Simon T. M. Allard

Epimerases: structure, function and mechanism

Abstract. Carbohydrates are ideally suited for molecular recognition. By varying the stereochemistry of the hydroxyl substituents, the simple six-carbon, six-oxygen pyranose ring can exist as 10 different molecules. With the further addition of simple chemical changes, the potential for generating distinct molecular recognition surfaces far exceeds that of amino acids. This ability to control and change the stereochemistry of the hydroxyl substituents is very important in biology. Epimerases can be found in animals, plants and microorganisms where they participate in important metabolic pathways such as the Leloir pathway, which involves the conversion of galactose to glucose-1-phosphate. Bacterial epimerases are involved in the production of complex carbohydrate polymers that are used in their cell walls and envelopes and are recognised as potential therapeutic targets for the treatment of bacterial infection. Several distinct strategies have evolved to invert or epimerise the hydroxyl substituents on carbohydrates. In this review we group epimerisation by mechanism and discuss in detail the molecular basis for each group. These groups include enzymes which epimerise by a transient keto intermediate, those that rely on a permanent keto group, those that eliminate then add a nucleotide, those that break then reform carbon-carbon bonds and those that linearize and cyclize the pyranose ring. This approach highlights the quite different biochemical processes that underlie what is seemingly a simple reaction. What this review shows is that each position on the carbohydrate can be epimerised and that epimerisation is found in all organisms.

High Resolution X-ray Structure of dTDP-Glucose 4,6-Dehydratase from Streptomyces venezuelae

Desosamine is a 3-(dimethylamino)-3,4,6-trideoxyhexose found in some macrolide antibiotics. In Streptomyces venezuelae, there are seven genes required for the biosynthesis of this unusual sugar. One of the genes, desIV, codes for a dTDP-glucose 4,6-dehydratase, which is referred to as DesIV. The reaction mechanisms for these types of dehydratases are quite complicated with proton abstraction from the sugar 4′-hydroxyl group and hydride transfer to NAD+, proton abstraction at C-5, and elimination of the hydroxyl group at C-6 of the sugar, and finally return of a proton to C-5 and a hydride from NADH to C-6. Here we describe the cloning, overexpression, and purification, and high resolution x-ray crystallographic analysis to 1.44 Å of wild-type DesIV complexed with dTDP. Additionally, for this study, a double site-directed mutant protein (D128N/E129Q) was prepared, crystallized as a complex with NAD+ and the substrate dTDP-glucose and its structure determined to 1.35 Å resolution. In DesIV, the phenolate group of Tyr151 and Oγ of Thr127 lie at 2.7 and 2.6 Å, respectively from the 4′-hydroxyl group of the dTDP-glucose substrate. The side chain of Asp128 is in the correct position to function as a general acid for proton donation to the 6′-hydroxyl group while the side chain of Glu129 is ideally situated to serve as the general base for proton abstraction at C-5. This investigation provides further detailed information for understanding the exquisite chemistry that occurs in these remarkable enzymes.

The purification, crystallization and structural elucidation of dTDP-d-glucose 4,6-dehydratase (RmlB), the second enzyme of the dTDP-l-rhamnose synthesis pathway from Salmonella enterica serovar Typhimurium

dTDP-D-glucose 4,6-dehydratase (RmlB) is the second of four enzymes involved in the dTDP-L-rhamnose pathway and catalyzes the dehydration of dTDP-D-glucose to dTDP-4-keto-6-deoxy-D-glucose. The ultimate product of the pathway, dTDP-L-rhamnose, is the precursor of L-rhamnose, which is a key component of the cell wall of many pathogenic bacteria. RmlB from Salmonella enterica serovar Typhimurium has been overexpressed and purified, and crystals of the enzyme have been grown using the sitting-drop vapour-diffusion technique with lithium sulfate as precipitant. Diffraction data have been obtained to a resolution of 2.8 A on a single frozen RmlB crystal which belongs to space group P2(1), with unit-cell parameters a = 111.85, b = 87.77, c = 145.66 A, beta = 131.53 degrees. The asymmetric unit contains four monomers in the form of two RmlB dimers with a solvent content of 62%. A molecular-replacement solution has been obtained and the model is currently being refined.

The structure at 1.6 Å resolution of the protein product of the At4g34215 gene from Arabidopsis thaliana

The crystal structure of the At4g34215 protein of Arabidopsis thaliana was determined by molecular replacement and refined to an R factor of 14.6% (R(free) = 18.3%) at 1.6 Angstroms resolution. The crystal structure confirms that At4g34215 belongs to the SGNH-hydrolase superfamily of enzymes. The catalytic triad of the enzyme comprises residues Ser31, His238 and Asp235. In this structure the catalytic serine residue was found to be covalently modified, possibly by phenylmethylsulfonyl fluoride. The structure also reveals a previously undescribed variation within the active site. The conserved asparagine from block III, which provides a hydrogen bond for an oxyanion hole in the SGNH-hydrolase superfamily enzymes, is missing in At4g34215 and is functionally replaced by Gln30 from block I. This residue is positioned in a catalytically competent conformation by nearby residues, including Gln159, Gly160 and Glu161, which are fully conserved in the carbohydrate esterase family 6 enzymes.

X-ray crystal structures of the conserved hypothetical proteins from Arabidopsis thaliana gene loci At5g11950 and AT2g37210.

Introduction. The gene loci At5g11950 and At2g37210 from Arabidopsis thaliana encode highly conserved hypothetical proteins with unknown function. The gene products were annotated as putative lysine decarboxylase (LDC)-like proteins by genome analysis. The crystal structure of At5g11950 was determined by the singlewavelength anomalous dispersion method with an R factor of 15.9% (Rfree 1⁄4 21.3%) at 2.15 Å resolution, and the structure of At2g37210 was solved using molecular replacement with an R factor of 18.1% (Rfree 1⁄4 23.4%) at 1.95 Å resolution. The crystal structure of At5g11950 includes two monomers in the asymmetric unit, and the monomeric structure shows an a/b protein fold comprising eight a-helices and seven b-strands. The structure of At2g37210 is almost identical to that of At5g11950. The fully and highly conserved Arg98 and PGGxGTxxE motif, respectively, were identified by sequence alignment with members of the LDC-like proteins and were mapped onto the structure of At5g11950. A possible active site was suggested based upon the analysis of the location of invariant residues and the consensus motif on the structures of At5g11950 and At2g37210. The Center for Eukaryotic Structural Genomics (CESG) focuses on technology and methodology development for high-throughput X-ray or NMR structure determination of proteins from eukaryotic organisms. The goals of this project also include the identification of new or unique protein folds and characterization of proteins of unknown structure or function. Through a process of selecting targets that have no close amino acid sequence relationship to those in Protein Data Bank (PDB), CESG selected two open reading frames, At5g11950 and At2g37210, from Arabidopsis thaliana for structural characterization. These two genes encode highly conserved hypothetical proteins with molecular weights of 23.8 and 23.6 kDa, respectively. The biological functions of the At5g11950 and At2g37210 genes in A. thaliana are not yet established. Based on sequence similarities, the protein products of At5g11950 and At2g37210 are annotated as lysine decarboxylase (LDC)like proteins; however, no indication of the basis for this annotation can be found. In A. thaliana, at least 11 hypothetical proteins are annotated as LDC-like proteins by genome analysis. No biochemical evidence supporting this annotation is available. Here, we report the X-ray crystal structures of the proteins from A. thaliana gene loci At5g11950 and At2g37210 and describe the structural context of the characteristic motif of this protein family.

Structure at 1.6 Å resolution of the protein from gene locus At3g22680 from Arabidopsis thaliana

The gene product of At3g22680 from Arabidopsis thaliana codes for a protein of unknown function. The crystal structure of the At3g22680 gene product was determined by multiple-wavelength anomalous diffraction and refined to an R factor of 16.0% (Rfree = 18.4%) at 1.60 A resolution. The refined structure shows one monomer in the asymmetric unit, with one molecule of the non-denaturing detergent CHAPS {3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulfonate} tightly bound. Protein At3g22680 shows no structural homology to any other known proteins and represents a new fold in protein conformation space.

The structure at 1.7 Å resolution of the protein product of the At2g17340 gene from Arabidopsis thaliana

The crystal structure of the At2g17340 protein from A. thaliana was determined by the multiple-wavelength anomalous diffraction method and was refined to an R factor of 16.9% (Rfree = 22.1%) at 1.7 A resolution. At2g17340 is a member of the Pfam01937.11 protein family and its structure provides the first insight into the structural organization of this family. A number of fully and highly conserved residues defined by multiple sequence alignment of members of the Pfam01937.11 family were mapped onto the structure of At2g17340. The fully conserved residues are involved in the coordination of a metal ion and in the stabilization of loops surrounding the metal site. Several additional highly conserved residues also map into the vicinity of the metal-binding site, while others are clearly involved in stabilizing the hydrophobic core of the protein. The structure of At2g17340 represents a new fold in protein conformational space.

The structure at 2.4 A resolution of the protein from gene locus At3g21360, a putative Fe(II)/2-oxoglutarate-dependent enzyme from Arabidopsis thaliana.

The crystal structure of the gene product of At3g21360 from Arabidopsis thaliana was determined by the single-wavelength anomalous dispersion method and refined to an R factor of 19.3% (Rfree = 24.1%) at 2.4 A resolution. The crystal structure includes two monomers in the asymmetric unit that differ in the conformation of a flexible domain that spans residues 178-230. The crystal structure confirmed that At3g21360 encodes a protein belonging to the clavaminate synthase-like superfamily of iron(II) and 2-oxoglutarate-dependent enzymes. The metal-binding site was defined and is similar to the iron(II) binding sites found in other members of the superfamily.

The structure at 2.5 Å resolution of human basophilic leukemia-expressed protein BLES03

The crystal structure of the human basophilic leukemia-expressed protein (BLES03, p5326, Hs.433573) was determined by single-wavelength anomalous diffraction and refined to an R factor of 18.8% (Rfree = 24.5%) at 2.5 A resolution. BLES03 shows no detectable sequence similarity to any functionally characterized proteins using state-of-the-art sequence-comparison tools. The structure of BLES03 adopts a fold similar to that of eukaryotic transcription initiation factor 4E (eIF4E), a protein involved in the recognition of the cap structure of eukaryotic mRNA. In addition to fold similarity, the electrostatic surface potentials of BLES03 and eIF4E show a clear conservation of basic and acidic patches. In the crystal lattice, the acidic amino-terminal helices of BLES03 monomers are bound within the basic cavity of symmetry-related monomers in a manner analogous to the binding of mRNA by eIF4E. Interestingly, the gene locus encoding BLES03 is located between genes encoding the proteins DRAP1 and FOSL1, both of which are involved in transcription initiation. It is hypothesized that BLES03 itself may be involved in a biochemical process that requires recognition of nucleic acids.