Structural basis for the altered activity of Gly794 variants of Escherichia coli beta-galactosidase

The open-closed conformational switch in the active site of Escherichia coli beta-galactosidase was studied by X-ray crystallography and enzyme kinetics. Replacement of Gly794 by alanine causes the apoenzyme to adopt the closed rather than the open conformation. Binding of the competitive inhibitor isopropyl thio-beta-D-galactoside (IPTG) requires the mutant enzyme to adopt its less favored open conformation, weakening affinity relative to wild type. In contrast, transition-state inhibitors bind to the enzyme in the closed conformation, which is favored for the mutant, and display increased affinity relative to wild type. Changes in affinity suggest that the free energy difference between the closed and open forms is 1-2 kcal/mol. By favoring the closed conformation, the substitution moves the resting state of the enzyme along the reaction coordinate relative to the native enzyme and destabilizes the ground state relative to the first transition state. The result is that the rate constant for galactosylation is increased but degalactosylation is slower. The covalent intermediate may be better stabilized than the second transition state. The substitution also results in better binding of glucose to both the free and the galactosylated enzyme. However, transgalactosylation with glucose to produce allolactose (the inducer of the lac operon) is slower with the mutant than with the native enzyme. This suggests either that the glucose is misaligned for the reaction or that the galactosylated enzyme with glucose bound is stabilized relative to the transition state for transgalactosylation.     

Structural basis for proofreading during replication of the Escherichia coli chromosome

The epsilon subunit of the Escherichia coli replicative DNA polymerase III is the proofreading 3'-5' exonuclease. Structures of its catalytic N-terminal domain (epsilon186) were determined at two pH values (5.8 and 8.5) at resolutions of 1.7-1.8 A, in complex with two Mn(II) ions and a nucleotide product of its reaction, thymidine 5'-monophosphate. The protein structure is built around a core five-stranded beta sheet that is a common feature of members of the DnaQ superfamily. The structures were identical, except for differences in the way TMP and water molecules are coordinated to the binuclear metal center in the active site. These data are used to develop a mechanism for epsilon and to produce a plausible model of the complex of epsilon186 with DNA.     

Structural basis for the unusual specificity of Escherichia coli aminopeptidase N

Aminopeptidase N from Escherichia coli is a M1 class aminopeptidase with the active-site region related to that of thermolysin. The enzyme has unusual specificity, cleaving adjacent to the large, nonpolar amino acids Phe and Tyr but also cleaving next to the polar residues Lys and Arg. To try to understand the structural basis for this pattern of hydrolysis, the structure of the enzyme was determined in complex with the amino acids L-arginine, L-lysine, L-phenylalanine, L-tryptophan, and L-tyrosine. These amino acids all bind with their backbone atoms close to the active-site zinc ion and their side chain occupying the S1 subsite. This subsite is in the form of a cylinder, about 10 A in cross-section and 12 A in length. The bottom of the cylinder includes the zinc ion and a number of polar side chains that make multiple hydrogen-bonding and other interactions with the alpha-amino group and the alpha-carboxylate of the bound amino acid. The walls of the S1 cylinder are hydrophobic and accommodate the nonpolar or largely nonpolar side chains of Phe and Tyr. The top of the cylinder is polar in character and includes bound water molecules. The epsilon-amino group of the bound lysine side chain and the guanidinium group of arginine both make multiple hydrogen bonds to this part of the S1 site. At the same time, the hydrocarbon part of the lysine and arginine side chains is accommodated within the nonpolar walls of the S1 cylinder. This combination of hydrophobic and hydrophilic binding surfaces explains the ability of ePepN to cleave Lys, Arg, Phe, and Tyr. Another favored substrate has Ala at the P1 position. The short, nonpolar side chain of this residue can clearly be bound within the hydrophobic part of the S1 cylinder, but the reason for its facile hydrolysis remains uncertain.     

A thioredoxin from the hyperthermophilic archaeon Methanococcus jannaschii has a glutaredoxin-like fold but thioredoxin-like activities

A thioredoxin homologue (Mj0307) from the hyperthermophilic archaeon Methanococcus jannaschii (MjTRX) was cloned, produced in E. coli, and compared to the thioredoxin from E. coli (ETRX). The secondary structure profile of MjTRX obtained by NMR spectroscopy shows that it has four beta-sheets and three alpha-helices arranged in betaalphabetaalphabetabetaalpha, similar to that of glutaredoxin. However, MjTRX supports the growth of T7 bacteriophage in E. coli and is weakly reduced by the thioredoxin reductase from E. coli, indicating that MjTRX is functionally closer to a thioredoxin than a glutaredoxin. MjTRX has higher specific insulin reductase activity than ETRX and retained its full activity over 4 days at 95 degrees C, whereas ETRX lost its activity in 150 min. The standard state redox potential of MjTRX is about -277 mV, which is the lowest value thus far known among redox potentials of the thioredoxin superfamily. This indicates that the lower redox potential is necessary in keeping catalytic disulfide bonds reduced in the cytoplasm and in coping with oxidative stress in an anaerobic hyperthermophile.     

Structural basis for substrate specificity of Escherichia coli purine nucleoside phosphorylase

Purine nucleoside phosphorylase catalyzes reversible phosphorolysis of purine nucleosides and 2'-deoxypurine nucleosides to the free base and ribose (or 2'-deoxyribose) 1-phosphate. Whereas the human enzyme is specific for 6-oxopurine ribonucleosides, the Escherichia coli enzyme accepts additional substrates including 6-oxopurine ribonucleosides, 6-aminopurine ribonucleosides, and to a lesser extent purine arabinosides. These differences have been exploited in a potential suicide gene therapy treatment for solid tumors. In an effort to optimize this suicide gene therapy approach, we have determined the three-dimensional structure of the E. coli enzyme in complex with 10 nucleoside analogs and correlated the structures with kinetic measurements and computer modeling. These studies explain the preference of the enzyme for ribose sugars, show increased flexibility for active site residues Asp204 and Arg24, and suggest that interactions involving the 1- and 6-positions of the purine and the 4'- and 5'-positions of the ribose provide the best opportunities to increase prodrug specificity and enzyme efficiency.     

Structural basis for the interaction of protein S1 with the Escherichia coli ribosome

In Gram-negative bacteria, the multi-domain protein S1 is essential for translation initiation, as it recruits the mRNA and facilitates its localization in the decoding centre. In sharp contrast to its functional importance, S1 is still lacking from the high-resolution structures available for Escherichia coli and Thermus thermophilus ribosomes and thus the molecular mechanism governing the S1–ribosome interaction has still remained elusive. Here, we present the structure of the N-terminal S1 domain D1 when bound to the ribosome at atomic resolution by using a combination of NMR, X-ray crystallography and cryo-electron microscopy. Together with biochemical assays, the structure reveals that S1 is anchored to the ribosome primarily via a stabilizing π-stacking interaction within the short but conserved N-terminal segment that is flexibly connected to domain D1. This interaction is further stabilized by salt bridges involving the zinc binding pocket of protein S2. Overall, this work provides one hitherto enigmatic piece in the ′ribosome puzzle′, namely the detailed molecular insight into the topology of the S1–ribosome interface. Moreover, our data suggest novel mechanisms that have the potential to modulate protein synthesis in response to environmental cues by changing the affinity of S1 for the ribosome.     

Structural basis for the feedback regulation of Escherichia coli pantothenate kinase by coenzyme A

Pantothenate kinase (PanK) is a key regulatory enzyme in the coenzyme A (CoA) biosynthetic pathway and catalyzes the phosphorylation of pantothenic acid to form phosphopantothenate. CoA is a feedback inhibitor of PanK activity by competitive binding to the ATP site. The structures of the Escherichia coli enzyme, in complex with a nonhydrolyzable analogue of ATP, 5'-adenylimido-diphosphate (AMPPNP), or with CoA, were determined at 2.6 and 2.5 A, respectively. Both structures show that two dimers occupy an asymmetric unit; each subunit has a alpha/beta mononucleotide-binding fold with an extensive antiparallel coiled coil formed by two long helices along the dimerization interface. The two ligands, AMPPNP and CoA, associate with PanK in very different ways, but their phosphate binding sites overlap, explaining the kinetic competition between CoA and ATP. Residues Asp(127), His(177), and Arg(243) are proposed to be involved in catalysis, based on modeling of the pentacoordinate transition state. The more potent inhibition by CoA, compared with the CoA thioesters, is explained by a tight interaction of the CoA thiol group with the side chains of aromatic residues, which is predicted to discriminate against the CoA thioesters. The PanK structure provides the framework for a more detailed understanding of the mechanism of catalysis and feedback regulation of PanK.     

Structural basis for recognition of the translocated intimin receptor (Tir) by intimin from enteropathogenic Escherichia coli

Intimin is a bacterial adhesion molecule involved in intimate attachment of enteropathogenic and enterohaemorrhagic Escherichia coli to mammalian host cells. Intimin targets the translocated intimin receptor (Tir), which is exported by the bacteria and integrated into the host cell plasma membrane. In this study we localized the Tir-binding region of intimin to the C-terminal 190 amino acids (Int190). We have also determined the region's high-resolution solution structure, which comprises an immunoglobulin domain that is intimately coupled to a novel C-type lectin domain. This fragment, which is necessary and sufficient for Tir interaction, defines a new super domain in intimin that exhibits striking structural similarity to the integrin-binding domain of the Yersinia invasin and C-type lectin families. The extracellular portion of intimin comprises an articulated rod of immunoglobulin domains extending from the bacterium surface, conveying a highly accessible 'adhesive tip' to the target cell. The interpretation of NMR-titration and mutagenesis data has enabled us to identify, for the first time, the binding site for Tir, which is located at the extremity of the Int190 moiety.     

Structural basis for the 3''-5'' exonuclease activity of Escherichia coli DNA polymerase I: a two metal ion mechanism.

The refined crystal structures of the large proteolytic fragment (Klenow fragment) of Escherichia coli DNA polymerase I and its complexes with a deoxynucleoside monophosphate product and a single-stranded DNA substrate offer a detailed picture of an editing 3'-5' exonuclease active site. The structures of these complexes have been refined to R-factors of 0.18 and 0.19 at 2.6 and 3.1 A resolution respectively. The complex with a thymidine tetranucleotide complex shows numerous hydrophobic and hydrogen-bonding interactions between the protein and an extended tetranucleotide that account for the ability of this enzyme to denature four nucleotides at the 3' end of duplex DNA. The structures of these complexes provide details that support and extend a proposed two metal ion mechanism for the 3'-5' editing exonuclease reaction that may be general for a large family of phosphoryltransfer enzymes. A nucleophilic attack on the phosphorous atom of the terminal nucleotide is postulated to be carried out by a hydroxide ion that is activated by one divalent metal, while the expected pentacoordinate transition state and the leaving oxyanion are stabilized by a second divalent metal ion that is 3.9 A from the first. Virtually all aspects of the pretransition state substrate complex are directly seen in the structures, and only very small changes in the positions of phosphate atoms are required to form the transition state.     

Structural basis of translational control by Escherichia coli threonyl tRNA synthetase

Escherichia coli threonyl-tRNA synthetase (ThrRS) represses the translation of its own messenger RNA by binding to an operator located upstream of the initiation codon. The crystal structure of the complex between the core of ThrRS and the essential domain of the operator shows that the mRNA uses the recognition mode of the tRNA anticodon loop to initiate binding. The final positioning of the operator, upon which the control mechanism is based, relies on a characteristic RNA motif adapted to the enzyme surface. The finding of other thrS operators that have this conserved motif leads to a generalization of this regulatory mechanism to a subset of Gram-negative bacteria.     

不同来源标本中大肠埃希菌耐药率的比较分析

目的研究不同标本来源中大肠埃希菌的耐药谱,为临床用药提供治疗参考。方法对温州医学院附属第二医院2010年1月到12月患者送检的体液标本进行培养,用microcsan walkaway 96S微生物自动鉴定仪对菌种鉴定及药敏分析,结果用2分割法进行统计分析。结果分离出大肠埃希菌597株,其中痰液、脓液、血液、分泌物、引流液中分别分离出295、148、102、37、15株。所有分离株均对亚胺培南敏感,对氨苄西林、哌拉西林的耐药率最高。痰液分离株对氨曲南、氨苄西林、氨苄西林/舒巴坦、哌拉西林、妥布霉素及所有头孢类的耐药率显著高于血液分离株的耐药率(P<0.05);痰液分离株对氨曲南、氨苄西林/舒巴坦、除头孢他啶外的所有头孢类的耐药率显著高于脓液分离株的耐药率(P<0.05);脓液分离株对氨苄西林、哌拉西林、妥布霉素的耐药率明显高于血液分离株的耐药率(P<0.05)。痰液中ESBLs阳性率显著高于血液和脓液中ESBLs阳性率。产ESBLs的大肠埃希菌共316株,所占的比例为52.9%;痰液分离的ESBLs阳性株对四环素、环丙沙星、加替沙星、左氧氟沙星、甲氧苄啶/磺胺呷恶唑、阿米卡星的耐药率显著低于脓液分离株的耐药率(P<0.05)。结论痰液分离的大肠埃希菌对β-内酰胺类抗生素的耐药率普遍高于脓液和血液分离株的耐药率,同时认识到该院抗生素的耐药现象很严重,临床上更加合理的使用抗菌药物。     

Molecular basis of the antimutagenic activity of the house-cleaning inosine triphosphate pyrophosphatase RdgB from Escherichia coli

Inosine triphosphate pyrophosphatases, which are ubiquitous house-cleaning enzymes, hydrolyze noncanonical nucleoside triphosphates (inosine triphosphate (ITP) and xanthosine triphosphate (XTP)) and prevent the incorporation of hypoxanthine or xanthine into nascent DNA or RNA. Here we present the 1.5-Å-resolution crystal structure of the inosine triphosphate pyrophosphatase RdgB from Escherichia coli in a free state and in complex with a substrate (ITP + Ca^2 +) or a product (inosine monophosphate (IMP)). ITP binding to RdgB induced a large displacement of the α1 helix, closing the enzyme active site. This positions the conserved Lys13 close to the bridging oxygen between the α- and β-phosphates of the substrate, weakening the P_α-O bond. On the other side of the substrate, the conserved Asp69 is proposed to act as a base coordinating the catalytic water molecule. Our data provide insight into the molecular mechanisms of the substrate selectivity and catalysis of RdgB and other ITPases.     

Structural elucidation of the O-antigenic polysaccharides from Escherichia coli O21 and the enteroaggregative Escherichia coli strain 105.

The structure of the O-antigen polysaccharide of the lipopolysaccharide from an enteroaggregative Escherichia coli (strain 105) has been elucidated, using primarily one-dimensional and two-dimensional NMR experiments. The sequence of residues was deduced with heteronuclear multiple-bond correlation and NOESY experiments. The structure of the repeating unit of the polysaccharide from the enteroaggregative E. coli is as follows:[sequence: see text] The structure of the O-antigen from enteroaggregative E. coli strain 105 was shown to be identical with that of E. coli O21 by sugar and methylation analyses as well as by 1H-NMR and 13C-NMR spectroscopy.     

Structural studies of the O-polysaccharide from the Escherichia coli O77 lipopolysaccharide.

The structure of the O-antigen polysaccharide (PS) from Escherichia coli O77 has been determined. Sugar and methylation analysis together with 1H and 13C NMR spectroscopy were the main methods used. The PS is composed of tetrasaccharide repeating units with the following structure:-->2)-alpha-D-Manp-(1-->2)-beta-D-Manp-(1-->3)-alpha-D-GlcpNAc-(1-->6)-alpha-D-Manp-(1-->     

Structural basis of tropism of Escherichia coli to the bladder during urinary tract infection

The first step in the colonization of the human urinary tract by pathogenic Escherichia coli is the mannose-sensitive binding of FimH, the adhesin present at the tip of type 1 pili, to the bladder epithelium. We elucidated crystallographically the interactions of FimH with D-mannose. The unique site binding pocket occupied by D-mannose was probed using site-directed mutagenesis. All but one of the mutants examined had greatly diminished mannose-binding activity and had also lost the ability to bind human bladder cells. The binding activity of the mono-saccharide D-mannose was delineated from this of mannotriose (Man(alpha 1-3)[Man(alpha 1-6)]Man) by generating mutants that abolished D-mannose binding but retained mannotriose binding activity. Our structure/function analysis demonstrated that the binding of the monosaccharide alpha-D-mannose is the primary bladder cell receptor for uropathogenic E. coli and that this event requires a highly conserved FimH binding pocket. The residues in the FimH mannose-binding pocket were sequenced and found to be invariant in over 200 uropathogenic strains of E. coli. Only enterohaemorrhagic E. coli (EHEC) possess a sequence variation within the mannose-binding pocket of FimH, suggesting a naturally occurring mechanism of attenuation in EHEC bacteria that would prevent them from being targeted to the urinary tract.     

Inositol monophosphatase activity from the Escherichia coli suhB gene product.

The suhB gene is located at 55 min on the Escherichia coli chromosome and encodes a protein of 268 amino acids. Mutant alleles of suhB have been isolated as extragenic suppressors for the protein secretion mutation (secY24), the heat shock response mutation (rpoH15), and the DNA synthesis mutation (dnaB121) (K. Shiba, K. Ito, and T. Yura, J. Bacteriol. 160:696-701, 1984; R. Yano, H. Nagai, K. Shiba, and T. Yura, J. Bacteriol. 172:2124-2130, 1990; S. Chang, D. Ng, L. Baird, and C. Georgopoulos, J. Biol. Chem. 266:3654-3660, 1991). These mutant alleles of suhB cause cold-sensitive cell growth, indicating that the suhB gene is essential at low temperatures. Little work has been done, however, to elucidate the role of the product of suhB in a normal cell and the suppression mechanisms of the suhB mutations in the aforementioned mutants. The sequence similarity shared between the suhB gene product and mammalian inositol monophosphatase has prompted us to test the inositol monophosphatase activity of the suhB gene product. We report here that the purified SuhB protein showed inositol monophosphatase activity. The kinetic parameters of SuhB inositol monophosphatase (Km = 0.071 mM; Vmax = 12.3 mumol/min per mg) are similar to those of mammalian inositol monophosphatase. The ssyA3 and suhB2 mutations, which were isolated as extragenic suppressors for secY24 and rpoH15, respectively, had a DNA insertion at the 5' proximal region of the suhB gene, and the amount of SuhB protein within mutant cells decreased. The possible role of suhB in E. coli is discussed.