Molecular cloning of the Pseudomonas putida glyoxalase I gene in Escherichia coli

The glyoxalase I gene of Pseudomonas putida was cloned onto a vector plasmid pBR 322 as a 7.5 kilobase Sau 3AI fragment of chromosomal DNA and the hybrid plasmid was designated pGI 318. The gene responsible for the glyoxalase I activity in pGI 318 was recloned in pBR 322 as a 2.2 kilobase Hin dIII fragment and was designated pGI 423. The P. putida glyoxalase I gene on pGI 318 and pGI 423 was highly expressed in E. coli cells and the glyoxalase I activity level was increased more than 150 fold in the pGI 423 bearing strain compared with that of E. coli cells without pGI 423. The E. coli transformants harboring pGI 318 or pGI 423 could grow normally in the presence of methylglyoxal, although the E. coli cells without plasmid were inhibited to grow and showed the extremely elongated cell shape.     

The crystal structure of R-specific alcohol dehydrogenase from Lactobacillus brevis suggests the structural basis of its metal dependency

The crystal structure of the apo-form of an R-specific alcohol dehydrogenase from Lactobacillus brevis (LB-RADH) was solved and refined to 1.8A resolution. LB-RADH is a member of the short-chain dehydrogenase/reductase (SDR) enyzme superfamily. It is a homotetramer with 251 amino acid residues per subunit and uses NADP(H) as co-enzyme. NADPH and the substrate acetophenone were modelled into the active site. The enantiospecificity of the enzyme can be explained on the basis of the resulting hypothetical ternary complex. In contrast to most other SDR enzymes, the catalytic activity of LB-RADH depends strongly on the binding of Mg(2+). Mg(2+) removal by EDTA inactivates the enzyme completely. In the crystal structure, the Mg(2+)-binding site is well defined. The ion has a perfect octahedral coordination sphere and occupies a special position concerning crystallographic and molecular point symmetry, meaning that each RADH tetramer contains two magnesium ions. The magnesium ion is no direct catalytic cofactor. However, it is structurally coupled to the putative C-terminal hinge of the substrate-binding loop and, via an extended hydrogen bonding network, to some side-chains forming the substrate binding region. Therefore, the presented structure of apo-RADH provides plausible explanations for the metal dependence of the enzyme.     

Escherichia coli glyoxalase II is a binuclear zinc-dependent metalloenzyme

Cytotoxic methylglyoxal is detoxified by the two-enzyme glyoxalase system. Glyoxalase I (GlxI) catalyzes conversion of non-enzymatically produced methylglyoxal-glutathione hemithioacetal into its corresponding thioester. Glyoxalase II (Glx II) hydrolyzes the thioester into d-lactate and free glutathione. Glyoxalase I and II are metalloenzymes, which possess mononuclear and binuclear active sites, respectively. There are two distinct classes of GlxI; the first class is Zn2+-dependent and is composed of GlxI from mainly eukaryotic organisms and the second class is composed of non-Zn2+-dependent (but Ni2+ or Co2+-dependent) GlxI enzymes (mainly prokaryotic and leishmanial species). GlxII is typically Zn2+-activated, containing Zn2+ and either Fe3+/Fe2+ or Mn2+ at the active site depending upon the biological source. To address whether two classes of GlxII might exist, glyoxalase II from Escherichia coli was cloned and overexpressed and characterized. Unlike E. coli GlxI, which is non-Zn2+-dependent, Zn2+ activates the E. coli GlxII enzyme, with no evidence for Ni2+ metal utilization.     

Structure of Escherichia coli exonuclease I suggests how processivity is achieved

Exonuclease I (ExoI) from Escherichia coli is a monomeric enzyme that processively degrades single stranded DNA in the 3' to 5' direction and has been implicated in DNA recombination and repair. Determination of the structure of ExoI to 2.4 A resolution using X-ray crystallography verifies the expected correspondence between a region of ExoI and the exonuclease (or proofreading) domains of the DNA polymerases. The overall fold of ExoI also includes two other regions, one of which extends the exonuclease domain and another that can be described as an elaborated SH3 domain. These three regions combine to form a molecule that is shaped like the letter C, although it also contains a segment that effectively converts the C into an O-like shape. The structure of ExoI thus provides additional support for the idea that DNA metabolizing enzymes achieve processivity by completely enclosing the DNA.     

The crystal structure of YdcE,a 4-oxalocrotonate tautomerase homologue from Escherichia coli,confirms the structural basis for oligomer diversity

The tautomerase superfamily consists of three major families represented by 4-oxalocrotonate tautomerase (4-OT), 5-(carboxymethyl)-2-hydroxymuconate isomerase (CHMI), and macrophage migration inhibitory factor (MIF). The members of this superfamily are structurally homologous proteins constructed from a simple beta-alpha-beta fold that share a key mechanistic feature; they use an amino-terminal proline, which has an unusually low pK(a), as the general base in a keto-enol tautomerization. Several new members of the 4-OT family have now been identified using PSI-BLAST and categorized into five subfamilies on the basis of multiple-sequence alignments and the conservation of key catalytic and structural residues. The members of subfamily 5, which includes a hypothetical protein designated YdcE from Escherichia coli, are predicted not to form hexamers. The crystal structure of YdcE has been determined to 1.35 A resolution and confirms that it is a dimer. In addition, YdcE complexed with (E)-2-fluoro-p-hydroxycinnamate, identified as a potent competitive inhibitor of this enzyme, as well as N-(2-hydroxyethyl)piperazine-N'-2-ethanesulfonic acid (HEPES) and benzoate are also presented. These latter crystal structures reveal the location of the active site and suggest a mechanism for the observed YdcE-catalyzed tautomerization reaction. The dimeric arrangement of YdcE represents a new structure in the 4-OT family and demonstrates structural diversity within the 4-OT family not previously reported.     

The structural basis for the interaction between L-tryptophan and the Escherichia coli trp aporepressor

We have employed equilibrium dialysis to help study the mechanism by which the unliganded Escherichia coli trp aporepressor is activated by L-tryptophan to the liganded trp repressor. By measuring the relative affinity of L-tryptophan and various tryptophan analogues for the co-repressor's binding site, we have estimated the extent to which each of the functional groups of L-tryptophan contributes to the liganding process and discuss their role in the context of the crystal structures of the trp repressor and aporepressor. We have found that the indole ring and alpha carboxyl group of L-tryptophan are mainly responsible for its affinity to the aporepressor. The alpha amino group, however, has a small negative contribution to the affinity of L-tryptophan for the aporepressor which may be associated with its essential role in operator-specific binding.     

Crystal structure of the vinculin tail suggests a pathway for activation

Vinculin plays a dynamic role in the assembly of the actin cytoskeleton. A strong interaction between its head and tail domains that regulates binding to other cytoskeletal components is disrupted by acidic phospholipids. Here, we present the crystal structure of the vinculin tail, residues 879-1066. Five amphipathic helices form an antiparallel bundle that resembles exchangeable apolipoproteins. A C-terminal arm wraps across the base of the bundle and emerges as a hydrophobic hairpin surrounded by a collar of basic residues, adjacent to the N terminus. We show that the C-terminal arm is required for binding to acidic phospholipids but not to actin, and that binding either ligand induces conformational changes that may represent the first step in activation.     

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.     

Determination of the nucleotide sequence for the exonuclease I structural gene (sbcB) of Escherichia coli K12

The complete nucleotide sequence of the structural gene for Escherichia coli exonuclease I has been determined. The coding region corresponds to a 465-amino acid protein with molecular weight of 53,174. The partial amino acid sequence of purified exonuclease I agrees with that predicted by the DNA sequence. Two putative weak promoters have been localized by S1 nuclease analysis. The sbcB coding sequence contains many non-optimal codons, characteristic of many poorly expressed E. coli genes.     

The crystal structure of the Escherichia coli lipocalin Blc suggests a possible role in phospholipid binding

Lipocalins form a large multifunctional family of small proteins (15-25 kDa) first discovered in eukaryotes. More recently, several types of bacterial lipocalins have been reported, among which Blc from Escherichia coli is an outer membrane lipoprotein. As part of our structural genomics effort on proteins from E. coli, we have expressed, crystallized and solved the structure of Blc at 1.8 A resolution using remote SAD with xenon. The structure of Blc, the first of a bacterial lipocalin, exhibits a classical fold formed by a beta-barrel and a alpha-helix similar to that of the moth bilin binding protein. Its empty and open cavity, however, is too narrow to accommodate bilin, while the alkyl chains of two fatty acids or of a phospholipid could be readily modeled inside the cavity. Blc was reported to be expressed under stress conditions such as starvation or high osmolarity, during which the cell envelope suffers and requires maintenance. These data, together with our structural interpretation, suggest a role for Blc in storage or transport of lipids necessary for membrane repair or maintenance.     

Crystal structure of the outer membrane protease OmpT from Escherichia coli suggests a novel catalytic site

OmpT from Escherichia coli belongs to a family of highly homologous outer membrane proteases, known as omptins, which are implicated in the virulence of several pathogenic Gram-negative bacteria. Here we present the crystal structure of OmpT, which shows a 10-stranded antiparallel beta-barrel that protrudes far from the lipid bilayer into the extracellular space. We identified a putative binding site for lipopolysaccharide, a molecule that is essential for OmpT activity. The proteolytic site is located in a groove at the extracellular top of the vase-shaped beta-barrel. Based on the constellation of active site residues, we propose a novel proteolytic mechanism, involving a His-Asp dyad and an Asp-Asp couple that activate a putative nucleophilic water molecule. The active site is fully conserved within the omptin family. Therefore, the structure described here provides a sound basis for the design of drugs against omptin-mediated bacterial pathogenesis. Coordinates are in the Protein Data Bank (accession No. 1I78)     

The crystal structure of Escherichia coli MoaB suggests a probable role in molybdenum cofactor synthesis

The crystal structure of Escherichia coli MoaB was determined by multiwavelength anomalous diffraction phasing and refined at 1.6-A resolution. The molecule displayed a modified Rossman fold. MoaB is assembled into a hexamer composed of two trimers. The monomers have high structural similarity with two proteins, MogA and MoeA, from the molybdenum cofactor synthesis pathway in E. coli, as well as with domains of mammalian gephyrin and plant Cnx1, which are also involved in molybdopterin synthesis. Structural comparison between these proteins and the amino acid conservation patterns revealed a putative active site in MoaB. The structural analysis of this site allowed to advance several hypothesis that can be tested in further studies.     

The structure of SENP1-SUMO-2 complex suggests a structural basis for discrimination between SUMO paralogues during processing

eNOS (endothelial nitric oxide synthase) activity is post-translationally regulated in a complex fashion by acylation, protein-protein interactions, intracellular trafficking and phosphorylation, among others. Signalling pathways that regulate eNOS activity include phosphoinositide 3-kinase/Akt, cyclic nucleotide-dependent kinases [PKA (protein kinase A) and PKG], PKC, as well as ERKs (extracellular-signal-regulated kinases). The role of ERKs in eNOS activation remains controversial. In the present study, we have examined the role of ERK1/2 in eNOS activation in HUVEC-CS [transformed HUVEC (human umbilical-vein endothelial cells)] as well as a widely used model for eNOS study, transiently transfected COS-7 cells. U0126 pretreatment of HUVEC-CS potentiated ATP-stimulated eNOS activity, independent of changes in intracellular Ca2+ concentration ([Ca2+]i). In COS-7 cells transiently expressing ovine eNOS, U0126 potentiated A23187-stimulated eNOS activity, but inhibited ATP-stimulated activity. Compensatory changes in phosphorylation of five key eNOS residues did not account for changes in A23187-stimulated activity. However, in the case of ATP, altered phosphorylation and changes in [Ca2+]i may partially contribute to U0126 inhibition of activity. Finally, seven eNOS alanine mutants of putative ERK1/2 targets were generated and the effects of U0126 pretreatment on eNOS activity were gauged with A23187 and ATP treatment. T97A-eNOS was the only construct significantly different from wild-type after U0126 pretreatment and ATP stimulation of eNOS activation. In the present study, eNOS activity was either potentiated or inhibited in COS-7 cells, suggesting agonist dependence for MEK/ERK1/2 signalling [where MEK is MAPK (mitogen-activated protein kinase)/ERK kinase] to eNOS and a complex mechanism including [Ca2+]i, phosphorylation and, possibly, intracellular trafficking.     

Hsp31 of Escherichia coli K-12 is glyoxalase III

Hsp31 encoded by hchA is known as a heat‐inducible molecular chaperone. Although structure studies revealed that Hsp31 has a putative catalytic triad consisting of Asp‐214, His‐186 and Cys‐185, its enzymatic function, besides weak amino‐peptidase activity, is still unknown. We found that Hsp31 displays glyoxalase activity that catalyses the conversion of methylglyoxal (MG) to d ‐lactate without an additional cofactor. The glyoxalase activity was completely abolished in the hchA‐deficient strain, confirming the relationship between the hchA gene and its enzymatic activity in vivo. Hsp31 exhibits Michaelis–Menten kinetics for substrates MG with K_m and k_cat of 1.43 ± 0.12 mM and 156.9 ± 5.5 min^?1 respectively. The highest glyoxalase activity was found at 35–40°C and pH of 6.0–8.0, and the activity was significantly inhibited by Cu²⁺, Fe³⁺ and Zn²⁺. Mutagenesis studies based on our evaluation of conserved catalytic residues revealed that the Cys‐185 and Glu‐77 were essential for catalysis, whereas His‐186 was less crucial for enzymatic function, although it participates in the catalytic process. The stationary‐phase Escherichia coli cells became more susceptible to MG when hchA was deleted, which was complemented by an expression of plasmid‐encoded hchA. Furthermore, an accumulation of intracellular MG was observed in hchA‐deficient strains.     

Crystal structure of C5b-6 suggests structural basis for priming assembly of the membrane attack complex

The complement membrane attack complex (MAC) forms transmembrane pores in pathogen membranes. The first step in MAC assembly is cleavage of C5 to generate metastable C5b, which forms a stable complex with C6, termed C5b-6. C5b-6 initiates pore formation via the sequential recruitment of homologous proteins: C7, C8, and 12–18 copies of C9, each of which comprises a central MAC-perforin domain flanked by auxiliary domains. We recently proposed a model of pore assembly, in which the auxiliary domains play key roles, both in stabilizing the closed conformation of the protomers and in driving the sequential opening of the MAC-perforin β-sheet of each new recruit to the growing pore. Here, we describe an atomic model of C5b-6 at 4.2 Å resolution. We show that C5b provides four interfaces for the auxiliary domains of C6. The largest interface is created by the insertion of an interdomain linker from C6 into a hydrophobic groove created by a major reorganization of the α-helical domain of C5b. In combination with the rigid body docking of N-terminal elements of both proteins, C5b becomes locked into a stable conformation. Both C6 auxiliary domains flanking the linker pack tightly against C5b. The net effect is to induce the clockwise rigid body rotation of four auxiliary domains, as well as the opening/twisting of the central β-sheet of C6, in the directions predicted by our model to activate or prime C6 for the subsequent steps in MAC assembly. The complex also suggests novel small molecule strategies for modulating pathological MAC assembly.     

Crystal structure of the YgfZ protein from Escherichia coli suggests a folate-dependent regulatory role in one-carbon metabolism

The ygfZ gene product of Escherichia coli represents a large protein family conserved in bacteria to eukaryotes. The members of this family are uncharacterized proteins with marginal sequence similarity to the T-protein (aminomethyltransferase) of the glycine cleavage system. To assist with the functional assignment of the YgfZ family, the crystal structure of the E. coli protein was determined by multiwavelength anomalous diffraction. The protein molecule has a three-domain architecture with a central hydrophobic channel. The structure is very similar to that of bacterial dimethylglycine oxidase, an enzyme of the glycine betaine pathway and a homolog of the T-protein. Based on structural superposition, a folate-binding site was identified in the central channel of YgfZ, and the ability of YgfZ to bind folate derivatives was confirmed experimentally. However, in contrast to dimethylglycine oxidase and T-protein, the YgfZ family lacks amino acid conservation at the folate site, which implies that YgfZ is not an aminomethyltransferase but is likely a folate-dependent regulatory protein involved in one-carbon metabolism.     

Novel approach for structural identification of protein family: glyoxalase I

Glyoxalase is one of two enzymes of the glyoxalase detoxification system against methylglyoxal and other aldehydes, the metabolites derived from glycolysis. The glyoxalase system is found almost in all living organisms: bacteria, protozoa, plants, and animals, including humans, and is related to the class of ‘life essential proteins’. The enzyme belongs to the expanded Glyoxalase/Bleomycin resistance protein/Dioxygenase superfamily. At present the GenBank contains about 700 of amino acid sequences of this enzyme type, and the Protein Data Bank includes dozens of spatial structures. We have offered a novel approach for structural identification of glyoxalase I protein family, which is based on the selecting of basic representative proteins with known structures. On this basis, six new subfamilies of these enzymes have been derived. Most populated subfamilies A1 and A2 were based on representative human Homo sapiens and bacterial Escherichia coli enzymes. We have found that the principle feature, which defines the subfamilies’ structural differences, is conditioned by arrangement of N- and C-domains inside the protein monomer. Finely, we have deduced the structural classification for the glyoxalase I and assigned about 460 protein sequences distributed among six new subfamilies. Structural similarities and specific differences of all the subfamilies have been presented. This approach can be used for structural identification of thousands of the so-called hypothetical proteins with the known PDB structures allowing to identify many of already existing atomic coordinate entrees.