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PubMedDate not supplied

Incorporation of alkaline phosphatase into layer-by-layer polyelectrolyte films on the surface of affi-gel heparin beads: physicochemical characterization and evaluation of the enzyme stability.

The preparation of functionalized beads in the micrometer size range that can be used to probe the action of immobilized biomolecules on cell cultures during controlled periods of time is of fundamental importance in cell biology. However, the preparation and characterization of such particles is tedious because of their fast sedimentation. It is hence difficult to prepare such beads in a reproducible manner. This highlights the need to prepare an important batch of functionnalized particles and to store them under conditions where the loss of biological activity is minimized. The aim of this paper was to immobilize alkaline phosphatase (AP) as a model enzyme on the surface of Affi-gel heparin beads functionnalized by means of a layer-by-layer (LBL) film made of poly-l-glutamic (PGA) acid and poly-l-lysine (PLL). The enzyme has been adsorbed either on the top of the LBL film or embedded under five polyelectrolyte layers. When embedded, the enzyme was not released in buffer and retained more than 30% of its initial activity after 3 months of storage at 4 degrees C. However, when the enzyme was adsorbed on top of the LBL film, about 80% of the adsorbed enzyme was released in the buffer after a few days of storage. Longer storage did not lead to any further desorption and the remaining enzyme displayed the same evolution of its activity with time as the embedded enzyme. The time evolution of the enzyme activity on the beads is compared with that in solution alone and in the presence of PGA and PLL separately.

Alkaline Phosphatase↗
PubMed1998

Correlation between the substitution rate and rate variation among sites in protein evolution.

It is well known that the rate of amino acid substitution varies among different proteins and among different sites of a protein. It is, however, unclear whether the extent of rate variation among sites of a protein and the mean substitution rate of the protein are correlated. We used two approaches to analyze orthologous protein sequences of 51 nuclear genes of vertebrates and 13 mitochondrial genes of mammals. In the first approach, no assumptions of the distribution of the rate variation among sites were made, and in the second approach, the gamma distribution was assumed. Through both approaches, we found a negative correlation between the extent of among-site rate variation and the average substitution rate of a protein. That is, slowly evolving proteins tend to have a high level of rate variation among sites, and vice versa. We found this observation consistent with a simple model of the neutral theory where most sites are either invariable or neutral. We conclude that the correlation is a general feature of protein evolution and discuss its implications in statistical tests of positive Darwinian selection and molecular time estimation of deep divergences.

Animals↗
PubMed2000

Rates of evolution of pyridoxal-5'-phosphate-dependent enzymes.

The pyridoxal-5'-phosphate-dependent enzymes (B(6) enzymes), that operate in the metabolism of amino acids, are of multiple evolutionary origin. To estimate their rates of evolution, a total of 180 sequences of 21 B(6) enzymes from distantly related eukaryotic species were compared. The enzymes belong to all four evolutionarily independent families of B(6) enzymes with different folds, i.e., the large alpha family, the beta family, the d-alanine aminotransferase family, and the alanine racemase family. Their unit evolutionary periods, i.e., the time for a 1% sequence difference to accumulate between branches, ranged from 4.6 to 45.1 million years. Both, fastest changing serine pyruvate aminotransferase and most slowly changing glutamate decarboxylase are members of the alpha family. The evolutionary rates of the few enzymes belonging to the other three families were interspersed among those of the alpha family members. Enzymes that catalyze the same reaction, e.g., transamination or amino acid decarboxylation, with different substrates show widely varying rates. The absence of correlations of the rate of evolution with either protein fold or type of catalyzed reaction suggests that individual functional constraints have determined the differential rates of evolution of B(6) enzymes.

Animals↗
PubMed2004

Mechanistic analogies amongst carbohydrate modifying enzymes.

Carbohydrates are known to play essential roles in a myriad of biological processes. The enormous complexity of the various oligosaccharide structures found in biology is derived from a rational orchestration of the enzymatic formation and breakdown of glycosidic linkages achieved by glycosyltransferases, glycosidases and phosphorylases. A detailed understanding of the chemical mechanisms by which these classes of enzymes function not only provides a rational basis for their engineering and application in both the development and synthesis of new classes of therapeutic agents, but also provides insight into the role of convergence in the natural evolution of enzyme function.

Animals↗
PubMed2002

Molecular evolution of the lysine biosynthetic pathways.

Among the different biosynthetic pathways found in extant organisms, lysine biosynthesis is peculiar because it has two different anabolic routes. One is the diaminopimelic acid pathway (DAP), and the other over the a-aminoadipic acid route (AAA). A variant of the AAA route that includes some enzymes involved in arginine and leucine biosyntheses has been recently reported in Thermus thermophilus (Nishida et al. 1999). Here we describe the results of a detailed genomic analysis of each of the sequences involved in the two lysine anabolic routes, as well as of genes from other routes related to them. No evidence was found of an evolutionary relationship between the DAP and AAA enzymes. Our results suggest that the DAP pathway is related to arginine metabolism, since the lysC, asd, dapC, dapE, and lysA genes from lysine biosynthesis are related to the argB, argC, argD, argE, and speAC genes, respectively, whose products catalyze different steps in arginine metabolism. This work supports previous reports on the relationship between AAA gene products and some enzymes involved in leucine biosynthesis and the tricarboxylic acid cycle (Irvin and Bhattacharjee 1998; Miyazaki et al. 2001). Here we discuss the significance of the recent finding that several genes involved in the arginine (Arg) and leucine (Leu) biosynthesis participate in a new alternative route of the AAA pathway (Miyazaki et al. 2001). Our results demonstrate a clear relationship between the DAP and Arg routes, and between the AAA and Leu pathways.

Amino Acid Sequence↗
PubMed2005

The hybrid enzymes from alpha-aspartyl dipeptidase and L-aspartase.

With combinative functionalities as well as the improved activity and stability, the novel hybrid enzymes (HEs) from the heterogeneous enzymes of alpha-aspartyl dipeptidase (PepE, monomer) and l-aspartase (l-AspA, tetramer) were constructed successfully by gene random deletion strategy. The wild-type hybrid enzyme (WHE) and the evolved hybrid enzyme (EHE) were selected, respectively, upon the phenotype and the enzyme activity. The relative activity of the WHE tested was about 110% of the wild-type PepE and 26% of the wild-type l-AspA, whilst the activity of EHE was about 340% of the PepE and 87% of the l-AspA. In comparison to its individual wild-type enzymes, the EHE exhibited an improved thermostability, when examined at the enzyme concentration of 10(-7)mol/L, but the WHE showed a reduced thermostability. The activity of the EHE was about 3-fold compared to that of the WHE. The current results give a good example that the hybridization of enzymes could be attained between the monomer and multimer enzymes. In addition, they also indicate that construction hybrid enzyme from evolved enzymes is feasible.

Aspartate Ammonia-Lyase↗
PubMed2006

The tryptophan oxidation pathway in mosquitoes with emphasis on xanthurenic acid biosynthesis.

Oxidation of tryptophan to kynurenine and 3-hydroxykynurenine (3-HK) is the major catabolic pathway in mosquitoes. However, 3-HK is oxidized easily under physiological conditions, resulting in the production of reactive radical species. To overcome this problem, mosquitoes have developed an efficient mechanism to prevent 3-HK from accumulating by converting this chemically reactive compound to the chemically stable xanthurenic acid. Interestingly, 3-HK is a precursor for the production of compound eye pigments during the pupal and early adult stages; consequently, mosquitoes need to preserve and transport 3-HK for compound eye pigmentation in pupae and adults. This review summarizes the tryptophan oxidation pathway, compares and contrasts the mosquito tryptophan oxidation pathway with other model species, and discusses possible driving forces leading to the functional adaptation and evolution of enzymes involved in the mosquito tryptophan oxidation pathway.

Alanine Transaminase↗
PubMed2006

Recognition of a complex substrate by the KsgA/Dim1 family of enzymes has been conserved throughout evolution.

Ribosome biogenesis is a complicated process, involving numerous cleavage, base modification and assembly steps. All ribosomes share the same general architecture, with small and large subunits made up of roughly similar rRNA species and a variety of ribosomal proteins. However, the fundamental assembly process differs significantly between eukaryotes and eubacteria, not only in distribution and mechanism of modifications but also in organization of assembly steps. Despite these differences, members of the KsgA/Dim1 methyltransferase family and their resultant modification of small-subunit rRNA are found throughout evolution and therefore were present in the last common ancestor. In this paper we report that KsgA orthologs from archaeabacteria and eukaryotes are able to complement for KsgA function in bacteria, both in vivo and in vitro. This indicates that all of these enzymes can recognize a common ribosomal substrate, and that the recognition elements must be largely unchanged since the evolutionary split between the three domains of life.

Amino Acid Motifs↗
PubMed2001

Evidence for three distinct hydrogenase activities in Rhodospirillum rubrum.

Inducer, inhibitor, and mutant studies on three hydrogenase activities of Rhodospirillum rubrum indicate that they are mediated by three distinct hydrogenase enzymes. Uptake hydrogenase mediates H2 uptake to an unknown physiological acceptor or methylene blue and is maximally synthesized during autotrophic growth in light. Formate-linked hydrogenase is synthesized primarily during growth in darkness or when light becomes limiting, and links formate oxidation to H2 production. Carbon-monoxide-linked hydrogenase is induced whenever CO is present and couples CO oxidation to H2 evolution. The enzymes can be expressed singly or conjointly depending on growth conditions, and the inhibitor or inducer added. All three hydrogenases can use methyl viologen as the mediator for both the H2 evolution and H2 uptake reactions while displaying distinct pH optima, reversibility, and sensitivity to C2H2 gas. Yet, we present evidence that the CO-linked hydrogenase, unlike the uptake hydrogenase, does not link to methylene blue as the electron acceptor. These differences allow conditions to be established to quantitatively assay each hydrogenase independently of the others both in vivo and in vitro.

Acetylene↗
PubMed2001

Dynamic regulation of the tryptophan operon: a modeling study and comparison with experimental data.

A mathematical model for regulation of the tryptophan operon is presented. This model takes into account repression, feedback enzyme inhibition, and transcriptional attenuation. Special attention is given to model parameter estimation based on experimental data. The model's system of delay differential equations is numerically solved, and the results are compared with experimental data on the temporal evolution of enzyme activity in cultures of Escherichia coli after a nutritional shift (minimal + tryptophan medium to minimal medium). Good agreement is obtained between the numeric simulations and the experimental results for wild-type E. coli, as well as for two different mutant strains.

Anthranilate Synthase↗
PubMed2004

Enzymes in organic media. Forms, functions and applications.

Enzyme catalysis in low water containing organic solvents is finding an increasing number of applications in diverse areas. This review focuses on some aspects which have not been reviewed elsewhere. Different strategies for obtaining higher activity and stability in such media are described. In this context, the damaging role of lyophilization and the means of overcoming such effects are discussed. Ultrasonication and microwave assistance are two emerging approaches for enhancing reaction rates in low water media. Control of water activity and medium engineering are two crucial approaches in optimization of catalytic behaviour in nonaqueous enzymology. Organometallics and synthesis/modification of polymers are two areas where nonaqueous enzymology can play a greater role in the coming years. The greater understanding of enzyme behaviour in nonaqueous media is expected to lead to larger and even more diverse kinds of applications.

Catalysis↗
PubMed1999

Improving enzymes for cancer gene therapy.

New techniques now make it feasible to tailor enzymes for cancer gene therapy. Novel enzymes with desired properties can be created and selected from vast libraries of mutants containing random substitutions within catalytic domains. In this review, we first consider genes for the ablation of tumors, namely, genes that have been mutated (or potentially can be mutated) to afford enhanced activation of prodrugs and increased sensitization of tumors to specific chemotherapeutic agents. We then consider genes that have been mutated to provide better protection of normal host tissues, such as bone marrow, against the toxicity of specific chemotherapeutic agents. Expression of the mutant enzyme could render sensitive tissues, such as bone marrow, more resistant to specific cytotoxic agents.

Bone Marrow↗
PubMed2006

Natural genetic variation in whole-genome expression in Arabidopsis thaliana: the impact of physiological QTL introgression.

A long-standing and fundamental question in biology is how genes influence complex phenotypes. Combining near-isogenic line mapping with genome expression profiling offers a unique opportunity for exploring the functional relationship between genotype and phenotype and for generating candidate genes for future study. We used a whole-genome microarray produced with ink-jet technology to measure the relative expression level of over 21,500 genes from an Arabidopsis thaliana near-isogenic line (NIL) and its recurrent parent. The NIL material contained two introgressions (bottom of chromosome II and top of chromosome III) of the Cvi-1 ecotype in a Ler-2 ecotype genome background. Each introgression 'captures' a Cvi allele of a physiological quantitative trait loci (QTL) that our previous studies have shown increases transpiration and reduces water-use efficiency at the whole-plant level. We used a mixed model anova framework for assessing sources of expression variability and for evaluating statistical significance in our array experiment. We discovered 25 differentially expressed genes in the introgression at a false-discovery rate (FDR) cut-off of 0.20 and identified new candidate genes for both QTL regions. Several differentially expressed genes were confirmed with QRT-PCR (quantitative reverse transcription-polymerase chain reaction) assays. In contrast, we found no statistically significant differentially expressed genes outside of the QTL introgressions after controlling for multiple tests. We discuss these results in the context of candidate genes, cloning QTL, and phenotypic evolution.

Arabidopsis↗
PubMed1994

alpha/beta barrel evolution and the modular assembly of enzymes: emerging trends in the flavin oxidase/dehydrogenase family.

Alpha/beta barrels have an ill-defined origin. Evidence exists which favours their divergent evolution from a common ancestral barrel and convergent evolution to a stable fold. However, recent sequence and structural information for the flavin oxidase/dehydrogenase family of barrel enzymes indicate that sub-families of alpha/beta barrels have evolved divergently. The modular fusion of barrel domains with core structures from other gene families has also contributed to the evolution of related but catalytically distinct enzyme molecules within each sub-family of the flavin oxidases/dehydrogenases. An analysis of the structures and sequences of the flavin oxidases/dehydrogenases has now enabled studies focusing on the evolutionary origins and modular assembly of this important family of proteins to be initiated.

Amino Acid Sequence↗
PubMed2001

Glucose-6-phosphate dehydrogenase-6-phosphogluconolactonase. A novel bifunctional enzyme in malaria parasites.

Plasmodium falciparum glucose 6-phosphate dehydrogenase (Pf Glc6PD), compared to other Glc6PDs has an additional 300 amino acids at the N-terminus. They are not related to Glc6PD but are similar to a family of proteins (devb) of unknown function, some of which are encoded next to Glc6PD in certain bacteria. The human devb homologue has recently been shown to have 6-phosphogluconolactonase (6PGL) activity. This suggests Pf Glc6PD may be a bifunctional enzyme, the evolution of which has involved the fusion of adjacent genes. Further functional analysis of Pf Glc6PD has been hampered because parts of the gene could not be cloned. We have isolated and sequenced the corresponding Plasmodium berghei gene and shown it encodes an enzyme (Pb Glc6PD) with the same structure as the P. falciparum enzyme. Pb Glc6PD is 950 amino acids long with significant sequence similarity in both the devb and Glc6PD domains with the P. falciparum enzyme. The P. berghei enzyme does not have an asparagine-rich segment between the N and C halves and it contains an insertion at the same point in the Glc6PD region as the P. falciparum enzyme but the insertion in the P. berghei is longer (110 versus 62 amino acids) and unrelated in sequence to the P. falciparum insertion. Though expression of this enzyme in bacteria produced largely insoluble protein, conditions were found where the full-length enzyme was produced in a soluble form which was purified via a histidine tag. We show that this enzyme has both Glc6PD and 6PGL activities. Thus the first two steps of the pentose phosphate pathway are catalysed by a single novel bifunctional enzyme in these parasites.

Amino Acid Sequence↗
PubMedDate not supplied

Regional assignment of ADA and ITPA to mouse chromosome 2 (C1----ter). A demonstration of the conserved linkage of enzyme and proto-oncogene loci.

Similarity of G-band patterns between the long arm of Chinese hamster chromosome 6 and mouse chromosome 2, combined with the assignments of AK1, ADA, and ITPA to hamster chromosome 6 and AK1 to mouse chromosome 2, suggested mouse chromosome 2 also might contain ADA and ITPA. Here, concordant segregation analysis of enzyme loci and chromosomes in mouse spleen X CHO as well as mouse microcell X CHO somatic cell hybrids established the assignments of ADA and ITPA onto mouse chromosome 2 in the region between the first G-band and the terminus (C1----ter). This assignment presents a demonstration of the conservation and evolution of enzyme and proto-oncogene loci linkage since two cellular homologs of viral oncogenes--c-src and c-abl--also map to mouse chromosome 2. In humans c-src, ADA, and ITPA remain conserved on chromosome 20, whereas AK1 and c-abl are together on chromosome 9. These observations and concepts are discussed with respect to the role of proto-oncogenes in chromosomal evolution and suggest the long arm of chromosome 6 as a fruitful place to look for c-src and c-abl in the Chinese hamster.

Adenosine Deaminase↗
PubMed1994

On the evolution of arginases and related enzymes.

Sequence analysis of the arginase/agmatine ureohydrolase family, important enzymes in arginine/agmatine metabolism and the urea cycle, reveals the similarity of arginases to formiminoglutamate hydrolase (hutG) in Klebsiella aerogenes and to a previously unidentified open reading frame adjacent to the HMf locus of the archaebacterium Methanothermus fervidus. The gene structure and distribution of these homologous proteins across primary kingdoms suggest that this family is another example of a primordial enzyme possibly present in the universal common ancestor and that can be used as phylogenetic marker.

Amino Acid Sequence↗