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At least 253 records · Page 14Linked to original sources
PubMed2004

The artificial evolution of an enzyme by random mutagenesis: the development of formaldehyde dehydrogenase.

A unique variant of glutathione independent formaldehyde dehydrogenase of Pseudomonas putida was obtained by random mutagenesis using the PCR-reaction. This YM042 mutant, S318G, was a cold-adapted formaldehyde dehyrogenase. The activity at 29 degrees C of the variant was 1.7-fold higher than that of the wild type. The K(m) values of the mutant at 37 degrees C were 0.40 mM for NAD(+) and 2.5 mM for formaldehyde, while those of the wild-type were 0.18 mM for NAD(+) and 2.1 mM for formaldehyde. The catalytic efficiency for formaldehyde was about 1.5-fold greater in the mutant than in the wild-type enzyme. The optimum pHs and temperatures of the mutant and the wild-type enzyme were 7.5, and 8.0 and 37 degrees C, and 47 degrees C, respectively. The thermal stability of the mutant was lower than that of the wild type.

Aldehyde Oxidoreductases↗
PubMed1998

Shaping of Drosophila alcohol dehydrogenase through evolution: relationship with enzyme functionality.

Drosophilidae is a large, widely distributed family of Diptera including 61 genera, of which Drosophila is the most representative. Drosophila feeding is part of the saprophytic trophic chain, because of its dependence upon decomposing organic matter. Many species have adapted to fermenting fruit feeding or to artificial (man-made) fermentation habitats, such as cellars and breweries. Actually, the efficient exploitation of niches with alcohols is considered one of the reasons for the worldwide success of this genus. Drosophila alcohol dehydrogenase (ADH), a member of the short-chain dehydrogenase/reductase family (SDR), is responsible for the oxidation of alcohols, but its direct involvement in fitness, including alcohol tolerance and utilization, gives rise to much controversy. Thus, it remains unclear whether ADH differentiation through evolution is somehow associated with natural adaptation to new feeding niches, and thus maybe to Drosophila speciation, or if it is a simple reflection of neutral divergence correlated with time separation between species. To build a hypothesis which could shed light on this dilemma, we analyzed the amino acid variability found in the 57 protein ADH sequences reported up to now, identified the taxon-specific residues, and localized them in a three-dimensional ADH model. Our results define three regions whose shaping has been crucial for ADH differentiation and would be compatible with a contribution of ADH to Drosophila speciation.

Adaptation, Biological↗
PubMed1993

Evolution of an enzyme activity: crystallographic structure at 2-A resolution of cephalosporinase from the ampC gene of Enterobacter cloacae P99 and comparison with a class A penicillinase.

The structure of the class C ampC beta-lactamase (cephalosporinase) from Enterobacter cloacae strain P99 has been established by x-ray crystallography to 2-A resolution and compared to a class A beta-lactamase (penicillinase) structure. The binding site for beta-lactam (penicillinase) structure. The binding site for beta-lactam antibiotics is generally more open than that in penicillinases, in agreement with the ability of the class C beta-lactamases to better bind third-generation cephalosporins. Four corresponding catalytic residues (Ser-64/70, Lys-67/73, Lys-315/234, and Tyr-150/Ser-130 in class C/A) lie in equivalent positions within 0.4 A. Significant differences in positions and accessibilities of Arg-349/244 may explain the inability of clavulanate-type inhibitors to effectively inactivate the class C beta-lactamases. Glu-166, required for deacylation of the beta-lactamoyl intermediate in class A penicillinases, has no counterpart in this cephalosporinase; the nearest candidate, Asp-217, is 10 A from the reactive Ser-64. A comparison of overall tertiary folding shows that the cephalosporinase, more than the penicillinase, is broadly similar to the ancestral beta-lactam-inhibited enzymes of bacterial cell wall synthesis. On this basis, it is proposed that the cephalosporinase is the older of the two beta-lactamases, and, therefore, that a local refolding in the active site, rather than a simple point mutation, was required for the primordial class C beta-lactamase to evolve to the class A beta-lactamase having an improved ability to catalyze the deacylation step of beta-lactam hydrolysis.

Binding Sites↗
PubMed1999

The solution structure of VAT-N reveals a 'missing link' in the evolution of complex enzymes from a simple betaalphabetabeta element.

BACKGROUND: The VAT protein of the archaebacterium Thermoplasma acidophilum, like all other members of the Cdc48/p97 family of AAA ATPases, has two ATPase domains and a 185-residue amino-terminal substrate-recognition domain, VAT-N. VAT shows activity in protein folding and unfolding and thus shares the common function of these ATPases in disassembly and/or degradation of protein complexes. RESULTS: Using nuclear magnetic resonance (NMR) spectroscopy, we found that VAT-N is composed of two equally sized subdomains. The amino-terminal subdomain VAT-Nn (comprising residues Met1-Thr92) forms a double-psi beta-barrel whose pseudo-twofold symmetry is mirrored by an internal sequence repeat of 42 residues. The carboxy-terminal subdomain VAT-Nc (comprising residues Glu93-Gly185) forms a novel six-stranded beta-clam fold. Together, VAT-Nn and VAT-Nc form a kidney-shaped structure, in close agreement with results from electron microscopy. Sequence and structure analyses showed that VAT-Nn is related to numerous proteins including prokaryotic transcription factors, metabolic enzymes, the protease cofactors UFD1 and PrlF, and aspartic proteinases. These proteins map out an evolutionary path from simple homodimeric transcription factors containing a single copy of the VAT-Nn repeat to complex enzymes containing four copies. CONCLUSIONS: Our results suggest that VAT-N is a precursor of the aspartic proteinases that has acquired peptide-binding activity while remaining proteolytically incompetent. We propose that the binding site of the protein is similar to that of aspartic proteinases, in that it lies between the psi-loops of the amino-terminal beta-barrel and that it coincides with a crescent-shaped band of positive charge extending across the upper face of the molecule.

Adenosine Triphosphatases↗
PubMed2000

Light-modulated NADP-malate dehydrogenases from mossfern and green algae: insights into evolution of the enzyme's regulation.

Chloroplast NADP-dependent malate dehydrogenase is one of the best-studied light-regulated enzymes. In C3 plants, NADP-MDH is a part of the 'malate valve' that controls the export of reducing equivalents in the form of malate to the cytosol. NADP-MDH is completely inactive in the dark and is activated in the light with reduced thioredoxin. Compared with its permanently active NAD-linked counterparts, NADP-MDH exhibits N- and C-terminal sequence extensions, each bearing one regulatory disulphide. Upon reduction of the C-terminal disulphide, the enzyme active site becomes accessible for the substrate. Reduction of the N-terminal disulphide promotes a conformational change advantageous for catalysis. To trace the evolutionary development of this intricate regulation mechanism, we isolated cDNA clones for NADP-MDH from the mossfern Selaginella and from two unicellular green algae. While the NADP-MDH sequence from Selaginella demonstrates the classic cysteine pattern of the higher plant enzyme, the sequences from the green algae are devoid of the N-terminal regulatory disulphide. Phylogenetic analysis of new sequences and of those available in the databases led to the conclusion that the chloroplast NADP-MDH and the cytosolic NAD-dependent form arose via duplication of an ancestral eubacterial gene, which preceded the separation of plant and animal lineages. Redox-sensitive NADP-MDH activity was detected only in the 'green' plant lineage starting from the primitive prasinophytic algae but not in cyanobacteria, Cyanophora paradoxa, red algae and diatoms. The latter organisms therefore appear to utilize mechanisms other than the light-regulated 'malate valve' to remove from plastids excessive electrons produced by photosynthesis.

Chlorophyta↗
PubMed1997

Creating novel enzymes by applied molecular evolution.

Recent molecular techniques have made it feasible to simulate evolutionary processes and apply in vitro selection to evolve enzymes with novel properties that may have potential benefits for medical and industrial applications. The characterization of such mutants has also provided new insights into how molecular structure determines enzyme function.

Amino Acid Sequence↗
PubMed1988

Complete primary structure of human and rabbit lactase-phlorizin hydrolase: implications for biosynthesis, membrane anchoring and evolution of the enzyme.

We report the primary structures of human and rabbit brush border membrane beta-glycosidase complexes (pre-pro-lactase-phlorizin hydrolase, or pre-pro-LPH, EC 3.2.1.23-62), as deduced from cDNA sequences. The human and rabbit primary translation products contain 1927 and 1926 amino acids respectively. Based on the data, as well as on peptide sequences and further biochemical data, we conclude that the proteins comprise five domains: (i) a cleaved signal sequence of 19 amino acids; (ii) a large 'pro' portion of 847 amino acids (rabbit), none of which appears in mature, membrane-bound LPH; (iii) the mature LPH, which contains both the lactase and phlorizin hydrolase activities in a single polypeptide chain; (iv) a membrane-spanning hydrophobic segment near the carboxy terminus, which serves as membrane anchor; and (v) a short hydrophilic segment at the carboxy terminus, which must be cytosolic (i.e. the protein has an Nout-Cin orientation). The genes have a 4-fold internal homology, suggesting that they evolved by two cycles of partial gene duplication. This repetition also implies that parts of the 'pro' portion are very similar to parts of mature LPH, and hence that the 'pro' portion may be a water-soluble beta-glycosidase with another cellular location than LPH. Our results have implications for the decline of LPH after weaning and for human adult-type alactasia, and for the evolutionary history of LPH.

Amino Acid Sequence↗
PubMed1982

Evolution of gluconeogenic enzyme activities during starvation in liver and kidney of the rainbow trout (Salmo gairdneri).

1. There was a general increase in the activities of enzymes involved in gluconeogenesis in liver and kidney of rainbow trout, Salmo gairdneri, during the second month of starvation. 2. The need of gluconeogenesis during the first month of the starvation period was probably minimal because of the utilization of liver glycogen as a source of blood glucose. 3. The decline of fat was more pronounced than that of protein total content in absolute values, suggesting that lipid reserved were the main sources of energy during starvation.

Animals↗
PubMed2002

Evolution of extracellular enzyme activities during manure composting.

AIMS: The objectives of this work were to determine the extracellular enzyme profiles during composting, relate the activities of these enzymes to the changes in microbial population and compare the enzyme profiles between two manures. METHODS AND RESULTS: API ZYM assay was used to monitor the activities of 19 extracellular enzymes during poultry and pig manure composting. Results showed an overall increase in diversity and relative abundance of enzymes present. The relative abundance and activities of enzymes were higher in poultry manure than in pig manure. Among the 19 enzymes tested, esterase, valine amino-peptidase and alpha-galactosidase were the most abundant enzymes in poultry manure, whereas it was N-acetyl-beta-glucosaminidase for the pig manure. A number of these enzymes correlated with change in numbers of different microbial groups during composting. CONCLUSIONS: The composting process represented a combined activity of a wide succession of environments, as one enzyme/microbial group overlapped the other and each emerged gradually due to the continual change in temperature and progressive breakdown of complex compounds to simpler ones. SIGNIFICANCE AND IMPACT OF THE STUDY: The results presented here show the applicability of the API ZYM test not only in monitoring the quantitative and qualitative fluctuation of the available substrate during composting, but also in revealing differences in composts and compost maturity.

Actinomycetales↗
PubMed1988

Molecular evolution of lytic enzymes of Streptococcus pneumoniae and its bacteriophages.

A 2.9-kilobase Acc I fragment of the DNA of the pneumococcal bacteriophage Cp-1, containing the cpl gene, hybridizes with the lytA gene encoding the pneumococcal amidase. The nucleotide sequence of the cpl gene of Cp-1, encoding a muramidase (CPL), has been determined. The 3' regions of the cpl and lytA coding sequences show considerable nucleotide sequence homology and the carboxyl-terminal domains of the deduced amino acid sequences of these lysins are quite similar: 73 of the carboxyl-terminal 142 amino acid residues are identical, and of the 69 substitutions, 55 are conservative. Comparisons between CPL, the pneumococcal amidase, and the muramidase of the fungus Chalaropsis sp. (an enzyme that also degrades the pneumococcal cell wall) strongly suggest that the carboxyl-terminal domains of CPL and of the amidase might be responsible for the specific recognition of choline-containing cell walls, as well as for the noncompetitive inhibition of the catalytic activity of these enzymes by the pneumococcal lipoteichoic acid or by high concentrations of choline. In addition, the active center of these enzymes should be located in their amino-terminal domains. Our results suggest an evolutionary relationship between phage and host lysins.

Amidohydrolases↗
PubMed1998

Evolution of an enzyme active site: the structure of a new crystal form of muconate lactonizing enzyme compared with mandelate racemase and enolase.

Muconate lactonizing enzyme (MLE), a component of the beta-ketoadipate pathway of Pseudomonas putida, is a member of a family of related enzymes (the "enolase superfamily") that catalyze the abstraction of the alpha-proton of a carboxylic acid in the context of different overall reactions. New untwinned crystal forms of MLE were obtained, one of which diffracts to better than 2.0-A resolution. The packing of the octameric enzyme in this crystal form is unusual, because the asymmetric unit contains three subunits. The structure of MLE presented here contains no bound metal ion, but is very similar to a recently determined Mn2+-bound structure. Thus, absence of the metal ion does not perturb the structure of the active site. The structures of enolase, mandelate racemase, and MLE were superimposed. A comparison of metal ligands suggests that enolase may retain some characteristics of the ancestor of this enzyme family. Comparison of other residues involved in catalysis indicates two unusual patterns of conservation: (i) that the position of catalytic atoms remains constant, although the residues that contain them are located at different points in the protein fold; and (ii) that the positions of catalytic residues in the protein scaffold are conserved, whereas their identities and roles in catalysis vary.

Binding Sites↗
PubMed2005

Modelling the thermal evolution of enzyme-created bubbles in DNA.

The formation of bubbles in nucleic acids (NAs) is fundamental in many biological processes such as DNA replication, recombination, telomere formation and nucleotide excision repair, as well as RNA transcription and splicing. These processes are carried out by assembled complexes with enzymes that separate selected regions of NAs. Within the frame of a nonlinear dynamics approach, we model the structure of the DNA duplex by a nonlinear network of coupled oscillators. We show that, in fact, from certain local structural distortions, there originate oscillating localized patterns, that is, radial and torsional breathers, which are associated with localized H-bond deformations, reminiscent of the replication bubble. We further study the temperature dependence of these oscillating bubbles. To this aim, the underlying nonlinear oscillator network of the DNA duplex is brought into contact with a heat bath using the Nosé-Hoover method. Special attention is paid to the stability of the oscillating bubbles under the imposed thermal perturbations. It is demonstrated that the radial and torsional breathers sustain the impact of thermal perturbations even at temperatures as high as room temperature. Generally, for non-zero temperature, the H-bond breathers move coherently along the double chain, whereas at T=0 standing radial and torsional breathers result.

DNA↗