Hyperstability and Substrate Promiscuity in Laboratory Resurrections of Precambrian β-Lactamases

Risso, Valeria A.; Gavira, José A.; Mejia-Carmona, Diego F.; Gaucher, Eric A.; Sanchez-Ruiz, José M.

doi:10.1021/ja311630a

Cited by 235 publications

(324 citation statements)

References 32 publications

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“…Thus, Ignisphera aggregans (12) and Pyrococcus horikoshii (13) exhibit optimal growth temperature of 92°and 98°C, respectively, whereas another organism isolated from a hydrothermal vent has been reported to grow at 121°C (14). Moreover, reconstructions of ancestral proteins, with amino acid sequences inferred from the sequences of their modern descendants, have been shown to be remarkably thermostable, with melting temperatures ∼30°C higher than those of proteins from their modern descendants (15,16).…”

Section: Hiv-1 Proteasementioning

confidence: 99%

Cytosine deamination and the precipitous decline of spontaneous mutation during Earth's history

Lewis

Crayle

Zhou

et al. 2016

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

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The hydrolytic deamination of cytosine and 5-methylcytosine residues in DNA appears to contribute significantly to the appearance of spontaneous mutations in microorganisms and in human disease. In the present work, we examined the mechanism of cytosine deamination and the response of the uncatalyzed reaction to changing temperature. The positively charged 1,3-dimethylcytosinium ion was hydrolyzed at a rate similar to the rate of acid-catalyzed hydrolysis of 1-methylcytosine, for which it furnishes a satisfactory kinetic model and a probable mechanism. In agreement with earlier reports, uncatalyzed deamination was found to proceed at very similar rates for cytosine, 1-methylcytosine, cytidine, and cytidine 5′-phosphate, and also for cytosine residues in single-stranded DNA generated from a phagemid, in which we sequenced an insert representing the gene of the HIV-1 protease. Arrhenius plots for the uncatalyzed deamination of cytosine were linear over the temperature range from 90°C to 200°C and indicated a heat of activation (ΔH ‡ ) of 23.4 ± 0.5 kcal/mol at pH 7. Recent evidence indicates that the surface of the earth has been cool enough to support life for more than 4 billion years and that life has been present for almost as long. If the temperature at Earth's surface is assumed to have followed Newton's law of cooling, declining exponentially from 100°C to 25°C during that period, then half of the cytosine-deaminating events per unit biomass would have taken place during the first 0.2 billion years, and <99.4% would have occurred during the first 2 billion years. spontaneous mutation | heat mutagenesis | cytosine deamination | HIV-1 protease I n the absence of enzymes, most biological reactions take place so slowly that they can be followed conveniently only at elevated temperatures (1). Among those reactions are several that may result in spontaneous mutation, notably the hydrolytic deamination of cytosine and 5-methylcytosine, which generate uracil and thymine, respectively. These deamination reactions have been shown to account for many of the single-site mutations that lead to inherited diseases in humans (2) and for the marked bias of spontaneous mutation toward increased AT content in microorganisms (3, 4). Rates of mutation have long been known to increase with increasing temperature, a phenomenon that Drake has termed "heat mutagenesis" (5).There is widespread (6-8), if not universal (9), agreement that life originated when the earth was warmer-perhaps much warmer-than it is today. A recent isotopic analysis of carbon inclusions in zircons from the Jack Hills in Western Australia indicates that life may have emerged as early as 4.1 billion years ago (10), shortly after water first appeared at the surface in liquid form (11). Several present-day organisms thrive at temperatures near the boiling point of water. Thus, Ignisphera aggregans (12) and Pyrococcus horikoshii (13) exhibit optimal growth temperature of 92°and 98°C, respectively, whereas another organism isolated from a hydrothermal vent has be...

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Section: Hiv-1 Proteasementioning

confidence: 99%

Cytosine deamination and the precipitous decline of spontaneous mutation during Earth's history

Lewis

Crayle

Zhou

et al. 2016

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

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“…This information then can be used to engineer proteins with desired folding properties. ASR has already provided important insights into evolutionary trends in stability, specificity, and other biophysical properties (12)(13)(14)(15)(16)(17)(18)(19)(20).Previously, we used ASR to study the thermostability of the ribonuclease H (RNase H) family and demonstrated divergent trends in thermostability along mesophilic and thermophilic lineages (16). Here we examined the folding mechanism and associated rates of these reconstructed ancestral RNases H. The folding pathway of the two extant homologs, Escherichia coli RNase H (ecRNH) and Thermus thermophilus RNase H Significance Because protein folding is crucial to proper cellular function, there must be evolutionary pressures on how a protein achieves and maintains its folded structure.…”

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confidence: 99%

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confidence: 99%

“…As such, it is important to address whether uncertainty in ASR affects our measured biophysical parameters. Numerous ASR studies have addressed the effect of uncertainty by a variety of approaches, and in general, the measured parameters have proven robust (15)(16)(17)(18)(19)(28)(29)(30)(31). For the RNase H family, we generated alternative sequences of the last common ancestor, Anc1*, which had the greatest uncertainty of all of the reconstructed ancestors (16).…”

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confidence: 99%

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Evolutionary trend toward kinetic stability in the folding trajectory of RNases H

Lim

Hart

Harms

2016

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

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Proper folding of proteins is critical to producing the biological machinery essential for cellular function. The rates and energetics of a protein's folding process, which is described by its energy landscape, are encoded in the amino acid sequence. Over the course of evolution, this landscape must be maintained such that the protein folds and remains folded over a biologically relevant time scale. How exactly a protein's energy landscape is maintained or altered throughout evolution is unclear. To study how a protein's energy landscape changed over time, we characterized the folding trajectories of ancestral proteins of the ribonuclease H (RNase H) family using ancestral sequence reconstruction to access the evolutionary history between RNases H from mesophilic and thermophilic bacteria. We found that despite large sequence divergence, the overall folding pathway is conserved over billions of years of evolution. There are robust trends in the rates of protein folding and unfolding; both modern RNases H evolved to be more kinetically stable than their most recent common ancestor. Finally, our study demonstrates how a partially folded intermediate provides a readily adaptable folding landscape by allowing the independent tuning of kinetics and thermodynamics.protein folding | energy landscape | protein evolution | ancestral sequence reconstruction B ecause most proteins need to fold to function, there must be selective pressures for efficient folding and maintenance of structure. Although many studies have investigated the evolution of protein function, a detailed understanding of the evolutionary constraints on protein folding is lacking.The amino acid sequence of a protein encodes its energy landscape, which determines its folding trajectory-its mechanism, rates, and energetics (1). Thus, the energy landscape defines a protein's ability to fold in a biologically reasonable time scale, avoid misfolding, and prevent frequent unfolding-all of which are likely important for the overall fitness of an organism. Indeed, numerous studies have proposed how protein folding might affect molecular evolution (2-8), and there is growing evidence that folding pathways and their associated kinetics and energetics are evolutionarily constrained. To date, however, there is little experimental evidence detailing how folding properties have changed throughout evolution. Are there trends in the folding mechanism or rates? Which aspects of the folding mechanism have been conserved, and how have they played a role in the evolution of modern-day homologs? We might naively expect folding to optimize, so that modern proteins generally fold faster than their ancestors. Or perhaps the energetics of the folding landscape evolved to avoid kinetic traps or misfolded states. If maintaining the folded state is important for fitness, then we might expect an improvement in kinetic stability over time, although a folded state that is too stable might be detrimental for conformational dynamics. These insights, and others, are critical for our und...

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“…This technique has recently been used by researchers to reconstruct ancient α,β-hydrolases that were found to be more promiscuous than present enzymes [30]. In another study, the reconstruction of extinct β-lactamases afforded highly thermostable scaffolds [32]. As we were previously unable to identify enzyme-assisted hydrogen bond acceptors in esterases belonging to the α,β-hydrolases [26], we reasoned that analyzing alternative and existing enzyme folds would be a relevant second strategy for the identification of promiscuous esterase scaffolds that could serve as a starting point for further engineering (represented by the forward gear, Figure 1, left).…”

Section: Introductionmentioning

confidence: 99%

Mechanism-Guided Discovery of an Esterase Scaffold with Promiscuous Amidase Activity

2016

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Abstract:The discovery and generation of biocatalysts with extended catalytic versatilities are of immense relevance in both chemistry and biotechnology. An enhanced atomistic understanding of enzyme promiscuity, a mechanism through which living systems acquire novel catalytic functions and specificities by evolution, would thus be of central interest. Using esterase-catalyzed amide bond hydrolysis as a model system, we pursued a simplistic in silico discovery program aiming for the identification of enzymes with an internal backbone hydrogen bond acceptor that could act as a reaction specificity shifter in hydrolytic enzymes. Focusing on stabilization of the rate limiting transition state of nitrogen inversion, our mechanism-guided approach predicted that the acyl hydrolase patatin of the α/β phospholipase fold would display reaction promiscuity. Experimental analysis confirmed previously unknown high amidase over esterase activity displayed by the first described esterase machinery with a protein backbone hydrogen bond acceptor to the reacting NH-group of amides. The present work highlights the importance of a fundamental understanding of enzymatic reactions and its potential for predicting enzyme scaffolds displaying alternative chemistries amenable to further evolution by enzyme engineering.

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Hyperstability and Substrate Promiscuity in Laboratory Resurrections of Precambrian β-Lactamases

Cited by 235 publications

References 32 publications

Cytosine deamination and the precipitous decline of spontaneous mutation during Earth's history

Cytosine deamination and the precipitous decline of spontaneous mutation during Earth's history

Evolutionary trend toward kinetic stability in the folding trajectory of RNases H

Mechanism-Guided Discovery of an Esterase Scaffold with Promiscuous Amidase Activity

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