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1.
Gloria Yang Charlotte M. Miton Nobuhiko Tokuriki 《Protein science : a publication of the Protein Society》2020,29(8):1724-1747
New enzyme functions often evolve through the recruitment and optimization of latent promiscuous activities. How do mutations alter the molecular architecture of enzymes to enhance their activities? Can we infer general mechanisms that are common to most enzymes, or does each enzyme require a unique optimization process? The ability to predict the location and type of mutations necessary to enhance an enzyme's activity is critical to protein engineering and rational design. In this review, via the detailed examination of recent studies that have shed new light on the molecular changes underlying the optimization of enzyme function, we provide a mechanistic perspective of enzyme evolution. We first present a global survey of the prevalence of activity‐enhancing mutations and their distribution within protein structures. We then delve into the molecular solutions that mediate functional optimization, specifically highlighting several common mechanisms that have been observed across multiple examples. As distinct protein sequences encounter different evolutionary bottlenecks, different mechanisms are likely to emerge along evolutionary trajectories toward improved function. Identifying the specific mechanism(s) that need to be improved upon, and tailoring our engineering efforts to each sequence, may considerably improve our chances to succeed in generating highly efficient catalysts in the future. 相似文献
2.
《Bioscience, biotechnology, and biochemistry》2013,77(9):1922-1927
Actinomycete cytochrome P450 from Nonomuraea recticatena NBRC 14525 (P450moxA) catalyzes the hydroxylation of a broad range of substrates, including fatty acids, steroids, and various aromatic compounds. Hence, the enzyme is potentially useful in medicinal applications, but the activity is insufficient for practical use. Here we applied directed evolution to enhance the activity. A random mutagenesis library was screened using 7-ethoxycoumarin as a substrate to retrieve 17 variants showing >2-fold activities. Twenty-five amino acid substitutions were found in the variants, of which five mutations were identified to have the largest effects (Q87W, T115A, H132L, R191W, and G294D). These mutations additively increased the activity; the quintet mutant had 20-times the activity of the wildtype. These five single mutations also increased in activity toward structurally distinct substrates (diclofenac and naringenin). Based on the three-dimensional structure of the enzyme, we discerned that mutations in the substrate recognition site improved the activity, which was substrate dependent; mutations apart from the active site improved the activity as well as the substrates did. 相似文献
3.
The aldehyde dehydrogenase from Thermoplasma acidophilum, which was previously implemented as a key enzyme in a synthetic cell-free reaction cascade for the production of alcohols, was optimized by directed evolution. Improvements have been made to enhance reaction velocity and solubility. Using a random approach followed by site-directed and saturation mutagenesis, three beneficial amino acid mutations were found after screening of ca. 20,000 variants. Mutation Y399C enhanced the protein solubility after recombinant expression in Escherichia coli 6-fold. Two further mutations, F34M and S405N, enhanced enzyme activity with the cofactor NAD+ by a factor of eight. Impacts on enzyme stability and substrate specificity were negligible. 相似文献
4.
The utility of engineering enzyme activity is expanding with the development of biotechnology. Conventional methods have limited applicability as they require high-throughput screening or three-dimensional structures to direct target residues of activity control. An alternative method uses sequence evolution of natural selection. A repertoire of mutations was selected for fine-tuning enzyme activities to adapt to varying environments during the evolution. Here, we devised a strategy called sequence co-evolutionary analysis to control the efficiency of enzyme reactions (SCANEER), which scans the evolution of protein sequences and direct mutation strategy to improve enzyme activity. We hypothesized that amino acid pairs for various enzyme activity were encoded in the evolutionary history of protein sequences, whereas loss-of-function mutations were avoided since those are depleted during the evolution. SCANEER successfully predicted the enzyme activities of beta-lactamase and aminoglycoside 3′-phosphotransferase. SCANEER was further experimentally validated to control the activities of three different enzymes of great interest in chemical production: cis-aconitate decarboxylase, α-ketoglutaric semialdehyde dehydrogenase, and inositol oxygenase. Activity-enhancing mutations that improve substrate-binding affinity or turnover rate were found at sites distal from known active sites or ligand-binding pockets. We provide SCANEER to control desired enzyme activity through a user-friendly webserver. 相似文献
5.
Evolutionary developmental biology and the problem of variation 总被引:11,自引:0,他引:11
Stern DL 《Evolution; international journal of organic evolution》2000,54(4):1079-1091
Abstract. One of the oldest problems in evolutionary biology remains largely unsolved. Which mutations generate evolutionarily relevant phenotypic variation? What kinds of molecular changes do they entail? What are the phenotypic magnitudes, frequencies of origin, and pleiotropic effects of such mutations? How is the genome constructed to allow the observed abundance of phenotypic diversity? Historically, the neo‐Darwinian synthesizers stressed the predominance of micromutations in evolution, whereas others noted the similarities between some dramatic mutations and evolutionary transitions to argue for macromutationism. Arguments on both sides have been biased by misconceptions of the developmental effects of mutations. For example, the traditional view that mutations of important developmental genes always have large pleiotropic effects can now be seen to be a conclusion drawn from observations of a small class of mutations with dramatic effects. It is possible that some mutations, for example, those in cis‐regulatory DNA, have few or no pleiotropic effects and may be the predominant source of morphological evolution. In contrast, mutations causing dramatic phenotypic effects, although superficially similar to hypothesized evolutionary transitions, are unlikely to fairly represent the true path of evolution. Recent developmental studies of gene function provide a new way of conceptualizing and studying variation that contrasts with the traditional genetic view that was incorporated into neo‐Darwinian theory and population genetics. This new approach in developmental biology is as important for micro‐evolutionary studies as the actual results from recent evolutionary developmental studies. In particular, this approach will assist in the task of identifying the specific mutations generating phenotypic variation and elucidating how they alter gene function. These data will provide the current missing link between molecular and phenotypic variation in natural populations. 相似文献
6.
Hamamatsu N Suzumura A Nomiya Y Sato M Aita T Nakajima M Husimi Y Shibanaka Y 《Applied microbiology and biotechnology》2006,73(3):607-617
A biased mutation-assembling method—that is, a directed evolution strategy to facilitate an optimal accumulation of multiple mutations on the basis of additivity principles, was applied to the directed evolution of water-soluble PQQ glucose dehydrogenase (PQQGDH-B) to reduce its maltose oxidation activity, which can lead to errors in blood glucose determination. Mutations appropriate for the reduction without fatal deterioration of its glucose oxidation activity were developed by an error-prone PCR method coupled with a saturation mutagenesis method. Moreover, two types of incorporation frequency based on their contribution were assigned to the mutations: high (80%) and evens (50%), in constructing a multiple mutant library. The best mutant created showed a marked reduction in maltose oxidation activity, corresponding to 4% of that of the wild-type enzyme, with 35% retention of glucose oxidation activity. In addition, this mutant showed a reduction in galactose oxidation activity corresponding to 5% of that of the wild-type enzyme. In conclusion, we succeeded in developing the PQQGDH-B mutants with improved substrate specificity and validated our method coupled with optimized mutations and their contribution-based incorporation frequencies by applying it to the development.Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users. 相似文献
7.
Chaperones are proteins that help other proteins fold. They also affect the adaptive evolution of their client proteins by buffering the effect of deleterious mutations and increasing the genetic diversity of evolving proteins. We study how the bacterial chaperone GroE (GroEL+GroES) affects the evolution of green fluorescent protein (GFP). To this end, we subjected GFP to multiple rounds of mutation and selection for its color phenotype in four replicate Escherichia coli populations, and studied its evolutionary dynamics through high-throughput sequencing and mutant engineering. We evolved GFP both under stabilizing selection for its ancestral (green) phenotype, and to directional selection for a new (cyan) phenotype. We did so both under low and high expression of the chaperone GroE. In contrast to previous work, we observe that GroE does not just buffer but also helps purge deleterious (fluorescence reducing) mutations from evolving populations. In doing so, GroE helps reduce the genetic diversity of evolving populations. In addition, it causes phenotypic heterogeneity in mutants with the same genotype, helping to enhance their fluorescence in some cells, and reducing it in others. Our observations show that chaperones can affect adaptive evolution in more than one way. 相似文献
8.
A promiscuous activity of an existing enzyme can confer an evolutionary advantage by providing an immediate response to a new selection pressure and a starting point for the divergence of a new enzyme. This work seeks to examine how this process might take place. Human carbonic anhydrase II (hCAII) is an enzyme that evolved to catalyze the reversible hydration of CO(2) and performs this task at a remarkable rate (k(cat) approximately 10(6) s(-)(1)). hCAII also exhibits promiscuous activity toward highly activated esters such as 4-nitrophenyl acetate. We describe a much weaker esterase activity of hCAII toward the bulkier and much less activated ester substrate 2-naphthyl acetate (2NA). Directed evolution of hCAII produced a variant with 40-fold higher rates toward 2NA, owing to two mutations: one within the active site (Ala65Val) and one at its mouth (Thr200Ala). Structure-activity studies suggest that these mutations led to adaptation of the active site for bulkier substrates and for the catalysis of nonactivated esters. The mutations did not, however, significantly alter the native activity of hCAII. Our results support the notion that the evolution of a new function can be driven by mutations that increase a promiscuous function (which serves as the starting point for the evolutionary process) but do not harm the native function. 相似文献
9.
Xinyu Ge Lang Peng Alfried P. Vogler John C. Morse Lianfang Yang Changhai Sun Beixin Wang 《Systematic Entomology》2023,48(2):278-295
Mitochondrial genomes have been widely used for phylogenetic reconstruction and evolutionary analysis in various groups of Insecta. Gene rearrangements in the mitogenome can be informative characters for phylogenetic reconstruction and adaptive evolution. Trichoptera is one of the most important groups of aquatic insects. Prior to this study, complete mitogenomes from Trichoptera were restricted to eight families, resulting in a biased view of their mitogenome structure and evolution. Here, we assemble new mitogenomes for 66 species by high-throughput sequencing. The mitogenomes of 19 families and 47 genera are documented for the first time. Combined with 16 previously published mitogenomes of Trichoptera, we find 14 kinds of gene rearrangement patterns novel for Trichoptera, including rearrangement of protein-coding genes, tRNAs and control regions. Simultaneously, we provide evidence for the occurrence of tandem duplication and non-random loss events in the mitogenomes of three families. Phylogenetic analyses show that Hydroptilidae was recovered as a sister group to Annulipalpia. The increased nucleotide substitution rate and adaptive evolution may have affected the mitochondrial gene rearrangements in Trichoptera. Our study offers new insights into the mechanisms and patterns of mitogenome rearrangements in Insecta at large and into the usefulness of mitogenomic gene order as a phylogenetic marker within Trichoptera. 相似文献
10.
Protein evolution has occurred by successive fixation of individual mutations. The probability of fixation depends on the fitness of the mutation, and the arising variant can be deleterious, neutral, or beneficial. Despite its relevance, only few studies have estimated the distribution of fitness effects caused by random single mutations on protein function. The human immunodeficiency virus type 1 (HIV-1) protease was chosen as a model protein to quantify protein's tolerability to random single mutations. After determining the enzymatic activity of 107 single random mutants, we found that 86% of single mutations were deleterious for the enzyme catalytic efficiency and 54% lethal. Only 2% of the mutations significantly increased the catalytic efficiency of the enzyme. These data demonstrate the vulnerability of HIV-1 protease to single random mutations. When a second random mutagenesis library was constructed from an HIV-1 protease carrying a highly deleterious single mutation (D30N), a higher proportion of mutations with neutral or beneficial effect were found, 26% and 9%, respectively. Importantly, antagonist epistasis was observed between deleterious mutations. In particular, the mutation N88D, lethal for the wild-type protease, restored the wild-type catalytic efficiency when combined with the highly deleterious mutation D30N. The low tolerability to single random substitutions shown here for the wild-type HIV-1 protease contrasts with its in vivo ability to generate an adaptive variation. Thus, the antagonist epistasis between deleterious or lethal mutations may be responsible for increasing the protein mutational robustness and evolvability. 相似文献
11.
Palackal N Brennan Y Callen WN Dupree P Frey G Goubet F Hazlewood GP Healey S Kang YE Kretz KA Lee E Tan X Tomlinson GL Verruto J Wong VW Mathur EJ Short JM Robertson DE Steer BA 《Protein science : a publication of the Protein Society》2004,13(2):494-503
Directed evolution technologies were used to selectively improve the stability of an enzyme without compromising its catalytic activity. In particular, this article describes the tandem use of two evolution strategies to evolve a xylanase, rendering it tolerant to temperatures in excess of 90 degrees C. A library of all possible 19 amino acid substitutions at each residue position was generated and screened for activity after a temperature challenge. Nine single amino acid residue changes were identified that enhanced thermostability. All 512 possible combinatorial variants of the nine mutations were then generated and screened for improved thermal tolerance under stringent conditions. The screen yielded eleven variants with substantially improved thermal tolerance. Denaturation temperature transition midpoints were increased from 61 degrees C to as high as 96 degrees C. The use of two evolution strategies in combination enabled the rapid discovery of the enzyme variant with the highest degree of fitness (greater thermal tolerance and activity relative to the wild-type parent). 相似文献
12.
Improving enzyme properties: when are closer mutations better? 总被引:7,自引:0,他引:7
Study of mutations that improve enzyme properties reveals that in many, but not all, cases closer mutations are more effective than distant ones. For enantioselectivity, substrate selectivity and new catalytic activity (catalytic promiscuity) closer mutations improved enzymes more effectively than distant ones. However, both close and distant mutations can improve activity, thermal stability and also probably stability toward organic solvents. Typical random mutagenesis methods, such as error-prone PCR, create greater numbers of distant mutations than close mutations because enzymes contain more amino acids distant from the active site than close to the active site. This suggests that instead of mutating the entire enzyme, focusing mutations near the substrate-binding site might dramatically increase the success rate in many directed evolution experiments. 相似文献
13.
The reporter enzyme beta-glucuronidase was mutagenized and evolved for thermostability. After four cycles of screening the best variant was more active than the wild-type enzyme, and retained function at 70 degrees C, whereas the wild-type enzyme lost function at 65 degrees C. Variants derived from sequential mutagenesis were shuffled together, and re-screened for thermostability. The best variants retained activities at even higher temperatures (80 degrees C), but had specific activities that were now less than that of the wild-type enzyme. The mutations clustered near the tetramer interface of the enzyme, and many of the evolved variants showed much greater resistance to quaternary structure disruption at high temperatures, which is also a characteristic of naturally thermostable enzymes. Together, these results suggest a pathway for the evolution of thermostability in which enzymes initially become stable at high temperatures without loss of activity at low temperatures, while further evolution leads to enzymes that have kinetic parameters that are optimized for high temperatures. 相似文献
14.
Differences in the chitinolytic activity of mammalian chitinases on soluble and insoluble substrates
Benjamin A. Barad Lin Liu Roberto E. Diaz Ralp Basilio Steven J. Van Dyken Richard M. Locksley James S. Fraser 《Protein science : a publication of the Protein Society》2020,29(4):952-963
Chitin is an abundant polysaccharide used by many organisms for structural rigidity and water repulsion. As such, the insoluble crystalline structure of chitin poses significant challenges for enzymatic degradation. Acidic mammalian chitinase, a processive glycosyl hydrolase, is the primary enzyme involved in the degradation of environmental chitin in mammalian lungs. Mutations to acidic mammalian chitinase have been associated with asthma, and genetic deletion in mice increases morbidity and mortality with age. We initially set out to reverse this phenotype by engineering hyperactive acidic mammalian chitinase variants. Using a screening approach with commercial fluorogenic substrates, we identified mutations with consistent increases in activity. To determine whether the activity increases observed were consistent with more biologically relevant chitin substrates, we developed new assays to quantify chitinase activity with insoluble chitin, and identified a one‐pot fluorogenic assay that is sufficiently sensitive to quantify changes to activity due to the addition or removal of a carbohydrate‐binding domain. We show that the activity increases from our directed evolution screen were lost when insoluble substrates were used. In contrast, naturally occurring gain‐of‐function mutations gave similar results with oligomeric and insoluble substrates. We also show that activity differences between acidic mammalian chitinase and chitotriosidase are reduced with insoluble substrate, suggesting that previously reported activity differences with oligomeric substrates may have been driven by differential substrate specificity. These results highlight the need for assays against physiological substrates when engineering metabolic enzymes, and provide a new one‐pot assay that may prove to be broadly applicable to engineering glycosyl hydrolases. 相似文献
15.
16.
Defined order of evolutionary adaptations: experimental evidence 总被引:1,自引:0,他引:1
Organisms often adapt to new conditions by means of beneficial mutations that become fixed in the population. Often, full adaptation requires several different mutations in the same cell, each of which may affect a different aspect of the behavior. Can one predict order in which these mutations become fixed? To address this, we experimentally studied evolution of Escherichia coli in a growth medium in which the effects of different adaptations can be easily classified as affecting growth rate or the lag‐phase duration. We find that adaptations are fixed in a defined and reproducible order: first reduction of lag phase, and then an increase of the exponential growth rate. A population genetics theory explains this order, and suggests growth conditions in which the order of adaptations is reversed. We experimentally find this order reversal under the predicted conditions. This study supports a view in which the evolutionary path to adaptation in a new environment can be captured by theory and experiment. 相似文献
17.
Augustyniak W Brzezinska AA Pijning T Wienk H Boelens R Dijkstra BW Reetz MT 《Protein science : a publication of the Protein Society》2012,21(4):487-497
Previously, Lipase A from Bacillus subtilis was subjected to in vitro directed evolution using iterative saturation mutagenesis, with randomization sites chosen on the basis of the highest B-factors available from the crystal structure of the wild-type (WT) enzyme. This provided mutants that, unlike WT enzyme, retained a large part of their activity after heating above 65 °C and cooling down. Here, we subjected the three best mutants along with the WT enzyme to biophysical and biochemical characterization. Combining thermal inactivation profiles, circular dichroism, X-ray structure analyses and NMR experiments revealed that mutations of surface amino acid residues counteract the tendency of Lipase A to undergo precipitation under thermal stress. Reduced precipitation of the unfolding intermediates rather than increased conformational stability of the evolved mutants seems to be responsible for the activity retention. 相似文献
18.
A. Labas E. Szabo L. Mones M. Fuxreiter 《Biochimica et Biophysica Acta - Proteins and Proteomics》2013,1834(5):908-917
Understanding enzymatic evolution is essential to engineer enzymes with improved activities or to generate enzymes with tailor-made activities. The computationally designed Kemp eliminase KE07 carries out an unnatural reaction by converting of 5-nitrobenzisoxazole to cyanophenol, but its catalytic efficiency is significantly lower than those of natural enzymes. Three series of designed Kemp eliminases (KE07, KE70, KE59) were shown to be evolvable with considerable improvement in catalytic efficiency. Here we use the KE07 enzyme as a model system to reveal those forces, which govern enzymatic evolution and elucidate the key factors for improving activity. We applied the Empirical Valence Bond (EVB) method to construct the free energy pathway of the reaction in the original KE07 design and the evolved R7 1/3H variant. We analyzed catalytic effect of residues and demonstrated that not all mutations in evolution are favorable for activity. In contrast to the small decrease in the activation barrier, in vitro evolution significantly reduced the reorganization energy. We developed an algorithm to evaluate group contributions to the reorganization energy and used this approach to screen for KE07 variants with potential for improvement. We aimed to identify those mutations that facilitate enzymatic evolution, but might not directly increase catalytic efficiency. Computational results in accord with experimental data show that all mutations, which appear during in vitro evolution were either neutral or favorable for the reorganization energy. These results underscore that distant mutations can also play role in optimizing efficiency via their contribution to the reorganization energy. Exploiting this principle could be a promising strategy for computer-aided enzyme design. This article is part of a Special Issue entitled: The emerging dynamic view of proteins: Protein plasticity in allostery, evolution and self-assembly. 相似文献
19.
Molly K. Burke 《Proceedings. Biological sciences / The Royal Society》2012,279(1749):5029-5038
A major goal in evolutionary biology is to understand the origins and fates of adaptive mutations. Natural selection may act to increase the frequency of de novo beneficial mutations, or those already present in the population as standing genetic variation. These beneficial mutations may ultimately reach fixation in a population, or they may stop increasing in frequency once a particular phenotypic state has been achieved. It is not yet well understood how different features of population biology, and/or different environmental circumstances affect these adaptive processes. Experimental evolution is a promising technique for studying the dynamics of beneficial alleles, as populations evolving in the laboratory experience natural selection in a replicated, controlled manner. Whole-genome sequencing, regularly obtained over the course of sustained laboratory selection, could potentially reveal insights into the mutational dynamics that most likely occur in natural populations under similar circumstances. To date, only a few evolution experiments for which whole-genome data are available exist. This review describes results from these resequenced laboratory-selected populations, in systems with and without sexual recombination. In asexual systems, adaptation from new mutations can be studied, and results to date suggest that the complete, unimpeded fixation of these mutations is not always observed. In sexual systems, adaptation from standing genetic variation can be studied, and in the admittedly few examples we have, the complete fixation of standing variants is not always observed. To date, the relative frequency of adaptation from new mutations versus standing variation has not been tested using a single experimental system, but recent studies using Caenorhabditis elegans and Saccharomyces cerevisiae suggest that this a realistic future goal. 相似文献
20.
Brown NG Pennington JM Huang W Ayvaz T Palzkill T 《Journal of molecular biology》2010,404(5):832-32735
The introduction of extended-spectrum cephalosporins and β-lactamase inhibitors has driven the evolution of extended-spectrum β-lactamases (ESBLs) that possess the ability to hydrolyze these drugs. The evolved TEM ESBLs from clinical isolates of bacteria often contain substitutions that occur in the active site and alter the catalytic properties of the enzyme to provide an increased hydrolysis of extended-spectrum cephalosporins or an increased resistance to inhibitors. These active-site substitutions often result in a cost in the form of reduced enzyme stability. The evolution of TEM ESBLs is facilitated by mutations that act as global suppressors of protein stability defects in that they allow the enzyme to absorb multiple amino acid changes despite incremental losses in stability associated with the substitutions. The best-studied example is the M182T substitution, which corrects protein stability defects and is commonly found in TEM ESBLs or inhibitor-resistant variants from clinical isolates. In this study, a genetic selection for second-site mutations that could partially restore function to a severely destabilized primary mutant enabled the identification of A184V, T265M, R275Q, and N276D, which are known to occur in TEM ESBLs from clinical isolates, as suppressors of TEM-1 protein stability defects. Further characterization demonstrated that these substitutions increased the thermal stability of TEM-1 and were able to correct the stability defects of two different sets of destabilizing mutations. The acquisition of compensatory global suppressors of stability costs associated with active-site mutations may be a common mechanism for the evolution of novel protein function. 相似文献