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The MnmE-MnmG complex is involved in tRNA modification. We have determined the crystal structure of Escherichia coli MnmG at 2.4-[Angstrom] resolution, mutated highly conserved residues with putative roles in flavin adenine dinucleotide (FAD) or tRNA binding and MnmE interaction, and analyzed the effects of these mutations in vivo and in vitro. Limited trypsinolysis of MnmG suggests significant conformational changes upon FAD binding. doi: 10.1128/JB.00650-09

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.tibs.2008.11.001 Byline: Renato Morona (1), Leanne Purins (1), Ante Tocilj (2), Allan Matte (3), Miroslaw Cygler (2)(3) Abstract: Polysaccharides are ubiquitously distributed on the cell surface of bacteria. These polymers are involved in many processes, including immune avoidance and bacteria-host interactions, which are especially important for pathogenic organisms. In many instances, the lengths of these polysaccharides are not random, but rather distribute around some mean value, termed the modal length. A large family of proteins, called polysaccharide co-polymerases (PCPs), found in both Gram-negative and Gram-positive species regulate polysaccharide modal length. Recent crystal structures of Wzz proteins from Escherichia coli and Salmonella typhimurium provide the first atomic-resolution information for one family of PCPs, the PCP1 group. These crystal structures have important implications for the structures of other PCP families. Author Affiliation: (1) Australian Bacterial Pathogenesis Program, School of Molecular and Biomedical Science, Discipline of Microbiology and Immunology, University of Adelaide, North Terrace, Adelaide, SA 5005, Australia (2) Department of Biochemistry, McGill University, 3655 Promenade Sir William Osler, Montreal, Quebec H4P 2R2, Canada (3) Biotechnology Research Institute, National Research Council, 6100 Royalmount Avenue, Montreal, Quebec H4P 2R2, Canada

Three catabolic enzymes, UlaD, UlaE, and UlaF, are involved in a pathway leading to fermentation of L-ascorbate under anaerobic conditions. UlaD catalyzes a [beta]-keto acid decarboxylation reaction to produce L-xylulose-5-phosphate, which undergoes successive epimerization reactions with UlaE (L-xylulose-5-phosphate 3-epimerase) and UlaF (L-ribulose-5-phosphate 4-epimerase), yielding D-xylulose-5-phosphate, an intermediate in the pentose phosphate pathway. We describe here crystallographic studies of UlaE from Escherichia coli O157:H7 that complete the structural characterization of this pathway. UlaE has a triosephosphate isomerase (TIM) barrel fold and forms dimers. The active site is located at the C-terminal ends of the parallel [beta]-strands. The enzyme binds [Zn.sup.2+], which is coordinated by Glu155, Asp185, His211, and Glu251. We identified a phosphate-binding site formed by residues from the [beta]1/[alpha]1 loop and [alpha]3' helix in the N-terminal region. This site differs from the well-characterized phosphate-binding motif found in several TIM barrel superfamilies that is located at strands [beta]7 and [beta]8. The intrinsic flexibility of the active site region is reflected by two different conformations of loops forming part of the substrate-binding site. Based on computational docking of the L-xylulose 5-phosphate substrate to UlaE and structural similarities of the active site of this enzyme to the active sites of other epimerases, a metal-dependent epimerization mechanism for UlaE is proposed, and Glu155 and Glu251 are implicated as catalytic residues. Mutation and activity measurements for structurally equivalent residues in related epimerases supported this mechanistic proposal.

Hydrogenases are enzymes involved in hydrogen metabolism, utilizing [H.sub.2] as an electron source. [NiFe] hydrogenases are heterodimeric Fe-S proteins, with a large subunit containing the reaction center involving Fe and Ni metal ions and a small subunit containing one or more Fe-S clusters. Maturation of the [NiFe] hydrogenase involves assembly of nonproteinaceous ligands on the large subunit by accessory proteins encoded by the hyp operon. HypE is an essential accessory protein and participates in the synthesis of two cyano groups found in the large subunit. We report the crystal structure of Escherichia coli HypE at 2.0-[Angstrom] resolution. HypE exhibits a fold similar to that of PurM and ThiL and forms dimers. The C-terminal catalytically essential Cys336 is internalized at the dimer interface between the N- and C-terminal domains. A mechanism for dehydration of the thiocarbamate to the thiocyanate is proposed, involving Asp83 and Glu272. The interactions of HypE and HypF were characterized in detail by surface plasmon resonance and isothermal titration calorimetry, revealing a [K.sub.d] (dissociation constant) of ~400 nM. The stoichiometry and molecular weights of the complex were verified by size exclusion chromatography and gel scanning densitometry. These experiments reveal that HypE and HypF associate to forma stoichiometric, hetero-oligomeric complex predominantly consisting of a [[EF].sub.2] heterotetramer which exists in a dynamic equilibrium with the EF heterodimer. The surface plasmon resonance results indicate that a conformational change occurs upon heterodimerization which facilitates formation of a productive complex as part of the carbamate transfer reaction.

Enterobacterial common antigen (ECA) is a polysaccharide found on the outer membrane of virtually all gram-negative enteric bacteria and consists of three sugars, N-acetyl-D-glucosamine, N-acetyl-D-mannosaminuronic acid, and 4-acetamido-4, 6-dideoxy-D-galactose, organized into trisaccharide repeating units having the sequence [right arrow] 3)-[alpha]-D-Fuc4NAc-(1 [right arrow] 4)-[beta]-D-ManNAcA-(1 [right arrow] 4)-[alpha]-D-GlcNAc-(1-[right arrow]. While the precise function of ECA is unknown, it has been linked to the resistance of Shiga-toxin-producing Escherichia coil (STEC) O157:H7 to organic acids and the resistance of Salmonella enterica to bile salts. The final step in the synthesis of 4-acetamido-4,6-dideoxy-o-galactose, the acetyl-coenzyme A (CoA)-dependent acetylation of the 4-amino group, is carried out by TDP-fucosamine acetyltransferase (WecD). We have determined the crystal structure of WecD in apo form at a 1.95-[Angstrom] resolution and bound to acetyl-CoA at a 1.66-[Angstrom] resolution. WecD is a dimeric enzyme, with each monomer adopting the GNAT N-acetyltransferase fold, common to a number of enzymes involved in acetylation of histones, aminoglycoside antibiotics, serotonin, and sugars. The crystal structure of WecD, however, represents the first structure of a GNAT family member that acts on nucleotide sugars. Based on this cocrystal structure, we have used flexible docking to generate a WecD-bound model of the acetyl-CoA-TDP-fucosamine tetrahedral intermediate, representing the structure during acetyl transfer. Our structural data show that WecD does not possess a residue that directly functions as a catalytic base, although Tyr208 is well positioned to function as a general acid by protonating the thiolate anion of coenzyme A.

Heme oxygenases (HOs) catalyze the oxidation of heme to biliverdin, carbon monoxide (CO), and free iron. Iron acquisition is critical for invading microorganisms to enable survival and growth. Here we report the crystal structure of ChuS, which displays a previously uncharacterized fold and is unique compared with other characterized HOs. Despite only 19% sequence identity between the N- and C-terminal halves, these segments of ChuS represent a structural duplication, with a root-mean-square deviation of 2.1 [Angstrom] between the two repeats. ChuS is capable of using ascorbic acid or cytochrome P450 reductase-NADPH as electron sources for heme oxygenation. CO detection confirmed that ChuS is a HO, and we have identified it in pathogenic Escherichia coli O157:H7. Based on sequence analysis, this HO is present in many bacteria, although not in the E. coli K-12 strain. The N- and C-terminal halves of ChuS are each a functional HO. ChuS | carbon monoxide | structural repeat | heme degredation

Intracellular glucose in Escherichia coli cells imported by phosphoenolpyruvate-dependent phosphotransferase system-independent uptake is phosphorylated by glucokinase by using ATP to yield glucose-6-phosphate. Glucokinases (EC 2.7.1.2) are functionally distinct from hexokinases (EC 2.7.1.1) with respect to their narrow specificity for glucose as a substrate. While structural information is available for ADP-dependent glucokinases from Archaea, no structural information exists for the large sequence family of eubacterial ATP-dependent glucokinases. Here we report the first structure determination of a microbial ATP-dependent glucokinase, that from E. coli O157:H7. The crystal structure of E. coli glucokinase has been determined to a 2.3-[Angstrom] resolution (apo form) and refined to final [R.sub.work]/[R.sub.free] factors of 0.200/0.271 and to 2.2-[Angstrom] resolution (glucose complex) with final [R.sub.work]/[R.sub.free] factors of 0.193/0.265. E. coli GIK is a homodimer of 321 amino acid residues. Each monomer folds into two domains, a small [alpha]/[beta] domain (residues 2 to 110 and 301 to 321) and a larger [alpha]+[beta] domain (residues 111 to 300). The active site is situated in a deep cleft between the two domains. E. coli GlK is structurally similar to Saccharomyces cerevisiae hexokinase and human brain hexokinase I but is distinct from the ADP-dependent GlKs. Bound glucose forms hydrogen bonds with the residues Asn99, Asp100, Glu157, His160, and Glu187, all of which, except His160, are structurally conserved in human hexokinase 1. Glucose binding results in a closure of the small domains, with a maximal C[alpha] shift of ~10 [Angstrom]. A catalytic mechanism is proposed that is consistent with Asp100 functioning as the general base, abstracting a proton from the 06 hydroxyl of glucose, followed by nucleophilic attack at the [gamma]-phosphoryl group of ATP, yielding glucose-6-phosphate as the product.

The histidine biosynthetic pathway is an ancient one found in bacteria, archaebacteria, fungi, and plants that converts 5-phosphoribosyl 1-pyrophosphate to L-histidine in 10 enzymatic reactions. This pathway provided a paradigm for the operon, transcriptional regulation of gene expression, and feedback inhibition of a pathway. L-histidinol dehydrogenase (HisD, EC 1.1.1.23) catalyzes the last two steps in the biosynthesis of L-histidine: sequential NAD-dependent oxidations of L-histidinol to L-histidinaldehyde and then to L-histidine. HisD functions as a homodimer and requires the presence of one [Zn.sup.2+] cation per monomer. We have determined the three-dimensional structure of Escherichia coli HisD in the apo state as well as complexes with substrate, [Zn.sup.2+], and [NAD.sup.+] (best resolution is 1.7 A). Each monomer is made of four domains, whereas the intertwined dimer possibly results from domain swapping. Two domains display a very similar incomplete Rossmann fold that suggests an ancient event of gene duplication. Residues from both monomers form the active site. [Zn.sup.2+] plays a crucial role in substrate binding but is not directly involved in catalysis. The active site residue His-327 participates in acid-base catalysis, whereas Glu-326 activates a water molecule. [NAD.sup.+] binds weakly to one of the Rossmann fold domains in a manner different from that previously observed for other proteins having a Rossmann fold.

The three-dimensional structure of Escherichia coli 2-amino-3-ketobutyrate CoA ligase reveals that it is a member of the alpha-family of pyridoxal phosphate-dependent enzymes and is a dimer with two active sites per dimer. Data show that the monomers contribute side chains to each of the active substrate binding site.

Researchers describe four subsites in the chondroitin AC lyase from Flavobacterium heparinium complexed with oligosaccharides. Subsites

Two endoxylynases (1, 4-beta-D-xylanohydrolases) from Fibrobacter succinogenees S85 were purified and characterized. The enzymes were grown on crystalline cellulose and purified to apparent electrophoretic homogeneity by column chromatography. Results showed that endoxylanase 1 was a basic protein of 53.7 kDa and exhibited arabinose-debranching activity. On the other hand, endoxylanase 2 with 66. kDa showed xyland-hydrolyzing activity and significant activity towards carboxymethyl cellulose. Both enzymes were observed to have important functions in in vivo plant cell digestion by F. succinogenes.

The crystal structure of the urease maturation protein UreE from Helicobacter pylori is determined in its apo form, bound to [Cu.sup.2+] and [Ni.sup.2+]. The comparison of independent subunits from different crystal forms has shown changes in the relative arrangement of the N- and C-terminal domains in response to metal binding.

Several biophysical methods are employed to explain the structural and binding properties of the different ligand complexes of PEB3, a glycoprotein adhesion obtained from Campylobacter jejuni. PEB3 is shown to act as a transport protein and can be easily used for utilizing the phosphates from the host.

A study was conducted to determine the crystal structure and function of PgID enzyme in CoA-bound and unbound forms isolated from Campylobacter jejuni, highly unusual bacteria forming N-linked glycoproteins. The enzyme is found to be the member of L[beta]H family of complex sugar N-acetyltransferases which adopts an extended structure in the unbound form and folds to extend the [beta]-helix upon binding CoA.

The crystal structure of Escherichia coli CoA-Transferase (CaiB) in the apo form and in complex with bound CoA is reported. It is observed that complex with carnitinyl-CoA reveals the interactions between CaiB and the betainyl moiety of the CoA thioester.