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PubMed1997

Optimizing industrial enzymes by directed evolution.

Enzymes can be tailored for optimal performance in industrial applications by directing their evolution in vitro. This approach is particularly attractive for engineering industrial enzymes. We have created an efficient para-nitrobenzyl esterase over six generations of random point mutagenesis and recombination coupled with screening for improved variants. The best clones identified after four generations of sequential random mutagenesis and two generations of random recombination display more than 150 times the p-nitrobenzyl esterase activity of wild type towards loracarbef-p-nitrobenzyl ester in 15% dimethylformamide. Although the contributions of individual effective amino acid substitutions to enhanced activity are small (< 2-fold increases), the accumulation of multiple mutations by directed evolution allows significant improvement of the biocatalyst for reactions on substrates and under conditions not already optimized in nature. The positions of the effective amino acid substitutions have been identified in a pNB esterase structural model. None appear to interact directly with the antibiotic substrate, further underscoring the difficulty of predicting their effects in a 'rational' design effort.

Carboxylic Ester Hydrolases↗
PubMed2001

How enzymes adapt: lessons from directed evolution.

Enzymes that are adapted to widely different temperature niches are being used to investigate the molecular basis of protein stability and enzyme function. However, natural evolution is complex: random noise, historical accidents and ignorance of the selection pressures at work during adaptation all cloud comparative studies. Here, we review how adaptation in the laboratory by directed evolution can complement studies of natural enzymes in the effort to understand stability and function. Laboratory evolution experiments can attempt to mimic natural evolution and identify different adaptive mechanisms. However, laboratory evolution might make its biggest contribution in explorations of nonnatural functions, by allowing us to distinguish the properties nutured by evolution from those dictated by the laws of physical chemistry.

Algorithms↗
PubMed1984

The evolution of enzyme kinetic power.

Evolution of the kinetic potential of enzyme reactions is discussed. Quantitative assessment of the evolution of enzyme action has usually focused on optimization of the parametric ratio kcat./Km, which is the apparent second-order rate constant for the reaction of free substrate with free enzyme to give product. We propose that the general form kcat.[E]T/Km (where [E]T is total enzyme concentration), which is designated the 'kinetic power', is the real measure of kinetic/catalytic potential in situ. The standard paradigm of 'perfection' dictates the evolutionary maximum of 'kinetic power' to be k+s[E]T/2, where k+s is the diffusion-controlled rate constant for formation of the ES complex (and, hence, for the overall enzyme reaction). We discuss the role of protein conformational mobility in determining this state of 'perfection', via gating of substrate binding and determination of the catalytic configuration. Going beyond the level of the individual enzyme, we indicate the manner by which the organizational features of enzyme action in vivo may enhance the 'kinetic power'. Through evolutionary 'perfection' of the microenvironment, one finds that the 'kinetic power' of enzymes can be affected by alteration of [E]T as well as the unitary rate constants. At this level of complexity, we begin to realize that the 'kinetic' description of cell metabolism must be supplemented with thermodynamic concepts.

Biological Evolution↗
PubMed2001

Enzyme engineering: rational redesign versus directed evolution.

Enzyme engineering is undergoing the most profound and exciting transformation in its history (1). It promises unprecedented expansion in the scope and applications of modified or improved enzymes with desired physical and catalytic properties. Two complementary strategies are currently available: rational redesign (2-5) and directed evolution (6,7). Although both approaches have met with great success, each has limitations. In this article, the perspectives for these enzyme-engineering strategies are discussed briefly.

Enzymes↗
PubMed1991

Flavonoid evolution: an enzymic approach.

Flavonoid evolution in land plants is discussed from an enzymic point of view, based on the present day distribution of the major subgroups of flavonoids in bryophytes, lower and higher vascular plants. The importance of varied functions in the origin of pathways with a series of sequential steps leading to end-products is considered; it is argued that the initial function is that of an internal regulatory agent, rather than as a filter against ultraviolet irradiation. The basic synthases, hydroxylases, and reductases of flavonoid pathways are presumed to have evolved from enzymes of primary metabolism. A speculative scheme is presented of flavonoid evolution within a primitive group of algae derived from a Charophycean rather than a Chlorophycean line, as a land environment was invaded. Flavonoid evolution was preceded by that of the phenylpropanoid and malonyl-coenzyme A pathways, but evolved prior to the lignin pathway.

Journal Article↗
PubMed2006

Microsatellite length differences between humans and chimpanzees at autosomal Loci are not found at equivalent haploid Y chromosomal Loci.

When homologous microsatellites are compared between species, significant differences in mean length are often noted. A dominant cause of these length differences is ascertainment bias due to selection for maximum repeat number and repeat purity when the markers are being developed. However, even after ascertainment bias has been allowed for through reciprocal comparisons, significant length differences remain, suggesting that the average microsatellite mutation rate differs between species. Two classes of mechanism have been proposed: rapid evolution of enzymes involved in the generation and repair of slippage products (enzyme evolution model) and heterozygote instability, whereby interchromosomal events at heterozygous sites offer extra opportunities for mutations to occur (heterozygote instability model). To examine which of these hypotheses is most likely, we compared ascertainment bias and species length differences between humans and chimpanzees in autosomal and Y chromosomal microsatellites. We find that levels of ascertainment bias are indistinguishable, but that interspecies length differences are significantly greater for autosomal loci compared with haploid Y chromosomal loci. Such a pattern is consistent with predictions from the heterozygote instability model and is not expected under models of microsatellite evolution that do not include interchromosomal events such as the enzyme evolution model.

Animals↗
PubMed2004

Metabolic network analysis of the causes and evolution of enzyme dispensability in yeast.

Under laboratory conditions 80% of yeast genes seem not to be essential for viability. This raises the question of what the mechanistic basis for dispensability is, and whether it is the result of selection for buffering or an incidental side product. Here we analyse these issues using an in silico flux model of the yeast metabolic network. The model correctly predicts the knockout fitness effects in 88% of the genes studied and in vivo fluxes. Dispensable genes might be important, but under conditions not yet examined in the laboratory. Our model indicates that this is the dominant explanation for apparent dispensability, accounting for 37-68% of dispensable genes, whereas 15-28% of them are compensated by a duplicate, and only 4-17% are buffered by metabolic network flux reorganization. For over one-half of those not important under nutrient-rich conditions, we can predict conditions when they will be important. As expected, such condition-specific genes have a more restricted phylogenetic distribution. Gene duplicates catalysing the same reaction are not more common for indispensable reactions, suggesting that the reason for their retention is not to provide compensation. Instead their presence is better explained by selection for high enzymatic flux.

Biomass↗
PubMed2002

Evolution of enzymes in metabolism: a network perspective.

Several models have been proposed to explain the origin and evolution of enzymes in metabolic pathways. Initially, the retro-evolution model proposed that, as enzymes at the end of pathways depleted their substrates in the primordial soup, there was a pressure for earlier enzymes in pathways to be created, using the later ones as initial template, in order to replenish the pools of depleted metabolites. Later, the recruitment model proposed that initial templates from other pathways could be used as long as those enzymes were similar in chemistry or substrate specificity. These two models have dominated recent studies of enzyme evolution. These studies are constrained by either the small scale of the study or the artificial restrictions imposed by pathway definitions. Here, a network approach is used to study enzyme evolution in fully sequenced genomes, thus removing both constraints. We find that homologous pairs of enzymes are roughly twice as likely to have evolved from enzymes that are less than three steps away from each other in the reaction network than pairs of non-homologous enzymes. These results, together with the conservation of the type of chemical reaction catalyzed by evolutionarily related enzymes, suggest that functional blocks of similar chemistry have evolved within metabolic networks. One possible explanation for these observations is that this local evolution phenomenon is likely to cause less global physiological disruptions in metabolism than evolution of enzymes from other enzymes that are distant from them in the metabolic network.

Databases, Protein↗
PubMed2006

Directed evolution of enzymes and biosynthetic pathways.

Directed evolution is an important tool for overcoming the limitations of natural enzymes as biocatalysts. Recent advances have focused on applying directed evolution to a variety of enzymes, such as epoxide hydrolase, glyphosate N-acetyltransferase, xylanase and phosphotriesterase, in order to improve their activity, selectivity, stability and solubility. The focus has also shifted to manipulating biosynthetic pathways for the production of many naturally synthesized compounds, as well as the production of novel 'unnatural' compounds. A combined directed evolution and computational design approach is becoming increasingly important in exploring enzyme sequence-space and creating improved or novel enzymes. Fueled by recent breakthroughs in genomics and metagenomics, these developments should help expand the use of biocatalysts in industry.

Anti-Infective Agents↗
PubMed1981

Turnover of mouse intestinal brush border membrane proteins and enzymes in organ culture. A direct evaluation from studies on the evolution of enzyme activities during the culture.

The turnover of mouse intestinal brush border membrane enzymes has been studied by kinetic analysis of the evolution of enzyme activities during organ culture. By comparing the results obtained in these studies with the predictions from a mathematical model of enzyme synthesis and degradation in organ cultures, it has been possible to reach the following conclusions: (1) There is no degradation of brush border membrane enzymes during culture and the rate of synthesis of each enzyme is directly measurable from the kinetics of total enzyme accumulation (tissue + media). (2) Brush border membrane enzymes are released in culture media by two complementary processes. The first one involves a differential solubilization of enzymes but its exact nature cannot be exactly stated. The second one involves a microvesiculation of brush border membranes, the importance of which in vivo is seen in the possible conciliation between urinary membrane synthesis and heterogeneous turnover of membrane components.

Alkaline Phosphatase↗
PubMed2003

Directed evolution of enzymes for applied biocatalysis.

Directed evolution has rapidly emerged as a powerful new strategy for improving the characteristics of enzymes in a targeted manner. By coupling various protocols for generating large variant libraries of genes, together with high-throughput screens that select for specific properties of an enzyme, such as thermostability, catalytic activity and substrate specificity, it is now possible to optimize biocatalysts for specific applications. However, further work is required to broaden the range of screens that can be used, particularly in terms of reaction type, such as hydroxylation and carbon-carbon bond formation, and functional characteristics, such as enantioselectivity and regioselectivity, so that directed evolution can be used in a routine manner for biocatalyst development.

Biotechnology↗
PubMed2000

Cold adaptation of a mesophilic subtilisin-like protease by laboratory evolution.

Enzymes isolated from organisms native to cold environments generally exhibit higher catalytic efficiency at low temperatures and greater thermosensitivity than their mesophilic counterparts. In an effort to understand the evolutionary process and the molecular basis of cold adaptation, we have used directed evolution to convert a mesophilic subtilisin-like protease from Bacillus sphaericus, SSII, into its psychrophilic counterpart. A single round of random mutagenesis followed by recombination of improved variants yielded a mutant, P3C9, with a catalytic rate constant (k(cat)) at 10 degrees C 6.6 times and a catalytic efficiency (k(cat)/K(M)) 9.6 times that of wild type. Its half-life at 70 degrees C is 3.3 times less than wild type. Although there is a trend toward decreasing stability during the progression from mesophile to psychrophile, there is not a strict correlation between decreasing stability and increasing low temperature activity. A first generation mutant with a >2-fold increase in k(cat) is actually more stable than wild type. This suggests that the ultimate decrease in stability may be due to random drift rather than a physical incompatibility between low temperature activity and high temperature stability. SSII shares 77. 4% identity with the naturally psychrophilic protease subtilisin S41. Although SSII and S41 differ at 85 positions, four amino acid substitutions were sufficient to generate an SSII whose low temperature activity is greater than that of S41. That none of the four are found in S41 indicates that there are multiple routes to cold adaptation.

Adaptation, Physiological↗
PubMed1980

Experiments in microbial evolution: new enzymes, new metabolic activities.

Biological evolution has resulted in a richness and diversity of species. Among microorganisms this is most evident in the wealth and diversity of biochemical transformations. Evidence for evolutionary relationships may be obtained from comparative studies, but with microorganisms it is also possible to follow evolution in action. Microbial populations adapt rapidly to changes in the environment and the evolution of new metabolic activities can be observed in laboratory experiments. The enzymes of many catabolic pathways are synthesized in response to the presence of inducing substrates. New catabolic activities may be acquired by mutations in regulatory genes resulting in alterations in the specificity of induction, or in enzyme synthesis in the absence of inducer. Mutations in structural genes may given rise to enzymes with altered substrate specificities. In bacteria, catabolic genes may be carried on plasmids and the exchange of plasmids among bacterial populations increases the evolutionary potential. Experiments in microbial evolution have produced strains with novel catabolic activities involving regulatory or structural gene mutations, gene duplications and plasmid exchange. Enzymes studied in this way include amidase, ribitol dehydrogenase, evolved beta-galactosidase, and enzymes of the catabolic pathways for pentoses and pentitols and haloaromatic compounds.

Amidohydrolases↗
PubMed2003

Spatial dynamics and the evolution of enzyme production.

We re-examine the problem of the evolution of protein synthesis or enzyme production using a stochastic cellular automaton model, where the replicators are fixed in the sites of a two-dimensional square lattice. In contrast with the classical chemical kinetics or mean-field predictions, we show that a small colony of mutant, protein-mediated (enzymatic) replicators has an appreciable probability to take over a resident population of simpler, direct-template replicators. In addition, we argue that the threshold phenomenon corresponding to the onset of invasion can be described quantitatively within the physics framework of nonequilibrium phase transitions. We study also the invasion of a resident population of enzymatic replicators by more efficient replicators of the same kind, and show that although slightly more efficient mutants cannot invade, invasion is a likely event if the productivity advantage of the mutants is large. In this sense, the establishment of a population of enzymatic replicators is not a 'once-forever' evolutionary decision.

Animals↗
PubMed2002

Directed evolution of enzymes and pathways for industrial biocatalysis.

Directed evolution has become a powerful tool for developing enzyme and whole cell based biocatalysts. Significant recent advances include the creation of novel enzyme functions and the development of several new efficient directed evolution methods. The combination of directed evolution and rational design promises to accelerate the development of biocatalysts for applications in the pharmaceutical, chemical and food industries.

Catalysis↗
PubMed2002

Evolution of enzyme cascades from embryonic development to blood coagulation.

Recent delineation of the serine protease cascade controlling dorsal-ventral patterning during Drosophila embryogenesis allows this cascade to be compared with those controlling clotting and complement in vertebrates and invertebrates. The identification of discrete markers of serine protease evolution has made it possible to reconstruct the probable chronology of enzyme evolution and to gain new insights into functional linkages among the cascades. Here, it is proposed that a single ancestral developmental/immunity cascade gave rise to the protostome and deuterostome developmental, clotting and complement cascades. Extensive similarities suggest that these cascades were built by adding enzymes from the bottom of the cascade up and from similar macromolecular building blocks.

Animals↗
PubMed1999

Multifunctional enzymes and evolution of biosynthetic pathways: retro-evolution by jumps.

A likely scenario of evolution of biosynthetic pathways is believed to have occurred by retro-evolution through recruitment of existing enzymes rather than generation of de novo classes. It had been proposed that such retro-evolution occurred in steps as a response to depletion of an essential metabolite and availability of another related substance in the environment. In this article, I argue that because of instability of many such extant intermediates, it is unlikely that retro-evolution had occurred in steps. I further propose that such evolution in many cases has taken place by jumps, i.e., by recruitment of a multifunctional enzyme capable of catalyzing several steps at a time, albeit inefficiently. I further speculate that in some cases one primordial multienzyme may have catalyzed the whole sequence of reaction of a biosynthetic pathway, i.e., the pathway may have evolved by a single leap. Gene duplications and further evolution to more efficient enzymes led to extant pathways. Such a mechanism predicts that some or all enzymes of a pathway must have descended from a common ancestor. Sequence and structural homologies among extant enzymes of a biosynthetic pathway have been examined.

Amino Acids↗