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PubMed2004

The porcine taurochenodeoxycholic acid 6alpha-hydroxylase (CYP4A21) gene: evolution by gene duplication and gene conversion.

Porcine taurochenodeoxycholic acid 6alpha-hydroxylase, cytochrome P450 4A21 (CYP4A21), differs from other members of the CYP4A subfamily in terms of structural features and catalytic activity. CYP4A21 participates in the formation of hyocholic acid, a species-specific primary bile acid in the pig. The CYP4A21 gene was investigated and found to be approx. 13 kb in size and split into 12 exons. The intron-exon organization of the CYP4A21 gene corresponds to that of CYP4A fatty acid hydroxylase genes in other species. Comparison with a genomic segment of a pig CYP4A fatty acid hydroxylase gene ( CYP4A24 ) revealed a sequence identity with CYP4A21 that extends beyond the exons, indicating a common origin by gene duplication. A pronounced sequence identity was found also within the proximal 5'-flanking regions, whereas the patterns of mRNA expression of CYP4A21 and CYP4A fatty acid hydroxylases in pig liver differ. Sequence comparison aiming to elucidate the origin of the unique features of CYP4A21 revealed a region of decreased sequence identity from exon 6 to exon 8, strongly suggesting that gene conversion could have contributed to the evolution of CYP4A21.

5' Flanking Region↗

PubMed2003

Human DNA polymerase lambda diverged in evolution from DNA polymerase beta toward specific Mn(++) dependence: a kinetic and thermodynamic study.

The recently discovered human DNA polymerase lambda (DNA pol lambda) has been implicated in translesion DNA synthesis across abasic sites. One remarkable feature of this enzyme is its preference for Mn(2+) over Mg(2+) as the activating metal ion, but the molecular basis for this preference is not known. Here, we present a kinetic and thermodynamic analysis of the DNA polymerase reaction catalyzed by full length human DNA pol lambda, showing that Mn(2+) favors specifically the catalytic step of nucleotide incorporation. Besides acting as a poor coactivator for catalysis, Mg(2+) appeared to bind also to an allosteric site, resulting in the inhibition of the synthetic activity of DNA pol lambda and in an increased sensitivity to end product (pyrophosphate) inhibition. Comparison with the closely related enzyme human DNA pol beta, as well as with other DNA synthesising enzymes (mammalian DNA pol alpha and DNA pol delta, Escherichia coli DNA pol I, and HIV-1 reverse transcriptase) indicated that these features are unique to DNA pol lambda. A deletion mutant of DNA pol lambda, which contained the highly conserved catalytic core only representing the C-terminal half of the protein, showed biochemical properties comparable to the full length enzyme but clearly different from the close homologue DNA pol beta, highlighting the existence of important differences between DNA pol lambda and DNA pol beta, despite a high degree of sequence similarity.

Allosteric Site↗

PubMed2005

Directed evolution of biocatalytic processes.

The benefits of applying biocatalysts to organic synthesis, such as their high chemo-, regio-, and enantio-specificity and selectivity, must be seriously considered, especially where chemical routes are unavailable, complex or prohibitively expensive. In cases where a potential biocatalytic route is not yet efficient enough to compete with chemical synthesis, directed evolution, and/or process engineering could be implemented for improvements. While directed evolution has demonstrated great potential to enhance enzyme properties, there will always be some aspects of biocatalytic processes that it does not address. Even where it can be successfully applied, the resources required for its implementation must currently be weighed against the feasibility of, and resources available for developing a chemical synthesis route. Here, we review the potential of combining directed evolution with process engineering, and recent developments to improve their implementation. Favourable targets for the directed evolution of new biocatalysts are the syntheses of highly complex molecules, especially where chemistry, metabolic engineering or recombineering provide a partial solution. We also review some of the recent advances in the application of these approaches alongside the directed evolution of biocatalysts.

Animals↗

PubMed2006

CYP6B1 and CYP6B3 of the black swallowtail (Papilio polyxenes): adaptive evolution through subfunctionalization.

Gene duplication provides essential material for functional divergence of proteins and hence allows organisms to adapt to changing environments. Following duplication events, redundant paralogs may undergo different evolutionary paths via processes known as nonfunctionalization, neofunctionalization, or subfunctionalization. Studies of adaptive evolution at the molecular level have progressed rapidly by computationally analyzing nucleotide substitution patterns but such studies are limited by the absence of information relating to alterations of function of the encoded enzymes. In this respect, evolution of the Papilio polyxenes cytochrome P450 monooxygenases (P450s) responsible for the adaptation of this insect to furanocoumarin-containing host plants provides an excellent model for elucidating the evolutionary fate of duplicated genes. Evidence from sequence and functional analysis in combination with molecular modeling indicates that the paralogous CYP6B1 and CYP6B3 genes in P. polyxenes have probably evolved via subfunctionalization after the duplication event by which they arose. Both enzymes have been under independent purifying selection as evidenced by the low dN/dS ratio in both the coding region and substrate recognition sites. Both enzymes have maintained their ability to metabolize linear and angular furanocoumarins albeit at different efficiencies. Comparisons of molecular models developed for the CYP6B3 and CYP6B1 proteins highlight differences in their binding modes that account for their different activities toward linear and angular furanocoumarins. That P. polyxenes maintains these 2 furanocoumarin-metabolizing loci with somewhat different activities and expression patterns provides this species with the potential to acquire P450s with novel functions while maintaining those most critical to its exclusive feeding on its current range of host plants.

Adaptation, Biological↗

PubMed2005

A highly sensitive selection method for directed evolution of homing endonucleases.

Homing endonucleases are enzymes that catalyze DNA sequence specific double-strand breaks and can significantly stimulate homologous recombination at these breaks. These enzymes have great potential for applications such as gene correction in gene therapy or gene alteration in systems biology and metabolic engineering. However, homing endonucleases have a limited natural repertoire of target sequences, which severely hamper their applications. Here we report the development of a highly sensitive selection method for the directed evolution of homing endonucleases that couples enzymatic DNA cleavage with the survival of host cells. Using I-SceI as a model homing endonuclease, we have demonstrated that cells with wild-type I-SceI showed a high cell survival rate of 80-100% in the presence of the original I-SceI recognition site, whereas cells without I-SceI showed a survival rate <0.003%. This system should also be readily applicable for directed evolution of other DNA cleavage enzymes.

Deoxyribonucleases, Type II Site-Specific↗

PubMed2000

Directed evolution of beta -glucosidase A from Paenibacillus polymyxa to thermal resistance.

The beta-glucosidase encoded by the bglA gene from Paenibacillus polymyxa has a half-life time of 15 min at 35 degrees C and no detectable activity at 55 degrees C. We have isolated random mutations that enhance the thermoresistance of the enzyme. Following a directed evolution strategy, we have combined some of the isolated mutations to obtain a beta-glucosidase with a half-life of 12 min at 65 degrees C, in the range of resistance of thermophilic enzymes. No significant alteration of the kinetic parameters of the enzyme was observed. One of the mutants isolated in the screening for thermoresistant beta-glucosidase had the same resistance to denaturation as the wild type. This mutation caused the accumulation of enzyme in E. coli, probably due to its lower turnover. The structural changes responsible for the properties of the mutant enzymes have been analyzed. The putative causes increasing thermoresistance are as follows: the formation of an extra salt bridge, the replacement of an Asn residue exposed to the solvent, stabilization of the hydrophobic core, and stabilization of the quaternary structure of the protein.

Adaptation, Physiological↗

PubMed2002

HoxE--a subunit specific for the pentameric bidirectional hydrogenase complex (HoxEFUYH) of cyanobacteria.

NAD(P)(+)-reducing hydrogenases have been described to be composed of a diaphorase (HoxFU) and a hydrogenase (HoxYH) moiety. This study presents for the first time experimental evidence that in cyanobacteria, a fifth subunit, HoxE, is part of this bidirectional hydrogenase. HoxE exhibits sequence identities to NuoE of respiratory complex I of Escherichia coli. The subunit composition of the cyanobacterial bidirectional hydrogenase has been investigated. The oxygen labile enzyme complex was purified to close homogeneity under anaerobic conditions from Synechocystis sp. PCC 6803 and Synechococcus sp. PCC 6301. The 647-fold and 1290-fold enriched purified enzyme has a specific activity of 46 micromol H(2) evolved (min mg protein)(-1) and 15 micromol H(2) evolved (min mg protein)(-1), respectively. H(2)-evolution of the purified enzyme of S. sp. PCC 6803 is highest at 60 degrees C and pH 6.3. Immunoblot experiments, using a polyclonal anti-HoxE antibody, demonstrate that HoxE co-purifies with the hydrogenase activity in S. sp. PCC 6301. SDS-PAGE gels of the purified enzymes revealed six proteins, which were partially sequenced and identified, besides one nonhydrogenase component, as HoxF, HoxU, HoxY, HoxH and, remarkably, HoxE. The molecular weight of the native protein (375 kDa) indicates a dimeric assembly of the enzyme complex, Hox(EFUYH)(2).

Amino Acid Sequence↗

PubMed2003

Life-history evolution and the microevolution of intermediary metabolism: activities of lipid-metabolizing enzymes in life-history morphs of a wing-dimorphic cricket.

Although a considerable amount of information is available on the ecology, genetics, and physiology of life-history traits, much more limited data are available on the biochemical and genetic correlates of life-history variation within species. Specific activities of five enzymes of lipid biosynthesis and two enzymes of amino acid catabolism were compared among lines selected for flight-capable (LW[f]) versus flightless (SW) morphs of the cricket Gryllus firmus. These morphs, which exist in natural populations, differ genetically in ovarian growth (100-400% higher in SW) and aspects of flight capability including the size of wings and flight muscles, and the concentration of triglyceride flight fuel (40% greater in LW[f]). Consistently higher activity of each enzyme in LW(f) versus SW-selected lines, and strong co-segregation between morph and enzyme activity, demonstrated genetically based co-variance between wing morph and enzyme activity. Developmental profiles of enzyme activities strongly paralleled profiles of triglyceride accumulation during adulthood and previous measures of in vivo lipid biosynthesis. These data strongly imply that genetically based elevation in activities of lipogenic enzymes, and enzymes controlling the conversion of amino acids into lipids, is an important cause underlying the elevated accumulation of triglyceride in the LW(f) morph, a key biochemical component of the trade-off between elevated early fecundity and flight capability. Global changes in lipid and amino-acid metabolism appear to have resulted from microevolutionary alteration of regulators of metabolism. Finally, strong genotype x environment (diet) interactions were observed for most enzyme activities. Future progress in understanding the functional causes of life-history evolution requires a more detailed synthesis of the fields of life-history evolution and metabolic biochemistry. Wing polymorphism is a powerful experimental model in such integrative studies.

Adipose Tissue↗

PubMed1979

Fate of vasoactive biogenic amines and activity of enzyme phenylethanolamine-N-methyltransferase in rat adrenal gland during the course of pregnancy, parturition and postpartum.

Influence of pregnancy, parturition and postparturition upon natural evolution of adrenaline, dopamine and enzyme of adrenaline synthesis, phenylethanolamine-N-methyltransferase in the adrenal gland was studied. Adrenaline content of the adrenal gland showed a progressive decrease from the 14th to the 18th day postcoitum. The mean values for adrenaline remained lower than the value of day 14 throughout the course of pregnancy up to day 21 postcoitum. At parturition (0 to 4 h) adrenaline declined again to its minimum value but was accompanied by an increase in the adrenal adrenaline level 24 h postpartum. The activity of enzyme phenylethanolamine-N-methyltransferase followed a similar pattern of evolution as that of adrenaline during pregnancy and postpartum. Dopamine concentration of the adrenal gland showed nonsignificant increases during days 14--18 postcoitum. At day 20 postcoitum adrenal dopamine was decreased to its minimum value and this decrease persisted up to day 21 postcoitum. At day 22 postcoitum, adrenal dopamine increased sharply to its maximum value but was again back to the level of day 21 at 0 h parturition. At 24 h postpartum, dopamine content decreased maximally again. The results presented here suggest that the content of vasoactive biogenic amines and the enzyme of adrenaline synthesis go through important natural variations during pregnancy, parturition and postpartum from day 14 postcoitum to 24 h postpartum. This phenomenon does not seem to be merely a cyclic effect. All these variations can be attributed to modified endocrine activity of the pregnant animals by correlating natural variations in steroid hormone levels with the observed changes in biogenic amines, since it is well established that most of the hormonal and nonhormonal steroids affect catecholamine regulation. The observed changes in amine levels could have important influence for the termination of pregnancy.

Adrenal Glands↗

PubMed2005

Co-evolution of tRNA 3' trailer sequences with 3' processing enzymes in bacteria.

Maturation of the tRNA 3' terminus is a complicated process in bacteria. Usually, it is initiated by an endonucleolytic cleavage carried out by RNase E and Z in different bacteria. In Escherichia coli, RNase E cleaves AU-rich sequences downstream of tRNA, producing processing intermediates with a few extra residues at the 3' end; these are then removed by exoribonuclease trimming to generate the mature 3' end. Here we show that essentially all E. coli tRNA precursors contain a potential RNase E cleavage site, the AU-rich sequence element (AUE), in the 3' trailer. This suggests that RNase E cleavage and exonucleolytic trimming is a general pathway for tRNA maturation in this organism. Remarkably, the AUE immediately downstream of each tRNA is selectively conserved in bacteria having RNase E and tRNA-specific exoribonucleases, suggesting that this pathway for tRNA processing is also commonly used in these bacteria. Two types of RNase E-like proteins are identified in actinobacteria and the alpha-subdivision of proteobacteria. The tRNA 3' proximal AUE is conserved in bacteria with only one type of E-like protein. Selective conservation of the AUE is usually not observed in bacteria without RNase E. These results demonstrate a novel example of co-evolution of RNA sequences with processing activities.

Bacteria↗

PubMed2006

Directed evolution of Bacillus subtilis lipase A by use of enantiomeric phosphonate inhibitors: crystal structures and phage display selection.

Phage display can be used as a protein-engineering tool for the selection of proteins with desirable binding properties from a library of mutants. Here we describe the application of this method for the directed evolution of Bacillus subtilis lipase A, an enzyme that has important properties for the preparation of the pharmaceutically relevant chiral compound 1,2-O-isopropylidene-sn-glycerol (IPG). PCR mutagenesis with spiked oligonucleotides was employed for saturation mutagenesis of a stretch of amino acids near the active site. After expression of these mutants on bacteriophages, dual selection with (S)-(+)- and (R)-(-)-IPG stereoisomers covalently coupled to enantiomeric phosphonate suicide inhibitors (SIRAN Sc and Rc inhibitors, respectively) was used for the isolation of variants with inverted enantioselectivity. The mutants were further characterised by determination of their Michaelis-Menten parameters. The 3D structures of the Sc and Rc inhibitor-lipase complexes were determined and provided structural insight into the mechanism of enantioselectivity of the enzyme. In conclusion, we have used phage display as a fast and reproducible method for the selection of Bacillus lipase A mutant enzymes with inverted enantioselectivity.

Bacillus subtilis↗

PubMedDate not supplied

Evolution of metabolisms: a new method for the comparison of metabolic pathways using genomics information.

The abundance of information provided by completely sequenced genomes defines a starting point for new insights in the multilevel organization of organisms and their evolution. At the lowest level enzymes and other protein complexes are formed by aggregating multiple polypeptides. At a higher level enzymes group conceptually into metabolic pathways as part of a dynamic information-processing system, and substrates are processed by enzymes yielding other substrates. A method based on a combination of sequence information with graph topology of the underlying pathway is presented. With this approach pathways of different organisms are related to each other by phylogenetic analysis, extending conventional phylogenetic analysis of individual enzymes. The new method is applied to pathways related to electron transfer and to the Krebs citric acid cycle. In addition to providing a more comprehensive understanding of similarities and differences between organisms, this method indicates different evolutionary rates between substrates and enzymes.

Animals↗

PubMed2004

Comparative genomics of rice and Arabidopsis. Analysis of 727 cytochrome P450 genes and pseudogenes from a monocot and a dicot.

Data mining methods have been used to identify 356 Cyt P450 genes and 99 related pseudogenes in the rice (Oryza sativa) genome using sequence information available from both the indica and japonica strains. Because neither of these genomes is completely available, some genes have been identified in only one strain, and 28 genes remain incomplete. Comparison of these rice genes with the 246 P450 genes and 26 pseudogenes in the Arabidopsis genome has indicated that most of the known plant P450 families existed before the monocot-dicot divergence that occurred approximately 200 million years ago. Comparative analysis of P450s in the Pinus expressed sequence tag collections has identified P450 families that predated the separation of gymnosperms and flowering plants. Complete mapping of all available plant P450s onto the Deep Green consensus plant phylogeny highlights certain lineage-specific families maintained (CYP80 in Ranunculales) and lineage-specific families lost (CYP92 in Arabidopsis) in the course of evolution.

Arabidopsis↗

PubMed1999

Stability and sulfur-reduction activity in non-aqueous phase liquids of the hydrogenase from the hyperthermophile Pyrococcus furiosus.

Hydrogenase from the hyperthermophilic archaeon, Pyrococcus furiosus, catalyzes the reversible activation of H(2) gas and the reduction of elemental sulfur (S degrees ) at 90 degrees C and above. The pure enzyme, modified with polyethylene glycol (PEG), was soluble (> 5 mg/mL) in toluene and benzene with t(1/2) values of more than 6 h at 25 degrees C. At 100 degrees C the PEG-modified enzyme was less stable in aqueous solution (t(1/2) approximately 10 min) than the native (unmodified) enzyme (t(1/2) approximately 1 h), but they exhibited comparable H(2) evolution, H(2) oxidation, and S degrees reduction activities at 80 degrees C. The H(2) evolution activity of the modified enzyme was twice that of the unmodified enzyme at 25 degrees C. The PEG-modified enzyme did not catalyze S degrees reduction (at 80 degrees C) in pure toluene unless H(2)O was added. The mechanism by which hydrogenase produces H(2)S appears to involve H(2)O as the proton source and H(2) as the electron source. The inability of the modified hydrogenase to catalyze S degrees reduction in a homogeneous non-aqueous phase complicates potential applications of this enzyme.

Biotechnology↗

PubMed2007

In silico panning for a non-competitive peptide inhibitor.

BACKGROUND: Peptide ligands have tremendous therapeutic potential as efficacious drugs. Currently, more than 40 peptides are available in the market for a drug. However, since costly and time-consuming synthesis procedures represent a problem for high-throughput screening, novel procedures to reduce the time and labor involved in screening peptide ligands are required. We propose the novel approach of 'in silico panning' which consists of a two-stage screening, involving affinity selection by docking simulation and evolution of the peptide ligand using genetic algorithms (GAs). In silico panning was successfully applied to the selection of peptide inhibitor for water-soluble quinoprotein glucose dehydrogenase (PQQGDH). RESULTS: The evolution of peptide ligands for a target enzyme was achieved by combining a docking simulation with evolution of the peptide ligand using genetic algorithms (GAs), which mimic Darwinian evolution. Designation of the target area as next to the substrate-binding site of the enzyme in the docking simulation enabled the selection of a non-competitive inhibitor. In all, four rounds of selection were carried out on the computer; the distribution of the docking energy decreased gradually for each generation and improvements in the docking energy were observed over the four rounds of selection. One of the top three selected peptides with the lowest docking energy, 'SERG' showed an inhibitory effect with Ki value of 20 microM. PQQGDH activity, in terms of the Vmax value, was 3-fold lower than that of the wild-type enzyme in the presence of this peptide. The mechanism of the SERG blockage of the enzyme was identified as non-competitive inhibition. We confirmed the specific binding of the peptide, and its equilibrium dissociation constant (KD) value was calculated as 60 microM by surface plasmon resonance (SPR) analysis. CONCLUSION: We demonstrate an effective methodology of in silico panning for the selection of a non-competitive peptide inhibitor from small virtual peptide library. This study is the first to demonstrate the usefulness of in silico evolution using experimental data. Our study highlights the usefulness of this strategy for structure-based screening of enzyme inhibitors.

Acinetobacter calcoaceticus↗

PubMed2003

Origin, evolution, and metabolic role of a novel glycolytic GAPDH enzyme recruited by land plant plastids.

NAD-specific glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a cytosolic marker enzyme of eukaryotes (GapC; EC 1.2.1.12). Land plants possess an additional NADP+-dependent enzyme (EC 1.2.1.13) within their chloroplasts which is composed of two subunits, GapA and GapB. Another plastid GAPDH enzyme (GapCp) was recently discovered in gymnosperms and ferns. This novel GapCp is closely related to cytosolic GapC and displays glycolytic NAD+ cosubstrate specificity. Here we show that this new gene GapCp is also present and actively expressed in angiosperms, mosses, and liverworts. Phylogenetic analyses of the available GapC and GapCp sequences suggest that the gene duplication giving rise to GapCp occurred in ancestral charophyte algae. The data are also consistent with a monophyletic origin of charophytes and land plants and further support the view that land plants arose from a Coleochaete-like green alga. Northern hybridizations were employed to study the expression of the genes GapCp, GapC, GapA, and GapB in green and nongreen tissues from pepper (Capsicum annuum). The results demonstrate that GapCp mRNAs are mainly expressed in red pepper fruit and roots, in which the transcript levels of photosynthetic GapA and GapB are downregulated. This suggests that in flowering plants GapCp plays a specific role in glycolytic energy production of nongreen plastids such as chromoplasts and leukoplasts and that angiosperms may be the only land plants where glycolysis is absent in green chloroplasts.

Amino Acid Sequence↗

PubMed2006

A filter paper-based assay for laboratory evolution of hydrolases and dehydrogenases.

Industrially important enzyme classes such as hydrolases and dehydrogenases are often not amenable to laboratory evolution methods due to a lack of sensitive and reliable high-throughput screening (HTS) systems. We developed a conceptually novel and technically simple high-throughput screening system based on detection of volatile aldehydes with the sensitive reagent Purpald (4-amino-3-hydrazino-5-mercapto-1,2,4-triazole). The aldehyde detection takes place on a filter-paper that is pre-soaked with Purpald and covers the microtiter plate. The filter paper-based Purpald assay separates aldehyde detection from biocatalytical conversion and thereby avoids interferences from biological materials with assay components. This screening principle allows, to our knowledge, for the first time to determine the synthetic activity of hydrolases such as lipases and esterases in organic solvents in a 96-well whole-cell format. Its simplicity and cost-effectiveness make the reported HTS system suitable as fast pre-screen in laboratory evolution experiments and for semi-quantitative assays of improved mutants.

Alcohol Dehydrogenase↗

PubMed1995

Divergent evolution of a beta/alpha-barrel subclass: detection of numerous phosphate-binding sites by motif search.

Study of the most conserved region in many beta/alpha-barrels, the phosphate-binding site, revealed a sequence motif in a few beta/alpha-barrels with known tertiary structure, namely glycolate oxidase (GOX), cytochrome b2 (Cyb2), tryptophan synthase alpha subunit (TrpA), and the indoleglycerolphosphate synthase (TrpC). Database searches identified this motif in numerous other enzyme families: (1) IMP dehydrogenase (IMPDH) and GMP reductase (GuaC); (2) phosphoribosylformimino-5-aminoimidazol carboxamide ribotide isomerase (HisA) and the cyclase-producing D-erythro-imidazole-glycerolphosphate (HisF) of the histidine biosynthetic pathway; (3) dihydroorotate dehydrogenase (PyrD); (4) glutamate synthase (GltB); (5) ThiE and ThiG involved in the biosynthesis of thiamine as well as related proteins; (6) an uncharacterized open reading frame from Erwinia herbicola; and (7) a glycerol uptake operon antiterminator regulatory protein (GlpP). Secondary structure predictions of the different families mentioned above revealed an alternating order of beta-strands and alpha-helices in agreement with a beta/alpha-barrel-like topology. The putative phosphate-binding site is always found near the C-terminus of the enzymes, which are all at least about 200 amino acids long. This is compatible with its assumed location between strand 7 and helix 8. The identification of a significant motif in functionally diverse enzymes suggests a divergent evolution of at least a considerable fraction of beta/alpha-barrels. In addition to the known accumulation of beta/alpha-barrels in the tryptophan biosynthetic pathway, we observe clusters of these enzymes in histidine biosynthesis, purine metabolism, and apparently also in thiamine biosynthesis. The substrates are mostly heterocyclic compounds.(ABSTRACT TRUNCATED AT 250 WORDS)

Alcohol Oxidoreductases↗