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Results for “enzyme evolution”
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Digging up the roots of life.
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Enzymes: by chance, or by design?
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Chemical technology: all together now.
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Directed evolution of a fungal peroxidase.
The Coprinus cinereus (CiP) heme peroxidase was subjected to multiple rounds of directed evolution in an effort to produce a mutant suitable for use as a dye-transfer inhibitor in laundry detergent. The wild-type peroxidase is rapidly inactivated under laundry conditions due to the high pH (10.5), high temperature (50 degrees C), and high peroxide concentration (5-10 mM). Peroxidase mutants were initially generated using two parallel approaches: site-directed mutagenesis based on structure-function considerations, and error-prone PCR to create random mutations. Mutants were expressed in Saccharomyces cerevisiae and screened for improved stability by measuring residual activity after incubation under conditions mimicking those in a washing machine. Manually combining mutations from the site-directed and random approaches led to a mutant with 110 times the thermal stability and 2.8 times the oxidative stability of wild-type CiP. In the final two rounds, mutants were randomly recombined by using the efficient yeast homologous recombination system to shuffle point mutations among a large number of parents. This in vivo shuffling led to the most dramatic improvements in oxidative stability, yielding a mutant with 174 times the thermal stability and 100 times the oxidative stability of wild-type CiP.
The single cell as a microplate well.
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Pharmacogenetics: its biologic roots and the medical challenge.
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Genetic polymorphisms in subterranean mammals (Spalax ehrenbergi superspecies) in the near east revisited: patterns and theory.
Allozyme diversity in the superspecies Spalax ehrenbergi has been revisited by studying 36 gene loci in 241 subterranean mole rats from 22 populations and nine chromosomal species, four from Turkey (2n = 52E (east), 52W (west), 56 and 58), four from Israel (2n = 52, 54, 58 and 60), and one from Egypt (2n = 60). The following results were indicated. (1) Genetic patterns: 11 of the 36 loci analysed (30.5 per cent) were monomorphic across the range, fifteen (41.7 per cent) were weakly polymorphic and the remaining 10 loci (27.8 per cent) were strongly polymorphic. (2) Heterozygosity: the average H was 0.051, range 0.00-0.098. In Israel, H increased with aridity and climatic unpredictability towards the northern Negev Desert, and was remarkably high in small steppic semi-isolates and desert isolates. (3) Species discrimination: some of the S. ehrenbergi species can be discriminated qualitatively. (4) Genetic distances (D): between species these values averaged 0.077, range 0.001-0.269, with the highest D between the ancestor Turkish and descendant Israeli and Egyptian species. The phylogenetic tree supports the Turkish origin of the Israeli Spalax ehrenbergi species, and the recent speciation of the Egyptian Spalax. (5) Genetic diversity is mostly (58 per cent) within populations. (6) Allozyme correlates: allozyme diversity was significantly correlated with the external physical (both climatic and edaphic) and biotic (parasite infection and plant cover) environment. (7) Spatial autocorrelation of allozyme frequencies suggests that migration is not influential. (8) Gametic phase disequilibria were significant in four out of five species tested, and were associated with climatic and edaphic factors. These results support the environmental selection hypothesis of genetic diversity including the niche-width variation hypothesis in space and time. Natural selection appears to play a major role in genetic differentiation of proteins in adaptive radiation and speciation.
The intelligent design of evolution.
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The genome of Cryptosporidium hominis.
Cryptosporidium species cause acute gastroenteritis and diarrhoea worldwide. They are members of the Apicomplexa--protozoan pathogens that invade host cells by using a specialized apical complex and are usually transmitted by an invertebrate vector or intermediate host. In contrast to other Apicomplexans, Cryptosporidium is transmitted by ingestion of oocysts and completes its life cycle in a single host. No therapy is available, and control focuses on eliminating oocysts in water supplies. Two species, C. hominis and C. parvum, which differ in host range, genotype and pathogenicity, are most relevant to humans. C. hominis is restricted to humans, whereas C. parvum also infects other mammals. Here we describe the eight-chromosome approximately 9.2-million-base genome of C. hominis. The complement of C. hominis protein-coding genes shows a striking concordance with the requirements imposed by the environmental niches the parasite inhabits. Energy metabolism is largely from glycolysis. Both aerobic and anaerobic metabolisms are available, the former requiring an alternative electron transport system in a simplified mitochondrion. Biosynthesis capabilities are limited, explaining an extensive array of transporters. Evidence of an apicoplast is absent, but genes associated with apical complex organelles are present. C. hominis and C. parvum exhibit very similar gene complements, and phenotypic differences between these parasites must be due to subtle sequence divergence.
Fancy footwork in the sequence space shuffle.
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The chemical elements of life.
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A new method for the preparation of human parathyroid hormone 1-34 peptides.
An engineered Escherichia coli strain, BL21 (DE3)/pGEX-4T-human parathyroid hormone (hPTH) (1-34), was constructed by oligonucleotide annealing and PCR amplification of the target gene, and then by ligating it with the pGEX-4T-3 vector and transferring into the BL21 host. The soluble glutathione S-transferase (GST) fusion protein GST-hPTH (1-34), expressed from BL21 (DE3)/pGEX-4T-hPTH (1-34), was harvested after fermentation and purification by affinity chromatography. Following double cleavage by thrombin and prolyl endopeptidase, about 0.6 g/l intact hPTH (1-34) was harvested. The product was checked by HPLC MS and N-terminal sequence analysis. The purified recombinant hPTH (1-34) stimulates adenylate cyclase in rabbit renal cortical cell membranes to exactly the same extent as synthetic hPTH standards, indicating that the recombinant product has full biological activity.
The comparative structure of mammalian glyceraldehyde 3-phosphate dehydrogenases.
1. The amino acid sequences around the thiol groups of glyceraldehyde 3-phosphate dehydrogenase from badger and monkey skeletal muscle were compared with the sequences around the thiol groups in the enzyme isolated from other organisms. 2. Preliminary evidence of the existence of isoenzymes in the badger was obtained. Only the major form, however, could be purified completely. 3. The monkey enzyme contains only three cysteine residues per polypeptide chain compared with the four found in all the other mammalian enzymes so far examined, including that of badger, and the two in yeast. The missing thiol group in monkey was identified as residue 281 in the corresponding sequence of the pig enzyme. 4. These experiments rule out any essential role for cysteine-281 in the function of the mammalian enzymes. 5. Further evidence of the remarkable conservation of amino acid sequence in this enzyme during evolution is presented and discussed.
Intracellular serine proteinase of Bacillus subtilis strain Marburg 168. Comparison with the homologous enzyme from Bacillus subtilis strain A-50.
Intracellular serine proteinase was isolated from sporulating cells of Bacillus subtilis Marburg 168 by gramicidin S-Sepharose 4B affinity chromatography. The enzymological characteristics, the amino acid composition and the 19 residues of the N-terminal sequence of the enzyme are reported. The isolated proteinase was closely related to, but not completely identical with, the intracellular serine proteinase of B. subtilis A-50. The divergence between these two intracellular enzymes was less than that between the corresponding extracellular serine proteinases (subtilisins) of types Carlsberg and BPN', produced by these bacterial strains. This may be connected with the more strict selection constraints imposed in intracellular enzymes during evolution.
The PLEES proteins: a family of structurally related enzymes widely distributed from bacteria to humans.
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Contrasting evolutionary forces driving population structure at expressed sequence tag polymorphisms, allozymes and quantitative traits in white spruce.
Patterns of variation in quantitative characters and genetic markers were compared among six regional populations of white spruce [Picea glauca (Moench) Voss]. Although some phenotypic characters were correlated with latitude (r = 0.791), longitude (r = -0.796) and precipitation during the growing season (r = 0.789), variability at genetic markers was not correlated with geographical or bioclimatic variables, and followed neutral expectations. Estimates of genetic diversity and population differentiation for 14 allozymes (translated regions of coding genes) were essentially indistinguishable from those observed for 11 expressed sequence tag polymorphisms (ESTPs) from untranslated regions of coding genes. Variation among populations for quantitative traits such as eighth year height (Q(ST) = 0.082), thirteenth year height (Q(ST) = 0.069), total wood density (Q(ST) = 0.102) and date of budset (Q(ST) = 0.246), was greater than for allozymes (G(ST) = 0.014) and ESTPs (G(ST) = 0.019). These trends suggest a strong adaptive response in quantitative traits, contrasting to allozymes and ESTPs where no selective response could be detected and where populations appeared to be essentially in a migration-drift equilibrium.