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At least 469 records · Page 26Linked to original sources
PubMed2005

Genome-wide analysis of restriction-modification system in unicellular and filamentous cyanobacteria.

Cyanobacteria are an ancient group of gram-negative bacteria with strong genome size variation ranging from 1.6 to 9.1 Mb. Here, we first retrieved all the putative restriction-modification (RM) genes in the draft genome of Spirulina and then performed a range of comparative and bioinformatic analyses on RM genes from unicellular and filamentous cyanobacterial genomes. We have identified 6 gene clusters containing putative Type I RMs and 11 putative Type II RMs or the solitary methyltransferases (MTases). RT-PCR analysis reveals that 6 of 18 MTases are not expressed in Spirulina, whereas one hsdM gene, with a mutated cognate hsdS, was detected to be expressed. Our results indicate that the number of RM genes in filamentous cyanobacteria is significantly higher than in unicellular species, and this expansion of RM systems in filamentous cyanobacteria may be related to their wide range of ecological tolerance. Furthermore, a coevolutionary pattern is found between hsdM and hsdR, with a large number of site pairs positively or negatively correlated, indicating the functional importance of these pairing interactions between their tertiary structures. No evidence for positive selection is found for the majority of RMs, e.g., hsdM, hsdS, hsdR, and Type II restriction endonuclease gene families, while a group of MTases exhibit a remarkable signature of adaptive evolution. Sites and genes identified here to have been under positive selection would provide targets for further research on their structural and functional evaluations.

Bacterial Proteins↗
PubMed2007

A general method of terminal truncation, evolution, and re-elongation to generate enzymes of enhanced stability.

Improving enzyme stability is a highly desirable design step in generating enzymes able to function under extreme conditions, such as elevated temperatures, while having the additional benefit of being less susceptible to cleavage by proteases. For these reasons, many different approaches and techniques have been devised in constructing such proteins, but the results to date have been of mixed success. Here, we present a robust method involving the terminal truncation, random mutagenesis and fragmentation, recombination, elongation, and finally, selection at physiological temperatures, to generate an enzyme with improved stability. Three cycles of directed evolution comprising of random mutagenesis, DNA shuffling, and selection at 37 degrees C were used, using the bacterial enzyme TEM-1 beta-lactamase as a model protein to yield deletion mutants with in vivo ampicillin resistance levels comparable to wild-type (wt) enzyme. Kinetic studies demonstrate the selected mutant to have a significantly improved thermostability relative to its wt counterpart. Elongation of this mutant to the full-length gene resulted in a beta-lactamase variant with dramatically increased thermostability. This technique was so fruitful that the evolved enzyme retained its maximum catalytic activity even 20 degrees C above its wt parent protein optimum. Thus, structural perturbation by terminal truncation and subsequent compensation by directed evolution at physiological temperatures is a fast, efficient, and highly effective way to improve the thermostability of proteins without the need for selecting at elevated temperatures.

Amino Acid Sequence↗
PubMed2001

Novel enzyme activities and functional plasticity revealed by recombining highly homologous enzymes.

BACKGROUND: Directed evolution by DNA shuffling has been used to modify physical and catalytic properties of biological systems. We have shuffled two highly homologous triazine hydrolases and conducted an exploration of the substrate specificities of the resulting enzymes to acquire a better understanding of the possible distributions of novel functions in sequence space. RESULTS: Both parental enzymes and a library of 1600 variant triazine hydrolases were screened against a synthetic library of 15 triazines. The shuffled library contained enzymes with up to 150-fold greater transformation rates than either parent. It also contained enzymes that hydrolyzed five of eight triazines that were not substrates for either starting enzyme. CONCLUSIONS: Permutation of nine amino acid differences resulted in a set of enzymes with surprisingly diverse patterns of reactions catalyzed. The functional richness of this small area of sequence space may aid our understanding of both natural and artificial evolution.

Aminohydrolases↗
PubMed2026

Identification and Catalytic Optimization of Pinene Oxidases in Paeoniflorin Biosynthetic Pathway.

Paeoniflorin is a pharmacologically important cage-like monoterpene glycoside characteristic of Paeonia plants, yet its biosynthetic pathway has remained largely unresolved, hindering sustainable production. Here, we confirmed that paeoniflorin biosynthesis originates from α-pinene and identified three novel cytochrome P450 enzymes that catalyze pinene oxidation. CYP71AN126 catalyzes the hydroxylation of α-pinene at positions C4 and C10, followed by further oxidation of the alcohol to a ketone at C4, whereas CYP76A225/226 exclusively catalyze C10 hydroxylation. Virus-induced gene silencing (VIGS) assays demonstrated that silencing CYP71AN126, but not CYP76A225 and CYP76A226, significantly reduced the paeoniflorin content, indicating that C4 hydroxylation plays an important role in paeoniflorin biosynthesis, whereas C10 hydroxylation is not. Through the analysis of natural sequence and activity divergence among CYP71AN126 and CYP76A225/226, combined with protein structure prediction and site-directed mutagenesis, we identified L493 as a critical residue involved in regulating catalytic site specificity and substrate specificity of CYP71AN126. Mutation of L493 reduced or eliminated the formation of undesired C10 hydroxylation side-product and enhanced substrate specificity. These findings establish C4 oxidation of α-pinene as the critical committed step in paeoniflorin biosynthesis. Our study lays a foundation for elucidating the complete biosynthetic pathway of paeoniflorin in Paeonia and provides a target for enzyme engineering of CYP71AN126 aimed at the efficient production of paeoniflorin via synthetic biology approaches.

Paeonia genus↗
PubMed1995

Molecular evolution in bacteria.

Recent advances in microbiology and molecular biology have a unifying influence on our understanding of genetic diversity/similarity and evolutionary relationships in microorganisms. This article attempts to unify information from diverse areas such as microbiology, molecular biology, microbial physiology, clay crystal genes, metals-microbe-clay interactions and bacterial DNA restriction-modification systems (R-M) as they may apply to molecular evolution of bacteria. The possibility is discussed that the first informational molecules may have been catalytic RNA (micro-assembler) not DNA (now the master copy) and these first micro-assemblers may have been precursors of ribosomes.

Adenosine Triphosphate↗
PubMed1996

Evolution of anthocyanin biosynthesis in maize kernels: the role of regulatory and enzymatic loci.

Understanding which genes contribute to evolutionary change and the nature of the alterations in them are fundamental challenges in evolution. We analyzed regulatory and enzymatic genes in the maize anthocyanin pathway as related to the evolution of anthocyanin-pigmented kernels in maize from colorless kernels of its progenitor, teosinte. Genetic tests indicate that teosinte possesses functional alleles at all enzymatic loci. At two regulatory loci, most teosintes possess alleles that encode functional proteins, but ones that are not expressed during kernel development and not capable of activating anthocyanin biosynthesis there. We investigated nucleotide polymorphism at one of the regulatory loci, cl. Several observations suggest that cl has not evolved in a strictly neutral manner, including an exceptionally low level of polymorphism and a biased representation of haplotypes in maize. Curiously, sequence data show that most of our teosinte samples possess a promoter element necessary for the activation of the anthocyanin pathway during kernel development, although genetic tests indicate that teosinte cl alleles are not active during kernel development. Our analyses suggest that the evolution of the purple kernels resulted from changes in cis regulatory elements at regulatory loci and not changes in either regulatory protein function nor the enzymatic loci.

Alleles↗
PubMed2003

Evolution of the protein repertoire.

Most proteins have been formed by gene duplication, recombination, and divergence. Proteins of known structure can be matched to about 50% of genome sequences, and these data provide a quantitative description and can suggest hypotheses about the origins of these processes.

Amino Acid Sequence↗
PubMed1993

Structural similarity of the binding sites of cyclophilin A-cyclosporin A and FKBP-FK506 systems.

Two enzymes, cyclophilin A and FK506-binding protein, with similar cis-trans isomerization catalytic activities but no similarity on the amino acid sequence level were compared in their three-dimensional structure by computer modelling. Cyclophilin A and FK506-binding protein proved to have similar arrangements at nine of the amino acids at their active site pockets. Two inhibitory peptides, cyclosporin A and FK506, also have structural similarities at their contact regions to the active sites. The studied systems may be another example of convergent evolution of enzyme catalytic site.

Amino Acid Isomerases↗
PubMed2003

Assembly of cell regulatory systems through protein interaction domains.

The sequencing of complete genomes provides a list that includes the proteins responsible for cellular regulation. However, this does not immediately reveal what these proteins do, nor how they are assembled into the molecular machines and functional networks that control cellular behavior. The regulation of many different cellular processes requires the use of protein interaction domains to direct the association of polypeptides with one another and with phospholipids, small molecules, or nucleic acids. The modular nature of these domains, and the flexibility of their binding properties, have likely facilitated the evolution of cellular pathways. Conversely, aberrant interactions can induce abnormal cellular behavior and disease. The fundamental properties of protein interaction domains are discussed in this review and in detailed reviews on individual domains at Science's STKE at http://www.sciencemag.org/cgi/content/full/300/5618/445/DC1.

Amino Acid Motifs↗
PubMed1988

Site-directed alteration of serine 82 causes nonproductive chain cleavage in prohistidine decarboxylase.

Prohistidine decarboxylase from Lactobacillus 30a normally autoactivates by cleavage of the Ser-81-Ser-82 peptide bond, converting Ser-82 to a pyruvoyl moiety which serves as the enzymatic cofactor. We have used site-directed methods to make two conservative mutations, converting Ser-82 to cysteine (S82C) and threonine (S82T). Both mutant proteins autoactivate, although dramatically (20- to 80-fold), more slowly than wild type. Roughly 55% of the mutant protein in each case undergoes a nonproductive chain cleavage which does not result in cofactor production. This finding suggests that an important feature in the enzyme's evolution has been development of an activation scheme which minimizes nonproductive side reactions. Catalytic constants are also affected by the mutations, particularly kcat which drops 8-fold in S82C and 450-fold in S82T. In addition, the S82T protein activates to produce a novel alpha-ketobutyroyl cofactor.

Carboxy-Lyases↗
PubMed2005

Directed coevolution of stability and catalytic activity in calcium-free subtilisin.

We have coevolved high activity and hyperstability in subtilisin by sequentially randomizing 12 amino acid positions in calcium-free subtilisin. The optimal amino acid for each randomized site was chosen based on stability and catalytic properties and became the parent clone for the next round of mutagenesis. Together, the 12 selected mutations increased the half-life of calcium-free subtilisin at elevated temperature by 15,000-fold. The catalytic properties of the mutants were examined against a range of substrates. In general, only mutations occurring at or near the substrate-binding surface have measurable effects on catalytic constants. No direct influence of stability on catalytic properties was observed. A high-stability mutant, Sbt140, was a more efficient enzyme in terms of k(cat)/K(m) than a commercial version of subtilisin across a range of substrates but had a lower k(cat) against tight-binding substrates. The reason for this behavior was discerned by examining microscopic rate constants for the hydrolysis of a tight-binding peptide substrate. Burst kinetics were observed for this substrate, indicating that acylation is not rate-limiting. Although acylation occurs at the rate of substrate binding, k(cat) is attenuated by the slow release of the N-terminal product. Natural evolution appears to have optimized catalytic activity against a range of sequences by achieving a balance between substrate binding and the rate of release of the N-terminal product.

Acylation↗
PubMed2005

A hypothetical pathway from the RNA to the DNA world.

If the DNA world was preceded by a RNA world as widely suggested a rational pathway should be discernable to link the two. This report uses as a starting point a membrane-enclosed ribozyme capable of polymerising itself and its counterpart copy. As molecular complexity increased, it is suggested that a consortia of the initial ribozyme polymerase and chaperone molecules formed a complex specifically for RNA replication. A mutation in one of several copy-genomes coding for these replication machines then led in step-wise fashion to a proto-ribosome that increasingly inserted specific amino acids instead of nucleotides into a growing RNA chain, the driving force being selection for improved or new function. Eventually the nucleotides would be entirely displaced in this proto-ribosome, after which the ribose-phosphate linkage would be replaced by peptide linkage. The final steps would be the formation of DNA from the RNA genomic material via reverse transcriptase, coupled with the evolution of enzymes for DNA polymerisation and transcription. At this point the original RNA-replicator machinery would be redundant and eliminated, the RNA genomic material would become mRNA and the present-day function of the ribosome would be fixed. In the scenario described a mechanism for the selection for L: -amino acids becomes evident.

Amino Acids↗
PubMed1994

The subunit interfaces of oligomeric enzymes are conserved to a similar extent to the overall protein sequences.

It is well established that, within families of homologous enzymes, amino acid residues that are involved in the chemistry of the reaction are highly conserved. To determine if residues at the subunit interface of oligomeric enzymes with shared active sites are also conserved, comparative analysis of five enzyme families was undertaken. For the chosen enzyme families, sequence data were available for a large number of proteins and a three-dimensional structure was known for at least two members of each family. The analysis indicates that the subunit interface and the hydrophobic core of proteins from all five families have diverged to a similar extent to the overall protein sequences.

Biological Evolution↗
PubMed2006

A systematic analysis of lineage-specific evolution in metabolic pathways.

In a search for the lineage-specific evolution of pathways between human, chimpanzee, mouse, and rat, orthologous gene families were generated from genome sequences. For each family, a model-based ratio of nonsynonymous to synonymous nucleotide substitution rates was calculated. Where the free-ratio model of individual ratios on each branch was supported, these families were mapped to two databases of metabolic pathways (KEGG and BioCyc) and the lineage-specific evolution of pathways was evaluated. The most similar pathway evolution was seen between mouse and rat, while the evolutionary pattern between human and chimpanzee was less correlated. Individual pathways in the human lineage were observed to evolve in a faster, lineage-specific manner, including the pathway involving arachidonic acid metabolism (identified through the KEGG analysis) and pyrimidine metabolism (identified through both analyses).

Animals↗
PubMed2002

Molecular basis of cold adaptation.

Cold-adapted, or psychrophilic, organisms are able to thrive at low temperatures in permanently cold environments, which in fact characterize the greatest proportion of our planet. Psychrophiles include both prokaryotic and eukaryotic organisms and thus represent a significant proportion of the living world. These organisms produce cold-evolved enzymes that are partially able to cope with the reduction in chemical reaction rates induced by low temperatures. As a rule, cold-active enzymes display a high catalytic efficiency, associated however, with a low thermal stability. In most cases, the adaptation to cold is achieved through a reduction in the activation energy that possibly originates from an increased flexibility of either a selected area or of the overall protein structure. This enhanced plasticity seems in turn to be induced by the weak thermal stability of psychrophilic enzymes. The adaptation strategies are beginning to be understood thanks to recent advances in the elucidation of the molecular characteristics of cold-adapted enzymes derived from X-ray crystallography, protein engineering and biophysical methods. Psychrophilic organisms and their enzymes have, in recent years, increasingly attracted the attention of the scientific community due to their peculiar properties that render them particularly useful in investigating the possible relationship existing between stability, flexibility and specific activity and as valuable tools for biotechnological purposes.

Acclimatization↗
PubMed1993

Salmonella reference collection B (SARB): strains of 37 serovars of subspecies I.

A reference collection of 72 strains representing 37 serovars of Salmonella subspecies I has been established for use in research on genetic and phenotypic variation in natural populations. Included are isolates of the host-adapted serovars S. choleraesuis, S. dublin, S. gallinarum, S. paratyphi A, S. paratyphi B, S. paratyphi C, S. pullorum, S. sendai, S. typhi and S. typhisuis, as well as strains of S. enteritidis, S. typhimurium, and other commonly recovered serovars with broad host ranges. The isolates were characterized by enzyme electrophoresis for allelic variation in 25 chromosomal genes and represent 71 distinctive multilocus genotypes (electrophoretic types or ETs). Genetic relationships among the ETs are indicated in an evolutionary tree constructed by the neighbour-joining method from a matrix of Nei's standard genetic distance.

Alleles↗
PubMed1999

Molecular phylogenetics and ecological diversification of the transisthmian fish genus Centropomus (Perciformes: Centropomidae).

Phylogenetic relationships among the 12 recognized fish species in the New World genus Centropomus (Pisces, Centropomidae) were analyzed using allozyme electrophoresis and 618 bp of the mitochondrial DNA 16S ribosomal RNA (rRNA) gene. Molecular phylogenetic trees were generally consistent with previously published partial hypotheses based on morphological evidence. However, previously undefined sister group relationships between major species groups were resolved using molecular data, and phylogenetic hypotheses for Centropomus based on 16S rRNA sequences were better supported than were allozyme-based hypotheses. The high level of congruence among the trees inferred from the nuclear and mitochondrial characters provided a firm phylogenetic basis for analysis of ecological diversification and molecular evolution in the genus. Compared to basal Centropomus species, members of the most nested species group were significantly larger in body size and occupied a marine niche only peripherally utilized by their congeners. We also observed substitution rate heterogeneity among 16S rRNA lineages; in contrast to expectations based on "metabolic rate" and "generation interval" models, relative substitution rates were faster than expected for the group of large-bodied snooks. Using the Pliocene rise of the Central American isthmian marine barrier to calibrate rates of 16S ribosomal gene evolution in Centropomus, we found that the rates for the genus were similar to those reported for higher vertebrates. Analysis of the three sets of transisthmian geminate taxa in Centropomus indicated that two of the pairs were probably formed during the Pliocene rise of the isthmus while the third pair diverged several million years earlier.

Animals↗