Insights into the interaction of human liver arginase with tightly and weakly bound manganese ions by chemical modification and site-directed mutagenesis studies

Diethyl pyrocarbonate (DEPC) caused a loss in the ability of inactive subunits of wild-type and H141F mutant human liver arginase (EC 3.5.3.1) to be reactivated by Mn(2+). The effect was reversed by hydroxylamine and involved a residue with a pK(a) of 6.5+/-0.1. Half activation with Mn(2+) was sufficient for total resistance of H141F and full activation was not impeded by a previous incubation of the half-active species with DEPC. The H101N and H126N mutants expressed 60 and 82% of the wild-type activity, respectively, without changes in K(m) for arginine or K(i) for lysine inhibition. After dialysis against EDTA, H126N was inactive in the absence of added Mn(2+) and contained <0.1 Mn(2+)/subunit, whereas H101N was half active and contained 1.2+/-0.1 Mn(2+)/subunit. Results support the concept that a weakly bound metal ion is needed only for conversion of active species to a more active active state.     

Role of the RecF gene product in UV mutagenesis of lambda phage

Summary E. coli recF mutants have a greatly reduced capacity for Weigle mutagenesis of ultraviolet light-irradiated lambda phage. A recF 332::Tn3 mutation was introduced into an E. coli recA441 lexA51 strain which constitutively expresses SOS functions. Weigle mutagenesis of phage lambda could occur in the resulting strain in the absence of host cell irradiation, and was increased when the recA441 (tif) allele was activated by increased temperature and excess adenine. The inability of recF strains to support Weigle mutagenesis can therefore be ascribed to a defect in expression of SOS functions after irradiation.     

Production of lysozyme and lysozyme-superoxide dismutase dimers bound by a ditryptophan cross-link in carbonate radical-treated lysozyme

Despite extensive investigation of the irreversible oxidations undergone by proteins in vitro and in vivo, the products formed from the oxidation of Trp residues remain incompletely understood. Recently, we characterized a ditryptophan cross-link produced by the recombination of hSOD1-tryptophanyl radicals generated from attack of the carbonate radical produced during the bicarbonate-dependent peroxidase activity of the enzyme. Here, we examine whether the ditryptophan cross-link is produced by the attack of the carbonate radical on proteins other than hSOD1. To this end, we treated hen egg white lysozyme with photolytically and enzymatically generated carbonate radical. The radical yields were estimated and the lysozyme modifications were analyzed by SDS-PAGE, western blot, enzymatic activity and MS/MS analysis. Lysozyme oxidation by both systems resulted in its inactivation and dimerization. Lysozyme treated with the photolytic system presented monomers oxidized to hydroxy-tryptophan at Trp²⁸ and Trp¹²³ and N-formylkynurenine at Trp²⁸, Trp⁶² and Trp¹²³. Lysozyme treated with the enzymatic system rendered monomers oxidized to N-formylkynurenine at Trp²⁸. The dimers were characterized as lysozyme-Trp²⁸-Trp²⁸-lysozyme and lysozyme-Trp²⁸-Trp³²-hSOD1. The results further demonstrate that the carbonate radical is prone to causing biomolecule cross-linking and hence, may be a relevant player in pathological mechanisms. The possibility of exploring the formation of ditryptophan cross-links as a carbonate radical biomarker is discussed.     

Incorporation of a lambda phage recombination system and EGFP detection to simplify mutagenesis of Herpes simplex virus bacterial artificial chromosomes

Background  

Targeted mutagenesis of the herpesvirus genomes has been facilitated by the use of bacterial artificial chromosome (BAC) technology. Such modified genomes have potential uses in understanding viral pathogenesis, gene identification and characterization, and the development of new viral vectors and vaccines. We have previously described the construction of a herpes simplex virus 2 (HSV-2) BAC and the use of an allele replacement strategy to construct HSV-2 recombinants. While the BAC mutagenesis procedure is a powerful method to generate HSV-2 recombinants, particularly in the absence of selective marker in eukaryotic culture, the mutagenesis procedure is still difficult and cumbersome.     

Quantitative studies of the non-productive binding of lysozyme to partially N-acetylated chitosans: Binding of large ligands to a one-dimensional binary lattice studied by a modified McGhee and von Hippel model

This paper considers the non-productive (inhibitory) binding of chitosans to lysozyme from chicken egg white. Chitosans are linear, binary heteropolysaccharides consisting of 2-acetamido-2-deoxy-β-d-glucose (GlcNAc; A-unit) and 2-amino-2-deoxy-β-d-glucose (GlcN, D-unit). The active site cleft of lysozyme can bind six consecutive sugar residues in subsites named A–F, and specific binding of chitosan sequences to lysozyme occurs with A-units in subsite C. Chitosans with different fractions of A-units (F_A) induced nearly identical changes in the ¹H NMR spectrum of lysozyme upon binding, and the concentration of bound lysozyme could be determined. The data were analysed using a modified version of the McGhee and von Hippel model for binding of large ligands to one-dimensional homogeneous lattices. The average value of the dissociation constant for different sequences that may bind to lysozyme (K^ave_D) was estimated, as well as the number of chitosan units covered by lysozyme upon binding. K^ave_D decreased with increasing F_A-values at pH* 3 and 4.5, while the opposite was true at pH* 5.5. Contributions from different hexamer sequences to K^ave_D of the chitosans were considered, and the data revealed interesting features with respect to binding of lysozyme to partially N-acetylated chitosans. The relevance of the present data with respect to understanding lysozyme degradation kinetics of chitosans is discussed.     

Dissection of the stepwise mechanism to beta-lactam formation and elucidation of a rate-determining conformational change in beta-lactam synthetase

Clavulanic acid is a widely used beta-lactamase inhibitor whose key beta-lactam core is formed by beta-lactam synthetase. beta-Lactam synthetase exhibits a Bi-Ter mechanism consisting of two chemical steps, acyl-adenylation followed by beta-lactam formation. 32PPi-ATP exchange assays showed the first irreversible step of catalysis is acyl-adenylation. From a small, normal solvent isotope effect (1.38 +/- 0.04), it was concluded that beta-lactam synthesis contributes at least partially to kcat. Site-specific mutation of Lys-443 identified this residue as the ionizable group at pKa approximately 8.1 apparent in the pH-kcat profile that stabilizes the beta-lactam-forming step. Viscosity studies demonstrated that a protein conformational change was also partially rate-limiting on kcat attenuating the observed solvent isotope effect on beta-lactam formation. Adherence to Kramers' theory gave a slope of 1.66 +/- 0.08 from a plot of log(o kcat/kcat) versus log(eta/eta(o)) consistent with opening of a structured loop visible in x-ray data preceding product release. Internal "friction" within the enzyme contributes to a slope of > 1 in this analysis. Correspondingly, earlier in the catalytic cycle ordering of a mobile active site loop upon substrate binding was manifested by an inverse solvent isotope effect (0.67 +/- 0.15) on kcat/Km. The increased second-order rate constant in heavy water was expected from ordering of this loop over the active site imposing torsional strain. Finally, an Eyring plot displayed a large enthalpic change accompanying loop movement (DeltaH approximately 20 kcal/mol) comparable to the chemical barrier of beta-lactam formation.     

Structural basis for the recognition of lysozyme by MliC, a periplasmic lysozyme inhibitor in Gram-negative bacteria

Lysozymes are an important component of the innate immune system of animals that hydrolyze peptidoglycan, the major bacterial cell wall constituent. Many bacteria have contrived various means of dealing with this bactericidal enzyme, one of which is to produce lysozyme inhibitors. Recently, a novel family of bacterial lysozyme inhibitors was identified in various Gram-negative bacteria, named MliC (membrane bound lysozyme inhibitor of C-type lysozyme). Here, we report the crystal structure of Pseudomonas aeruginosa MliC in complex with chicken egg white lysozyme. Combined with mutational study, the complex structure demonstrates that the invariant loop of MliC plays a crucial role in the inhibition of the lysozyme by its insertion to the active site cleft of the lysozyme, where the loop forms hydrogen and ionic bonds with the catalytic residues. Since MliC family members have been implicated as putative colonization or virulence factors, the structures and mechanism of action of MliC will be of relevance to the control of bacterial growth in animal hosts.     

Site-directed mutagenesis of ricin A-chain and implications for the mechanism of action

Ricin A-chain is an N-glycosidase that attacks ribosomal RNA at a highly conserved adenine residue. The enzyme is representative of a large family of medically significant proteins used in the design of anticancer agents and in the treatment of HIV infection. The x-ray structure has been used as a guide to create several active site mutations by directed mutagenesis of the cloned gene. Glu177 is a key catalytic residue, and conversion to Gln reduces activity 180-fold. Asn209 is shown to participate in substrate binding by kinetic analysis. Conversion to Ser increases Km sixfold but has no effect on kcat. Conversion of Tyr80 and Tyr123 to Phe decreases activity by 15- and 7-fold respectively. A mechanism of action is proposed that involves binding of the substrate adenine in a syn configuration that resembles the transition state; the putative oxycarbonium ion is probably stabilized by interaction with Glu177.     

Alanine-scanning mutagenesis of the beta-sheet region of phage T4 lysozyme suggests that tertiary context has a dominant effect on beta-sheet formation

In general, alpha-helical conformations in proteins depend in large part on the amino acid residues within the helix and their proximal interactions. For example, an alanine residue has a high propensity to adopt an alpha-helical conformation, whereas that of a glycine residue is low. The sequence preferences for beta-sheet formation are less obvious. To identify the factors that influence beta-sheet conformation, a series of scanning polyalanine mutations were made within the strands and associated turns of the beta-sheet region in T4 lysozyme. For each construct the stability of the folded protein was reduced substantially, consistent with removal of native packing interactions. However, the crystal structures showed that each of the mutants retained the beta-sheet conformation. These results suggest that the structure of the beta-sheet region of T4 lysozyme is maintained to a substantial extent by tertiary interactions with the surrounding parts of the protein. Such tertiary interactions may be important in determining the structures of beta-sheets in general.     

Transition temperatures in the sensitivity of escherichia coli B/r to lysozyme

Lysozyme attacked Escherichia coli B/r in the absence of EDTA or imposed osmotic shocks when the cells were rapidly cooled below specific temperatures. Cells subjected to lysozyme while being cooled to below 20°C began to lose ability to subsequently form colonies. This sensitivity increased with decreasing temperatures and almost all cells cooled to 0°C were affected. Slightly hypertonic solutions did not improve survival. Cells cooled first to as low as 5°C and then subjected to lysozyme while cool did not lose their ability to form colonies subsequent to rewarming. However, 70% of the cells cooled first to 0°C and subjected to lysozyme lost their colony-forming ability. Cell lysis also began when treated near 5°C, but even when treated at 0°C about 50% of the cells maintained their rod shape in the presence of lysozyme. These results are discussed in terms of a possible phase transition in a portion of the cell envelope and/or a transient osmotic swelling as a results of metabolic pumps failing at the low temperatures.     

The glutamine/amino acid transporter (ASCT2) reconstituted in liposomes: Transport mechanism, regulation by ATP and characterization of the glutamine/glutamate antiport

The glutamine/amino acid transporter solubilized from rat renal apical plasma membrane (brush-border membrane) with C₁₂E₈ and reconstituted into liposomes has been previously identified as the ASCT2 transporter. The reconstituted transporter catalyses an antiport reaction in which external glutamine and Na⁺ are cotransported in exchange with internal glutamine (or other amino acids). The glutamine-Na⁺ cotransport occurred with a 1:1 stoichiometry. The concentration of Na⁺ did not influence the Km for glutamine and vice versa. Experimental data obtained by a bi-substrate analysis of the glutamine-Na⁺ cotransport, together with previous report on the glutamine_ex/glutamine_in pseudo bi-reactant analysis, indicated that the transporter catalyses a three-substrate transport reaction with a random simultaneous mechanism. The presence of ATP in the internal compartment of the proteoliposomes led to an increase of the Vmax of the transport and to a decrease of the Km of the transporter for external Na⁺. The reconstituted glutamine/amino acid transporter was inhibited by glutamate; the inhibition was more pronounced at acidic pH. A kinetic analysis revealed that the inhibition was competitive with respect to glutamine. Glutamate was also transported in exchange with glutamine. The external Km of the transporter for glutamate (13.3 mM) was slightly higher than the internal one (8.3 mM). At acidic pH the external but not the internal Km decreased. According with the Km values, glutamate should be transported preferentially from inside to outside in exchange for external glutamine and Na⁺.     

Development of a short-term, in vivo mutagenesis assay: the effects of methylation on the recovery of a lambda phage shuttle vector from transgenic mice.

Transgenic mice suitable for the in vivo assay of suspected mutagens at the chromosome level have been constructed by stable integration of a lambda phage shuttle vector. The shuttle vector, which contains a beta-galactosidase (beta-gal) target gene, can be rescued from genomic DNA with in vitro packaging extracts. Mutations in the target gene are detected by a change in lambda phage plaque color on indicator agar plates. Initial rescue efficiencies of less than 1 plaque forming unit (pfu)/100 micrograms of genomic DNA were too low for mutation analysis. We determined the cause of the low rescue efficiencies by examining primary fibroblast cultures prepared from fetuses of lambda transgenic animals. The rescue efficiency of 5-azacytidine-treated cells increased 50-fold over non-treated controls indicating that methylation was inhibiting rescue. The inhibitory role of methylation was supported by the observation that mcr deficient E. coli plating strains and mcr deficient lambda packaging extracts further improved lambda rescue efficiency. Present rescue efficiencies of greater than 2000 pfu/copy/micrograms of genomic DNA represent a 100,000-fold improvement over initial rescue efficiencies, permitting quantitative mutational analysis. The background mutagenesis rate was estimated at 1 x 10(-5) in two separate lineages. Following treatment with the mutagen N-ethyl-N-nitrosourea (EtNU), a dose dependent increase in the mutation rate was observed in DNA isolated from mouse spleen, with significant induction also observed in mouse testes DNA.     

Studies on the reaction mechanism of a microbial lipase/acyltransferase using chemical modification and site-directed mutagenesis

Aeromonas hydrophila releases a protein which is a member of the lipase superfamily, similar in reaction mechanism to the important mammalian plasma enzyme lecithin-cholesterol acyltransferase. We have used chemical modification and site-directed mutagenesis of the protein to identify amino acids which may be involved in catalysis. The enzyme was unaffected by phenylmethylsulfonyl fluoride, but it was almost completely inhibited by another serine-reactive compound, diethyl p-nitrophenyl phosphate. A serine selectively modified by this reagent was identified by sequencing the amino-terminal region of the protein. It was located at position 16, in the short consensus sequence shared by the enzyme with other lipases. When this serine was changed to asparagine the product was an enzymatically inert protein which nevertheless retained the surface activity of the wild-type enzyme, suggesting its ability to bind to substrate was not changed. Diethylpyrocarbonate treatment drastically reduced the rate of acyl transfer by the native enzyme, but this did not appear to be due to modification of an essential histidine, since inhibition was not reversed by addition of hydroxylamine. We have shown that only two of the histidines in the enzyme can be involved in catalysis (Hilton, S., McCubbin, W. D., Kay, C.M., and Buckley, J. T. (1990) Biochemistry, 29, 9072-9078). Replacing both of these with asparagines had little or no effect on enzyme activity. These results indicate that, in apparent contrast to other lipases, histidine does not participate in the reaction catalyzed by the microbial enzyme. Since catalysis was not inhibited by sulfhydryl reagents, we conclude that a free cysteine is also not required for activity. This may distinguish the microbial enzyme from the mammalian acyltransferase.     

A large conformational change couples the ATP binding site of SecA to the SecY protein channel

In bacteria, the SecYEG protein translocation complex employs the cytosolic ATPase SecA to couple the energy of ATP binding and hydrolysis to the mechanical force required to push polypeptides through the membrane. The molecular basis of this energy transducing reaction is not well understood. A peptide-binding array has been employed to identify sites on SecYEG that interact with SecA. These results along with fluorescence spectroscopy have been exploited to characterise a long-distance conformational change that connects the nucleotide-binding fold of SecA to the transmembrane polypeptide channel in SecY. These movements are driven by binding of non-hydrolysable ATP analogues to a monomer of SecA in association with the SecYEG complex. We also determine that interaction with SecYEG simultaneously decreases the affinity of SecA for ATP and inhibitory magnesium, favouring a previously identified active state of the ATPase. Mutants of SecA capable of binding but not hydrolysing ATP do not elicit this conformationally active state, implicating residues of the Walker B motif in the early chain of events that couple ATP binding to the mobility of the channel.     

A point mutation in the Nul gene of bacteriophage lambda facilitates phage growth in Escherichia coli with himA and gyrB mutations

Summary A mutant of was isolated that grows in the Escherichia coli himA/gyrB-him320(Ts) double mutant at 42°C; conditions which are non-permissive for wild-type growth. The responsible mutation, ohm1, alters the 40th codon of the Nul reading frame. The Nul and A gene products comprise the terminase protein which cleaves concatameric DNA into unit-length phage genomes during DNA packaging. The Nul-ohm1 gene product acts in trans to support growth in the double himA/gyrB mutant, and cos154 growth in the single himA mutant. The observation that an alteration in Nul suppresses the inhibition of growth in the double himA/gyrB mutant implicates DNA gyrase, as well as integration host factor, in the DNA: protein interactions that occur at the initiation of packaging.     

Overexpression of the phage lambda lysozyme cloned in Escherichia coli: use of a degenerative mixture of synthetic ribosome binding sites and increase of the protein stability in vivo.

The R gene of the phage lambda coding for a lysozyme expressed at the end of an infection cycle in Escherichia coli has been cloned in a series of vector plasmids. Two methods for improving the efficiency of translation have been tested. First, the use of a bicistronic construction in which the ribosome binding site (RBS) of the first cistron is that of a highly expressed gene or the use of a degenerate mixture of synthetic oligonucleotides for the optimization of a RBS. The second strategy is more efficient: the analysis of a number of clones reveals that the LaL expression levels are increased by a factor between 3 and 6 times compared with the clone using the natural RBS. The expression levels are described by an approximately Gaussian histogram. The translation promoter that was found to afford the best expression (PL) is under the control of a thermolabile repressor. Under the expression conditions, the protein is partially proteolysed. The proteolysis is significantly decreased by adding salt to the growth medium. After optimization, an increase in expression by a factor of 40 is obtained compared with the initial conditions. An efficient purification protocol is described.     

Binding of cGMP to the transducin-activated cGMP phosphodiesterase, PDE6, initiates a large conformational change involved in its deactivation

Retinal photoreceptor phosphodiesterase (PDE6), a key enzyme for phototransduction, consists of a catalytic subunit complex (Pαβ) and two inhibitory subunits (Pγs). Pαβ has two noncatalytic cGMP-binding sites. Here, using bovine PDE preparations, we show the role of these cGMP-binding sites in PDE regulation. Pαβγγ and its transducin-activated form, Pαβγ, contain two and one cGMP, respectively. Only Pαβγ shows [(3)H]cGMP binding with a K(d) ～ 50 nM and Pγ inhibits the [(3)H]cGMP binding. Binding of cGMP to Pαβγ is suppressed during its formation, implying that cGMP binding is not involved in Pαβγγ activation. Once bound to Pαβγ, [(3)H]cGMP is not dissociated even in the presence of a 1000-fold excess of unlabeled cGMP, binding of cGMP changes the apparent Stokes' radius of Pαβγ, and the amount of [(3)H]cGMP-bound Pαβγ trapped by a filter is spontaneously increased during its incubation. These results suggest that Pαβγ slowly changes its conformation after cGMP binding, i.e. after formation of Pαβγ containing two cGMPs. Binding of Pγ greatly shortens the time to detect the increase in the filter-trapped level of [(3)H]cGMP-bound Pαβγ, but alters neither the level nor its Stokes' radius. These results suggest that Pγ accelerates the conformational change, but does not add another change. These observations are consistent with the view that Pαβγ changes its conformation during its deactivation and that the binding of cGMP and Pγ is crucial for this change. These observations also imply that Pαβγγ changes its conformation during its activation and that release of Pγ and cGMP is essential for this change.