The role of amino-acid residues Q39 and E451 in the determination of substrate specificity of the Spodoptera frugiperda beta-glycosidase.

The design of beta-glycosidases with planed substrate specificity for biotechnological application has received little attention. This is mostly a consequence of the lack of data on the molecular basis of the beta-glycosidase specificity, namely data on the energy of the noncovalent interactions in the enzyme-transition state complex. In an attempt to fill this gap, site-directed mutagenesis and enzyme steady-state kinetic experiments with different substrates were conducted, using as model a digestive beta-glycosidase (glycoside hydrolase family 1) from Spodoptera frugiperda (Lepidoptera) (Sfbetagly50). The active site of this enzyme was modeled based on its sequence and on crystallographic data of similar enzymes. Energy of noncovalent interactions in transition state between Sfbetagly50 amino acids and glycone hydroxyls was determined. Sfbetagly50 residue E451 seems to be a key residue in determining beta-glycosidase preference for glucosides vs. galactosides based on the following data: (a) The energy of the noncovalent interaction between glycone equatorial hydroxyl 4 with E451 in the transition state is about 60% higher than its interaction with Q39. (b) The energy of the E451-hydroxyl 4 interaction decreases more than the Q39-hydroxyl 4 interaction when hydroxyl 4 is changed from equatorial to axial position. (c) A Sfbetagly50 mutant, where E451 was substituted by A, hydrolyzes galactosides faster than glucosides. It was also shown that glycone hydroxyl 6 interacts favorably with Q39, but not with E451, probably due to steric hindrance. These interactions result in the beta-glycosidase hydrolyzing fucosides (6-deoxygalactosides) faster than glucosides and galactosides.     

Molecular basis of substrate specificity in family 1 glycoside hydrolases

ss-glycosidases are active upon a large range of substrates. Besides this, subtle changes in the substrate structure may result in large modifications on the ss-glycosidase activity. The characterization of the molecular basis of ss-glycosidases substrate preference may contribute to the comprehension of the enzymatic specificity, a fundamental property of biological systems. ss-glycosidases specificity for the monosaccharide of the substrate nonreducing end (glycone) is controlled by a hydrogen bond network involving at least 5 active site amino acid residues and 4 substrate hydroxyls. From these residues, a glutamate, which interacts with hydroxyls 4 and 6, seems to be a key element in the determination of the preference for fucosides, glucosides and galactosides. Apart from this, interactions with the hydroxyl 2 are essential to the ss-glycosidase activity. The active site residues forming these interactions and the pattern of the hydrogen bond network are conserved among all ss-glycosidases. The region of the ss-glycosidase active site that interacts with the moiety (called aglycone) which is bound to the glycone is formed by several subsites (1 to 3). However, the majority of the non-covalent interactions with the aglycone is concentrated in the first one, which presents a variable spatial structure and amino acid composition. This structural variability is in accordance with the high diversity of aglycones recognized by ss-glycosidases. Hydrophobic interactions and hydrogen bonds are formed with the aglycone, but the manner in which they control the ss-glycosidase specificity still remains to be determined.     

The role of residues R97 and Y331 in modulating the pH optimum of an insect beta-glycosidase of family 1.

The activity of the digestive beta-glycosidase from Spodoptera frugiperda (Sfbetagly50, pH optimum 6.2) depends on E399 (pKa = 4.9; catalytic nucleophile) and E187 (pKa = 7.5; catalytic proton donor). Homology modelling of the Sfbetagly50 active site confirms that R97 and Y331 form hydrogen bonds with E399. Site-directed mutagenesis showed that the substitution of R97 by methionine or lysine increased the E399 pKa by 0.6 or 0.8 units, respectively, shifting the pH optima of these mutants to 6.5. The substitution of Y331 by phenylalanine increased the pKa of E399 and E187 by 0.7 and 1.6 units, respectively, and displaced the pH optimum to 7.0. From the observed deltapKa it was calculated that R97 and Y331 contribute 3.4 and 4.0 kJ.mol(-1), respectively, to stabilization of the charged E399, thus enabling it to be the catalytic nucleophile. The substitution of E187 by D decreased the pKa of residue 187 by 0.5 units and shifted the pH optimum to 5.8, suggesting that an electrostatic repulsion between the deprotonated E399 and E187 may increase the pKa of E187, which then becomes the catalytic proton donor. In short the data showed that a network of noncovalent interactions among R97, Y331, E399 and E187 controls the Sfbetagly50 pH optimum. As those residues are conserved among the family 1 beta-glycosidases, it is proposed here that similar interactions modulate the pH optimum of all family 1 beta-glycosidases.     

The role in the substrate specificity and catalysis of residues forming the substrate aglycone-binding site of a beta-glycosidase

The relative contributions to the specificity and catalysis of aglycone, of residues E190, E194, K201 and M453 that form the aglycone-binding site of a beta-glycosidase from Spodoptera frugiperda (EC 3.2.1.21), were investigated through site-directed mutagenesis and enzyme kinetic experiments. The results showed that E190 favors the binding of the initial portion of alkyl-type aglycones (up to the sixth methylene group) and also the first glucose unit of oligosaccharidic aglycones, whereas a balance between interactions with E194 and K201 determines the preference for glucose units versus alkyl moieties. E194 favors the binding of alkyl moieties, whereas K201 is more relevant for the binding of glucose units, in spite of its favorable interaction with alkyl moieties. The three residues E190, E194 and K201 reduce the affinity for phenyl moieties. In addition, M453 favors the binding of the second glucose unit of oligosaccharidic aglycones and also of the initial portion of alkyl-type aglycones. None of the residues investigated interacted with the terminal portion of alkyl-type aglycones. It was also demonstrated that E190, E194, K201 and M453 similarly contribute to stabilize ES(double dagger). Their interactions with aglycone are individually weaker than those formed by residues interacting with glycone, but their joint catalytic effects are similar. Finally, these interactions with aglycone do not influence glycone binding.     

Binding specificity and reactivity studies on a broad-specificity beta-glycosidase from porcine kidney

A broad-specificity beta-glycosidase from porcine kidney was purified to homogeneity. Sodium dodecyl sulfate - polyacrylamide gel electrophoresis showed that it had a monomeric molecular weight of 55,000-60,000. Gel filtration showed native molecular weight of about 115,000. These data imply that the native enzyme is a dimer. The enzyme can catalyze the hydrolysis of beta bonds between glycosides and 4-methylumbelliferone or nitrophenol yielding D-fucopyranose, D-galactopyranose, D-glucopyranose, D-xylopyranose, and D-mannopyranose and of alpha bonds to yield L-arabinopyranose. This is the first study that shows a mammalian broad-specificity cytosolic beta-glycosidase carrying out a reaction with a beta-D-mannopyranoside. The nature of the broad specificity was studied with inhibitors. Similar inhibitor constants were found regardless of whether the substrate was a beta-D-glucopyranoside or a beta-D-galactopyranoside, so the enzyme probably has only one binding site with a broad specificity. The enzyme prefers to bind compounds with an axial hydroxyl at the 2 position and an equatorial hydroxyl at the 4 position; the 3 position does not affect binding significantly. The hydroxyl at the 6 position affects binding, but binding at that position depends on the configurations at the 2 and 4 positions. Thus, there must be some interactions between these three positions (2, 4, and 6). Lactones are also good inhibitors and this may relate to strain effects.     

Subsite mapping of human salivary alpha-amylase and the mutant Y151M

This study characterizes the substrate-binding sites of human salivary alpha-amylase (HSA) and its Y151M mutant. It describes the first subsite maps, namely, the number of subsites, the position of cleavage sites and apparent subsite energies. The product pattern and cleavage frequencies were determined by high-performance liquid chromatography, utilizing a homologous series of chromophore-substituted maltooligosaccharides of degree of polymerization 3-10 as model substrates. The binding region of HSA is composed of four glycone and three aglycone-binding sites, while that of Tyr151Met is composed of four glycone and two aglycone-binding sites. The subsite maps show that Y151M has strikingly decreased binding energy at subsite (+2), where the mutation has occurred (-2.6 kJ/mol), compared to the binding energy at subsite (+2) of HSA (-12.0 kJ/mol).     

Beta-glycosidase of Thermus thermophilus KNOUC202: gene and biochemical properties of the enzyme expressed in Escherichia coli

The beta-glycosidase gene of Thermus thermophilus KNOUC202 was cloned, expressed in Escherichia coli JM109(DE3), and the enzyme was purified and characterized. The gene (KNOUC202/beta-gly) was composed of 1296 bp encoding a beta-glycosidase (KNOUC202beta-glycosidase) of 431 a.a., belonging to the family 1 of glycosyl hydrolase. The gene was expressed as monomer of 430 a.a. with amino terminal methionine excised in E. coli JM109(DE3). The enzyme hydrolyzed beta-glycosides whose glycone are galactose, glucose and fucose well, however showed no or very low activity on beta-D-glycosides whose glycone are disaccharides and xylose. kcat of the enzyme for the hydrolysis of p-Nph-beta-D-Glcp was lower than those for p-Nph-beta-D-Galp and ONPG, however K(m) for p-Nph-beta-D-Glcp was highly lower than those for p-Nph-beta-D-Galp and ONPG resulting in the catalytic efficiency(k(cat)/K(m)) for the hydrolysis of p-Nph-beta-D-Glcp much higher than those for p-Nph-beta-D-Galp and ONPG. Optimum pH and optimum temperature of the enzyme were pH 5.4 and 90 degrees C. The enzyme has high thermostability, not losing its activity at 80 degrees C for 2 h in 0.05 M Na-phosphate buffer of pH 6.8 with T(m) of 100.0 +/- 0.031 degrees C in 0.02 M Tris-HCl buffer of pH 8.2. The beta-glycosidase produced a disaccharide composed of galactose as transglycosylation byproduct during hydrolysis of lactose.     

Amino acid residues involved in substrate binding and catalysis in an insect digestive beta-glycosidase

A beta-glycosidase (M(r) 50000) from Spodoptera frugiperda larval midgut was purified, cloned and sequenced. It is active on aryl and alkyl beta-glucosides and cellodextrins that are all hydrolyzed at the same active site, as inferred from experiments of competition between substrates. Enzyme activity is dependent on two ionizable groups (pK(a1)=4.9 and pK(a2)=7.5). Effect of pH on carbodiimide inactivation indicates that the pK(a) 7.5 group is a carboxyl. k(cat) and K(m) values were obtained for different p-nitrophenyl beta-glycosides and K(i) values were determined for a range of alkyl beta-glucosides and cellodextrins, revealing that the aglycone site has three subsites. Binding data, sequence alignments and literature beta-glycosidase 3D data supported the following conclusions: (1) the groups involved in catalysis were E(187) (proton donor) and E(399) (nucleophile); (2) the glycone moiety is stabilized in the transition state by a hydrophobic region around the C-6 hydroxyl and by hydrogen bonds with the other equatorial hydroxyls; (3) the aglycone site is a cleft made up of hydrophobic amino acids with a polar amino acid only at its first (+1) subsite.     

The conformational properties of the glycosidic linkage

Stereochemical properties of the glycosidic linkage have been studied by the quantum-chemical PCILO method, using 2-methoxytetrahydropyran as a model. Calculations of the two-dimensional, conformational (Φ, Ψ) maps showed that the rotation around the C-1---O-1 bond is more hindered than that around the O-1---C-6 bond, and that there are differences in the shape of the energy curve for the axial and equatorial forms of 2-methoxytetrahydropyran. The observed population of the five stable conformers at equilibrium (GG:GT:TG¹:TG₂:TT = 70.8:6.0:19.9:2.0:1.3) is consistent with the prediction of the anomeric and exo-anomeric effects. The calculated abundance (76.8%) of the axial form of 2-methoxytetrahydropyran is comparable with experimental results (77–80%) obtained by n.m.r. measurements in non-polar solvents. The energies found for individual conformers made it possible to calculate the magnitude of the anomeric effect (3 kJ/mol) and to determine, for the first time, the values of the exo-anomeric effect for axial (6 kJ/mol) and equatorial 2-methoxytetrahydropyran (7 kJ/mol). The calculated variations of the geometry arising from rotation around the C-1---O-1 bond are consistent with results obtained by statistical analysis of experimental data for - and β-glycosides. The results obtained, indicating that the energy, geometry, and electronic structure of glycosides are largely affected by the conformation of the acetal segment, are discussed from the point of view of conformational analysis of oligo- and poly-saccharides.     

Engineering the substrate specificity of Escherichia coli asparaginase. II. Selective reduction of glutaminase activity by amino acid replacements at position 248

The use of Escherichia coli asparaginase II as a drug for the treatment of acute lymphoblastic leukemia is complicated by the significant glutaminase side activity of the enzyme. To develop enzyme forms with reduced glutaminase activity, a number of variants with amino acid replacements in the vicinity of the substrate binding site were constructed and assayed for their kinetic and stability properties. We found that replacements of Asp248 affected glutamine turnover much more strongly than asparagine hydrolysis. In the wild-type enzyme, N248 modulates substrate binding to a neighboring subunit by hydrogen bonding to side chains that directly interact with the substrate. In variant N248A, the loss of transition state stabilization caused by the mutation was 15 kJ mol(-1) for L-glutamine compared to 4 kJ mol(-1) for L-aspartic beta-hydroxamate and 7 kJ mol(-1) for L-asparagine. Smaller differences were seen with other N248 variants. Modeling studies suggested that the selective reduction of glutaminase activity is the result of small conformational changes that affect active-site residues and catalytically relevant water molecules.     

Characterization of a beta-N-acetylhexosaminidase and a beta-N-acetylglucosaminidase/beta-glucosidase from Cellulomonas fimi

The gram-positive soil bacterium Cellulomonas fimi is shown to produce at least two intracellular beta-N-acetylglucosaminidases, a family 20 beta-N-acetylhexosaminidase (Hex20), and a novel family 3-beta-N-acetylglucosaminidase/beta-glucosidase (Nag3), through screening of a genomic expression library, cloning of genes and analysis of their sequences. Nag3 exhibits broad substrate specificity for substituents at the C2 position of the glycone: kcat/Km values at 25 degrees C were 0.066 s(-1) x mM(-1) and 0.076 s(-1) x mM(-1) for 4'-nitrophenyl beta-N-acetyl-D-glucosaminide and 4'-nitrophenyl beta-D-glucoside, respectively. The first glycosidase with this broad specificity to be described, Nag3, suggests an interesting evolutionary link between beta-N-acetylglucosaminidases and beta-glucosidases of family 3. Reaction by a double-displacement mechanism was confirmed for Nag3 through the identification of a glycosyl-enzyme species trapped with the slow substrate 2',4'-dinitrophenyl 2-deoxy-2-fluoro-beta-D-glucopyranoside. Hex20 requires the acetamido group at C2 of the substrate, being unable to cleave beta-glucosides, since its mechanism involves an oxazolinium ion intermediate. However, it is broad in its specificity for the D-glucosyl/D-galactosyl configuration of the glycone: Km and kcat values were 53 microM and 482.3 s(-1) for 4'-nitrophenyl beta-N-acetyl-D-glucosaminide and 66 microM and 129.1 s(-1) for 4'-nitrophenyl beta-N-acetyl-D-galactosaminide.     

Biochemical characterization of the glutamate transport in Trypanosoma cruzi

The role of amino acids in trypanosomatids goes beyond protein synthesis, involving processes such as differentiation, osmoregulation and energy metabolism. The availability of the amino acids involved in those functions depends, among other things, on their transport into the cell. Here we characterize a glutamate transporter from the human protozoan parasite Trypanosoma cruzi. Kinetic data show a single saturable system with a Km of 0.30 mM and a maximum velocity of 98.34 pmoles min(-1) per 2 x 10(7) cells for epimastigotes and 20 pmoles min(-1) per 2 x 10(7) cells for trypomastigotes. Transport was not affected by parasite nutrient starvation for up to 3h. Aspartate, alanine, glutamine, asparagine, methionine, oxaloacetate and alpha-ketoglutarate competed with the substrate in 10-fold excess concentrations. Glutamate uptake was strongly dependent on pH, but not on Na+ or K+ concentrations in the extracellular medium. These data were consistent with the sensitivity of the system to the H+ ionophore carbonyl cyanide p-trifluoromethoxyphenylhydrazone, suggesting that transport is driven by H+ concentration gradient across the cytoplasmic membrane. The glutamate transport increased linearly with temperature in a range from 15 to 40 degrees C, allowing the calculation of an activation energy of 52.38 kJ/mol.     

A heterodimerizing leucine zipper coiled coil system for examining the specificity of a position interactions: amino acids I,V, L,N, A,and K

We use a heterodimerizing leucine zipper system to examine the contribution of the interhelical a-a' interaction to dimer stability for six amino acids (A, V, L, I, K, and N). Circular dichroism (CD) spectroscopy monitored the thermal denaturation of 36 heterodimers that generate six homotypic and 30 heterotypic a-a' interactions. Isoleucine (I-I) is the most stable homotypic a-a' interaction, being 9.2 kcal/mol per dimer more stable than the A-A interaction and 4.0 kcal/mol per dimer more stable than either the L-L or V-V interaction, and 7.0 kcal/mol per dimer more stable than the N-N interaction. Only lysine was less stable than alanine. An alanine-based double-mutant thermodynamic cycle calculated coupling energies between the a and a' positions in the heterodimer. The aliphatic amino acids L, V, and I prefer to form homotypic interactions with coupling energies of -0.6 to -0.9 kcal/mol per dimer, but the heterotypic aliphatic interactions have positive coupling energies of <1.0 kcal/mol per dimer. The asparagine homotypic interaction has a coupling energy of -0.5 kcal/mol per dimer, while heterotypic interactions with the aliphatic amino acids produce coupling energies ranging from 2.6 to 4.9 kcal/mol per dimer. The homotypic K-K interaction is 2.9 kcal/mol per dimer less stable than the A-A interaction, but the coupling energy is only 0.3 kcal/mol per dimer. Heterotypic interactions with lysine and either asparagine or aliphatic amino acids produce similar coupling energies ranging from -0.2 to -0.7 kcal/mol per dimer. Thus, of the amino acids that were examined, asparagine contributes the most to dimerization specificity because of the large positive coupling energies in heterotypic interactions with the aliphatic amino acids which results in the N-N homotypic interaction.     

Thermodynamic analysis of ligand binding to winged bean (Psophocarpus tetragonolobus) acidic agglutinin reveals its specificity for terminally monofucosylated H-reactive sugars

The sugar-specific binding of N-dansylgalactosamine to WBA II (n = 2; Ka = 5.6 x 10(3) M-1; delta H = -21 kJ.mol-1; delta S = -21.3 J.mol-1.K-1) was utilized in substitution titrations for evaluating the association constants for the interaction of sugars with the lectin. An axial hydroxyl at C-4 and equatorial hydroxyls at C-3 and C-6 as in D-galacto configuration are crucial for binding. Both axial and equatorial hydroxyls are tolerated at C-2. Conformationally akin disaccharides such as lactose, N-acetyllactosamine, Gal beta 1-3GlcNAc, and Gal beta 1-3GalNAc show similar affinities. 2'-Fucosyllactose and H-disaccharide display 146 and 13 times stronger affinity over lactose and galactose, yet fucose by itself is devoid of activity. An interesting feature, noted for the first time, in protein-sugar interactions is the positive entropy change for the binding of 2'-fucosyllactose, suggesting that nonpolar interactions play an important role in stabilization of the lectin-sugar complex. 3-Fucosyllactose, lactodifucotetraose, lacto-N-fucopentaose II and III are inactive, whereas lacto-N-fucopentaose I has 14-fold lower affinity as compared with 2'-fucosyllactose. Conformational analysis indicates that the substitution at subterminal glucose or GlcNAc by L-fucose in either alpha 1-3 or alpha 1-4 linkage leads to its projection so as to sterically hinder the access of 3'-fucosyllactose, lactodifucotetraose, and lacto-N-fucopentaose II and III to the binding site of winged bean agglutinin II. Similarly the projection of alpha 1-3 linked Gal/GalNAc also leads to steric hindrance and hence prevents the binding of blood group A and B reactive sugars. Considering its unique specificity winged bean agglutinin II should be useful in the isolation and characterization of terminally monofucosylated H-reactive oligosaccharides from those that are difucosylated or internally fucosylated.     

Studies on the aminopeptidase activity of rat cathepsin H.

Three synthetic substrates H-Arg-NH-Mec, Bz-Arg-NH-Mec and H-Cit-NH-Mec (Bz, Benzoyl; NH-Mec, 4-methylcoumaryl-7-amide; Cit, citrulline) were used to characterize specificity requirements for the P1-S1 interaction of cathepsin H from rat liver. From rapid equilibrium kinetic studies it was shown that Km, kcat and the specificity constants kcat/Km are quite similar for substrates with a free alpha-amino group. In contrast, a 25-fold decrease of kcat/Km was observed for the N-terminal-blocked substrate Bz-Arg-NH-Mec. The activation energies for H-Arg-NH-Mec and Bz-Arg-NH-Mec were determined to be 37 kJ/mol and 55 kJ/mol, respectively, and the incremental binding energy delta delta Gb of the charged alpha-amino group was estimated to -8.1 kJ/mol at pH 6.8. The shown preference of cathepsin H for the unblocked substrates H-Arg-NH-Mec and H-Cit-NH-Mec was further investigated by inspection of the pH dependence of kcat/Km. The curves of the two substrates with a charged alpha-amino group showed identical bell-shaped profiles which both exhibit pKa1 and pKa2 values of 5.5 and 7.4, respectively, at 30 degrees C. The residue with a pKa1 of 5.5 in the acid limb of the activity profile of H-Arg-NH-Mec was identified by its ionization enthalpy delta Hion = 21 kJ/mol as a beta-carboxylate or gamma-carboxylate of the enzyme, whereas the residue with a pKa2 of 7.4 was assigned to the free alpha-amino group of the substrate with a delta Hion of 59 kJ/mol. Bz-Arg-NH-Mec showed a different pH-activity profile with a pKa1 of 5.4 and a pKa2 of 6.6 at 30 degrees C. Cathepsin H exhibits no preference for a basic P1 side chain as has been shown by the similar kinetics of H-Arg-NH-Mec and the uncharged, isosteric substrate H-Cit-NH-Mec. In summary, specific interactions of an anionic cathepsin H active site residue with the charged alpha-amino group of substrates caused transition state stabilization which proves the enzyme to act preferentially as an aminopeptidase.     

The primary acid product of DPNH

Analysis of the proton magnetic resonance spectra obtained at 220 MHz confirms the axial conformation of the C-6 hydroxyl in the model primary acid product 1-n-(2,6-dichlorobenzyl)-6-hydroxy-1,4,5,6-tetrahydronicotinamide. In the primary acid product of DPNH however the reaction occurs stereospecifically with the substitution at the C-6 position equatorial and on the B-side of the pyridine ring and the C-4A proton axial. A cyclic structure α,O^2′-6B cyclotetrahydronicotinamide is proposed for the primary acid product of DPNH, formed by epimerization of βDPNH to the α configuration followed by protonation at C-5 and subsequent attack of the ribose C-2′-OH on the C-6 position forming a new five membered ring.     

Interactions of cytochrome c with DNA at glassy carbon surface

Cyclic voltammetry (CV) was used to investigate the interactions of Cytochrome c (Cyt c) with deoxyribonucleic acid (DNA) at glassy carbon (GC) electrodes. The results indicate that there are strong interactions between Cyt c and DNA. The binding constant (k(A)) and binding free energy (Delta(r)G) of Cyt c with dsDNA are (1.69+/-0.38) x 10(5) L.mol(-1) and -(29.76+/-0.56) kJ.mol(-1), respectively; and those of Cyt c with ssDNA are (3.35+/-0.50) x 10(5) L.mol(-1) and -(31.49+/-0.37) kJ.mol(-1), respectively. The binding sites are achieved to be 3.3 bp per Cyt c molecule with dsDNA and 4.0 nucleotides (ssDNA) binding one Cyt c molecule. This experiment affords a valid method for investigating the interactions between DNA and proteins by electrochemical techniques.     

Altered -3 substrate specificity of Escherichia coli signal peptidase 1 mutants as revealed by screening a combinatorial peptide library

Signal peptidase functions to cleave signal peptides from preproteins at the cell membrane. It has a substrate specificity for small uncharged residues at -1 (P1) and aliphatic residues at the -3 (P3) position. Previously, we have reported that certain alterations of the Ile-144 and Ile-86 residues in Escherichia coli signal peptidase I (SPase) can change the specificity such that signal peptidase is able to cleave pro-OmpA nuclease A in vitro after phenylalanine or asparagine residues at the -1 position (Karla, A., Lively, M. O., Paetzel, M. and Dalbey, R. (2005) J. Biol. Chem. 280, 6731-6741). In this study, screening of a fluorescence resonance energy transfer-based peptide library revealed that the I144A, I144C, and I144C/I86T SPase mutants have a more relaxed substrate specificity at the -3 position, in comparison to the wild-type SPase. The double mutant tolerated arginine, glutamine, and tyrosine residues at the -3 position of the substrate. The altered specificity of the I144C/I86T mutant was confirmed by in vivo processing of pre-beta-lactamase containing non-canonical arginine and glutamine residues at the -3 position. This work establishes Ile-144 and Ile-86 as key P3 substrate specificity determinants for signal peptidase I and demonstrates the power of the fluorescence resonance energy transfer-based peptide library approach in defining the substrate specificity of proteases.     

Transformation of Lotus corniculatus Plants with Escherichia coli Asparagine Synthetase A: Effect on Nitrogen Assimilation and Plant Development

Asparagine and glutamine are major forms of nitrogen in the phloem sap of many higher plants. In vascular plants, glutamine-dependent asparagine synthetase (AS) is the primary source of asparagine. In Escherichia coli, asparagine is synthesized by the action of two distinct enzymes, AS-A which utilizes ammonia as a nitrogen donor, and AS-B which utilizes both glutamine and ammonia as substrates, but with a preference for glutamine. In this study, the possibility to endow plants with ammonia-dependent AS activity was investigated by heterologous expression of E. coli asnA gene with the aim to introduce a new ammonium assimilation pathway in plants. The bacterial gene is placed under the control of light-dependent promoters, and introduced by transformation into Lotus corniculatus plants. Analysis of transgenic plants has revealed a phenomenon of transgene silencing which has prevented asnA expression in several transgenics. The asnA-expressing plants are characterized by premature flowering and reduced growth. A significant reduction of the total free amino acid accumulation in transgenic plants is observed. Surprisingly, the content of asparagine in wild-type plants is about 2.5-fold higher than that of transgenic plants. While glutamine levels in transgenic plants are about 3–4-fold higher than those in wild-type plants, aspartate levels are significantly lower. Transformation with asnA also induced a significant reduction of photosynthesis when measured under saturated light and ambient CO₂ conditions.     

Creating new DNA binding specificities in the yeast transcriptional activator GCN4 by combining selected amino acid substitutions.

The specificity of the GCN4/DNA complex is mediated by a complicated network of interactions between the basic regions of both GCN4 monomers and their target halfsites. According to X-ray analyses (1, 2) one particular thymine of the target sequence is recognized by serine -11 and alanine -15 (we define the leucine in the first d-position of the heptad repeats as +1). We replaced serine -11 or alanine -15 with all other amino acids and analysed the DNA binding properties of the resulting stable GCN4 derivatives by electrophoretic mobility shift assays. Among these, mutants with tryptophan in position -11, or glutamic acid and glutamine in position -15, differ significantly from GCN4 in their DNA binding specificities. We then constructed selected double mutants, which differ from GCN4 in positions -11, -15 or -14 (3) of the basic region. The double mutants with tryptophan in position -11 and asparagine or serine in position -14 show drastically altered DNA binding specificities, presumably due to additive effects.