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PubMed2004

Controlling the enantioselectivity of enzymes by directed evolution: practical and theoretical ramifications.

A fundamentally new approach to asymmetric catalysis in organic chemistry is described based on the in vitro evolution of enantioselective enzymes. It comprises the appropriate combination of gene mutagenesis and expression coupled with an efficient high-throughput screening system for evaluating enantioselectivity (enantiomeric excess assay). Several such cycles lead to a "Darwinistic" process, which is independent of any knowledge concerning the structure or the mechanism of the enzyme being evolved. The challenge is to choose the optimal mutagenesis methods to navigate efficiently in protein sequence space. As a first example, the combination of error-prone mutagenesis, saturation mutagenesis, and DNA-shuffling led to a dramatic enhancement of enantioselectivity of a lipase acting as a catalyst in the kinetic resolution of a chiral ester. Mutations at positions remote from the catalytically active center were identified, a surprising finding, which was explained on the basis of a novel relay mechanism. The scope and limitations of the method are discussed, including the prospect of directed evolution of stereoselective hybrid catalysts composed of robust protein hosts in which transition metal centers have been implanted.

Directed Molecular Evolution↗
PubMed1995

Alcohol dehydrogenase of class I: kiwi liver enzyme, parallel evolution in separate vertebrate lines, and correlation with 12S rRNA patterns.

Alcohol dehydrogenase class I from kiwi liver has been purified, analyzed, and compared with that of other alcohol dehydrogenases. The results show that several avian and mammalian forms of the enzyme exhibit parallel evolutionary patterns in two independent lineages of a single protein, establishing a pattern in common. Furthermore, the data correlate the enzyme evolutionary pattern with that of 12S rRNA. Biologically, the patterns complement those on ratite and other avian relationships. Functionally, the enzyme has a low Km with ethanol and a branched-chain residue at position 141, like the mammalian enzymes but in contrast to the other characterized ratite enzyme (with Ala-141 and a higher Km). This pattern of natural variability suggests a frequent but not fully complete correlation between a large residue size at position 141 and tight ethanol binding.

Alcohol Dehydrogenase↗
PubMed2006

Three rounds (1R/2R/3R) of genome duplications and the evolution of the glycolytic pathway in vertebrates.

BACKGROUND: Evolution of the deuterostome lineage was accompanied by an increase in systematic complexity especially with regard to highly specialized tissues and organs. Based on the observation of an increased number of paralogous genes in vertebrates compared with invertebrates, two entire genome duplications (2R) were proposed during the early evolution of vertebrates. Most glycolytic enzymes occur as several copies in vertebrate genomes, which are specifically expressed in certain tissues. Therefore, the glycolytic pathway is particularly suitable for testing theories of the involvement of gene/genome duplications in enzyme evolution. RESULTS: We assembled datasets from genomic databases of at least nine vertebrate species and at least three outgroups (one deuterostome and two protostomes), and used maximum likelihood and Bayesian methods to construct phylogenies of the 10 enzymes of the glycolytic pathway. Through this approach, we intended to gain insights into the vertebrate specific evolution of enzymes of the glycolytic pathway. Many of the obtained gene trees generally reflect the history of two rounds of duplication during vertebrate evolution, and were in agreement with the hypothesis of an additional duplication event within the lineage of teleost fish. The retention of paralogs differed greatly between genes, and no direct link to the multimeric structure of the active enzyme was found. CONCLUSION: The glycolytic pathway has subsequently evolved by gene duplication and divergence of each constituent enzyme with taxon-specific individual gene losses or lineage-specific duplications. The tissue-specific expression might have led to an increased retention for some genes since paralogs can subdivide the ancestral expression domain or find new functions, which are not necessarily related to the original function.

Animals↗
PubMed1977

Regulation of newly evolved enzymes. III Evolution of the ebg repressor during selection for enhanced lactase activity.

The evolution of lactose utilization by lacZ deletion strains of E. coli occurs via mutations in the ebg genes. We show that one kind of mutation in the regulatory gene ebgR results in a repressor which retains the ability to repress synthesis of ebg enzymes, but which permits 4.5-fold more ebg enzyme synthesis during lactose induction than does the wild-type repressor. A comparison between the growth rate of various ebg+ strains on lactose and the amount of ebg enzyme synthesized by these strains shows that the rate of enzyme synthesis permitted by the wild-type repressor is insufficient for growth on lactose as a sole carbon source by a cell with the most active ebg lactase yet isolated. We conclude, therefore, that the evolution of lactose utilization requires both a structural and a regulatory mutation.

Biological Evolution↗
PubMed1992

Eggs, enzymes, and evolution: natural genetic variants change insect fecundity.

Phosphoglucose isomerase genotypes in the butterfly Colias differ dramatically in biochemical properties. These differences were evaluated earlier, using metabolic network theory, to predict, successfully, their effects on glycolytic metabolism and hence on Colias flight capacity and several consequent fitness components in the wild. Female egg-laying, not previously studied, also depends on flight, so female fecundity is now predicted to differ among these genotypes. An experimental design incorporating the thermal ecology of Colias confirms these predictions in a cool habitat. Thus female fecundity differences among animal enzyme polymorphs have now been found. Quantitative reconstruction of the selection regime for phosphoglucose isomerase genotypes in Colias can now begin. The most heat-stable genotypes are the least fecund, suggesting that global warming, if it occurs, may have severe impacts, through population genetics, on demography of thermally sensitive creatures.

Analysis of Variance↗
PubMed1989

Molecular evolution of enzyme structure: construction of a hybrid hamster/Escherichia coli aspartate transcarbamoylase.

Aspartate transcarbamoylase (ATCase, EC 2.1.3.2) is the first unique enzyme common to de novo pyrimidine biosynthesis and is involved in a variety of structural patterns in different organisms. In Escherichia coli, ATCase is a functionally independent, oligomeric enzyme; in hamster, it is part of a trifunctional protein complex, designated CAD, that includes the preceding and subsequent enzymes of the biosynthetic pathway (carbamoyl phosphate synthetase and dihydroorotase). The complete complementary DNA (cDNA) nucleotide sequence of the ATCase-encoding portion of the hamster CAD gene is reported here. A comparison of the deduced amino acid sequences of the hamster and E. coli catalytic peptides revealed an overall 44% amino acid similarity, substantial conservation of predicted secondary structure, and complete conservation of all the amino acids implicated in the active site of the E. coli enzyme. These observations led to the construction of a functional hybrid ATCase formed by intragenic fusion based on the known tertiary structure of the bacterial enzyme. In this fusion, the amino terminal half (the "polar domain") of the fusion protein was provided by a hamster ATCase cDNA subclone, and the carboxyl terminal portion (the "equatorial domain") was derived from a cloned pyrBI operon of E. coli K-12. The recombinant plasmid bearing the hybrid ATCase was shown to satisfy growth requirements of transformed E. coli pyrB- cells. The functionality of this E. coli-hamster hybrid enzyme confirms conservation of essential structure-function relationships between evolutionarily distant and structurally divergent ATCases.

Amino Acid Sequence↗
PubMed2006

Evolution of enzymes in a series is driven by dissimilar functional demands.

That distinct enzyme activities in an unbranched metabolic pathway are evolutionarily tuned to a single functional requirement is a pervasive assumption. Here we test this assumption by examining the activities of two consecutively acting enzymes in human erythrocytes with an approach to quantitative evolutionary design that avoids the above-mentioned assumption. We previously found that avoidance of NADPH depletion during the pulses of oxidative load to which erythrocytes are normally exposed is the main functional requirement mediating selection for high glucose-6-phosphate dehydrogenase activity. In the present study, we find that, in contrast, the maintenance of oxidized glutathione at low concentrations is the main functional requirement mediating selection for high glutathione reductase activity. The results in this case show that, contrary to the assumption of a single functional requirement, natural selection for the normal activities of the distinct enzymes in the pathway is mediated by different requirements. On the other hand, the results agree with the more general principles that underlie our approach. Namely, that (i) the values of biochemical parameters evolve so as to fulfill the various performance requirements that are relevant to achieve high fitness, and (ii) these performance requirements can be inferred from quantitative systems theory considerations, informed by knowledge of specific aspects of the biochemistry, physiology, genetics, and ecology of the organism.

Erythrocytes↗
PubMed2003

Dispelling the myths--biocatalysis in industrial synthesis.

Biocatalysis has emerged as an important tool in the industrial synthesis of bulk chemicals, pharmaceutical and agrochemical intermediates, active pharmaceuticals, and food ingredients. However, the number and diversity of the applications are modest, perhaps in part because of perceived or real limitations of biocatalysts, such as limited enzyme availability, substrate scope, and operational stability. Recent scientific breakthroughs in genomics, directed enzyme evolution, and the exploitation of biodiversity should help to overcome these limitations. As a result, we expect many new industrial applications of biocatalysis to be realized, from single-step enzymatic conversions to customized multistep microbial synthesis by means of metabolic pathway engineering.

Biotechnology↗
PubMed2005

Laboratory evolution of catabolic enzymes and pathways.

The laboratory evolution of environmentally relevant enzymes and proteins has resulted in the generation of optimized and stabilized enzymes, as well as enzymes with activity against new substrates. Numerous methods, including random mutagenesis, site-directed mutagenesis and DNA shuffling, have been widely used to generate variants of existing enzymes. These evolved catabolic enzymes have application for improving biodegradation pathways, generating engineered pathways for the degradation of particularly recalcitrant compounds, and for the development of biocatalytic processes to produce useful compounds. Regulatory proteins associated with catabolic pathways have been utilized to generate biosensors for the detection of bioavailable concentrations of environmentally relevant chemicals.

Bacteria↗
PubMed1996

Stereospecificity for the hydrogen transfer and molecular evolution of pyridoxal enzymes.

We here describe the stereochemical aspects of the reactions of pyridoxal 5'-phosphate (PLP)-dependent enzymes, and the relationship between the stereochemistry of the enzyme reaction and molecular evolution of the enzyme. The reactions of PLP-dependent enzymes proceed through the formation of an anionic Schiff base intermediate between the substrate and the coenzyme. Three stereochemical possibilities exist for the formation and cleavage of bonds in the intermediate: the reaction occurs stereospecifically on either the si- or the re-face of the planar intermediate, or alternatively, non-stereospecifically on both faces. The stereospecificities for hydrogen transfer between C-4' of the cofactor and substrate in the transamination catalyzed by various PLP-dependent enzymes have been studied. The stereospecificities reflect the active-site structures of the enzymes, especially the topographical situation of a coenzyme-substrate Schiff base and a catalytic base for the hydrogen transfer. The aminotransferases and other PLP-enzymes catalyzing the transamination as a side-reaction so far studied catalyze only the si-face specific hydrogen transfer. This suggests that these PLP enzymes have similar active-site structures and are evolved divergently from a common ancestral protein. We recently established a new method for the identification of stereospecificity for the hydrogen transfer, and found that D-amino acid aminotransferase and branched chain L-amino acid aminotransferase, which have significant sequence similarity to each other, catalyze the re-face hydrogen transfer on the intermediate. The X-ray crystallographic studies of D-amino acid aminotransferase showed that the relative arrangement of the catalytic base of the enzyme active center to the C4' of the bound cofactor is opposite to that of other aminotransferases catalyzing the si-face hydrogen transfer. The folding of D-amino acid aminotransferase is also different from those of the other aminotransferase so far studied. Therefore, the classifications of the aminotransferases based on their primary structures, three dimensional structures, and stereochemistry of their hydrogen transfer coincide with one another. We also found that PLP-dependent amino acid racemases, the primary structures of which are similar to none of the other PLP-enzymes, catalyze the non-stereospecific hydrogen transfer on both faces of the planar intermediate. Stereospecificities for the hydrogen transfer suggest convergent evolution of the PLP-dependent enzymes. The stereochemical aspects of the enzyme reactions give a clue to the molecular evolution of the enzymes as well as the primary structures and three-dimensional structures of the enzymes.

Amino Acids↗
PubMed1995

[The origin and early development of biological catalysts].

Biochemical processes are based on the catalytic activity of enzymes. Highly ordered stereometry of protein macromolecules perfectly adapted to fyziological function of enzymes is the result of a long evolution. Catalytic processes, e.g. inorganic catalysis on the surface of cosmic dust grains, or on the surface of clay particles on the Earth, occurred as early as during chemical evolution. Enzyme predecessors in prebiotic systems-probably, thermal polymers of amino acids (proteinoids)-were polyfunctional catalysts. Low-effective proteinoids have been replaced by the highly efficient and specific enzymes in the course of evolution; spatial and temporal co-ordination of the individual enzymes has developed instead of the proteinoid polyfunctionality. The comprehensive effect of the mixtures of hydrolytic enzymes in systemic enzyme therapy can be regarded as a as a sort of reflection of these evolutionary trends.

Catalysis↗