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PubMed1990

Plasmodium falciparum aldolase: gene structure and localization.

A genomic clone was isolated which codes for the fructose bisphosphate aldolase of Plasmodium falciparum. The aldolase gene is interrupted by one intron which divides the coding region into two exons. The first one codes for one amino acid only, the initiation methionine, while the second one encodes the residual 368 amino acids of the protein. The gene, which is represented only once in the genome, is transcribed at high rates as a 2.4-kb mRNA in the P. falciparum blood stage. The aldolase gene encodes a protein of 40,105 Da, which is 61-68% homologous to known eukaryotic aldolases. The protein was expressed in Escherichia coli cells in an unfused and enzymatically active form. Antisera raised against amino acids 9-96 recognize a 41-kDa protein band previously shown to protect monkeys against a P. falciparum infection. These antisera cross-react with aldolases of different species, which confirms the strong conservation of this enzyme during evolution. The aldolase could be localized in the cytoplasm of the parasite as an active and soluble form. An inactive form was found to be associated with the membrane fraction. Digestion data with phospholipase C suggest a membrane association of this polypeptide via a glycosylphosphatidylinositol anchor.

Amino Acid Sequence↗
PubMed1986

Is there a single origin of life?

The emergence of the first life on the earth is now established as an early event, and closely related to the evolving earth. Laboratory experiments examining possible chemical events have revealed a multitude of plausible pathways. Lack of knowledge of the primitive terrestrial conditions contemporary with the evolving prebolic organic chemistry limits reconstruction techniques. The primitive earth's aqueous history is essential to unraveling this problem. Based on our current knowledge of other planets of the solar system, we do not expect close analogues to the early earth. We still do not know if there was a second origin or if only earth has life. This may depend upon the question of the survival of information bearing chemical systems in a dynamic or chaotic environment and the chemical protection afforded within such a system. Water is the central molecule of controversy: the blessing and the curse of the chemist. New and novel chemical mechanisms and systems abound.

Enzymes↗
PubMed1991

Homochirality and stereospecific activity: evolutionary aspects.

The problem discussed in this paper is the connection between the unique property of biopolymers (proteins, DNA and RNA), i.e. homochirality, and their main functional property, i.e. self-replication. Our approach is based on an analysis of the conditions for the origination of the mechanism of self-replication of chiral polymers. It is demonstrated that self-replication could originate only on the basis of homochiral structures, possessing stereospecific (enzymatic) activity. It is also shown that complete breaking of the mirror symmetry of the organic medium is required both at the stage of polymeric takeover and at the stage of formation of structures possessing stereospecific activity. This requirement is satisfied only in the framework of the mechanism of spontaneous symmetry breaking i.e. the mechanism of non-equilibrium phase transition from the racemic state of the organic medium to the chirally pure one. The results obtained suggest that homochirality is a necessary condition for the origination of biological specificity and plays a fundamental role in the formation of structures capable of self-replication.

Biological Evolution↗
PubMed1991

Effect of varying the concentration of phenobarbital and its duration of treatment on the evolution of carcinogen induced enzyme-altered foci in rat liver.

The present study was aimed to investigate whether the promoting activity of phenobarbital in rodent liver is related to its daily dose level and duration of treatment or rather to its total dose administered. For this purpose groups of female Wistar rats were treated for 5 consecutive days with an initiating dose of 10 mg/kg body weight N-nitrosodiethylamine. Subsequently, rats were given phenobarbital-sodium (PB) in their drinking water at concentrations of 20, 50, 100 and 200 mg/l for varying lengths of time, such that the total dose of xenobiotic was very similar throughout the different treatment groups ranging from approximately 950 to 1100 mg/kg body weight. The number and volume fraction of lesions negative for the marker enzyme adenosine triphosphatase in liver were subsequently scored as a means to determine the carcinogenic response in this organ. Slight promoting effects of PB were only seen at the lowest concentration of 20 mg/l, whereas no significant effects were observed at 50 and 100 mg/l. At the highest concentration of 200 mg/l an inhibition of carcinogenic response was obtained. Although the effects seen in this study were only moderate, our data favour the idea that the promoting effects of PB depend on the actual concentration of the compound and the duration of treatment rather than on the total dose administered.

Adenosine Triphosphatases↗
PubMed1992

Allozymic and morphological differentiation among three South American frogs, genus Eupsophus (E. roseus, E. insularis and E. contulmoensis).

1. Three species of the frog, genus Eupsophus, were examined using electrophoresis at 21 loci, and nine morphological characters employing discriminant and principal components analyses. 2. The allozymic and morphological differentiation patterns do not agree. 3. It is suggested that selective pressures are responsible for the incongruence of both sets of characters.

Animals↗
PubMed1995

Phage display: protein engineering by directed evolution.

Phage display of proteins has become an important tool for protein engineering. Over the past year, the versatility of the technology has expanded to include the development of DNA-binding proteins with novel specificities, energetics of protein folding and directed evolution of antibodies. In addition, display of expressed cDNA libraries opens an exciting opportunity for studying protein-protein interactions.

Antibodies↗
PubMed1992

Evolution by acquisition: the case for horizontal gene transfers.

One of the most debated questions in the field of molecular evolution is the possible role of horizontal transfer in evolution. Of all the claims that have been made over the years, those reporting transfers between eukaryotes and prokaryotes are the most controversial. Here we present the cases for and against several such possible gene acquisitions.

Animals↗
PubMed2006

Enzyme promiscuity: evolutionary and mechanistic aspects.

The past few years have seen significant advances in research related to the 'latent skills' of enzymes - namely, their capacity to promiscuously catalyze reactions other than the ones they evolved for. These advances regard (i) the mechanism of catalytic promiscuity - how enzymes, that generally exert exquisite specificity, promiscuously catalyze other, and sometimes barely related, reactions; (ii) the evolvability of promiscuous functions - namely, how latent activities evolve further, and in particular, how promiscuous activities can firstly evolve without severely compromising the original activity. These findings have interesting implications on our understanding of how new enzymes evolve. They support the key role of catalytic promiscuity in the natural history of enzymes, and suggest that today's enzymes diverged from ancestral proteins catalyzing a whole range of activities at low levels, to create families and superfamilies of potent and highly specialized enzymes.

Animals↗
PubMed2005

Fluorescence resonance energy transfer as a probe of peptide cyclization catalyzed by nonribosomal thioesterase domains.

Macrocyclization of synthetic peptides by thioesterase (TE) domains excised from nonribosomal peptide synthetases (NRPS) has been limited to peptides that contain TE-specific recognition elements. To alter substrate specificity of these enzymes by evolution efforts, macrocyclization has to be detected under high-throughput conditions. Here we describe a method to selectively detect cyclic peptides by fluorescence resonance energy transfer (FRET). Using this method, picomolar detection limits were easily realized, providing novel entry for kinetic studies of catalyzed macrocyclization. Application of this method also provides an ideal tool to track TE-mediated peptide cyclization in real time. The general utility of FRET-assisted detection of cyclopeptides was demonstrated for two cyclases, namely tyrocidine (Tyc) TE and calcium-dependent antibiotic (CDA) TE. For the latter cyclase, this approach was combined with site-directed affinity labeling, opening the possibility for high-throughput enzymatic screening.

Affinity Labels↗
PubMed2006

A set of UV-inducible autolytic vectors for high throughput screening.

A high throughput screening scheme is often a prerequisite for directed evolution of enzymes or metagenomic analysis of DNA samples. For assaying intracellular enzymes of interest (e.g. when Escherichia coli is used), it requires cell lysis in many cases, chemical or enzymatic, which can be tedious and cost-consuming. In this study, a set of UV-inducible autolytic vectors was constructed to offer a simpler means of cell lysis that is free of additional liquid handling. The SRRz lysis gene cassette from bacteriophage Lambda was cloned downstream of a UV-inducible promoter, the recA promoter or the umuDC promoter, and further inserted into the backbone of pUC18, and transformed into E. coli BL21 cells. The SRRz expression and cell lysis was induced by UV irradiation. For both the recA and umuDC promoters, at 30 degrees C the lysis efficiency was found to be consistent and above 60% as measured using beta-galactosidase as the reporter. However, at 37 degrees C the lysis profiles were found to be erratic. UV lysis in 96-well plates also produced consistent lysis results that were comparable to those obtained by lysozyme treatment, demonstrating the utility of these autolytic vectors in high throughput screening. This set of artificial SRRz autolysis units should be transferable to other vectors. Surprisingly, it was found that the E. coli BL21(DE3) was also partially disrupted under UV irradiation, with a lysis efficiency of 44.5% at 30 degrees C, and 22.5% at 37 degrees C.

Bacteriolysis↗
PubMed2006

Stability and activity improvement of cephalosporin esterase EstB from Burkholderia gladioli by directed evolution and structural interpretation of muteins.

Esterase EstB from Burkholderia gladioli, which belongs to a family of esterases related to beta-lactamases and DD-peptidases was evolved for increased stability and simultaneously maintaining high cephalosporin C deacetylation activity. Random mutagenesis PCR was used to generate up to 5 aa substitutions per gene. A newly designed colony filter-screening assay, which was based on pH change after deacetylation of cephalosporin C in presence of DMF was established. In a first evolution round employing random mutagenesis, which included about 10(6) mutants, a set of interesting mutants was isolated. Distinct mutations identified as significant for stability were combined by a rational recombination step and the resulting recombinant was further evolved by an additional random mutagenesis round. After screening an additional 10(5) clones, it was possible to isolate a variant of EstB having more than 100-fold better activity in reactions containing 35% DMF. This mutant also showed a high increase in temperature stability (T(m) was raised by 13 degrees C) and retained high activity towards cephalosporin C under standard assay conditions. The molecular effects of mutations found in random mutants are discussed in view of the three-dimensional structure of wild-type EstB.

Amino Acid Sequence↗
PubMed2004

Interconverting the catalytic activities of (betaalpha)(8)-barrel enzymes from different metabolic pathways: sequence requirements and molecular analysis.

The (betaalpha)(8)-barrel enzymes N'-[(5'-phosphoribosyl)formimino]-5-aminoimidazole-4-carboxamide ribonucleotide isomerase (tHisA) and imidazole glycerol phosphate synthase (tHisF) from Thermotoga maritima catalyze two successive reactions in the biosynthesis of histidine. In both enzymes, aspartate residues at the C-terminal end of beta-strand 1 (Asp8 in tHisA and Asp11 in tHisF) and beta-strand 5 (Asp127 in tHisA and Asp130 in tHisF) are essential for catalytic activity. It was demonstrated earlier that in tHisA the substitution of Asp127 by valine (tHisA-D127V) generates phosphoribosylanthranilate isomerase (TrpF) activity, a related (betaalpha)(8)-barrel enzyme participating in tryptophan biosynthesis. It is shown here that in tHisF the corresponding substitution of Asp130 by valine (tHisF-D130V) also generates TrpF activity. To determine the effectiveness of individual amino acid exchanges in these conversions, each of the 20 standard amino acid residues was introduced at position 127 of tHisA and 130 of tHisF by saturation random mutagenesis. The tHisA-D127X and tHisF-D130X variants with TrpF activity were identified by selection in vivo, and the proteins purified and characterized. The results obtained show that removal of the negatively charged carboxylate side-chain at the C-terminal end of beta-strand 5 is sufficient to establish TrpF activity in tHisA and tHisF, presumably because it allows the binding of the negatively charged TrpF substrate, phosphoribosylanthranilate. In contrast, the double mutants tHisA-D8N+D127V and tHisF-D11N+D130V did not show detectable activity, demonstrating that the aspartate residues at the C-terminal end of beta-strand 1 are essential for catalysis of the TrpF reaction. The ease with which TrpF activity can be established on both the tHisA and tHisF scaffolds supports the evolutionary relationship of these three enzymes and highlights the functional plasticity of the (betaalpha)(8)-barrel enzyme fold.

Aldose-Ketose Isomerases↗
PubMed2005

Minimization and stabilization of the Mycobacterium tuberculosis recA intein.

Many naturally occurring inteins consist of two functionally independent domains, a protein-splicing domain and an endonuclease domain. In a previous study, a 168 amino acid residue mini-intein was generated by removal of the central endonuclease domain of the 440 residue Mycobacterium tuberculosis (Mtu) recA intein. In addition, directed evolution experiments identified a mutation, V67L, that improved the activity of the mini-intein significantly. A recent crystal structure shows that the loop connecting two beta-strands from the N-terminal and C-terminal intein subdomains of the mini-intein is disordered. The goals of the present study were to generate smaller mini-intein derivatives and to understand the basis for reversal of the splicing defect by the V67L mutation. Guided by the structural information, we generated a number of derivatives 135 to 152 residues in length, with V67 or L67. All of the new minimal inteins are functional in splicing. In vivo selection experiments for function showed that by removal of the loop region, 137 residues may be the lower limit for full protein-splicing activity. In addition, the activation effect of the V67L mutation was observed to be universal for mini-inteins longer than 137 residues. Structural and functional analyses indicate that the role of the mutation is in stabilization of the mini-intein core.

Directed Molecular Evolution↗
PubMed2004

Reduction of intrinsic kinetic and thermodynamic barriers for enzyme-catalysed proton transfers from carbon acid substrates.

Many enzymes catalyse the heterolytic abstraction of the alpha-proton from a carbon acid substrate. Gerlt and Gassman have applied Marcus formalism to such proton transfer reactions to argue that transition states for concerted general acid-general base catalysed enolization at enzyme active sites occur late on the reaction coordinate (J. Am. Chem. Soc. 115 (1993) 11552). We postulate that as an enzyme evolves, it may decrease deltaG++ for a proton transfer step associated with substrate enolization by following the path of steepest descent on the two-dimensional surface corresponding to deltaG++, as defined by Marcus formalism. We show that for an enzyme that has decreased deltaG++ following the path of steepest descent, the values of the intrinsic kinetic (deltaG++(int,E)) and thermodynamic (deltaG(E)0) barriers for proton transfer reactions on the enzyme may be predicted from the known values of deltaG++(int,N) and deltaG(N)0 for the corresponding non-enzymic reaction and the free energy of activation on the enzyme (deltaG++(E)). In addition, the enzymic transition state will occur later on the reaction coordinate than the corresponding non-enzymic transition state (i.e. x++(E)>x++(N)) if the condition (6 - square root 2)/8 2deltaG++(int,N).

Animals↗
PubMed2004

Transcription factors as tools for metabolic engineering in plants.

The functions of an increasing number of plant transcription factors are being elucidated, and many of these factors have been found to impact flux through metabolic pathways. Because transcription factors, as opposed to most structural genes, tend to control multiple pathway steps, they have emerged as powerful tools for the manipulation of complex metabolic pathways in plants. The review describes the highlights of recent experiments that have targeted transcription factors that control plant metabolic pathways, and discusses their potential as tools for metabolic engineering.

Enzymes↗