A Covalent Enzyme-Substrate Intermediate with Saccharide Distortion in a Mutant T4 Lysozyme

Kuroki, Ryota; Weaver, L.H.; Matthews, Brian W.

doi:10.1126/science.8266098

Cited by 197 publications

(231 citation statements)

References 34 publications

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“…With the notable exception of M106K, each of the variants constructed here has activity within about a factor of two equal to that of WT*. In an adduct of T4 lysozyme with an extended peptidoglycan cell-wall fragment, the side chain of Met 106 makes van der Waals contacts with the peptidic part of the fragment ( Kuroki et al, 1993). In the structure of M106K (Fig.…”

Section: Discussionmentioning

confidence: 85%

“…4B) the €-amino group of Lys 106 occupies a position that would potentially interfere with bound substrate. Possible steric interference was checked by super-imposing the structure of M106K on the lysozyme-peptidoglycan complex described by Kuroki et al (1993). ence between M106K and bound substrate could explain the reduction in activity for this mutant.…”

Section: Discussionmentioning

confidence: 99%

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Context‐dependent protein stabilization by methionine‐to‐leucine substitution shown in T4 lysozyme

et al. 1998

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The substitution of methionines with leucines within the interior of a protein is expected to increase stability both because of a more favorable solvent transfer term as well as the reduced entropic cost of holding a leucine side chain in a defined position. Together, these two terms are expected to contribute about 1.4 kcal/mol to protein stability for each Met + Leu substitution when fully buried. At the same time, this expected beneficial effect may be offset by steric factors due to differences in the shape of leucine and methionine. To investigate the interplay between these factors, all methionines in T4 lysozyme except at the amino-terminus were individually replaced with leucine. Of these mutants, M106L and M120L have stabilities 0.5 kcal/mol higher than wild-type T4 lysozyme, while M6L is significantly destabilized (-2.8 kcal/mol). M102L, described previously, is also destabilized (-0.9 kcal/mol). Based on this limited sample it appears that methionine-to-leucine substitutions can increase protein stability but only in a situation where the methionine side chain is fully or partially buried, yet allows the introduction of the leucine without concomitant steric interference. The variants, together with methionine-to-lysine substitutions at the same sites, follow the general pattern that substitutions at rigid, internal sites tend to be most destabilizing, whereas replacements at more solvent-exposed sites are better tolerated.

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Section: Discussionmentioning

confidence: 85%

Section: Discussionmentioning

confidence: 99%

Context‐dependent protein stabilization by methionine‐to‐leucine substitution shown in T4 lysozyme

et al. 1998

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“…As concerns the slightly twisted boat conformation adopted by the castanospermine in the MutB⅐castanospermine structure (Fig. 5C), it may be the precursor of the transition state, assumed to be in a half-chair conformation (51,52). In the latter the glycosidic linkage at the anomeric carbon would be in an axial position, which is needed for the terminal glycosyl residue to be completely enclosed in the pocket with the nucleophilic Asp 200 adjacent to C1 and to allow the aglycon to depart following bond cleavage.…”

Section: Discussionmentioning

confidence: 99%

Trehalulose Synthase Native and Carbohydrate Complexed Structures Provide Insights into Sucrose Isomerization

Ravaud

Robert

Watzlawick

et al. 2007

Journal of Biological Chemistry

137

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Various diseases related to the overconsumption of sugar make a growing need for sugar substitutes. Because sucrose is an inexpensive and readily available D-glucose donor, the industrial potential for enzymatic synthesis of the sucrose isomers trehalulose and/or isomaltulose from sucrose is large. The product specificity of sucrose isomerases that catalyze this reaction depends essentially on the possibility for tautomerization of sucrose, which is required for trehalulose formation. For optimal use of the enzyme, targeting controlled synthesis of these functional isomers, it is necessary to minimize the side reactions. This requires an extensive analysis of substrate binding modes and of the specificity-determining sites in the structure. The 1.6 -2.2-Å resolution three-dimensional structures of native and mutant complexes of a trehalulose synthase from Pseudomonas mesoacidophila MX-45 mimic successive states of the enzyme reaction. Combined with mutagenesis studies they give for the first time thorough insights into substrate recognition and processing and reaction specificities of these enzymes. Among the important outcomes of this study is the revelation of an aromatic clamp defined by Phe 256 and Phe 280 playing an essential role in substrate recognition and in controlling the reaction specificity, which is further supported by mutagenesis studies. Furthermore, this study highlights essential residues for binding the glucosyl and fructosyl moieties. The introduction of subtle changes informed by comparative three-dimensional structural data observed within our study can lead to fundamental modifications in the mode of action of sucrose isomerases and hence provide a template for industrial catalysts.

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“…The tolerance of the enzyme's function to several nonconservative substitutions (Asp 20-Cys and Asp 20-Ala), as well as the conservative substitution Asp 20-Glu (Anand et al, 1988;Rennell et al, 1991), indicated a less critical role for the Asp 20 side-chain carboxylate. A recent structure of a quasi-stable covalent adduct between a peptidoglycan substrate and several T4 lysozyme variants has led to a revision of the role for the Asp 20 carboxylate (Kuroki et al, 1993(Kuroki et al, , 1995. This residue seems to optimize the enzyme's binding of the transition state, rather than playing a pivotal catalytic role.…”

Section: Suppressible Deleterious Priman Substitutionsmentioning

confidence: 99%

Alteration of T4 lysozyme structure by second‐site reversion of deleterious mutations

et al. 1997

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Mutations that suppress the defects introduced into T4 lysozyme by single amino acid substitutions were isolated and characterized. Among 53 primary sites surveyed, 8 yielded second-site revertants; a total of 18 different mutants were obtained. Most of the restorative mutations exerted global effects, generally increasing lysozyme function in a number of primary mutant contexts. Six of them were more specific, suppressing only certain specific deleterious primary substitutions, or diminishing the function of lysozymes bearing otherwise nondeleterious primary substitutions. Some variants of proteins bearing primary substitutions at the positions of Asp 20 and Ala 98 are inferred to have significantly altered structures.Keywords: bacteriophage; mutations; second-site revertants; T4 lysozymeThe lysozyme encoded by bacteriophage T4 is a small, globular, monomeric protein that has been the subject of extensive structural and genetic studies (for reviews, see Potkte & Hardy, 1994;Matthews, 1995). The native structure of the protein has been determined to 1.7 8, resolution by X-ray crystallography (Weaver & Matthews, 1987).Systematic probing of the T4 lysozyme structure has shown that 74 of the 164 positions in its sequence are sensitive to single amino acid substitutions; that is, at least one single amino acid substitution at one of these positions results in at least a 50-fold reduction in function (Rennell et al., 1991). Not surprisingly, the most critical amino acid residues in the protein were found to be either buried, or else solvent-exposed, but in the active site cleft.A second level of genetic probing of protein structure consists of isolating and characterizing intragenic second-site revertants of mutant proteins. A survey of second-site suppressors could be expected to reveal interactions between residues in the folded (or possibly, folding) structure. Some of the resulting multiply mutant proteins might be expected to have significantly altered structures, the solution of which could lead to insights on the protein folding problem.An early study of second-site revertants of temperature-sensitive mutants of staphylococcal nuclease (Shortle & Lin, 1985) Bouvier & Poteete, 1996) uncovered structural variants in addition to global stabilizers.Here we report the results of a systematic survey of second-site revertants in the T4 lysozyme gene. Presumptively significant structural variants are described. ResultsIn a previous study (Rennell et al., 1991), we examined the effects of single amino acid substitutions in the T4 lysozyme gene by the use of amber suppressors. A bacteriophage P22 hybrid bearing the T4 lysozyme gene in place of its P22 counterpart was constructed, amber mutations were introduced into its lysozyme gene, and the mutant phages were tested for ability to form plaques on a collection of 13 Salmonella amber suppressor strains that each insert a different amino acid in response to the amber codon. Of 163 amber mutant phages tested in this way, 74 failed to form plaques, or formed tiny p...

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A Covalent Enzyme-Substrate Intermediate with Saccharide Distortion in a Mutant T4 Lysozyme

Cited by 197 publications

References 34 publications

Context‐dependent protein stabilization by methionine‐to‐leucine substitution shown in T4 lysozyme

Context‐dependent protein stabilization by methionine‐to‐leucine substitution shown in T4 lysozyme

Trehalulose Synthase Native and Carbohydrate Complexed Structures Provide Insights into Sucrose Isomerization

Alteration of T4 lysozyme structure by second‐site reversion of deleterious mutations

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