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Results for “enzyme evolution”

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PubMed1999

Directed evolution of the surface chemistry of the reporter enzyme beta-glucuronidase.

The use of the Escherichia coli enzyme beta-glucuronidase (GUS) as a reporter in gene expression studies is limited due to loss of activity during tissue fixation by glutaraldehyde or formaldehyde. We have directed the evolution of a GUS variant that is significantly more resistant to both glutaraldehyde and formaldehyde than the wild-type enzyme. A variant with eight amino acid changes was isolated after three rounds of mutation, DNA shuffling, and screening. Surprisingly, although glutaraldehyde is known to modify and cross-link free amines, only one lysine residue was mutated. Instead, amino acid changes generally occurred near conserved lysines, implying that the surface chemistry of the enzyme was selected to either accept or avoid glutaraldehyde modifications that would normally have inhibited function. We have shown that the GUS variant can be used to trace cell lineages in Xenopus embryos under standard fixation conditions, allowing double staining when used in conjunction with other reporters.

Animals↗

PubMed1985

Structural and immunological relationships among mammalian arylsulfatase A enzymes.

Structural and immunological properties of numerous arylsulfatase A enzymes (EC 3.1.6) were examined in order to assess the relationships among these enzymes in animals. Arylsulfatase A enzymes from all animals bind to a Concanavalin A-Sepharose column, consistent with the conclusion that they are all glycoproteins. At pH 7.5 the apparent mol. wts of the enzymes are 80-182 kDa, while at pH 4.5 the mammalian arylsulfatase A enzymes dimerize and exhibit apparent mol. wts in the range of 297-348 kDa, but the enzymes from opossum and other lower classes of animals do not aggregate at pH 4.5. The mammalian arylsulfatase A enzymes, which aggregate at pH 4.5, also bind to rabbit liver arylsulfatase A monomers immobilized on an Affi-Gel 10 matrix. The arylsulfatase A enzymes that were studied all exhibit the anomalous kinetic behavior regarded as characteristic of these enzymes. However, not all of the inactivated enzymes are reactivated by sulfate ions. Goat antiserum raised against homogeneous rabbit liver arylsulfatase A cross-reacts with all of the mammalian enzymes in Ouchterlony gel diffusion experiments, whereas the enzymes from lower classes of animals do not cross-react. Quantitative immunoprecipitation experiments demonstrate that the mammalian enzymes are very similar to each other, with greater than 60% primary sequence homology indicated, while arylsulfatase A from opossum and other lower classes of animals show only a partial immunological similarity with the mammalian enzymes. Taken together, the data suggest that the active site of the enzyme and the structural features of the protein are highly conserved during the evolution of the enzyme molecule.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed2006

Molecular mechanisms of enzyme-catalysed halogenation.

Since their discovery, halogenated metabolites have been somewhat of a biological peculiarity and it is only now that we are beginning to realize the full extent of their medicinal value. With the exception of the well characterized haloperoxidases, most of the biosynthetic enzymes and mechanisms responsible for the halogenations have remained elusive. The crystal structures of two functionally diverse halogenases have been recently solved, providing us with new and exciting mechanistic detail. This new insight has the potential to be used both in the development of biomimetic halogenation catalysts and in engineering halogenases, and related enzymes, to halogenate new substrates. Interestingly, these new structures also illustrate how the evolution of these enzymes mirrors that of the monooxygenases, where the cofactor is selected for its ability to generate a powerful oxygenating species. In this highlight article we will examine the proposed catalytic mechanisms of the halogenases and how these relate to their structures. In addition, we will consider how this chemistry might be harnessed and developed to produce novel enzymatic activity.

Animals↗

PubMed2004

Co-evolution of steroidogenic and steroid-inactivating enzymes and adrenal and sex steroid receptors.

Receptors for the adrenal and sex steroids arose by a series of gene duplications from an ancestral nuclear receptor in a primitive vertebrate, at least 540 million years ago. Sequence analysis indicates many steroidogenic and steroid-inactivating enzymes, including cytochrome P450s and hydroxysteroid dehydrogenases (HSDs), arose at the same time. The estrogen receptor (ER) appears to be the ancestral steroid receptor. Initially, the redundant duplicated ER had a low specificity for its new ligand. This raises the question: "How was specificity for responses to different steroids regulated early in the evolution of steroid receptors?" Selective expression of these steroid-metabolizing enzymes provided specificity for different steroid responses in primitive vertebrates. 17 beta-Hydroxysteroid dehydrogenase-type 1 (17 beta-HSD-type 1) and 17 beta-HSD-type 2, which preferentially catalyze the reduction and oxidation at C17 of androgens and estrogens, respectively, provide an example of this mechanism. Selective expression of either 17 beta-HSD-type 1 or 17 beta-HSD-type 2 can regulate synthesis or inactivation of androgens or estrogens in specific cells. Steroids also were important in the evolution of land animals, which began about 400 million years ago. Steroidogenic and steroid-inactivating enzymes were recruited to regulate steroid-mediated responses as organ function became more complex. For example, in the kidney 11 beta-HSD-type 2 prevents binding of glucocorticoids to the mineralocorticoid receptor (MR), which is crucial for aldosterone-mediated regulation of electrolyte transport in the distal tubule. We propose that Delta 5 steroids, such as dehydroepiandrosterone and its metabolites, were the ligands for the ancestral ER. Understanding the actions of Delta 5 steroids in amphioxus and lamprey may shed light on adrenarche and neurosteroid actions in humans.

Adrenal Cortex Hormones↗

PubMed2006

Flavocytochrome P450 BM3 and the origin of CYP102 fusion species.

Flavocytochrome P450 (cytochrome P450) BM3 is an intensively studied model system within the P450 enzyme superfamily, and is a natural fusion of a P450 to its P450 reductase redox partner. The fusion arrangement enables efficient electron transfer within the enzyme and a catalytic efficiency that cannot be matched in P450 systems from higher organisms. P450 BM3's potential for industrially relevant chemical transformations is now recognized, and variants with biotechnological applications have been constructed. Simultaneously, structural and mechanistic studies continue to reveal the intricate mechanistic details of this enzyme, including its dimeric organization and the relevance of this quaternary structure to catalysis. Homologues of BM3 have been found in several bacteria and fungi, indicating important physiological functions in these microbes and enabling first insights into evolution of the enzyme family. This short paper deals with recent developments in our understanding of structure, function, evolution and biotechnological applications of this important P450 system.

Bacterial Proteins↗

PubMed2003

In vitro evolution of an RNA-cleaving DNA enzyme into an RNA ligase switches the selectivity from 3'-5' to 2'-5'.

Deoxyribozymes that ligate RNA expand the scope of nucleic acid catalysis and allow preparation of site-specifically modified RNAs. Previously, deoxyribozymes that join a 5'-hydroxyl and a 2',3'-cyclic phosphate were identified by in vitro selection from random DNA pools. Here, the alternative strategy of in vitro evolution was used to transform the 8-17 deoxyribozyme that cleaves RNA into a family of DNA enzymes that ligate RNA. The parent 8-17 DNA enzyme cleaves native 3'-5' phosphodiester linkages but not 2'-5' bonds. Surprisingly, the new deoxyribozymes evolved from 8-17 create only 2'-5' linkages. Thus, reversing the direction of the DNA-mediated process from ligation to cleavage also switches the selectivity in forming the new phosphodiester bond. The same change in selectivity was observed upon evolution of the 10-23 RNA-cleaving deoxyribozyme into an RNA ligase. The DNA enzymes previously isolated from random pools also create 2'-5' linkages. Therefore, deoxyribozyme-mediated formation of a non-native 2'-5' phosphodiester linkage from a 5'-hydroxyl and a 2',3'-cyclic phosphate is strongly favored in many different contexts.

Cloning, Molecular↗

PubMed1995

Emergence of nylon oligomer degradation enzymes in Pseudomonas aeruginosa PAO through experimental evolution.

Through selective cultivation with 6-aminohexanoate linear dimer, a by-product of nylon-6 manufacture, as the sole source of carbon and nitrogen, Pseudomonas aeruginosa PAO, which initially has no enzyme activity to degrade this xenobiotic compound, was successfully expanded in its metabolic ability. Two new enzyme activities, 6-aminohexanoate cyclic dimer hydrolase and 6-aminohexanoate dimer hydrolase, were detected in the adapted strains.

Amidohydrolases↗

PubMed1995

Molecular evolution of plant beta-glucan endohydrolases.

The evolutionary relationships of two classes of plant beta-glucan endohydrolases have been examined by comparison of their substrate specificities, their three-dimensional conformations and the structural features of their corresponding genes. These comparative studies provide compelling evidence that the (1-->3)-beta-glucanases and (1-->3,1-->4)-beta-glucanases from higher plants share a common ancestry and, in all likelihood, that the (1-->3,1-->4)-beta-glucanases diverged from the (1-->3)-beta-glucanases during the appearance of the graminaceous monocotyledons. The evolution of (1-->3,1-->4)-beta-glucanases from (1-->3)-beta-glucanases does not appear to have invoked 'modular' mechanisms of change, such as those caused by exon shuffling or recombination. Instead, the shift in specificity has been acquired through a limited number of point mutations that have resulted in amino acid substitutions along the substrate-binding cleft. This is consistent with current theories that the evolution of new enzymic activity is often achieved through duplication of the gene encoding an existing enzyme which is capable of performing the required chemistry, in this case the hydrolysis of a glycosidic linkage, followed by the mutational alteration and fine-tuning of substrate specificity. The evolution of a new specificity has enabled a dramatic shift in the functional capabilities of the enzymes. (1-->3)-beta-Glucanases that play a major role, inter alia, in the protection of the plant against pathogenic microorganisms through their ability to hydrolyse the (1-->3)-beta-glucans of fungal cell walls, appear to have been recruited to generate (1-->3,1-->4)-beta-glucanases, which quite specifically hydrolyse plant cell wall (1-->3,1-->4)-beta-glucans in the graminaceous monocotyledons during normal wall metabolism. Thus, one class of beta-glucan endohydrolase can degrade beta-glucans in fungal walls, while the other hydrolyses structurally distinct beta-glucans of plant cell walls. Detailed information on the three-dimensional structures of the enzymes and the identification of catalytic amino acids now present opportunities to explore the precise molecular and atomic details of substrate-binding, catalytic mechanisms and the sequence of molecular events that resulted in the evolution of the substrate specificities of the two classes of enzyme.

Amino Acid Sequence↗

PubMed2000

Evolution in the test tube as a means to create enantioselective enzymes for use in organic synthesis.

A novel method for the creation of mutant enzymes showing increased levels of enantioselectivity in a given synthetic organic reaction is reviewed. It makes use of directed evolution and is therefore independent of structural or mechanistic aspects. Accordingly, known molecular biological methods for random mutagenesis coupled with the proper expression system and high-throughput screening methods for enantioselectivity form the basis of this new approach. An example is the lipase-catalyzed hydrolytic kinetic resolution of a chiral ester in which the original enantioselectivity of 2% ee is increased to > 90% ee in just a few rounds of mutagenesis.

Directed Molecular Evolution↗

PubMed2006

Diversification and specialization of HIV protease function during in vitro evolution.

Our goal is to understand how enzymes adapt to utilize novel substrates. We and others have shown that directed evolution tends to generate enzyme variants with broadened substrate specificity. Broad-specificity enzymes are generally deleterious to living cells, so this observed trend might be an artifact of the most commonly employed high throughput screens. Here, we demonstrate a more natural and effective screening strategy for directed evolution. The gene encoding model enzyme HIV protease was randomly mutated, and the resulting library was expressed in Escherichia coli cells to eliminate cytotoxic broad-specificity variants. The surviving variants were screened for clones with activity against a reporter enzyme. The wild-type human immunodeficiency virus type I protease (HIV PR) is cytotoxic and exhibits no detectable activity in reactions with beta-galactosidase (BGAL). In contrast, the selected variants were nontoxic and exhibited greater activity and specificity against BGAL than did the wild-type HIV PR in reactions with any substrate. A single round of whole gene random mutagenesis and conventional high-throughput screening does not usually effect complete inversions of substrate specificity. This suggests that a combination of positive and purifying selection engenders more rapid adaptation than positive selection alone.

Adaptation, Biological↗

PubMed1995

Modelling the three-dimensional structure and the electrostatic potential field of two Cu,Zn superoxide dismutase variants from tomato leaves.

The three-dimensional structure of tomato P31 and T10 Cu,Zn superoxide dismutases (SODs) were computer modelled using the structure of the bovine enzyme as a template. The structure-essential residues retain in the models the position occupied in the other Cu,Zn SODs of known 3D structure and the overall packing of the beta-barrel is maintained. Formation of 'aromatic pairs' occurs between newly inserted aromatic residues. The number of total charges changes in the two variants and some charged residues located in the proximity of the active site in most Cu,Zn SODs disappear in tomato enzymes. Calculation of the electrostatic potential field, carried out by numerically solving the Poisson-Boltzmann equation, indicates that in both variants a negative potential field surrounds all the protein surface except the active site areas, characterized by positive potential values, as already observed in the bovine enzyme. This result confirms that coordinated mutations of charged residues have occurred in the evolution of this enzyme giving rise to a peculiar electrostatic potential distribution common to all members of this protein family.

Amino Acid Sequence↗

PubMed1999

High temperature cDNA synthesis by AMV reverse transcriptase improves the specificity of PCR.

The enzyme avian myeloblastosis virus reverse transcriptase (AMV-RT) is routinely used for cDNA synthesis, which is generally carried out at temperatures between 37 degrees C and 42 degrees C. We show that this enzyme can support cDNA synthesis, at temperatures as high as 70 degrees C. We have utilized this property of the AMV-RT to improve the specificity of polymerase chain reaction (PCR). Furthermore, this apparently thermophilic property of the enzyme, which is an important constituent of a mesophilic organism, raises intriguing questions regarding evolution of the enzyme structure.

Avian Myeloblastosis Virus↗

PubMed1991

Structural conservation in parallel beta/alpha-barrel enzymes that catalyze three sequential reactions in the pathway of tryptophan biosynthesis.

Three successive steps in tryptophan biosynthesis are catalyzed by single-domain proteins, each folded as a parallel beta/alpha-barrel, as observed in the crystal structures of the bienzyme (phosphoribosyl)-anthranilate isomerase:indoleglycerolphosphate synthase from Escherichia coli [Priestle, J.P., Grütter, M. G., White, J. L., Vincent, M. G., Kania, M., Wilson, E., Jardetzky, T. S., Kirschner, K., & Jansonius, J. N. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 5690-5694] and the alpha-subunit of the tetrameric bienzyme tryptophan synthase from Salmonella typhimurium [Hyde, C. C., Ahmed, S. A., Padlan, E. A., Miles, E. W., & Davies, D. R. (1988) J. Biol. Chem. 263, 17857-17871]. Recent refinement of the crystal structures of these enzymes at atomic resolution revealed that they contain a common phosphate group binding site in the beta/alpha-barrel, created by residues of the loop between beta-strand 7 and alpha-helix 7 and the N-terminus of an additional helix 8'. The close similarities of their beta/alpha-barrel structures permitted the alignment of 50-75% of their respective amino acid sequences. Considerable sequence similarity was detected in the regions spanning the phosphate binding sites, whereas the percentage of identical residues was barely significant for the remaining parts of the enzymes. These observations suggest divergent evolution of these three beta/alpha-barrel enzymes involved in tryptophan biosynthesis. The same phosphate binding site was also observed in six other beta/alpha-barrel enzymes that are functionally unrelated to those involved in tryptophan biosynthesis: triosephosphate isomerase, ribulose-1,5-bisphosphate carboxylase/oxygenase, glycolate oxidase, flavocytochrome b2, trimethylamine dehydrogenase, and tentatively also fructosebisphosphate aldolase.(ABSTRACT TRUNCATED AT 250 WORDS)

Aldose-Ketose Isomerases↗

PubMed1994

Evolution of transcription-regulating proteins by enzyme recruitment: molecular models for nitrogen metabolite repression and ethanol utilisation in eukaryotes.

Studies on the quinic acid utilisation gene (qut) cluster in Aspergillus nidulans showed that the genes encoding transcriptional activator and repressor proteins evolved by co-opting duplicated copies of genes encoding metabolic enzymes. In order to test the hypothesis that this was a general route for the genesis of regulatory proteins, the origins of the major control protein mediating nitrogen metabolite repression (an example of inter-pathway regulation) and ethanol utilisation (an example of intra-pathway regulation) in filamentous fungi were sought. The regulatory proteins mediating nitrogen metabolite repression were deduced to have originated in a duplication of genes encoding the anthranilate synthase complex which is active in the shikimate pathway. The major protein regulating ethanol utilisation was deduced to have its origin in the fusion of duplicated genes encoding the aldehyde and alcohol dehydrogenases (ALDA and ALCA). These data strongly support the view that transcriptional regulatory proteins evolve by the recruitment of functional domains provided by metabolic enzymes.

Aspergillus nidulans↗

PubMedDate not supplied

[Periodic classification of DNA sequences recognized by restrictases: properties of symmetry and regularity].

A circular periodic map of short palindromic DNA sequences is constructed using the sequence homology and symmetry. It is applied to compare sequences recognised by class II restriction and modification enzymes with other similar DNA sequences. All known restriction sites have two strong properties: they are enriched by GC pairs, and clustered (purine-purine, pyrimidine-pyrimidine) bonds predominant upon alternating ones. The preference of AT/GC alternation is only slight. These properties were compared quantitatively with the help of suggested numerical methods among different groups of restriction enzymes. The map is applied for prediction of new specificities of restriction modification systems. Possible mechanism of DNA sequence recognition by these enzymes and their evolution are discussed.

Base Sequence↗

PubMed2003

Molecular evolution and genetic engineering of C4 photosynthetic enzymes.

The majority of terrestrial plants, including many important crops such as rice, wheat, soybean, and potato, are classified as C(3) plants that assimilate atmospheric CO(2) directly through the C(3) photosynthetic pathway. C(4) plants, such as maize and sugarcane, evolved from C(3) plants, acquiring the C(4) photosynthetic pathway in addition to the C(3) pathway to achieve high photosynthetic performance and high water- and nitrogen-use efficiencies. Consequently, the transfer of C(4) traits to C(3) plants is one strategy being adopted for improving the photosynthetic performance of C(3) plants. The recent application of recombinant DNA technology has made considerable progress in the molecular engineering of photosynthetic genes in the past ten years. It has deepened understanding of the evolutionary scenario of the C(4) photosynthetic genes. The strategy, based on the evolutionary scenario, has enabled enzymes involved in the C(4) pathway to be expressed at high levels and in desired locations in the leaves of C(3) plants. Although overproduction of a single C(4) enzyme can alter the carbon metabolism of C(3) plants, it does not show any positive effects on photosynthesis. Transgenic C(3) plants overproducing multiple enzymes are now being produced for improving the photosynthetic performance of C(3) plants.

Evolution, Molecular↗

PubMed1985

Superoxide dismutase: an evolutionary puzzle.

We have obtained the complete amino acid sequence of copper/zinc-containing superoxide dismutase (SOD, superoxide:superoxide oxidoreductase, EC 1.15.1.1) from Drosophila melanogaster. The sequence of this enzyme is also known for man, horse, cow, and the yeast Saccharomyces cerevisiae. The rate of evolution of this enzyme is far from constant. The number of amino acid substitutions per 100 residues per 100 million years is 30.9 when the three mammals are compared to each other, 10.6 when Drosophila is compared to the three mammals, and 5.8 when the yeast is compared to the four animals. The first value represents one of the fastest evolutionary rates for any protein, the second is similar to the globin rate, and the third is similar to some cytochromes and other slowly evolving proteins. Hence, SOD is not an acceptable evolutionary clock. Another peculiarity of this enzyme is that a two-amino-acid deletion must have occurred independently in the lineages going to the cow and to Drosophila. We conclude that using the primary structure of a single gene or protein to time evolutionary events or to reconstruct phylogenetic relationships is potentially fraught with error.

Amino Acid Sequence↗

PubMed1980

Analytical solutions of a simple enzyme kinetic problem by a perturbative procedure.

A systematic perturbative procedure (the method of singular perturbation) is developed to follow the time evolution of an enzyme catalyzed reaction with one intermediate product over the entire time domain of interest. The perturbation parameter is the ratio of the enzyme concentration to the Michaelis-Menten constant. The treatment leads to a meaningful definition of the so-called quasi-steady state often invoked in the description of enzyme catalyzed reactions. The legitimacy and the domain of validity of this assumption are examined in the context of both the reversible and irreversible Michaelis-Menten kinetics.

Chemical Phenomena↗