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PubMed2006

Alternative mutations of a positively selected residue elicit gain or loss of functionalities in enzyme evolution.

All molecular species in an organism are connected physically and functionally to other molecules. In evolving systems, it is not obvious to what extent functional properties of a protein can change to selective advantage and leave intact favorable traits previously acquired. This uncertainty has particular significance in the evolution of novel pathways for detoxication, because an organism challenged with new xenobiotics in the environment may still require biotransformation of previously encountered toxins. Positive selection has been proposed as an evolutionary mechanism for facile adaptive responses of proteins to changing conditions. Here, we show, by saturation mutagenesis, that mutations of a hypervariable residue in human glutathione transferase M2-2 can differentially change the enzyme's substrate-activity profile with alternative substrates and, furthermore, enable or disable dissimilar chemical reactions. Crystal structures demonstrate that activity with epoxides is enabled through removal of steric hindrance from a methyl group, whereas activities with an orthoquinone and a nitroso donor are maintained in the variant enzymes. Given the diversity of cellular activities in which a single protein can be engaged, the selective transmutation of functional properties has general significance in molecular evolution.

Enzyme Stability↗
PubMed2000

[ST-segment and myocardial enzymes evolution during myocardial infarction after fibrinolysis therapy and its relation with postinfarction angina, Killip class and mortality in intensive unit care].

INTRODUCTION: The prognostic value of enzyme peaks, the sum of the ST segment and reperfusion arrythmias during myocardial infarction remains unclear. AIM: The aim of this study was to relate the early enzymatic peaks, the sum of the ST segment and reperfusion arrythmias after thrombolytic therapy with postinfarction angina, Killip class and mortality during the acute phase of myocardial infarction. PATIENTS AND METHODS: Of 187 patients receiving fibrinolytic therapy, 169 were consecutively and prospectively included in the study. The following myocardial enzymes were determined: CK, MB, TGO, LDH. Electrocardiograms were performed prior to and 2, 6, 12 and 24 hours after the administration of the fibrinolytic drug. RESULTS: The mean age of the patients was 60.12 +/- 11.3 years, with 138 (81.7%) being males. Myocardial infarction was anterior in 74 (43.7%) and inferior in 95 cases (56.3%). Reperfusion arrythmias were observed in 65 cases (38.5%). One hundred thirty-one (77.5%) were Killip class I, 12 (7.1%) presented postinfarction angina, and 8 (4.7%) died. A peak was observed in MB at 6 hours in cases of anterior myocardial infarction and the sum of the ST segment decreased less than 50% in the first 2 hours. No statistically significant correlation was observed between the enzymatic peaks, the reperfusion arrythmias, Killip class, postinfarction angina or early mortality. The greater the ST segment sum, the greater the severity according to the Killip class. On multivariate analysis no model was found to be related to postinfarction angina. However, age was related to mortality and sex and age were associated with heart failure. CONCLUSIONS: In our population, the variables studied were not found to be useful to determine the prognosis during the early phase of acute myocardial infarction.

Aged↗
PubMed1977

Growth of Klebsiella aerogenes on xylitol: implications for bacterial enzyme evolution.

When Klebsiella aerogenes was grown in continuous culture with xylitol. an unnatural pentitol, as the growth limiting substrate, the structural gene which codes for ribitol dehydrogenase, an enzyme which gratuitously catalyzes the oxidation of xylitol to D-xylulose, was duplicated. It appears that the duplication mechansim only duplicates the gene which is subjected to selective pressure and not any of the other closely linked genes. The degree to which the ribitol dehydrogenase gene is duplicated does not appear to be strictly correlated with the ability to grow faster on xylitol. Duplication mutants do, in fact, grow faster than their parent strain, but when challenged to grow at even higher growth rates there is a catabolic repression of enzyme activity. Thus a situation is created in which a structural gene is duplicated in response to selective pressure; these mutants can grow faster on the new substrate, but faster growth results in a "silencing" of a portion of the genes by catabolite repression.

Alcohol Oxidoreductases↗
PubMed2004

Directed enzyme evolution guided by multidimensional analysis of substrate-activity space.

The directed evolution of protein function frequently involves identification of mutants with improved properties from a population of variants obtained by mutagenesis. The selection of clones to parent the subsequent generation is crucial to the continued creation of superior progeny. In the present study, multivariate analysis guided the evolution of human glutathione transferase (GST) T1-1 to 65-fold enhanced alkyltransferase activity. Six alternative substrates monitored the substrate-activity space that characterized a mutant library of enzymes, obtained by recombination of DNA and heterologous expression in Escherichia coli. A subset of mutants was identified by their proximity in the targeted region of six-dimensional factor space. DNA from these mutants was recombined to create a new generation of GST variants from which an improved enzyme was isolated. The multidimensional cluster analysis is applicable to quantitative properties in any population of molecules undergoing evolution and can guide the tailoring of proteins, nucleic acids and other chemical structures to novel and improved functions.

Alkyl and Aryl Transferases↗
PubMed1972

Enzyme evolution in the Enterobacteriaceae.

An immunological approach has been used for the study of alkaline phosphatase evolution in bacteria of the family Enterobacteriaceae. Antisera were prepared against alkaline phosphatase from Escherichia coli and Klebsiella aerogenes and tested against the unpurified alkaline phosphatases of 32 strains of enterobacteria by double diffusion and quantitative micro-complement fixation. The immunological relationships detected among the alkaline phosphatases of enterobacteria agree approximately with those reported for five other enzymes, as well as with the tryptic peptide pattern similarities found for two other enzymes, and with the relationships detected by interspecific deoxyribonucleic acid hybridization tests.

Alkaline Phosphatase↗
PubMed1998

Enzyme evolution and cancer: hypothesis why natural carcinogens are more potent than synthetic ones.

A great deal of evidence shows that carcinogen induced mutations in human cancers point towards natural rather than man-made agents. Here, we propose a model based on the premise that the evolutionary pressure of nature renders natural carcinogens more potent than artificial ones, present in equal concentration, by suitably modifying kinetic parameters of carcinogen metabolizing enzymes. Enzymes are evolved to bind the transition state of substrates more strongly than substrates themselves, thus obtaining more elevate values of the specificity constant kcat/Km (Ksp). Natural selection optimizing the catalytic power at the proper substrate concentration by suitable raising the Km values, reduce the Gibbs standard activation energy (G(0#)), accelerating the conversion of natural precarcinogens to potent carcinogens. Conversely, "man-made" carcinogens, since the last century in the biosphere, are converted to active metabolites at a lower rate than natural chemicals and the slower rate of activation would allow protective enzymes and DNA repair machinery more time to clean up the damage.

Carcinogens↗
PubMed1979

Probable mechanism of enzyme evolution: how did EBG of E. coli originate?

A mechanism is proposed for the formation of Ebg-evolved beta galactosidase-of E. coli based on the following assumptions: 1. In the presence of lactose, certain proteins being translated bind to their m-RNA-ribosome complexes; 2. This binding interferes with the release of m-RNA from the bacterial chromosome, marking the gene; 3. Thereupon a cytosine specific methylase and methyl cytosine deaminase pair, modify - mutate - the marked gene; 4. The result, after five or so mutations, is a new gene capable of coding for a different protein which can split lactose; 5. I propose that this enzyme pair has evolved to produce mutations internally, when need arises, as is the case here; 6. This may be a general mechanism through which drug resistance and detoxification of a novel chemical, could be achieved in bacteria; 7. All of these ideas are experimentally testable.

Bacterial Proteins↗
PubMed2002

Insights into enzyme evolution revealed by the structure of methylaspartate ammonia lyase.

Methylaspartate ammonia lyase (MAL) catalyzes the magnesium-dependent reversible alpha,beta-elimination of ammonia from L-threo-(2S,3S)-3-methylaspartic acid to mesaconic acid. The 1.3 A MAD crystal structure of the dimeric Citrobacter amalonaticus MAL shows that each subunit comprises two domains, one of which adopts the classical TIM barrel fold, with the active site at the C-terminal end of the barrel. Despite very low sequence similarity, the structure of MAL is closely related to those of representative members of the enolase superfamily, indicating that the mechanism of MAL involves the initial abstraction of a proton alpha to the 3-carboxyl of (2S,3S)-3-methylasparic acid to yield an enolic intermediate. This analysis resolves the conflict that had linked MAL to the histidine and phenylalanine ammonia lyase family of enzymes.

Amino Acid Sequence↗
PubMed1976

Enzyme evolution in a microbial community growing on the herbicide Dalapon.

A seven-membered microbial community capable of utilising the herbicide Dalapon has been isolated by continuous-flow enrichment culture. The composition of this community has remained remarkably stable over thousands of hours in a Dalapon-limited chemostat. During this period, however, one member of the community, Pseudomonas putida, acquired the ability to grow on Dalapon through the evolution of an extant dehalogenase.

Biological Evolution↗
PubMed1992

Molecular cloning and characterization of Na,K-ATPase from Hydra vulgaris: implications for enzyme evolution and ouabain sensitivity.

We have used molecular and biochemical techniques to analyze Na,K-ATPase from a simple metazoan, Hydra vulgaris. First we isolated and characterized cDNA clones encoding the Na,K-ATPase alpha subunit from a Hydra lambda gt11 cDNA library. The open reading frame predicts a protein of 1031 amino acids that bears a high degree of primary sequence and secondary structure similarity to mammalian, avian, and arthropod alpha subunits. The predicted Hydra alpha subunit contains charged residues at the termini of the H1-H2 extracellular domain, suggesting that the Hydra alpha subunit may be resistant to cardiac glycoside inhibition. Biochemical analysis of partially purified Hydra Na,K-ATPase reveals both high- and low-affinity components of ouabain-inhibitable ATPase activity. Our results suggest that the evolutionary ancestor of all metazoans possessed a Na,K-ATPase alpha subunit that was highly conserved with respect to its vertebrate counterparts. Further, expression of a ouabain-resistant Na,K-ATPase activity in Hydra suggests that cardiac glycoside resistance arose randomly during evolution of the Na,K-ATPase.

Amino Acid Sequence↗
PubMed1988

Organization of the oxygen-evolution enzyme complex studied by butanol/water phase partitioning of spinach photosystem II particles.

The oxygen-evolving photosystem II particles prepared from spinach chloroplasts with brief sonication and Triton X-100 treatment were subjected to butanol/water phase partitioning. Three peripheral proteins of photosystem II having relative molecular masses of 33,000, 24,000, and 18,000 daltons and a part of the manganese atoms associated with photosystem II were partitioned into the aqueous phase, depending on the concentration of salt which was included in the suspension of the photosystem II particles. Quantitative analysis of the phase partitioning of the photosystem II particles under the various ionic conditions at pH 6.5 suggested the following: (a) two of the four atoms of manganese associated with photosystem II are located at a relatively hydrophilic environment and easily extracted from the membrane; (b) one of these "hydrophilic manganese atoms" is structurally in close proximity to the protein of the relative molecular mass of 33,000 daltons and stabilized by the protein specifically; (c) the protein of the relative molecular mass of 24,000 daltons as well as that of 33,000 daltons is involved in the stabilization of the other "hydrophilic manganese" in the membrane; (d) each of the three proteins has an independent binding site on the membrane and organizes a specific catalytic domain where oxidation of water is carried out efficiently in collaboration with the reaction center of photosystem II.

Butanols↗
PubMed2006

Evolution of enzyme superfamilies.

Enzyme evolution is often constrained by aspects of catalysis. Sets of homologous proteins that catalyze different overall reactions but share an aspect of catalysis, such as a common partial reaction, are called mechanistically diverse superfamilies. The common mechanistic steps and structural characteristics of several of these superfamilies, including the enolase, Nudix, amidohydrolase, and haloacid dehalogenase superfamilies have been characterized. In addition, studies of mechanistically diverse superfamilies are helping to elucidate mechanisms of functional diversification, such as catalytic promiscuity. Understanding how enzyme superfamilies evolve is vital for accurate genome annotation, predicting protein functions, and protein engineering.

Binding Sites↗