Long-range structural effects in a second-site revertant of a mutant dihydrofolate reductase.

Brown, Katherine A.; Howell, Elizabeth E.; Kraut, Joseph

doi:10.1073/pnas.90.24.11753

Cited by 28 publications

(32 citation statements)

References 20 publications

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“…Although our calculated structures are not as reliable as those obtained from crystal structures, they do have some advantages such as accessibility to distribution of conformations and hydrogen bonding. Based on those parameters, we reach the same conclusion as Brown et al (10), namely that mutations can lead to structural perturbations far beyond the region that is local to the mutation.…”

Section: Resultssupporting

confidence: 58%

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Correlated motion and the effect of distal mutations in dihydrofolate reductase

Rod

Radkiewicz²,

Brooks³

2003

Proc. Natl. Acad. Sci. U.S.A.

215

289

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Dihydrofolate reductase (DHFR) catalyzes the reduction of dihydrofolate to tetrahydrofolate. The catalytic rate in this system has been found to be significantly affected by mutations far from the site of chemical activity in the enzyme [Rajagopalan, P. T. R, Lutz, S., and Benkovic, S. J. (2002) Biochemistry 41, 12618 -12628]. On the basis of extensive computer simulations for wild-type DHFR from Escherichia coli and four mutants (G121S, G121V, M42F, and M42F͞ G121S), we show that key parameters for catalysis are changed. The parameters we study are relative populations of different conformations sampled and hydrogen bonds. We find that the mutations result in long-range structural perturbations, rationalizing the effects that the mutations have on the kinetics of the enzyme. Such perturbations also provide a rationalization for the reported nonadditivity effect for double mutations. We finally examine the role a structural perturbation will have on the hydride transfer step. On the basis of our new findings, we discuss the role of coupled motions between distant regions in the enzyme, which previously was reported by Radkiewicz and Brooks.

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Section: Resultssupporting

confidence: 58%

“…This finding has been argued to support the notion that communication occurs between the two (sequentially and spatially distant) residues (7,8). Other studies have also shown the existence of long-range and nonadditive effects (9,10).…”

supporting

confidence: 78%

Correlated motion and the effect of distal mutations in dihydrofolate reductase

Rod

Radkiewicz²,

Brooks³

2003

Proc. Natl. Acad. Sci. U.S.A.

215

289

View full text Add to dashboard Cite

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“…In the case of structural perturbations caused by the single mutations, it is likely that the effect of the double mutation is non-additive due to different structural perturbations. This principle has been demonstrated for some residues of dihydrofolate reductase (53,54).…”

Section: Discussionmentioning

confidence: 98%

Laboratory-evolved Vanillyl-alcohol Oxidase Produces Natural Vanillin

Heuvel

Berg

Rovida

et al. 2004

Journal of Biological Chemistry

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The flavoenzyme vanillyl-alcohol oxidase was subjected to random mutagenesis to generate mutants with enhanced reactivity to creosol (2-methoxy-4-methylphenol). The vanillyl-alcohol oxidase-mediated conversion of creosol proceeds via a two-step process in which the initially formed vanillyl alcohol (4-hydroxy-3-methoxybenzyl alcohol) is oxidized to the widely used flavor compound vanillin (4-hydroxy-3-methoxybenzaldehyde). The first step of this reaction is extremely slow due to the formation of a covalent FAD N-5-creosol adduct. After a single round of error-prone PCR, seven mutants were generated with increased reactivity to creosol. The single-point mutants I238T, F454Y, E502G, and T505S showed an up to 40-fold increase in catalytic efficiency (k cat /K m ) with creosol compared with the wild-type enzyme. This enhanced reactivity was due to a lower stability of the covalent flavin-substrate adduct, thereby promoting vanillin formation. The catalytic efficiencies of the mutants were also enhanced for other ortho-substituted 4-methylphenols, but not for p-cresol (4-methylphenol). The replaced amino acid residues are not located within a distance of direct interaction with the substrate, and the determined three-dimensional structures of the mutant enzymes are highly similar to that of the wild-type enzyme. These results clearly show the importance of remote residues, not readily predicted by rational design, for the substrate specificity of enzymes.The increased use of enzymes and other proteins in the pharmaceutical, chemical, and agricultural industry has generated considerable interest in the design of proteins with new or improved properties. Two different but complementary technologies have been applied to this goal: (i) rational design, which relies on the availability of the three-dimensional structure and knowledge about the relationship between sequence, structure, and mechanism, and (ii) directed evolution methods, which use random mutagenesis of the gene encoding the protein or recombination of gene fragments to create diversity and then experimental screening of the libraries generated for the desired properties. Rational design has been used to elucidate and change enzyme mechanism, substrate and product specificity, enantioselectivity, cofactor specificity, and protein stability (1-3). Directed evolution has been applied to increase catalytic activity; to invert or improve enantioselectivity; and to alter substrate and product specificity, protein stability, pH optimum, and tolerance against organic solvents (1, 4 -7). An obvious advantage of directed evolution methods over sitedirected mutagenesis is that enzymes can be tailored for the production of (intermediate) products without detailed knowledge of protein structure and structure-function relationships.In this study, we engineered the flavoenzyme vanillyl-alcohol oxidase (VAO) 1 by random mutagenesis such that the evolved mutants are capable of producing natural vanillin (4-hydroxy-3-methoxybenzaldehyde) from the precursor creosol (2-methoxy...

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“…Intramolecular compensatory mutations in proteins can sometimes occur in residues located far away from the primary mutation but appear to have a tendency to involve spatially close residues (23,33,56,57). In a few cases, detailed structure-function studies have provided some insight into the molecular mechanisms of compensatory effects (5,6,7,12,21,(27)(28)(29)37). The bases of the compensatory effects range from near-additive, nonspecific (global) effects on a single property of the protein, such as stability, to complex, nonadditive, multifactorial effects including altered interaction patterns and propagated conformational rearrangements.…”

mentioning

confidence: 99%

Deterministic, Compensatory Mutational Events in the Capsid of Foot-and-Mouth Disease Virus in Response to the Introduction of Mutations Found in Viruses from Persistent Infections

Mateo

Mateu

2007

J Virol

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The evolution of foot-and-mouth disease virus (FMDV) (biological clone C-S8c1) in persistently infected cells led to the emergence of a variant (R100) that displayed increased virulence, reduced stability, and other modified phenotypic traits. Some mutations fixed in the R100 genome involved a cluster of highly conserved residues around the capsid pores that participate in interactions with each other and/or between capsid protomers. We have investigated phenotypic and genotypic changes that occurred when these replacements were introduced into the C-S8c1 capsid. The C3007V and M3014L mutations exerted no effect on plaque size or viral yield during lytic infections, or on virion stability, but led to a reduction in biological fitness; the D3009A mutation caused drastic reductions in plaque size and viability. Remarkably, competition of the C3007V mutant with the nonmutated virus invariably resulted in the fixation of the D3009A mutation in the C3007V capsid. In turn, the presence of the D3009A mutation invariably led to the fixation of the M3014L mutation. In both cases, two individually disadvantageous mutations led, together, to an increase in fitness, as the double mutants outcompeted the nonmutated genotype. The higher fitness of C3007V/D3009A was related to a faster multiplication rate. These observations provide evidence for a chain of linked, compensatory mutational events in a defined region of the FMDV capsid. Furthermore, they indicate that the clustering of unique amino acid replacements in viruses from persistent infections may also occur in cytolytic infections in response to changes caused by previous mutations without an involvement of the new mutations in the adaptation to a different environment.

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Long-range structural effects in a second-site revertant of a mutant dihydrofolate reductase.

Cited by 28 publications

References 20 publications

Correlated motion and the effect of distal mutations in dihydrofolate reductase

Correlated motion and the effect of distal mutations in dihydrofolate reductase

Laboratory-evolved Vanillyl-alcohol Oxidase Produces Natural Vanillin

Deterministic, Compensatory Mutational Events in the Capsid of Foot-and-Mouth Disease Virus in Response to the Introduction of Mutations Found in Viruses from Persistent Infections

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