two mechanisms. The electron transfer mechanism is consistent with the structural data, not surprisingly since it makes fewer demands on the precise positioning of atoms. A model based on a covalent adduct FAD requires repositioning of the enzyme active site and would appear to require that the isoalloxazine ring of FADH(−) to buckle in a particular way. However, the FADH(−) structure reveals that the isoalloxazine ring buckles in the opposite sense, this apparently requires the covalent adduct to trigger profound conformational changes in the protein or to buckle the FADH(−) opposite to that seen in the apo structure

Design of a hyperstable 60-subunit protein icosahedron

The icosahedron and the dodecahedron are the largest of the Platonic solids, and icosahedral protein structures are widely utilized in biological systems for packaging and transport(1,2). There has been considerable interest in repurposing such structures(3–5), for example, virus-like particles for the targeted delivery and vaccine design. The ability to design proteins that self assemble into precisely specified, highly ordered icosahedral structures would open the door to a new generation of protein 'containers' that could exhibit properties custom-made for various applications. In this manuscript, we describe the computational design of an icosahedral nano-cage that self-assembles from trimeric building blocks. Electron microscopy images of the designed protein expressed in E. coli reveals a homogenous population of icosahedral particles nearly identical to the design model. The particles are stable in 6.7 M guanidine hydrochloride at up to 80 °C, and undergo extremely abrupt, but reversible, disassembly between 2 M and 2.25 M guanidinium thiocyanate. The icosahedron is robust to genetic fusions: one or two copies of superfolder GFP can be fused to each of the 60 subunits to create highly fluorescent standard candles for light microscopy, and a designed protein pentamer can be placed in the center of each of the twenty pentameric faces to potentially gate macromolecule access to the nanocage interior. Such robust designed nanocages should have considerable utility for targeted drug delivery(6), vaccine design(7), and synthetic biology(8)