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A recent analysis of protein sequences from diverse organisms has estimated that all extant species share a common ancestor that lived only 2 billion years ago; but how can this be squared with the fossil evidence that complex cells existed up to 3.5 billion years ago?
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Patterns of genetic variation in Schilbe intermedius were investigated due to morphological differences and taxonomic uncertainties regarding the Southern African schilbeids. A total of three populations, two Southern populations representing the former Eutropius depressirostris and a Northern population representing S. mystus, were electrophoretically analysed to determine the extent of genetic differentiation among these populations. The Northern and Southern populations were fixed for different alleles at the G3PDH-2 protein coding locus and allozyme differentiation between populations, using the 0.95 criterion, were also encountered at the PGDH-2 locus. Genetic distance values indicate greater genetic differentiation between the Northern and Southern populations compared to the two Southern populations. DNA sequence analysis of 900-1000 nucleotides of the cytochrome b gene revealed distances of 3.2-3.5% between the Schilbe/Eutropius complex. This finding, together with ingroup and outgroup analysis of evolutionary relationships, is congruent with the results from the electrophoretic analysis of the taxa. Sufficient differentiation exist between the Northern and Southern populations to regard them as distinct species.
A selection of World Wide Web sites relevant to papers published in this issue of Current Opinion in Chemical Biology.
Directed evolution is being used increasingly in academic and industrial laboratories to modify and improve important biocatalysts. Significant advances during this period of review include compartmentalization of genes and the in vitro translation apparatus in emulsions, as well as several impressive demonstrations of catalyst improvement. Shuffling of homologous genes offers a new way to utilize natural diversity in the evolution of novel catalysts.
Cytochrome c peroxidase (CCP) from Saccharomyces cerevisiae was subjected to directed molecular evolution to generate mutants with increased activity against the classical peroxidase substrate guaiacol, thus changing the substrate specificity of CCP from the protein cytochrome c to a small organic molecule. After three rounds of DNA shuffling and screening, mutants were isolated which possessed a 300-fold increased activity against guaiacol and an up to 1000-fold increased specificity for this substrate relative to that for the natural substrate. In all of the selected mutants, the distal arginine (Arg48), which is fully conserved in the superfamily of peroxidases, was mutated to histidine, showing that this mutation plays a key role in the significant increase in activity against phenolic substrates. The results suggest that, in addition to stabilizing the reactive intermediate compound I, the distal arginine plays an important role as a gatekeeper in the active site of CCP, controlling the access to the ferryl oxygen and the distal histidine. Other isolated mutations increase the general reactivity of the peroxidase or increase the intracellular concentration of the active holo form, allowing their selection under the employed screening conditions. The results illustrate the ability of directed molecular evolution technologies to deliver solutions to biochemical problems that would not be readily predicted by rational design.
Electrostatic interactions play a key role in enzyme catalytic function. At long range, electrostatics steer the incoming ligand/substrate to the active site, and at short distances, electrostatics provide the specific local interactions for catalysis. In cases in which electrostatics determine enzyme function, orthologs should share the electrostatic properties to maintain function. Often, electrostatic potential maps are employed to depict how conserved surface electrostatics preserve function. We expand on previous efforts to explain conservation of function, using novel electrostatic sequence and structure analyses of four enzyme families and one enzyme superfamily. We show that the spatial charge distribution is conserved within each family and superfamily. Conversely, phylogenetic analysis of key electrostatic residues provide the evolutionary origins of functionality.
D-Pantonohydrolase has attracted increasing attention as a biocatalyst for stereospecific production of D-pantoic acid. The Fusarium moniliforme D-pantonohydrolase was selected for directed evolution through error-prone Polymerase Chain Reaction (PCR) combined with DNA shuffling for improved activity and pH stability using a convenient two-step high-throughput screening method based on the product formation and pH indicator. After three sequential error-prone PCRs and two rounds of DNA shuffling followed by screening, about 60 positive mutants were produced and a best mutant, Mut H-1287, with improved activity and pH stability was obtained. As compared to wild-type D-pantonohydrolase, Mut H-1287 showed a 10.5-fold higher specific activity; moreover, it could retain 85% of its original activity after incubation under low pH. Gene analysis indicated that the Mut H-1287 had D63H, K118Q, and V241I substitutions. The wild-type and evolved D-pantonohydrolase (Mut H-1287) was purified in three steps. The activities and characteristics of purified wild-type and evolved D-pantonohydrolase were also studied and compared.
A computational method to identify residues important in creating a protein promoting vibration (PPV) in enzymes was previously developed and applied to horse liver alcohol dehydrogenase (HLADH), resulting in the identification of eight important residues. From these residues, we define a sequence motif, the PPV generating sequence, and find it to be unique and general to a larger group of alcohol dehydrogenases from diverse sources, demonstrating that nature has selected for the PPV generating sequence.
Cytogenetic studies were performed in four species of two scorpaeniform teleost families: Scorpaenidae and Triglidae. The karyotypes of Trigla lucerna, Trigloporus lastoviza (Triglidae), Scorpaena porcus and S. notata (Scorpaenidae) were analysed using various banding methods and in situ hybridization with a telomeric probe. In the two Scorpaena species, modest morphological divergence corresponded to considerable karyotype reorganization, while in the two Triglidae substantial phenotypical divergence corresponded to limited chromosomal changes. These data stress the need for a taxonomical re-evaluation of these teleosts based on characters independent of morphology.
The bean shoot borer, Epinotia aporema (Lep. Tortricidae), is an economically important pest of legume crops in South America. Recently, a granulovirus (EpapGV) was isolated from E. aporema larvae, and evaluated as a potential biological control agent. In order to generate a restriction map and to investigate the gene organisation of EpapGV genome, DNA isolated from occlusion bodies as well as a set of cloned genomic fragments were analysed using combinations of restriction endonucleases and Southern blot analyses that lead to a first version of the physical map. It was subsequently confirmed and refined by sequencing the termini of the cloned fragments and assessing their contiguity by comparing the sequences with databases to identify putative ORFs spanning neighbour fragments. This was also aided by PCR amplifications with primers that pointed outwards of the cloned viral DNA. The granulin gene was positioned on the physical map, cloned and sequenced. Its 747-nucleotide-long ORF encodes a predicted protein of 29 kDa and the core of the baculovirus very late promoter ATAAG was found 29 nucleotides upstream the initiation codon. In addition, 27 putative ORFs were located on the map and used to explore the genome organisation by GeneParityPlot against the fully sequenced granulovirus genomes. These data, taken together with the phylogenetic tree generated by alignment of the major occlusion proteins, indicate that EpapGV is closely related to CpGV, but has a distinct gene organisation.
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Explore the source record for details and available documents.
The close correspondence often observed between the taxonomy of parasites and their hosts has led to Fahrenholz's rule, which postulates that parasites and their hosts speciate in synchrony. This leads to the prediction that phylogenetic trees of parasites and their hosts should be topologically identical. We report here a test of this prediction which involves the construction of phylogenetic trees for rodents and their ectoparasites using protein electrophoretic data. We find a high degree of concordance in the branching patterns of the trees which suggests that there is a history of cospeciation in this host-parasite assemblage. In several cases where the branching patterns were identical in the host and parasite phylogenies, the branch lengths were also very similar which, given the assumptions of molecular clock theory, strongly suggests that the speciation of these hosts and ectoparasites was roughly contemporaneous and causally related.
Aminoacyl transfer RNA synthetases catalyse the first step of protein synthesis and establish the rules of the genetic code through the aminoacylation of tRNAs. There is a distinct synthetase for each of the 20 amino acids and throughout evolution these enzymes have been divided into two classes of ten enzymes each. These classes are defined by the distinct architectures of their active sites, which are associated with specific and universal sequence motifs. Because the synthesis of aminoacyl-tRNAs containing each of the twenty amino acids is a universally conserved, essential reaction, the absence of a recognizable gene for cysteinyl tRNA synthetase in the genomes of Archae such as Methanococcus jannaschii and Methanobacterium thermoautotrophicum has been difficult to interpret. Here we describe a different cysteinyl-tRNA synthetase from M. jannaschii and Deinococcus radiodurans and its characterization in vitro and in vivo. This protein lacks the characteristic sequence motifs seen in the more than 700 known members of the two canonical classes of tRNA synthetase and may be of ancient origin. The existence of this protein contrasts with proposals that aminoacylation with cysteine in M. jannaschii is an auxiliary function of a canonical prolyl-tRNA synthetase.