Coli Enzyme Research Articles

Drosophila methionine sulfoxide reductase A (MSRA) lacks methionine oxidase activity

Mouse, human, and E. coli methionine sulfoxide reductase A (MSRA) stereospecifically catalyze both the reduction of S-methionine sulfoxide to methionine and the oxidation of methionine to S-methionine sulfoxide. Calmodulin has 9 methionine residues, but only Met77 is oxidized by MSRA, and this is completely reversed when MSRA operates in the reductase direction. Given the powerful genetic tools available for Drosophila, we selected this model organism to identify the in vivo calmodulin targets regulated by redox modulation of Met77. The active site sequences of mammalian and Drosophila MSRA are identical, and both contain two cysteine residues in their carboxy terminal domains. We produced recombinant Drosophila MSRA and studied its biochemical and biophysical properties. The enzyme is active as a methionine sulfoxide reductase, but it cannot function as a methionine oxidase. The first step in the mammalian oxidase reaction is formation of a sulfenic acid at the active site, and the second step is the reaction of the sulfenic acid with a carboxy terminal domain cysteine to form a disulfide bond. The third step regenerates the active site through a disulfide exchange reaction with a second carboxy terminal domain cysteine. Drosophila MSRA carries out the first and second steps, but it cannot regenerate the active site in the third step. Thus, unlike the E. coli and mammalian enzymes, Drosophila MSRA catalyzes only the reduction of methionine sulfoxide and not the oxidation of methionine.

Pathological macromolecular crystallographic data affected by twinning, partial-disorder and exhibiting multiple lattices for testing of data processing and refinement tools

Twinning is a crystal growth anomaly, which has posed a challenge in macromolecular crystallography (MX) since the earliest days. Many approaches have been used to treat twinned data in order to extract structural information. However, in most cases it is usually simpler to rescreen for new crystallization conditions that yield an untwinned crystal form or, if possible, collect data from non-twinned parts of the crystal. Here, we report 11 structures of engineered variants of the E. coli enzyme N-acetyl-neuraminic lyase which, despite twinning and incommensurate modulation, have been successfully indexed, solved and deposited. These structures span a resolution range of 1.45–2.30 Å, which is unusually high for datasets presenting such lattice disorders in MX and therefore these data provide an excellent test set for improving and challenging MX data processing programs.

Synthesis and Evaluation of Fluoroalkyl Phosphonyl Analogues of 2- C-Methylerythritol Phosphate as Substrates and Inhibitors of IspD from Human Pathogens.

Targeting essential bacterial processes beyond cell wall, protein, nucleotide, and folate syntheses holds promise to reveal new antimicrobial agents and expand the potential drugs available for combination therapies. The synthesis of isoprenoid precursors, isopentenyl diphosphate (IDP) and dimethylallyl diphosphate (DMADP), is vital for all organisms; however, humans use the mevalonate pathway for production of IDP/DMADP while many pathogens, including Plasmodium falciparum and Mycobacterium tuberculosis, use the orthogonal methylerythritol phosphate (MEP) pathway. Toward developing novel antimicrobial agents, we have designed and synthesized a series of phosphonyl analogues of MEP and evaluated their abilities to interact with IspD, both as inhibitors of the natural reaction and as antimetabolite alternative substrates that could be processed enzymatically to form stable phosphonyl analogues as potential inhibitors of downstream MEP pathway intermediates. In this compound series, the S-monofluoro MEP analogue displays the most potent inhibitory activity against Escherichia coli IspD and is the best substrate for both the E. coli and P. falciparum IspD orthologues with a Km approaching that of the natural substrate for the E. coli enzyme. This work represents a first step toward the development of phosphonyl MEP antimetabolites to modulate early isoprenoid biosynthesis in human pathogens.

Functional importance of Glutamate-445 and Glutamate-99 in proton-coupled electron transfer during oxygen reduction by cytochrome bd from Escherichia coli

The recent X-ray structure of the cytochrome bd respiratory oxygen reductase showed that two of the three heme components, heme d and heme b595, have glutamic acid as an axial ligand. No other native heme proteins are known to have glutamic acid axial ligands. In this work, site-directed mutagenesis is used to probe the roles of these glutamic acids, E445 and E99 in the E. coli enzyme. It is concluded that neither glutamate is a strong ligand to the heme Fe and they are not the major determinates of heme binding to the protein. Although very important, neither glutamate is absolutely essential for catalytic function. The close interactions between the three hemes in cyt bd result in highly cooperative properties. For example, mutation of E445, which is near heme d, has its greatest effects on the properties of heme b595 and heme b558. It is concluded that 1) O2 binds to the hydrophilic side of heme d and displaces E445; 2) E445 forms a salt bridge with R448 within the O2 binding pocket, and both residues play a role to stabilize oxygenated states of heme d during catalysis; 3) E445 and E99 are each protonated accompanying electron transfer to heme d and heme b595, respectively; 4) All protons used to generate water within the heme d active site come from the cytoplasm and are delivered through a channel that must include internal water molecules to assist proton transfer: [cytoplasm] → E107 → E99 (heme b595) → E445 (heme d) → oxygenated heme d.

Studying polyphosphorylation, a novel PTM, in mammalian cell lines

Polyphosphates are long chains of phosphates attached to one another via high‐energy phosphoanhydride bonds. The literature reports their implication in a variety of medically related functions, such as apoptosis, blood coagulation, and inflammation. The mechanisms involved with polyphosphate and its functions have been studied in bacteria and in yeast, but the synthesis pathway of polyphosphate in mammals is still unknown. Moreover, the concentrations of polyphosphate in mammalian cells (<1mM) are much lower than the ones found in yeast (100–200mM). This makes it challenging to study polyphosphate biology and polyphosphorylation in mammalian cell lines. Recently, Azevedo, et al. (2015) identified a new PTM in yeast called polyphosphorylation and two targets of this PTM: Nsr1 and Top1. It consists of the non‐enzymatic covalent attachment of polyphosphate chains to lysine residues found in a poly‐acidic serine and lysine‐rich (PASK) motif. Our lab has since identified 24 novel yeast targets being polyphosphorylated. Our next aim was to test if human proteins can also be polyphosphorylated. In order to modulate the concentrations of polyphosphate in the cell, we transfected mammalian cells with PPK1, an E. coli enzyme that synthesizes polyphosphate. This ectopic expression allowed us to perform a small “screen” and identify 6 human proteins that can be polyphosphorylated: nucleolin, hNop56, Mesd chaperone, DEK, eIF5B and UPF3B. We will present our latest work focused on determining the molecular function of polyphosphorylation in mammalian cells.Support or Funding InformationThis project is funded by the Canadian Institutes of Health Research (CIHR).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

A Structural View of the Dissociation of Escherichia Coli Tryptophanase

Tryptophanase (Trpase) is a pyridoxal 5′-phosphate (PLP)-dependent homotetrameric enzyme which catalyzes the degradation of l-tryptophan. Trpase is known for its cold lability, i.e., a reversible loss of activity at low temperature (2°C), associated with tetramer dissociation. Escherichia coli Trpase dissociates into dimers, while Proteus vulgaris Trpase dissociates into monomers. Accordingly, Trpase is an appropriate model to study the quaternary structure of proteins. We aimed at understanding the differences in the mode of dissociation between the E. coli and P. vulgaris Trpases. In particular, the effect of mutations along the molecular axes of homotetrameric Trpase on its dissociation was studied. Two groups of mutants of the E. coli enzyme were created to resemble the amino-acid sequence of P. vulgaris Trpase. In one, residues 15 and 59 that are located along the molecular axis R (termed the noncatalytic axis) were mutated. The second group included a mutation at position 298, located along the molecular axis Q (termed the catalytic axis). Replacing amino-acid residues along the R axis resulted in dissociation of the tetramers into monomers, similar to the P. vulgaris Trpase, while replacing amino-acid residues along the Q axis resulted in dissociation into dimers only. The crystal structure of the V59M mutant of E. coli Trpase was also determined in its apo form and was found to be similar to that of the wild type. This study suggests that in E. coli Trpase hydrophobic interactions along the R axis hold the two monomers together more strongly, preventing the dissociation of the dimers into monomers. Mutation of position 298 along the Q axis to a charged residue resulted in tetramers that are less susceptible to dissociation. The results indicate that dissociation of E. coli Trpase into dimers occurs along the molecular Q axis.

Characterization of the catalytic disulfide bond in E. coli 4-thiouridine synthetase to elucidate its functional quaternary structure.

4-Thiouridine at position 8 in prokaryotic tRNA serves as a photosensor for near-UV light, and the posttranscriptional conversion of uridine to 4-thiouridine is catalyzed by the 4-thiouridine synthetases (s(4) US, also named ThiI), which fall into two classes that differ in the presence of a C-terminal rhodanese homology domain. A cysteine residue in this domain first bears a persulfide group and then forms a disulfide bond with a cysteine residue that is conserved in both classes of s(4) US. Recent crystal structures suggest that s(4) US dimerizes in the presence of RNA substrate with domains from each subunit contributing to the binding and reaction of one RNA molecule, which raises the question of whether the catalytic disulfide bond in the longer class of s(4) US is formed within or between subunits. The E. coli enzyme is the best-characterized member of the longer class of s(4) US, and it was examined after quantitative installation of the disulfide bond during a single catalytic turnover. Gel electrophoresis and proteolysis/MALDI-MS results strongly imply that the disulfide bond forms within a single subunit, which provides a vital constraint for the structural modeling of the class of s(4) US with an appended rhodanese homology domain and the design and interpretation of experiments to probe the dynamics of the domains during catalysis.

Substrate Recognition and Activity Regulation of the Escherichia coli mRNA Endonuclease MazF

Escherichia coli MazF (EcMazF) is the archetype of a large family of ribonucleases involved in bacterial stress response. The crystal structure of EcMazF in complex with a 7-nucleotide substrate mimic explains the relaxed substrate specificity of the E. coli enzyme relative to its Bacillus subtilis counterpart and provides a framework for rationalizing specificity in this enzyme family. In contrast to a conserved mode of substrate recognition and a conserved active site, regulation of enzymatic activity by the antitoxin EcMazE diverges from its B. subtilis homolog. Central in this regulation is an EcMazE-induced double conformational change as follows: a rearrangement of a crucial active site loop and a relative rotation of the two monomers in the EcMazF dimer. Both are induced by the C-terminal residues Asp-78-Trp-82 of EcMazE, which are also responsible for strong negative cooperativity in EcMazE-EcMazF binding. This situation shows unexpected parallels to the regulation of the F-plasmid CcdB activity by CcdA and further supports a common ancestor despite the different activities of the MazF and CcdB toxins. In addition, we pinpoint the origin of the lack of activity of the E24A point mutant of EcMazF in its inability to support the substrate binding-competent conformation of EcMazF.

Structural and Biochemical Investigations on the Catalytic Mechanism of Pyridoxal Kinase (PdxK) from Salmonella Typhimurium and its Interactions with PLP-Dependent Enzymes

Pyridoxal kinase (PdxK), a member of ribokinase superfamily of enzymes, is involved in pyridoxal 5’ phosphate (PLP) synthesis by a salvage pathway. PdxK ensures availability of PLP to a large number of enzymes involved in amino acid and sugar metabolism that use PLP as a cofactor and is regarded as a potential drug target. We have determined the crystal structure of PdxK from Salmonella typhimurium (stPLK) in its unliganded form as well as in complex with Mg ATP and Mg ATP-PLP at 2.6A, 1.9A and 2.5A resolutions, respectively. The protomeric structure of the dimeric enzyme consisting of three layered αβα structure is similar to those of other ribokinase family proteins. A segment of residues 134-142 constituting a flexible loop undergoes a large conformational change from an open form to a closed form upon ligand binding and guards the active site from solvent exposure and prevents premature hydrolysis of ATP. During catalysis, the substrate PL moves by a distance of ∼6A to interact with the γ phosphate of ATP bound near an anion hole constituted by residues 234-237 (GTGD). As reported for the E. coli enzyme, kinetic studies show that stPLK has higher activity in the presence of Mg2+ when compared to other divalent metal ions. High concentration of PLP was found to inhibit stPLK and the structure of crystals obtained in presence of excess PLP reveals that PLP is covalently attached as an internal aldimine to Lys233. Surface Plasmon resonance (SPR ) and ELISA studies show that stPLK specifically interacts with diaminopropionate ammonia lyase, a fold type II PLP dependent enzyme, suggesting probable direct transfer of the product PLP from PdxK to the apo form of PLP-dependent enzymes.

Isofunctional Enzymes PAD1 and UbiX Catalyze Formation of a Novel Cofactor Required by Ferulic Acid Decarboxylase and 4-Hydroxy-3-polyprenylbenzoic Acid Decarboxylase

The decarboxylation of antimicrobial aromatic acids such as phenylacrylic acid (cinnamic acid) and ferulic acid by yeast requires two enzymes described as phenylacrylic acid decarboxylase (PAD1) and ferulic acid decarboxylase (FDC). These enzymes are of interest for various biotechnological applications, such as the production of chemical feedstocks from lignin under mild conditions. However, the specific role of each protein in catalyzing the decarboxylation reaction remains unknown. To examine this, we have overexpressed and purified both PAD1 and FDC from E. coli. We demonstrate that PAD1 is a flavin mononucleotide (FMN)-containing protein. However, it does not function as a decarboxylase. Rather, PAD1 catalyzes the formation of a novel, diffusible cofactor required by FDC for decarboxylase activity. Coexpression of FDC and PAD1 results in the production of FDC with high levels cofactor bound. Holo-FDC catalyzes the decarboxylation of phenylacrylic acid, coumaric acid and ferulic acid with apparent kcat ranging from 1.4-4.6 s(-1). The UV-visible and mass spectra of the cofactor indicate that it appears to be a novel, modified form of reduced FMN; however, its instability precluded determination of its structure. The E. coli enzymes UbiX and UbiD are related by sequence to PAD1 and FDC respectively and are involved in the decarboxylation of 4-hydroxy-3-octaprenylbenzoic acid, an intermediate in ubiquinone biosynthesis. We found that endogenous UbiX can also activate FDC. This implies that the same cofactor is required for decarboxylation of 4-hydroxy-3-polyprenylbenzoic acid by UbiD and suggests a wider role for this cofactor in metabolism.

Enzyme dynamics from NMR spectroscopy.

ConspectusBiological activities of enzymes, including regulation or coordination of mechanistic stages preceding or following the chemical step, may depend upon kinetic or equilibrium changes in protein conformations. Exchange of more open or flexible conformational states with more closed or constrained states can influence inhibition, allosteric regulation, substrate recognition, formation of the Michaelis complex, side reactions, and product release. NMR spectroscopy has long been applied to the study of conformational dynamic processes in enzymes because these phenomena can be characterized over multiple time scales with atomic site resolution. Laboratory-frame spin-relaxation measurements, sensitive to reorientational motions on picosecond–nanosecond time scales, and rotating-frame relaxation-dispersion measurements, sensitive to chemical exchange processes on microsecond–millisecond time scales, provide information on both conformational distributions and kinetics. This Account reviews NMR spin relaxation studies of the enzymes ribonuclease HI from mesophilic (Escherichia coli) and thermophilic (Thermus thermophilus) bacteria, E. coli AlkB, and Saccharomyces cerevisiae triosephosphate isomerase to illustrate the contributions of conformational flexibility and dynamics to diverse steps in enzyme mechanism.Spin relaxation measurements and molecular dynamics (MD) simulations of the bacterial ribonuclease H enzymes show that the handle region, one of three loop regions that interact with substrates, interconverts between two conformations. Comparison of these conformations with the structure of the complex between Homo sapiens ribonuclease H and a DNA:RNA substrate suggests that the more closed state is inhibitory to binding. The large population of the closed conformation in T. thermophilus ribonuclease H contributes to the increased Michaelis constant compared with the E. coli enzyme. NMR spin relaxation and fluorescence spectroscopy have characterized a conformational transition in AlkB between an open state, in which the side chains of methionine residues in the active site are disordered, and a closed state, in which these residues are ordered. The open state is highly populated in the AlkB/Zn(II) complex, and the closed state is highly populated in the AlkB/Zn(II)/2OG/substrate complex, in which 2OG is the 2-oxoglutarate cosubstrate and the substrate is a methylated DNA oligonucleotide. The equilibrium is shifted to approximately equal populations of the two conformations in the AlkB/Zn(II)/2OG complex. The conformational shift induced by 2OG ensures that 2OG binds to AlkB/Zn(II) prior to the substrate. In addition, the opening rate of the closed conformation limits premature release of substrate, preventing generation of toxic side products by reaction with water. Closure of active site loop 6 in triosephosphate isomerase is critical for forming the Michaelis complex, but reopening of the loop after the reaction is (partially) rate limiting. NMR spin relaxation and MD simulations of triosephosphate isomerase in complex with glycerol 3-phosphate demonstrate that closure of loop 6 is a highly correlated rigid-body motion. The MD simulations also indicate that motions of Gly173 in the most flexible region of loop 6 contribute to opening of the active site loop for product release. Considered together, these three enzyme systems illustrate the power of NMR spin relaxation investigations in providing global insights into the role of conformational dynamic processes in the mechanisms of enzymes from initial activation to final product release.

Purification of bacterial RNA polymerase: tools and protocols.

Bacterial RNA polymerase is the first point of gene expression and a validated target for antibiotics. Studied for several decades, the Escherichia coli transcriptional apparatus is by far the best characterized, with numerous RNA polymerase mutants and auxiliary factors isolated and analyzed in great detail. Since the E. coli enzyme was refractory to crystallization, structural studies have been focused on Thermus RNA polymerases, revealing atomic details of the catalytic center and RNA polymerase interactions with nucleic acids, antibiotics, and regulatory proteins. However, numerous differences between these enzymes, including resistance of Thermus RNA polymerases to some antibiotics, underscored the importance of the E. coli enzyme structures. Three groups published these long awaited structures in 2013, enabling functional and structural studies of the same model system. This progress was made possible, in large part, by the use of multicistronic vectors for expression of the E. coli enzyme in large quantities and in a highly active form. Here we describe the commonly used vectors and procedures for purification of the E. coli RNA polymerase.

Development of asparaginase Erwinia chrysanthemi for the treatment of acute lymphoblastic leukemia.

Since it was identified in 1963 as the antileukemic agent in guinea pig serum, L-asparaginase (ASNase) has become an integral component of chemotherapy protocols to treat patients with acute lymphoblastic leukemia (ALL). Escherichia coli and Erwinia chrysanthemi provide the sources of ASNase used clinically today. From the time ASNase was first introduced into treatment protocols, the 5-year survival rate has increased significantly, particularly in children and adolescents. E. coli-derived ASNase was approved in 1978 to be used as part of a multiagent chemotherapy to treat ALL. However, the development of hypersensitivity in 10-30% of patients often leads to treatment discontinuation. E. chrysanthemi-derived ASNase (referred to herein as ASNase Erwinia chrysanthemi) is immunologically distinct from E. coli ASNase and therefore does not cross-react with the E. coli enzyme. In 2011, ASNase Erwinia chrysanthemi was approved in the United States for patients who develop hypersensitivity to E. coli-derived ASNase. When indicated, a switch from ASNase E. coli to ASNase E. chrysanthemi allows patients to continue to receive treatment and maintain therapeutic levels of ASNase activity. Therapeutic drug monitoring may help ensure that therapeutic enzyme levels are maintained. Pegylated recombinant ASNase Erwinia chrysanthemi is currently being developed to improve pharmacokinetic properties and reduce immunogenicity.

Reconstitution of peptidoglycan cross-linking leads to improved fluorescent probes of cell wall synthesis.

The peptidoglycan precursor, Lipid II, produced in the model Gram-positive bacterium Bacillus subtilis differs from Lipid II found in Gram-negative bacteria such as Escherichia coli by a single amidation on the peptide side chain. How this difference affects the cross-linking activity of penicillin-binding proteins (PBPs) that assemble peptidoglycan in cells has not been investigated because B. subtilis Lipid II was not previously available. Here we report the synthesis of B. subtilis Lipid II and its use by purified B. subtilis PBP1 and E. coli PBP1A. While enzymes from both organisms assembled B. subtilis Lipid II into glycan strands, only the B. subtilis enzyme cross-linked the strands. Furthermore, B. subtilis PBP1 catalyzed the exchange of both d-amino acids and d-amino carboxamides into nascent peptidoglycan, but the E. coli enzyme only exchanged d-amino acids. We exploited these observations to design a fluorescent d-amino carboxamide probe to label B. subtilis PG in vivo and found that this probe labels the cell wall dramatically better than existing reagents.

Inhibition of the Ribonuclease H Activity of HIV-1 Reverse Transcriptase by GSK5750 Correlates with Slow Enzyme-Inhibitor Dissociation

Compounds that efficiently inhibit the ribonuclease (RNase) H activity of the human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) have yet to be developed. Here, we demonstrate that GSK5750, a 1-hydroxy-pyridopyrimidinone analog, binds to the enzyme with an equilibrium dissociation constant (K(d)) of ~400 nM. Inhibition of HIV-1 RNase H is specific, as DNA synthesis is not affected. Moreover, GSK5750 does not inhibit the activity of Escherichia coli RNase H. Order-of-addition experiments show that GSK5750 binds to the free enzyme in an Mg(2+)-dependent fashion. However, as reported for other active site inhibitors, binding of GSK5750 to a preformed enzyme-substrate complex is severely compromised. The bound nucleic acid prevents access to the RNase H active site, which represents a possible biochemical hurdle in the development of potent RNase H inhibitors. Previous studies suggested that formation of a complex with the prototypic RNase H inhibitor β-thujaplicinol is slow, and, once formed, it dissociates rapidly. This unfavorable kinetic behavior can limit the potency of RNase H active site inhibitors. Although the association kinetics of GSK5750 remains slow, our data show that this compound forms a long lasting complex with HIV-1 RT. We conclude that slow dissociation of the inhibitor and HIV-1 RT improves RNase H active site inhibitors and may circumvent the obstacle posed by the inability of these compounds to bind to a preformed enzyme-substrate complex.

Organization of the Escherichia coli aerobic enzyme complexes of oxidative phosphorylation in dynamic domains within the cytoplasmic membrane.

The Escherichia coli cytoplasmic membrane contains the enzyme complexes of oxidative phosphorylation (OXPHOS). Not much is known about their supramolecular organization and their dynamics within the membrane in this model organism. In mitochondria and other bacteria, it was demonstrated by nondenaturing electrophoretic methods and electron microscopy that the OXPHOS complexes are organized in so-called supercomplexes, stable assemblies with a defined number of the individual enzyme complexes. To investigate the organization of the E. coli enzyme complexes of aerobic OXPHOS in vivo, we established fluorescent protein fusions of the NADH:ubiquinone oxidoreductase, the succinate:ubiquinone oxidoreductase, the cytochrome bd-I, and the cytochrome bo3 terminal oxidases, and the FoF1 ATP-synthase. The fusions were integrated in the chromosome to prevent artifacts caused by protein overproduction. Biochemical analysis revealed that all modified complexes were fully assembled, active, and stable. The distribution of the OXPHOS complexes in living cells was determined using total internal reflection fluorescence microscopy. The dynamics within the membrane were detected by fluorescence recovery after photobleaching. All aerobic OXPHOS complexes showed an uneven distribution in large mobile patches within the E. coli cytoplasmic membrane. It is discussed whether the individual OXPHOS complexes are organized as clustered individual complexes, here called “segrazones.”

Circular dichroism and fluorescence spectroscopy of cysteinyl-tRNA synthetase from Halobacterium salinarum ssp. NRC-1 demonstrates that group I cations are particularly effective in providing structure and stability to this halophilic protein.

Proteins from extremophiles have the ability to fold and remain stable in their extreme environment. Here, we investigate the presence of this effect in the cysteinyl-tRNA synthetase from Halobacterium salinarum ssp. NRC-1 (NRC-1), which was used as a model halophilic protein. The effects of salt on the structure and stability of NRC-1 and of E. coli CysRS were investigated through far-UV circular dichroism (CD) spectroscopy, fluorescence spectroscopy, and thermal denaturation melts. The CD of NRC-1 CysRS was examined in different group I and group II chloride salts to examine the effects of the metal ions. Potassium was observed to have the strongest effect on NRC-1 CysRS structure, with the other group I salts having reduced strength. The group II salts had little effect on the protein. This suggests that the halophilic adaptations in this protein are mediated by potassium. CD and fluorescence spectra showed structural changes taking place in NRC-1 CysRS over the concentration range of 0–3 M KCl, while the structure of E. coli CysRS was relatively unaffected. Salt was also shown to increase the thermal stability of NRC-1 CysRS since the melt temperature of the CysRS from NRC-1 was increased in the presence of high salt, whereas the E. coli enzyme showed a decrease. By characterizing these interactions, this study not only explains the stability of halophilic proteins in extremes of salt, but also helps us to understand why and how group I salts stabilize proteins in general.

Investigation of Osmolyte Effects on FolM: Comparison with Other Dihydrofolate Reductases

A weak association between osmolytes and dihydrofolate (DHF) decreases the affinity of the substrate for the Escherichia coli chromosomal and R67 plasmid dihydrofolate reductase (DHFR) enzymes. To test whether the osmolyte-DHF association also interferes with binding of DHF to FolM, an E. coli enzyme that possesses weak DHFR activity, ligand binding was monitored in the presence of osmolytes. The affinity of FolM for DHF, measured by kcat/Km(DHF), was decreased by the addition of an osmolyte. Additionally, binding of the antifolate drug, methotrexate, to FolM was weakened by the addition of an osmolyte. The changes in ligand binding with water activity were unique for each osmolyte, indicating preferential interaction between the osmolyte and folate and its derivatives; however, additional evidence provided support for further interactions between FolM and osmolytes. Binding of the reduced nicotinamide adenine dinucleotide phosphate (NADPH) cofactor to FolM was monitored by isothermal titration calorimetry as a control for protein-osmolyte association. In the presence of betaine (proposed to be the osmolyte most excluded from protein surfaces), the NADPH Kd decreased, consistent with dehydration effects. However, other osmolytes did not tighten binding to the cofactor. Rather, dimethyl sulfoxide (DMSO) had no effect on the NADPH Kd, while ethylene glycol and polyethylene glycol 400 weakened cofactor binding. Differential scanning calorimetry of FolM in the presence of osmolytes showed that both DMSO and ethylene glycol decreased the stability of FolM, while betaine increased the stability of the protein. These results suggest that some osmolytes can destabilize FolM by preferentially interacting with the protein. Further, these weak attractions can impede ligand binding. These various contributions have to be considered when interpreting osmotic pressure results.

E. coli sabotages the in vivo production of O-linked β-N-acetylglucosamine-modified proteins

The O-linked β-N-acetylglucosamine (O-GlcNAc) post-translational modification is an important, regulatory modification of cytosolic and nuclear enzymes. To date, no 3-dimensional structures of O-GlcNAc-modified proteins exist due to difficulties in producing sufficient quantities with either in vitro or in vivo techniques. Recombinant co-expression of substrate protein and O-GlcNAc transferase in Escherichia coli was used to produce O-GlcNAc-modified domains of human cAMP responsive element-binding protein (CREB1) and Abelson tyrosine-kinase 2 (ABL2). Recombinant expression in E. coli is an advantageous approach, but only small quantities of insoluble O-GlcNAc-modified protein were produced. Adding β-N-acetylglucosaminidase inhibitor, O-(2-acetamido-2-dexoy-d-glucopyranosylidene)amino-N-phenylcarbamate (PUGNAc), to the culture media provided the first evidence that an E. coli enzyme cleaves O-GlcNAc from proteins in vivo. With the inhibitor present, the yields of O-GlcNAc-modified protein increased. The E. coli β-N-acetylglucosaminidase was isolated and shown to cleave O-GlcNAc from a synthetic O-GlcNAc-peptide in vitro. The identity of the interfering β-N-acetylglucosaminidase was confirmed by testing a nagZ knockout strain. In E. coli, NagZ natively cleaves the GlcNAc-β1,4-N-acetylmuramic acid linkage to recycle peptidoglycan in the cytoplasm and cleaves the GlcNAc-β-O-linkage of foreign O-GlcNAc-modified proteins in vivo, sabotaging the recombinant co-expression system.

Spotlighting motors and controls of single FoF1-ATP synthase

Subunit rotation is the mechanochemical intermediate for the catalytic activity of the membrane enzyme FoF1-ATP synthase. smFRET (single-molecule FRET) studies have provided insights into the step sizes of the F1 and Fo motors, internal transient elastic energy storage and controls of the motors. To develop and interpret smFRET experiments, atomic structural information is required. The recent F1 structure of the Escherichia coli enzyme with the ϵ-subunit in an inhibitory conformation initiated a study for real-time monitoring of the conformational changes of ϵ. The present mini-review summarizes smFRET rotation experiments and previews new smFRET data on the conformational changes of the CTD (C-terminal domain) of ϵ in the E. coli enzyme.