PubMed Health⌕ Search

SEARCH · PubMed Health

Results for “enzyme evolution”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 919 records · Page 51Linked to original sources
PubMed2005

Multiple mutagenesis of P450 isoflavonoid synthase reveals a key active-site residue.

The leguminous isoflavonoid skeleton is constructed by P450 2-hydroxyisoflavanone synthase (CYP93C). Two active-site residues of CYP93C2, Ser 310 and Lys 375, are critical for unusual aryl migration of the flavanone substrate. Leu 371 is located near the substrate in a homology model, and mutant proteins regarding this residue were expressed in recombinant yeast microsomes. The single mutant, L371V, yielded only inactive P420, but multiple mutants incorporating K375T restored the P450 fold: the S310T-L371V-K375T triple mutant showed four times higher P450 level than the wild type. L371V-K375T and S310T-L371V-K375T produced a mixture of major 3beta-hydroxyflavanone and minor flavone, and 100% flavone, respectively, from a flavanone. Thus, Leu 371 appeared to control the substrate accommodation in favor of hydrogen abstraction from C-3 of the flavanone molecule and contribute to the P450 fold under the presence of Lys 375, the residue responsible for aryl migration. The molecular evolution of CYP93 enzymes is discussed.

Binding Sites↗
PubMed2005

Plasmalogen metabolism-related enzymes in rat brain during aging: influence of n-3 fatty acid intake.

Plasmalogens (Pls) are phospholipids containing a vinyl-ether bond at the sn-1 position of the glycerol backbone. They represent between 1/2 and 2/3 of the ethanolamine phospholipids in the brain. During aging, the Pls content in human brain falls down. However, the role of Pls metabolism-related enzymes in the regulation of Pls levels remains to be determined. Dihydroxyacetone phosphate acyltransferase (DHAP-AT) is the enzyme involved in the first step of Pls biosynthesis. In the brain, a phospholipase A2, which selectively acts on Pls, has been isolated (Pls-PLA2s). In this work, we aimed to evaluate the impact of DHAP-AT (a key enzyme of Pls biosynthesis) and Pls-PLA2 (a specific Pls degradation enzyme) on the evolution of Pls content in the rat brain during aging. The influence of n-3 fatty acid intake was also evaluated. Littermates from two generations of n-3 deficient rats were fed an equilibrated diet containing either alpha-LNA alone or with two doses of DHA. After weaning, 3, 9 or 21 months of diet, rats were sacrificed. Enzymatic assays were performed, Pls levels were assessed and the sn-2 position of ethanolamine Pls was analyzed. DHAP-AT activity significantly increased between weaning and 3 months with a concomitant increase of brain Pls, which reached maximal levels after 9 months. Then, Pls levels and DHAP-AT activity significantly decreased while Pls-PLA2s activity significantly increased. Dietary n-3 fatty acids had no effect on DHAP-AT activity and on Pls levels. In conclusion, the increase of brain Pls content in the first part of the life may be related to the high increase of DHAP-AT activity, probably stimulated by DHA. In aged animals, the decrease of Pls levels may mainly be caused to an increase of their degradation by Pls-PLA2. Dietary DHA may not oppose the physiologic aging.

Acyltransferases↗
PubMed2004

Inactive enzyme-homologues find new function in regulatory processes.

Although the catalytic center of an enzyme is usually highly conserved, there have been a few reports of proteins with substitutions at essential catalytic positions, which convert the enzyme into a catalytically inactive form. Here, we report a large-scale analysis of substitutions at enzymes' catalytic sites in order to gain insight into the function and evolution of inactive enzyme-homologues. Our analysis revealed that inactive enzyme-homologues are not an exception only found in single enzyme families, but that they are represented in a large variety of enzyme families and conserved among metazoan species. Even though they have lost their catalytic activity, they have adopted new functions and are now mainly involved in regulatory processes, as shown by several case studies. This modification of existing modules is an efficient mechanism to evolve new functions. The invention of inactive enzyme-homologues in metazoa has thereby led to an enhancement of complexity of regulatory networks.

Enzymes↗
PubMed2006

Role of energetic coenzyme pools in the production of L-carnitine by Escherichia coli.

The aim of this work was to understand the steps controlling the biotransformation of trimethylammonium compounds into L(-)-carnitine by Escherichia coli. The high-cell density reactor steady-state levels of carbon source (glycerol), biotransformation substrate (crotonobetaine), acetate (anaerobiosis product) and fumarate (as an electron acceptor) were pulsed by increasing them fivefold. Following the pulse, the evolution of the enzyme activities involved in the biotransformation process of crotonobetaine into L(-)-carnitine (crotonobetaine hydration), in the synthesis of acetyl-CoA (ACS: acetyl-CoA synthetase and PTA: ATP: acetate phosphotransferase) and in the distribution of metabolites for the tricarboxylic acid (ICDH: isocitrate dehydrogenase) and glyoxylate (ICL: isocitrate lyase) cycles was monitored. In addition, the levels of carnitine, the cell ATP content and the NADH/NAD(+) ratio were measured in order to assess the importance and participation of these energetic coenzymes in the catabolic system. The results provided an experimental demonstration of the important role of the glyoxylate shunt during biotransformation and the need for high levels of ATP to maintain metabolite transport and biotransformation. Moreover, the results obtained for the NADH/NAD(+) pool indicated that it is correlated with the biotransformation process at the NAD(+) regeneration and ATP production level in anaerobiosis. More importantly, a linear correlation between the NADH/NAD(+) ratio and the levels of the ICDH and ICL (carbon and electron flows) and the PTA and ACS (acetate and ATP production and acetyl-CoA synthesis) activity levels was assessed. The main metabolic pathway operating during cell metabolic perturbation with a pulse of glycerol and acetate in the high-cell density membrane reactor was that related to ICDH and ICL, both regulating the carbon metabolism, together with PTA and ACS enzymes (regulating ATP production).

Acetates↗
PubMed2002

A hot story from comparative genomics: reverse gyrase is the only hyperthermophile-specific protein.

I have looked for proteins that are present in all hyperthermophile genomes, but absent from all mesophile or thermophile genomes by using the phylogenetic pattern search program of the COG database. Surprisingly, this search retrieved only one such hyperthermophile-specific protein: reverse gyrase. This result emphasizes the importance of reverse gyrase in the adaptation of life to very high temperatures, and strengthens the idea that evolution of this enzyme was crucial in the origin of hyperthermophiles.

Archaea↗
PubMed1975

[Control of glycogen biosynthesis in fetal rat liver].

We have studied through the late foetal period of the rat, the evolution of two enzymes involved in glycogen metabolism of the liver : UDPG glycogen synthetase (a and b forms) and branching enzyme. The activities were measured during the development of normal foetuses and of foetuses experimentally deprived of corticosteroids. In normal foetus the activity of glycogen synthetase and the branching enzyme increased progressively between days 18 and 21 ; but the percentage of glycogen synthetase a increased promptly between days 18 and 19. This variation coincides with the beginning of glycogen accumulation in the liver. In foetuses submitted to corticosteroid shortage, the activity of each enzyme, and the amount of glycogen in the liver, were reduced at term. Cortisol given to the decapited foetus restores subnormal glycogen storage and normal activity of branching enzyme. The activity of synthetase a was slightly increased but remained very low ; only synthetase b was restored to normal. It seems that there is no relationship between the synthesis of glycogen, in the foetal rat liver, and the activity of synthetase a.

Adrenal Cortex Hormones↗
PubMed1975

Amino acid sequence of two functional sites in yeast glycogen phosphorylase.

The structure of two functional sites in baker's yeast (Saccharomyces cerevisiae) glycogen phosphorylase (EC 2.4 1.1) was determined as part of a study on the evolution of regulatory enzymes. S-Carboxymethylated, MaBH4-reduced 32-P-labeled yeast phosphorylase a was cleaved with CNBr, thermolysin, and pepsin. Peptides labeled with 32-P or carrying the fluorescent pyridoxyl marker were isolated and purified using ion-exchange chromatography and gel filtration. CNBr cleavage yielded a single radioactive phosphopeptide (42 residues long) and one small fluorescent peptide with the unique sequence epsilon-Pxy-Lys-Phe-Val-Met. Thermolysin digestion gave rise to one radioactive octapeptide and two fluorescent peptides, 15 and 2 residues long, respectively. From a combination of substractive Edman degradations and digestion with yeast protease C, the sequence of the 32-P-labeled octapeptide was established. Phosphothreonine was identified as the sole phosphorylated amino acid, giving the following structure for the site involved in the covalent regulation of yeast phosphorylase: Leu-Thr(P) -Gly-Phe-Leu-Pro-Gln-Glu. The two fluorescent thermolytic peptides, together with two additional pyridoxyl peptides isolated after peptic digestion of the enzyme yielded the following sequence around the site binding pyridoxal-5'-P, the cofactor essential for phosphorylase activity: Ile-Ser-Thr-Ala-Gly-Thr-Glu-Ala-Ser-Gly-Thr-Ser-Asn-Met-Lys(P Pxy)-Phe-Val-Met. While the phosphorylated site bears no resemblance to the site of covalent control in vertebrate phosphorylases, the pyridoxal-P binding site in the yeast enayme displays remarkable homologies with its animal counterparts; the finding that 14 out of 18 amino acids are identical strongly suggests that the cofactor must be directly involved in catalysis.

Amino Acid Sequence↗
PubMed2002

Organization of an efficient carbonic anhydrase: implications for the mechanism based on structure-function studies of a T199P/C206S mutant.

Substitution of Pro for Thr199 in the active site of human carbonic anhydrase II (HCA II)(1) reduces its catalytic efficiency about 3000-fold. X-ray crystallographic structures of the T199P/C206S variant have been determined in complex with the substrate bicarbonate and with the inhibitors thiocyanate and beta-mercaptoethanol. The latter molecule is normally not an inhibitor of wild-type HCA II. All three ligands display novel binding interactions to the T199P/C206S mutant. The beta-mercaptoethanol molecule binds in the active site area with its sulfur atom tetrahedrally coordinated to the zinc ion. Thiocyanate binds tetrahedrally coordinated to the zinc ion in T199P/C206S, in contrast to its pentacoordinated binding to the zinc ion in wild-type HCA II. Bicarbonate binds to the mutant with two of its oxygens at the positions of the zinc water (Wat263) and Wat318 in wild-type HCA II. The environment of this area is more hydrophilic than the normal bicarbonate-binding site of HCA II situated in the hydrophobic part of the cavity normally occupied by the so-called deep water (Wat338). The observation of a new binding site for bicarbonate has implications for understanding the mechanism by which the main-chain amino group of Thr199 acquired an important role for orientation of the substrate during the evolution of the enzyme.

Amino Acid Substitution↗
PubMed2003

Enhanced degradation of chemical warfare agents through molecular engineering of the phosphotriesterase active site.

The bacterial phosphotriesterase has been utilized as a template for the evolution of improved enzymes for the catalytic decomposition of organophosphate nerve agents. A combinatorial library of active site mutants was constructed by randomizing residues His-254, His-257, and Leu-303. The collection of mutant proteins was screened for the ability to hydrolyze a chromogenic analogue of the most toxic stereoisomer of the chemical warfare agent, soman. The mutant H254G/H257W/L303T catalyzed the hydrolysis of the target substrate nearly 3 orders of magnitude faster than the wild-type enzyme. The X-ray crystal structure was solved in the presence and absence of diisopropyl methyl phosphonate. The mutant enzyme was ligated to an additional divalent cation at the active site that was displaced upon the binding of the substrate analogue inhibitor. These studies demonstrate that substantial changes in substrate specificity can be achieved by relatively minor changes to the primary amino acid sequence.

Binding Sites↗
PubMed1978

The alpha-helix dipole and the properties of proteins.

Phosphate moieties bind frequently at N-termini of helices in proteins. It is shown that this corresponds with an optimal interaction of the helix dipole and the charged phosphate. This favourable arrangement may have been discovered several times during evolution. In some enzymes, the helix dipole might be used in catalysis.

Amino Acid Sequence↗
PubMed1980

Influence of the position of the double bond in steroid substrates on the efficiency of the proton-transfer reaction by Pseudomonas testosteroni 3-oxo-steroid delta 4-delta 5-isomerase.

Studies of the proton-transfer reaction by Pseudomonas testosteroni 3-oxo steroid Delta(4)-Delta(5)-isomerase with Delta(5(6))- and Delta(5(10))-steroid substrates demonstrate the importance of the position of the double bond for the efficiency of the isomerization process. Thus 3-oxo-Delta(5(6))-substrates have markedly high k(cat.) values, whereas those of 3-oxo-Delta(5(10))-substrates are very low and their apparent K(m) values approach equilibrium dissociation constants. The first step in the isomerization process is: [Formula: see text] which is governed by the k(-1)/k(+1) ratio and is shown to be very similar for the two classes of substrates (3-oxo-Delta(5(6))- and -Delta(5(10))-steroids). They therefore differ in the steps distal to the initial formation of the Michaelis-Menten complex. The use of the deuterated androst-5(6)-ene-3,17-dione substrate enabled us to calculate individual rate constants k(+1) and k(-1) as well as to determine the apparent rate-limiting step in the isomerization process. With the deuterated oestr-5(10)-ene-3,17-dione substrate, no significant isotope effect was observed suggesting that a different rate-limiting step may be operative in this isomerization process. Data are presented that indicate that under optimal concentrations of the efficient androst-5(6)-ene-3,17-dione substrate, the forward reaction for ES complex formation (as defined by k(+1)) is limited only by diffusion and the apparent K(m) does not approach the equilibrium constant, suggesting that the evolution of this enzyme has proceeded close to ;catalytic perfection'.

Androstenedione↗
PubMed1980

Duplicated cytosolic malate dehydrogenase genes in Zea mays.

Six inbred lines of Zea mays expressing different soluble (cytosolic) malate dehydrogenase (sMDH) zymogram phenotypes were analyzed genetically. sMDH was found to be coded for by unlinked duplicated loci in four of these inbred lines. The remaining two lines were found not to possess these duplicated loci. Furthermore, the duplicated loci, sMdh1 and sMdh2, have been found to be located on different chromosomes: sMdh1 on chromosome 1L linked to Amp1, and sMdh2 on chromosome 5S linked to Cat1 and Amp3. The importance of finding sMDH encoded by duplicated loci is discussed in relation to the role of chromosomal rearrangements, the relationship between the cytoplasmic and mitochondrial enzymes, and the evolution of Z. mays.

Journal Article↗
PubMed1996

Initiation of (-) strand DNA synthesis from tRNA(3Lys) on lentiviral RNAs: implications of specific HIV-1 RNA-tRNA(3Lys) interactions inhibiting primer utilization by retroviral reverse transcriptases.

Initiation of minus (-) strand DNA synthesis was examined on templates containing R, U5, and primer-binding site regions of the human immunodeficiency virus type 1 (HIV-1), feline immunodeficiency virus (FIV), and equine infectious anemia virus (EIAV) genomic RNA. DNA synthesis was initiated from (i) an oligoribonucleotide complementary to the primer-binding sites, (ii) synthetic tRNA(3Lys), and (iii) natural tRNA(3Lys), by the reverse transcriptases of HIV-1, FIV, EIAV, simian immunodeficiency virus, HIV type 2 (HIV-2), Moloney murine leukemia virus, and avian myeloblastosis virus. All enzymes used an oligonucleotide on wild-type HIV-1 RNA, whereas only a limited number initiated (-) strand DNA synthesis from either tRNA(3Lys). In contrast, all enzymes supported efficient tRNA(3Lys)-primed (-) strand DNA synthesis on the genomes of FIV and EIAV. This may be in part attributable to the observation that the U5-inverted repeat stem-loop of the EIAV and FIV genomes lacks an A-rich loop shown with HIV-1 to interact with the U-rich tRNA anticodon loop. Deletion of this loop in HIV-1 RNA, or disrupting a critical loop-loop complex by tRNA(3Lys) extended by 9 nt, restored synthesis of HIV-1 (-) strand DNA from primer tRNA(3Lys) by all enzymes. Thus, divergent evolution of lentiviruses may have resulted in different mechanisms to use the same host tRNA for initiation of reverse transcription.

Animals↗
PubMed2002

Novel [2Fe-2S]-type redox center C in SdhC of archaeal respiratory complex II from Sulfolobus tokodaii strain 7.

The SdhC subunit of the archaeal respiratory complex II (succinate:quinone oxidoreductase) from Sulfolobus tokodaii strain 7 has a novel cysteine rich motif and is also related to archaeal and bacterial heterodisulfide reductase subunits. We overexpressed the sdhC gene heterologously in Escherichia coli and characterized the gene product in greater detail. Low temperature resonance Raman and x-ray absorption spectroscopic investigation collectively demonstrate the presence of a [2Fe-2S] cluster core with complete cysteinyl ligation (Center C) and an isolated zinc site in the recombinant SdhC. The [2Fe-2S]2+ cluster core is sensitive to dithionite, resulting in irreversible breakdown of the Fe-Fe interaction. EPR analysis confirmed that the novel Center C is an inherent redox center in the archaeal complex II, showing unique EPR signals in the succinate-reduced state. Distinct subunit and cofactor arrangements in the S. tokodaii respiratory complex II, as compared with those in mitochondrial and some mesophilic bacterial enzymes, indicate modular evolution of this ubiquitous electron entry site in the respiratory chains of aerobic organisms.

Archaea↗
PubMed1998

Data decisiveness, data quality, and incongruence in phylogenetic analysis: an example from the monocotyledons using mitochondrial atp A sequences.

We examined three parallel data sets with respect to qualities relevant to phylogenetic analysis of 20 exemplar monocotyledons and related dicotyledons. The three data sets represent restriction-site variation in the inverted repeat region of the chloroplast genome, and nucleotide sequence variation in the chloroplast-encoded gene rbcL and in the mitochondrion-encoded gene atpA, the latter of which encodes the alpha-subunit of mitochondrial ATP synthase. The plant mitochondrial genome has been little used in plant systematics, in part because nucleotide sequence evolution in enzyme-encoding genes of this genome is relatively slow. The three data sets were examined in separate and combined analyses, with a focus on patterns of congruence, homoplasy, and data decisiveness. Data decisiveness (described by P. Goloboff) is a measure of robustness of support for most parsimonious trees by a data set in terms of the degree to which those trees are shorter than the average length of all possible trees. Because indecisive data sets require relatively fewer additional steps than decisive ones to be optimized on nonparsimonious trees, they will have a lesser tendency to be incongruent with other data sets. One consequence of this relationship between decisiveness and character incongruence is that if incongruence is used as a criterion of noncombinability, decisive data sets, which provide robust support for relationships, are more likely to be assessed as noncombinable with other data sets than are indecisive data sets, which provide weak support for relationships. For the sampling of taxa in this study, the atpA data set has about half as many cladistically informative nucleotides as the rbcL data set per site examined, and is less homoplastic and more decisive. The rbcL data set, which is the least decisive of the three, exhibits the lowest levels of character incongruence. Whatever the molecular evolutionary cause of this phenomenon, it seems likely that the poorer performance of rbcL than atpA, in terms of data decisiveness, is due to both its higher overall level of homoplasy and the fact that it is performing especially poorly at nonsynonymous sites.

Adenosine Triphosphatases↗
PubMed1992

Molecular evolution of phosphoenolpyruvate carboxylase for C4 photosynthesis in maize: comparison of its cDNA sequence with a newly isolated cDNA encoding an isozyme involved in the anaplerotic function.

Molecular events associated with the evolution of an enzyme for C4 photosynthesis were investigated. In maize, at least three isozymes of phosphoenolpyruvate carboxylase [EC 4.1.1.31] are known: the C4-form, the C2-form and the root-form, being named according to their physiological roles and pattern of tissue distribution [Ting, I.P. & Osmond, C.B. (1973) Plant Physiol. 51, 448-453]. A cDNA clone which presumably encodes the C3-form isozyme was newly isolated and analyzed. Comparison of the sequences of the C3-form and C4-form isozymes revealed that (i) the homologies in the nucleotide and deduced amino acid sequences were 71 and 77%, respectively, and (ii) the gene for the C4-form isozyme evolved under strong G/C pressure. The genes for these isozymes were found to be located apart on different chromosomes. A phylogenetic tree was constructed using 8 amino acid sequences of phosphoenolpyruvate carboxylases from various sources. The topology of the tree indicated that, at least in monocots such as maize and sorghum, the genes for the C4-form and C3-form isozymes diverged from their common ancestral gene earlier than the monocot-dicot divergence (about 2 x 10(8) yr ago), though the divergence of maize (C4 plant) from wheat (C3 plant) is supposed to have occurred much later (6 x 10(7) yr ago).

Amino Acid Sequence↗
PubMed2001

Polymorphism of the polyketide synthase gene phID in biocontrol fluorescent pseudomonads producing 2,4-diacetylphloroglucinol and comparison of PhID with plant polyketide synthases.

Many biocontrol fluorescent pseudomonads can protect plants from soilborne fungal pathogens through production of the antifungal secondary metabolite 2,4-diacetylphloroglucinol (Phl). One of the phl biosynthetic genes, phlD, encodes a polyketide synthase similar to plant chalcone synthases. Here, restriction analysis of phlD from 39 Phl+ biocontrol fluorescent pseudomonads yielded seven different banding patterns. The gene was sequenced in seven strains, representing the different restriction patterns. Cluster analysis of phlD restriction data or phlD sequences indicated that phlD polymorphism was high, and two main clusters were obtained when predicted PhlD sequences were compared. When the seven PhlD sequences were studied with those of other procaryotic polyketide synthases (gram-positive bacteria) and plant chalcone synthases, however, Phl+ pseudomonads, gram-positive bacteria, and plants clustered separately. Yet, sequence analysis of active site regions for PhlD and plant chalcone synthases revealed that PhlD can be considered a member of the chalcone synthase family, which may be interpreted as convergent evolution of key enzymes involved in secondary metabolism. For the 39 Phl+ pseudomonads, a relationship was found among phlD restriction patterns, phylogenetic groups defined by 16S rDNA restriction analysis (confirmed by 16S rDNA sequencing), and production levels of Phl in vitro.

Acyltransferases↗