Molecular dissection of a critical specificity determinant within the amino acid editing domain of leucyl-tRNA synthetase

A highly conserved threonine residue marks the amino acid binding pocket within the editing active site of leucyl-tRNA synthetases (LeuRSs). It is essential to substrate specificity for the Escherichia coli enzyme in that it blocks the cognate leucine amino acid from binding in the hydrolytic editing active site. We combined mutagenesis and computational approaches to elucidate the molecular role of the critical side chain of this threonine residue. Removal of the terminal methyl group of the threonine side chain by replacement with serine yielded a mutant LeuRS that hydrolyzes Leu-tRNA(Leu). Substitution of valine for the conserved threonine conferred similar activities to the wild-type enzyme. However, an additional substitution within the editing active site suggested synergistic interactions with the conserved threonine site that significantly affected amino acid editing. On the basis of our combined biochemical and computational data, we propose that the threonine 252 side chain not only sterically hinders the cognate charged leucine from binding for hydrolysis but also plays a critical role in maintaining an active site geometry that is required for the fidelity of LeuRS.     

Interaction between a twelve-residue segment of antifreeze protein type I,or its mutants,and water molecules

A molecular dynamics simulation has been carried out for water molecules with a rigid segment of antifreeze protein type I. The segment consists of nine alanine residues, two threonine residues and one asparagine residue. Mutant segments, in which the threonine residues are replaced with valine residues, or serine residues, are also used. It is predicted that the hydrogen site of asparagine residue, and that of threonine residue, play an important role in the hydrogen bond of water molecules in these sites. This hydrogen bond is not noticeable between water molecules and the valine residue, or serine residue. The existence of four hydrophilic sites enhances the mobility of water molecules close to the serine residue of the mutant segment. The difference in the zenith-angle fluctuations of the original segment and the valine-mutant segment is less noticeable in the case of 230 K. This is because the gathering of water molecules due to the hydrophobic hydration is predominant near the alanine residues of the segments at this temperature.     

Dissection of helix capping in T4 lysozyme by structural and thermodynamic analysis of six amino acid substitutions at Thr 59.

Threonine 59, a helix-capping residue at the amino terminus of the longest helix in T4 phage lysozyme, was substituted with valine, alanine, glycine, serine, asparagine, and aspartic acid. The valine, alanine, and glycine replacements were observed to be somewhat more destabilizing than serine, asparagine, and aspartic acid. The crystal structures of the different variants showed that changes in conformation occurred at the site of substitution, including Asp 61, which is nearby, as well as displacement of a solvent molecule that is hydrogen-bonded to the gamma-oxygen of Thr 59 in wild-type lysozyme. Neither the structures nor the stabilities of the mutant proteins support the hypothesis of Serrano and Fersht (1989) that glycine and alanine are better helix-capping residues than valine because a smaller-sized residue allows better hydration at the end of the helix. In the aspartic acid and asparagine replacements the substituted side chains form hydrogen bonds with the end of the helix, as does threonine and serine at this position. In contrast, however, the Asp and Asn side chains also make unusually close contacts with carbon atoms in Asp 61. This suggests a structural basis for the heretofore puzzling observations that asparagine is more frequently observed as a helix-capping residue than threonine [Richardson, J. S., & Richardson, D. C. (1988) Science 240, 1648-1652] yet Thr----Asn replacements at N-cap positions in barnase were found to be destabilizing [Serrano, L., & Fersht, A. R. (1989) Nature 342, 296-299].(ABSTRACT TRUNCATED AT 250 WORDS)     

Functional roles of amino acid residues involved in forming the alpha-helix-turn-alpha-helix operator DNA binding motif of Tet repressor from Tn10.

The Tn10 derived Tet repressor contains an amino acid segment with high homology to the alpha-helix-turn-alpha-helix motif (HTH) of other DNA binding proteins. The five most conserved amino acids in HTH are probably involved in structural formation of the motif. Their functional role was probed by saturation mutagenesis yielding 95 single amino acid replacement mutants of Tet repressor. Their binding efficiencies to tet operator were quantitatively determined in vivo. All functional mutants contain amino acid substitutions consistent with their proposed role in a HTH. In particular, only the two smallest amino acids (serine, glycine) can substitute a conserved alanine in the proposed first alpha-helix without loss of activity. The last position of the first alpha-helix, the second position in the turn, and the fourth position in the second alpha-helix require mostly hydrophobic residues. The proposed C-terminus of the first alpha-helix is supported by a more active asparagine compared to glutamine replacement mutant of the wt leucine residue. The turn is located close to the protein surface as indicated by functional lysine and arginine replacements for valine. A glycine residue at the first position in the turn can be replaced by any amino acid yielding mutants with at least residual tet operator affinity. A structural model of the HTH of Tet repressor is presented.     

Binding of penicillin to thiol-penicillin-binding protein 3 of Escherichia coli: identification of its active site

Summary In order to determine the active site of penicillin-binding protein 3 of Escherichia coli (PBP3), the serine residue at position 307 was replaced with alanine, threonine or cysteine by oligonucleotide-directed site-specific mutagenesis. Since a unique BanII site exists at the position corresponding to serine-307, BanII digestion of the plasmid DNA after mutagenesis resulted in significant enrichment of the mutant plasmids. For mutagenesis, the gene coding for PBP3 (ftsI) was inserted into the expression cloning vector pIN-IIB. The hybrid protein produced was able to bind penicillin while mutant PBP3 in which serine-307 was replaced with either alanine or threonine did not lead to any detectable binding. However, contrary to the report of Broome-Smith et al. (1985) thiol-penicillin-binding protein 3, in which serine-307 was replaced with cysteine, was still able to bind penicillin. Replacement of serine-445 with an alanine residue had no effect on penicillin binding to PBP3.     

Alteration of substrate specificity of valine dehydrogenase from Streptomyces albus

The catabolism of branched chain amino acids, especially valine, appears to play an important role in furnishing building blocks for macrolide and polyether antibiotic biosyntheses. To determine the active site residues of ValDH, we previously cloned, partially characterized, and identified the active site (lysine) of Streptomyces albus ValDH. Here we report further characterization of S. albus ValDH. The molecular weight of S. albus ValDH was determined to be 38 kDa by SDS-PAGE and 67 kDa by gel filtration chromatography indicating that the enzyme is composed of two identical subunits. Optimal pHs were 10.5 and 8.0 for dehydrogenase activity with valine and for reductive amination activity with -ketoisovaleric acid, respectively. Several chemical reagents, which modify amino-acid side chains, inhibited the enzyme activity. To examine the role played by the residue for enzyme specificity, we constructed mutant ValDH by substituting alanine for glycine at position 124 by site-directed mutagenesis. This residue was chosen because it has been considered to be important for substrate discrimination by phenylalanine dehydrogenase (PheDH) and leucine dehydrogenase (LeuDH). The Ala-124–Gly mutant enzyme displayed lower activities toward aliphatic amino acids, but higher activities toward L-phenylalanine, L-tyrosine, and L-methionine compared to the wild type enzyme suggesting that Ala-124 is involved in substrate binding in S. albus ValDH.     

Chemotaxis toward amino acids in Escherichia coli

Escherichia coli cells are shown to be attracted to the l-amino acids alanine, asparagine, aspartate, cysteine, glutamate, glycine, methionine, serine, and threonine, but not to arginine, cystine, glutamine, histidine, isoleucine, leucine, lysine, phenylalanine, tryptophan, tyrosine, or valine. Bacteria grown in a proline-containing medium were, in addition, attracted to proline. Chemotaxis toward amino acids is shown to be mediated by at least two detection systems, the aspartate and serine chemoreceptors. The aspartate chemoreceptor was nonfunctional in the aspartate taxis mutant, which showed virtually no chemotaxis toward aspartate, glutamate, or methionine, and reduced taxis toward alanine, asparagine, cysteine, glycine, and serine. The serine chemoreceptor was nonfunctional in the serine taxis mutant, which was defective in taxis toward alanine, asparagine, cysteine, glycine, and serine, and which showed no chemotaxis toward threonine. Additional data concerning the specificities of the amino acid chemoreceptors with regard to amino acid analogues are also presented. Finally, two essentially nonoxidizable amino acid analogues, alpha-aminoisobutyrate and alpha-methylaspartate, are shown to be attractants for E. coli, demonstrating that extensive metabolism of attractants is not required for amino acid taxis.     

Role of aspartate 400, arginine 262, and arginine 401 in the catalytic mechanism of human coproporphyrinogen oxidase

Coproporphyrinogen oxidase (CPO) is the sixth enzyme in the heme biosynthetic pathway, catalyzing two sequential oxidative decarboxylations of propionate moieties on coproporphyrinogen-III forming protoporphyrinogen-IX through a monovinyl intermediate, harderoporphyrinogen. Site-directed mutagenesis studies were carried out on three invariant amino acids, aspartate 400, arginine 262, and arginine 401, to determine residue contribution to substrate binding and/or catalysis by human recombinant CPO. Kinetic analyses were performed on mutant enzymes incubated with three substrates, coproporphyrinogen-III, harderoporphyrinogen, or mesoporphyrinogen-VI, in order to determine catalytic ability to perform the first and/or second oxidative decarboxylation. When Asp400 was mutated to alanine no divinyl product was detected, but the production of a small amount of monovinyl product suggested the K(m) value for coproporphyrinogen-III did not change significantly compared to the wild-type enzyme. Upon mutation of Arg262 to alanine, CPO was again a poor catalyst for the production of a divinyl product, with a catalytic efficiency <0.01% compared to wild-type, including a 15-fold higher K(m) for coproporphyrinogen-III. The efficiency of divinyl product formation for mutant enzyme Arg401Ala was approximately 3% compared to wild-type CPO, with a threefold increase in the K(m) value for coproporphyrinogen-III. These data suggest Asp400, Arg262, and Arg401 are active site amino acids critical for substrate binding and/or catalysis. Possible roles for arginine 262 and 401 include coordination of carboxylate groups of coproporphyrinogen-III, while aspartate 400 may initiate deprotonation of substrate, resulting in an oxidative decarboxylation.     

Mutation of active site residues of the puromycin-sensitive aminopeptidase: conversion of the enzyme into a catalytically inactive binding protein

The active site glutamate, Glu 309, of the puromycin-sensitive aminopeptidase was mutated to glutamine, alanine, and valine. These mutants were characterized with amino acid beta-naphthylamides as substrates and dynorphin A(1-9) as an alternate substrate inhibitor. Conversion of glutamate 309 to glutamine resulted in a 5000- to 15,000-fold reduction in catalytic activity. Conversion of this residue to alanine caused a 25,000- to 100,000-fold decrease in activity, while the glutamate to valine mutation was the most dramatic, reducing catalytic activity 300,000- to 500,000-fold. In contrast to the dramatic effect on catalysis, all three mutations produced relatively small (1.5- to 4-fold) effects on substrate binding affinity. Mutation of a conserved tyrosine, Y394, to phenylalanine resulted in a 1000-fold decrease in k(cat), with little effect on binding. Direct binding of a physiological peptide, dynorphin A(1-9), to the E309V mutant was demonstrated by gel filtration chromatography. Taken together, these data provide a quantitative assessment of the effect of mutating the catalytic glutamate, show that mutation of this residue converts the enzyme into an inactive binding protein, and constitute evidence that this residue acts a general acid/base catalyst. The effect of mutating tyrosine 394 is consistent with involvement of this residue in transition state stabilization.     

Amino acid fermentation and hydrogen transfer in mixed cultures

Abstract The degradation of the following amino acids was investigated in mixed cultures obtained from a waste water purification plant: aspartate, glutamate, serine, alanine, valine and leucine. Inhibition of sulfate-reducing bacteria in these mixed cultures by molybdate was found to inhibit amino acid degradation. The degradation of serine, alanine, valine and leucine was accelerated considerably by active sulfate reduction. The fermentation of aspartate and glutamate was not stimulated by the presence of sulfate-reducing bacteria. The existence of species which are able to ferment valine and leucine by coupling their oxidation to the reduction of exogenous acetate to butyrate was demonstrated.     

Characterization of the calmodulin binding domain of neuromodulin. Functional significance of serine 41 and phenylalanine 42.

Neuromodulin (also designated P-57, GAP-43, B-50) is a major presynaptic substrate for protein kinase C. Phosphorylation of neuromodulin decreases its affinity for calmodulin, suggesting that neuromodulin may function to bind and concentrate calmodulin at specific sites within neurons, releasing calmodulin locally in response to phosphorylation by protein kinase C (Alexander, K. A., Cimler, B. M., Meier, K. E., and Storm, D. R. (1987) J. Biol. Chem. 262, 6108-6113). In the present study, we have constructed and characterized several mutant neuromodulins to demonstrate that the amino acid sequence 39-56 is required for calmodulin binding, and that this domain contains the sole in vitro protein kinase C phosphorylation site at serine 41. We also demonstrate that the adjacent phenylalanine 42, interacts hydrophobically with calmodulin. These hydrophobic interactions may be disrupted by the introduction of negative charge at serine 41, and thereby regulate the neuromodulin/calmodulin binding interactions. The sensitivity of the neuromodulin/calmodulin binding interaction to negative charge at serine 41 was determined by substitution of serine 41 with an aspartate or an asparagine residue. The asparagine mutant retained its affinity for calmodulin-Sepharose while the aspartate mutant did not adsorb to calmodulin-Sepharose. We conclude that protein kinase C phosphorylation of neuromodulin abolishes calmodulin binding by introducing negative charges within the calmodulin binding domain at a position adjacent to the phenylalanine.     

Escherichia coli serine hydroxymethyltransferase. The role of histidine 228 in determining reaction specificity.

Serine hydroxymethyltransferase has a conserved histidine residue (His-228) next to the lysine residue (Lys-229) which forms the internal aldimine with pyridoxal 5'-phosphate. This histidine residue is also conserved at the equivalent position in all amino acid decarboxylases and tryptophan synthase. Two mutant forms of Escherichia coli serine hydroxymethyltransferase, H228N and H228D, were constructed, expressed, and purified. The properties of the wild type and mutant enzymes were studied with substrates and substrate analogs by differential scanning calorimetry, circular dichroism, steady state kinetics, and rapid reaction kinetics. The conclusions of these studies were that His-228 plays an important role in the binding and reactivity of the hydroxymethyl group of serine in the one-carbon-binding site. The mutant enzymes utilize substrates and substrate analogs more effectively for a variety of alternate non-physiological reactions compared to the wild type enzyme. As one example, the mutant enzymes cleave L-serine to glycine and formaldehyde when tetrahydropyteroylglutamate is replaced by 5-formyltetrahydropteroylglutamate. The released formaldehyde inactivates these mutant enzymes. The loss of integrity of the one-carbon-binding site with L-serine in the two mutant forms of the enzyme may be the result of these enzymes not undergoing a conformational change to a closed form of the active site when serine forms the external aldimine complex.     

Site-specific mutagenesis of aspartate transcarbamoylase. Replacement of tyrosine 165 in the catalytic chain by serine reduces enzymatic activity

Site-specific mutagenesis was used to modify an amino acid residue of the catalytic trimer of aspartate transcarbamoylase thought to be at the active site. Tyrosine 165 of the catalytic chain was replaced by a serine residue. This mutation substantially reduces but does not entirely abolish the catalytic activity of the holoenzyme and the isolated catalytic trimer. Km for aspartate for the mutant catalytic trimer is 12-fold higher than for the wild type. Vmax is reduced by a factor of 4 and Kd for carbamoylphosphate is increased 3-fold in the mutant. Although these results suggest that tyrosine 165 is at the active site, they demonstrate that the residue is not essential for catalysis.     

Role of two active site Glu residues in the molecular action of botulinum neurotoxin endopeptidase

Botulinum neurotoxin type A (BoNT/A) light chain (LC) is a zinc endopeptidase that causes neuroparalysis by blocking neurotransmitter release at the neuromuscular junctions. The X-ray crystal structure of the toxin reveals that His223 and His227 of the Zn(2+) binding motif HEXXH directly coordinate the active site zinc. Two Glu residues (Glu224 and Glu262) are also part of the active site, with Glu224 coordinating the zinc via a water molecule whereas Glu262 coordinates the zinc directly as the fourth ligand. In the past we have investigated the topographical role of Glu224 by replacing it with Asp thus reducing the side chain length by 1.4 A that reduced the endopeptidase activity dramatically [L. Li, T. Binz, H. Niemann, and B.R. Singh, Probing the role of glutamate residue in the zinc-binding motif of type A botulinum neurotoxin light chain, Biochemistry 39 (2000) 2399-2405]. In this study we have moved the Glu 224 laterally by a residue (HXEXH) to assess its positional influence on the endopeptidase activity, which was completely lost. The functional implication of Glu262 was investigated by replacing this residue with aspartate and glutamine using site-directed mutagenesis. Substitution of Glu262 with Asp resulted in a 3-fold decrease in catalytic efficiency. This mutation did not induce any significant structural alterations in the active site and did not interfere with substrate binding. Substitution of Glu262 with Gln however, dramatically impaired the enzymatic activity and this is accompanied by global alterations in the active site conformation in terms of topography of aromatic amino acid residues, zinc binding, and substrate binding, resulting from the weakened interaction between the active site zinc and Gln. These results suggest a pivotal role of the negatively charged carboxyl group of Glu262 which may play a critical role in enhancing the stability of the active site with strong interaction with zinc. The zinc may thus play structural role in addition to its catalytic role.     

Transient kinetic analysis of L-serine interaction with Escherichia coli D-3-phosphoglycerate dehydrogenase containing amino acid mutations in the hinge regions

In Escherichia colid-3-phosphoglycerate dehydrogenase, the amino acid sequences G294-G295 and G336-G337 are found between structural domains and appear to function as hinge regions. Mutagenesis studies of these sequences showed that bulky side chains had significant effects on the kinetic properties of the enzyme. Placement of a tryptophanyl residue near the serine binding site (W139F/E360W) allows serine binding to be monitored by fluorescence quenching analysis. Pre-steady-state analysis has demonstrated that serine binds to two forms of the free enzyme, E and E*. Conversion of Gly-336 to valine has its main effect on the Kd of serine binding to one form of the free enzyme (E) while maintaining the cooperativity of binding observed in the native enzyme. Conversion of Gly-294 to valine eliminates a rate limiting conformational change that follows serine binding to E. The conformational change between the two forms of free enzyme is maintained, but the Hill coefficient for cooperativity is significantly lowered. The data indicate that the cooperative transmission induced by serine binding is transmitted through the Gly294-Gly295 hinge region to the opposite serine binding interface and that this is most likely propagated by way of the substrate binding domain-regulatory domain interface. In the G294 mutant enzyme, both serine bound species, E·Ser and E*·Ser, are present in significant amounts indicating that cooperativity of serine binding does not result from the binding to two different forms. The data also suggest that the E* form may be inactive even when serine is not bound.     

Asparagine 23 and aspartate 305 are essential residues in the active site of UDP-N-acetylglucosamine enolpyruvyl transferase from Enterobacter cloacae

UDP-N-acetylglucosamine enolpyruvyl transferase (MurA) catalyzes the transfer of the intact enolpyruvyl moiety of phosphoenolpyruvate (PEP) to the 3'-hydroxyl group of UDP-N-acetylglucosamine (UDPNAG). This reaction constitutes the first committed step in the biosynthesis of the bacterial cell wall component peptidoglycan (murein). The transfer reaction involves the nucleophilic attack of the 3'-hydroxyl group of UDPNAG at the C-2 of PEP. The three-dimensional structure of MurA complexed with UDPNAG revealed an aspartate residue (D305 in the En. cloacae sequence) close to the 3'-hydroxyl group of UDPNAG, suggesting that it may act as an acid-base catalyst in the active center of the enzyme. In addition to aspartate 305, asparagine 23 also interacts with the 3'-hydroxyl group; however, its role in catalysis or binding of the UDPNAG substrate is unclear. To gain information on the role of these two amino acids in the MurA-catalyzed reaction we have exchanged D305 for alanine, cysteine, histidine, and glutamate, and N23 for alanine and serine using site-directed mutagenesis. While the D305 alanine, cysteine, and histidine mutant proteins do not have detectable enzymatic activity, the D305E mutant protein exhibits a low residual activity (ca. 0.1% of the wild-type enzyme). Unlike with wild-type MurA, no exothermic signal was obtained when the D305A and -E mutant proteins were titrated with UDPNAG, demonstrating that the affinity of the sugar nucleotide substrate is reduced as a result of the amino acid exchange. The reduced affinity to UDPNAG leads to a lower propensity of C115 to form either the O-phosphothioketal with PEP or the thioether with the antibiotic fosfomycin. These findings emphasize the dual role of D305 as a general base and an essential binding partner to UDPNAG in the active site of MurA. Similarly, the two N23 mutant proteins showed a much lower catalytic activity although binding of UDPNAG was not as much affected as in the case of the D305 mutant proteins. This result indicates that this amino acid residue is mainly involved in stabilization of transition states.     

The specificity of the S1 and S2 subsites of elastase

Esters of tetrapeptides of the general formula ethoxycarbonyl-prolyl-alanyl-X-Y where either X or Y was an alanine residue were synthesised and their cleavage by elastase studied. It was found that variation of the alcohol moiety between methyl, cyclohexyl and nitrophenyl residues had no effect on the catalytic rate constant for cleavage of ethoxycarbonyl-prolyl-dialanyl-alanine esters demonstrating that acylation is much faster than deacylation for this system and also that non-productive binding is not kinetically significant. The effect of changing the amino acid residue in position X was small compared with that of change in position Y. The presence of valine and serine residues in position Y produced the highest specificity constant but the highest catalytic rate constant was found for a leucine residue in this position. The results are discussed in terms of the binding of the substrate to the enzyme.     

Chlorophyllase as a serine hydrolase: identification of a putative catalytic triad

Chlorophyllases (Chlases), cloned so far, contain a lipase motif with the active serine residue of the catalytic triad of triglyceride lipases. Inhibitors specific for the catalytic serine residue in serine hydrolases, which include lipases effectively inhibited the activity of the recombinant Chenopodium album Chlase (CaCLH). From this evidence we assumed that the catalytic mechanism of hydrolysis by Chlase might be similar to those of serine hydrolases that have a catalytic triad composed of serine, histidine and aspartic acid in their active site. Thus, we introduced mutations into the putative catalytic residue (Ser162) and conserved amino acid residues (histidine, aspartic acid and cysteine) to generate recombinant CaCLH mutants. The three amino acid residues (Ser162, Asp191 and His262) essential for Chlase activity were identified. These results indicate that Chlase is a serine hydrolase and, by analogy with a plausible catalytic mechanism of serine hydrolases, we proposed a mechanism for hydrolysis catalyzed by Chlase.