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At least 253 records · Page 14Linked to original sources
PubMed2001

Functional evolution of two subtly different (similar) folds.

BACKGROUND: The function of proteins is a direct consequence of their three-dimensional structure. The structural classification of proteins describes the ways of folding patterns all proteins could adopt. Although, the protein folds were described in many ways the functional properties of individual folds were not studied. RESULTS: We have analyzed two beta-barrel folds generally adopted by small proteins to be looking similar but have different topology. On the basis of the topology they could be divided into two different folds named SH3-fold and OB-fold. There was no sequence homology between any of the proteins considered. The sequence diversity and loop variability was found to be important for various binding functions. CONCLUSIONS: The function of Oligonucleotide/oligosaccharide-binding (OB) fold proteins was restricted to either DNA/RNA binding or sugar binding whereas the Src homology 3 (SH3) domain like proteins bind to a variety of ligands through loop modulations. A question was raised whether the evolution of these two folds was through DNA shuffling.

Amino Acid Sequence↗
PubMed2003

Molecular basis for the subtype discrimination of the estrogen receptor-beta-selective ligand, diarylpropionitrile.

Although the two subtypes of the human estrogen receptor (ER), ERalpha and ERbeta, share only 56% amino acid sequence identity in their ligand binding domain (LBD), the residues that surround the ligand are nearly identical; nevertheless, subtype-selective ligands are known. To understand the molecular basis by which diarylpropionitrile (DPN), an ERbeta-selective ligand, is able to discriminate between the two ERs, we examined its activity on ER mutants and chimeric constructs generated by DNA shuffling. The N-terminal region of the ERbeta LBD (through helix 6) appears to be fully responsible for the ERbeta selectivity of DPN. In fact, a single ERalpha point mutation (L384M) was largely sufficient to switch the DPN response of this ER to that of the ERbeta type, but residues in helix 3 are also important in achieving the full ERbeta selectivity of DPN. Using molecular modeling, we found an energetically favorable fit for the S-DPN enantiomer in ERbeta, in which the proximal phenol mimics the A ring of estradiol, and the nitrile engages in stabilizing interactions with residues in the ligand-binding pocket of ERbeta. Our findings highlight that a limited number of critical interactions of DPN with the ERbeta ligand-binding pocket underlie its ER subtype-selective character.

Amino Acid Sequence↗
PubMed2006

Steady-state kinetic characterization of evolved biphenyl dioxygenase, which acquired novel degradation ability for benzene and toluene.

Biphenyl dioxygenase (Bph Dox) catalyzes initial oxygenation in the bacterial biphenyl degradation pathway. Bph Dox in Pseudomonas pseudoalcaligenes KF707 is a Rieske type three-component enzyme in which a large subunit (encoded by the bphA1 gene) plays an important role in the substrate specificity of Bph Dox. Steady-state kinetic assays using purified enzyme components demonstrated that KF707 Bph Dox had a kcat/Km of 33.1 x 10(3) (M(-1) s(-1)) for biphenyl. Evolved 1072 Bph Dox generated by the process of DNA shuffling (Suenaga, H. et al., J. Bacteriol., 184, 3682-3688 (2002)) exhibited enhanced degradation activity not only for biphenyl (kcat/Km of 62.2 x 10(3) [M(-1) s(-1)]) but also for benzene and toluene, compounds that are rarely attacked by KF707 Bph Dox. These results suggest that evolved 1072 Bph Dox acquires higher affinities and catalytic efficiencies for various substrates than the original KF707 enzyme.

Benzene↗
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↗
PubMed2006

Mutant library construction in directed molecular evolution: casting a wider net.

Directed molecular evolution imitates the natural selection process in the laboratory to find mutant proteins with improved properties in the expected aspects by exploring the encoding sequence space. The success of directed molecular evolution experiment depends on the quality of artificially prepared mutant libraries and the availability of convenient high-throughput screening methods. Well-prepared libraries promise the possibility of obtaining desired mutants by screening a library containing a relatively small number of mutants. This article summarizes and reviews the currently available methodologies widely used in directed evolution practices in the hope of providing a general reference for library construction. These methods include error-prone polymerase chain reaction (epPCR), oligonucleotide-based mutagenesis, and genetic recombination exemplified by DNA shuffling and its derivatives. Another designed method is also discussed, in which B-lymphocytes are fooled to mutate nonantibody foreign proteins through somatic hypermutation (SHM).

Animals↗
PubMed2002

Expanding the natural repertoire of protein structure and function.

This review considers chemical and genetic approaches to the modification of protein structure. The historical interest in chemical and site-directed modifications will be briefly covered. Current chemical modification strategies will be presented. Biosynthetic mutagenesis with unnatural aminoacyl-tRNAs and current synthetic peptide ligation technologies will be covered in greater detail. The application of combinatorial genetic methods (e.g. phage display, DNA shuffling) to protein engineering with unnatural amino acids will be briefly discussed, with emphasis on the in vitro evolution of new enzymatic function (i.e. aminoacyl-tRNA synthetases). Throughout the review, the powerful insights gained from the combined use of these technologies will be illustrated by examples that focus on the elucidation of protein-ligand interactions.

Amino Acid Sequence↗
PubMed2002

Designing proteins that work using recombinant technologies.

Therapeutic proteins have been engineered for a variety of purposes including reduced antigenicity, longer half-life, simplified process development, and increased affinity. Fusion proteins bring together functions from two different molecules creating therapeutics with completely novel activities. Protein engineering technologies have relied on rational design, directed evolution, DNA shuffling, RNA-peptide fusion, phage and ribosomal display methods to select out candidate protein forms with the desired therapeutic properties. Engineered site-specific pegylation and glycosylation strategies have improved circulation half-life, reduced immunogenicity and increased protein therapeutic stability. In this review we describe how protein engineering techniques have been used to select out, improve stability and clinical efficacy of protein therapeutics.

Animals↗
PubMed2000

Biochemical and genetic bases of microbial degradation of polychlorinated biphenyls (PCBs).

The microbial degradation of polychlorinated biphenyls (PCBs) has been extensively conducted by many workers, and the following general results have been obtained. (1) PCBs are degraded oxidatively by aerobic bacteria and other microorganisms such as white rot fungi. PCBs are also reductively dehalogenated by anaerobic microbial consortia. (2) The biodegradability of PCBs is highly dependent on chlorine substitution, i.e., number and position of chlorine. The degradation and dehalogenation capabilities are also highly strain dependent. (3) Biphenyl-utilizing bacteria can cometabolize many PCB congeners to chlorobenzoates by biphenl-catabolic enzymes. (4) Enzymes involved in the PCB degradation were purified and characterized. Biphenyl dioxygenase, ring-cleavage dioxygenase, and hydrolase are crystallized, and two ring-cleavage dioxygenases are being solved by x-ray crystallography. (5) The bph gene clusters responsible for PCB degradation are cloned from a variety of bacterial strains. The structure and function are analyzed with respect to the evolutionary relationship. (6) The molecular engineering of biphenyl dioxygenases is successfully performed by DNA shuffling, domain exchange, and subunit exchange. The evolved enzymes exhibit wide and enhanced degradation capacities for PCBs and other aromatic compounds.

Journal Article↗
PubMed2000

Directed evolution of a novel N-carbamylase/D-hydantoinase fusion enzyme for functional expression with enhanced stability.

Bifunctional enzymes find a wide application as a monitoring facility and a potential biocatalyst in molecular biology and biotechnology. Recombination of natural enzymes to a bifunctional fusion offers valuable tools, but the functional and structural instability of artificial fusion enzymes remains to be solved. Based on structural traits of microbial D-hydantoinase, we attempted to construct a bifunctional N-carbamylase/D-hydantoinase fusion enzyme that would be useful for the synthesis of nonnatural D-amino acids in a concerted fashion. The bifunctional ability of D-hydantoinase, as a fusion partner, was noticeable, but the resulting fusion enzyme was subjected to serious proteolysis in vivo, as generally encountered in the expression of large the multidomain polypeptide in E. coli. In an effort to improve the structural instability imposed by artificial linear fusion, directed evolution of the fusion enzyme was performed using DNA shuffling with a consensus primer to maintain a crucial domain for the enzyme activity. The evolved fusion enzyme, F11, was selected after repeated rounds, and this enzyme was found to show sixfold increased performance in the production of D-amino acid compared with the parent fusion enzyme, which was mainly due to the enhanced structural stability of the evolved fusion enzyme. This result is an example showing that directed evolution of the linearly fused polypeptide may broaden the opportunity to generate a fusion enzyme with greater potential.

Amidohydrolases↗
PubMed2000

[Molecular modification and artificial evolution of microbial enzymes].

There exists a remarkably progress in molecular modification and artificial evolution of microbial enzymes in recent years. Error-prone polymerase chain reaction and DNA shuffling have been developed and successfully applied and a lot of startling achievements have been obtained in artificial evolution of enzymes.

Bacteria↗
PubMed1988

Molecular basis for the defective expression of the mouse Ew17 beta gene.

Four of the eleven independent H-2 haplotypes of inbred mouse strains and approximately 15% of wild mouse chromosomes 17 fail to express the E alpha E beta class II histocompatibility (Ia) Ag. These E- haplotypes are defective in the expression of the E alpha and/or the E beta chain. None of the E beta defects has previously been described at the molecular level. In this study, we report the molecular basis for the defective expression of the E beta gene from the w17 haplotype of the H-2 congenic strain B10.CAS2, derived from wild Mus musculus castaneus. Comparison of the Ew17 beta genomic sequence to those of the functional Eb beta and Ed beta genes reveals a single base insertion in the RNA donor splice site of the first intron. By DNA shuffling, we have corrected the single base mutation, and we show by FACS analysis and 2-D PAGE of immunoprecipitates that the corrected Ew17 beta is expressed in L cells when co-transfected with an Ed alpha gene. Conversely, an Eb beta gene construct containing the mutant RNA splice site from Ew17 beta is not expressed. We conclude that the single base insertion in the first RNA splice donor site is the sole molecular defect in the Ew17 beta gene.

Amino Acid Sequence↗
PubMed2006

Family shuffling of soil DNA to change the regiospecificity of Burkholderia xenovorans LB400 biphenyl dioxygenase.

Previous work has shown that the C-terminal portion of BphA, especially two amino acid segments designated region III and region IV, influence the regiospecificity of the biphenyl dioxygenase (BPDO) toward 2,2'-dichlorobiphenyl (2,2'-CB). In this work, we evolved BPDO by shuffling bphA genes amplified from polychlorinated biphenyl-contaminated soil DNA. Sets of approximately 1-kb DNA fragments were amplified with degenerate primers designed to amplify the C-terminal portion of bphA. These fragments were shuffled, and the resulting library was used to replace the corresponding fragment of Burkholderia xenovorans LB400 bphA. Variants were screened for their ability to oxygenate 2,2'-CB onto carbons 5 and 6, which are positions that LB400 BPDO is unable to attack. Variants S100, S149, and S151 were obtained and exhibited this feature. Variant S100 BPDO produced exclusively cis-5,6-dihydro-5,6-dihydroxy-2,2'-dichlorobiphenyl from 2,2'-CB. Moreover, unlike LB400 BPDO, S100 BphA catalyzed the oxygenation of 2,2',3,3'-tetrachlorobiphenyl onto carbons 5 and 6 exclusively and it was unable to oxygenate 2,2',5,5'-tetrachlorobiphenyl. Based on oxygen consumption measurements, variant S100 oxygenated 2,2'-CB at a rate of 16 +/- 1 nmol min(-1) per nmol enzyme, which was similar to the value observed for LB400 BPDO. cis-5,6-Dihydro-5,6-dihydroxy-2,2'-dichlorobiphenyl was further oxidized by 2,3-dihydro-2,3-dihydroxybiphenyl dehydrogenase (BphB) and 2,3-dihydroxybiphenyl dioxygenase (BphC). Variant S100 was, in addition, able to oxygenate benzene, toluene, and ethyl benzene. Sequence analysis identified amino acid residues M237 S238 and S283 outside regions III and IV that influence the activity toward doubly ortho-substituted chlorobiphenyls.

Amino Acid Sequence↗
PubMed2001

A DNA vaccine based on a shuffled E7 oncogene of the human papillomavirus type 16 (HPV 16) induces E7-specific cytotoxic T cells but lacks transforming activity.

Vaccination with oncogene-derived DNA for anti-cancer treatment carries a risk of de-novo tumor induction triggered by the persisting recombinant DNA. We hypothesized that an oncoprotein whose primary sequence has been rearranged ('shuffled') to maintain all possible T cell epitopes still induces cytotoxic T cells against the authentic protein but is devoid of transforming properties. As a model antigen, we used the E7 oncoprotein of the human papillomavirus (HPV) type 16, the major cause of cervical cancer. We have generated an artificial E7 molecule in which four domains were rearranged and, in order to maintain all possible T cell epitopes, certain sequences were duplicated. Upon transfection of this shuffled E7 gene (E7SH) into RMA cells, presentation of an E7 Db-restricted T cell epitope was shown by an E7-specific CTL line in vitro. Immunization of C57BL/6 mice with E7SH DNA induced E7-specific CTL and also conveyed protection against E7-positive syngeneic tumor cells. No transforming activity of E7SH DNA in NIH3T3 cells was detected, as determined by focus formation, induction of S-phase under conditions of serum deprivation and degradation of endogenous pRB. Our results suggest that DNA shuffling may become a promising concept for DNA-based anti-cancer vaccines.

3T3 Cells↗
PubMed1994

Use of a photoactive derivative of actinomycin to investigate shuffling between binding sites on DNA.

A photoreactive analog of actinomycin (7-azidoactinomycin D) has been used in experiments to probe directly the shuffling hypothesis of Fox and Waring [Fox, K. R., & Waring, M. J. (1984) Nucleic Acids Res. 12, 9271-9285]. According to this theory, actinomycin D molecules initially interact with non-sequence-specific sites on DNA and subsequently "shuffle" along the polymer in a one-dimensional migratory fashion so as to locate their preferred sequence-dependent binding sites. In the study presented here, the drug-DNA complex was allowed to equilibrate (in the dark) for various periods of time, followed by photolysis which renders the complex irreversible and traps the ligand at its instantaneous binding sites. Visualization of the piperidine-labile sites and investigation of how the intensity of reaction at each site changes with time can provide direct confirmation of the shuffling hypothesis. The data reveal that actinomycin D does indeed engage in shuffling along the DNA. After only short equilibration times (20 s) actinomycin D is observed to bind to a variety of sites on the DNA, including many which are not regarded as canonical preferred binding sites at equilibrium. However, after longer periods of equilibration the intensity of reaction is shown to drop as a function of time at nonspecific sites, with corresponding increase at sequence-specific sites. In addition, base sequences which flank the intercalation sites can be seen to play a major role in influencing the binding and sequence specificity of actinomycin.

Base Sequence↗
PubMed1990

DNA sequences of genes encoding Acinetobacter calcoaceticus protocatechuate 3,4-dioxygenase: evidence indicating shuffling of genes and of DNA sequences within genes during their evolutionary divergence.

The DNA sequence of a 2,391-base-pair HindIII restriction fragment of Acinetobacter calcoaceticus DNA containing the pcaCHG genes is reported. The DNA sequence reveals that A. calcoaceticus pca genes, encoding enzymes required for protocatechuate metabolism, are arranged in a single transcriptional unit, pcaEFDBCHG, whereas homologous genes are arranged differently in Pseudomonas putida. The pcaG and pcaH genes represent separate reading frames respectively encoding the alpha and beta subunits of protocatechuate 3,4-dioxygenase (EC 1.13.1.3); previously a single designation, pcaA, had been used to represent DNA encoding this enzyme. The alpha and beta protein subunits appear to share common ancestry with each other and with catechol 1,2-dioxygenases from A. calcoaceticus and P. putida. Marked conservation of amino acid sequence is observed in a region containing two histidyl residues and two tyrosyl residues that appear to ligate iron within each oxygenase. In some regions within the aligned oxygenase sequences, DNA sequences appear to be conserved at a level beyond the extent that might have been demanded by selection at the level of protein. In other regions, divergence of DNA sequences appears to have been achieved by substitution of DNA sequence from one genetic segment into another. The results are interpreted to be the consequence of sequence exchange by gene conversion between slipped strands of DNA during evolutionary divergence; mismatch repair between slipped strands may contribute to the maintenance of DNA sequence in divergent genes.

Acinetobacter↗
PubMed1999

Genetic manipulation of Kluyveromyces lactis linear DNA plasmids: gene targeting and plasmid shuffles.

Genetic manipulation of yeast linear DNA plasmids, particularly of k1 and k2 from the non-conventional dairy yeast Kluyveromyces lactis, has been advanced by the recent establishment of DNA transformation-mediated one-step gene disruption and allele replacement techniques. These methods provide the basis for a strategy for the functional analysis of plasmid genes and DNA elements. By use of double selection regimens, these single-gene procedures have been extended to effect disruption of individual genes on plasmid k2 and transplacement of a functional copy onto plasmid k1, resulting in the production of yeast strains with an altered plasmid composition. This cytoplasmic gene shuffle system facilitates the introduction of specifically modified alleles into k1 or k2 in order to study the function, expression (from UCS promoters) and regulation of cytoplasmic linear plasmid genes. Additionally, identification, characterization and localization of plasmid gene products of interest are made possible by shuffling GFP-, epitope- or affinity purification-tagged alleles between k2 and k1. The gene shuffle approach can also be used for vector development and heterologous protein expression in order to exploit the biotechnical potential of the K. lactis k1/k2 system in yeast cell factory research.

Biological Evolution↗