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At least 343 records · Page 19Linked to original sources
PubMed2002

Evolution of the enzyme-linked immunosorbent spot assay for post-transplant alloreactivity as a potentially useful immune monitoring tool.

Post-transplant monitoring of cellular immunity has the potential to guide alterations in medical therapy. To this end, our laboratory has developed an enzyme-linked immunosorbent spot (ELISPOT) assay for detection of peripheral blood alloimmunity. Peripheral blood lymphocytes (PBLs) from normal volunteers and from renal allograft recipients were tested against donor stimulator cells for their ability to respond in 'one-way' cytokine ELISPOT assays. T cell depletion of donor spleen or PBLs eliminated donor cell cytokine secretion while preserving the ability of these cells to present allo-antigen to responding T cells. Alloreactive IFN-gamma-producing PBLs derive from the memory T cell pool and are readily detectable in recipients of renal allografts taking immunosuppressant medications. A significant expansion of IFN-gamma-producing donor-reactive memory PBLs was detectable at 4-6 months post-transplant in those who had experienced an acute rejection episode compared with those with a stable post-transplant course. The data demonstrate the feasibility of repeated post-transplant monitoring of allograft recipients, and provide the foundation for improving the care of human transplant recipients through rational clinical decision-making based on measures of immune function.

Antigen-Presenting Cells↗
PubMed2002

Oligomeric structure of proclavaminic acid amidino hydrolase: evolution of a hydrolytic enzyme in clavulanic acid biosynthesis.

During biosynthesis of the clinically used beta-lactamase inhibitor clavulanic acid, one of the three steps catalysed by clavaminic acid synthase is separated from the other two by a step catalysed by proclavaminic acid amidino hydrolase (PAH), in which the guanidino group of an intermediate is hydrolysed to give proclavaminic acid and urea. PAH shows considerable sequence homology with the primary metabolic arginases, which hydrolyse arginine to ornithine and urea, but does not accept arginine as a substrate. Like other members of the bacterial sub-family of arginases, PAH is hexameric in solution and requires Mn2+ ions for activity. Other metal ions, including Co2+, can substitute for Mn2+. Two new substrates for PAH were identified, N-acetyl-(L)-arginine and (3R)-hydroxy-N-acetyl-(L)-arginine. Crystal structures of PAH from Streptomyces clavuligerus (at 1.75 A and 2.45 A resolution, where 1 A=0.1 nm) imply how it binds beta-lactams rather than the amino acid substrate of the arginases from which it evolved. The structures also suggest how PAH selects for a particular alcohol intermediate in the clavam biosynthesis pathway. As observed for the arginases, each PAH monomer consists of a core of beta-strands surrounded by alpha-helices, and its active site contains a di-Mn2+ centre with a bridging water molecule responsible for hydrolytic attack on to the guanidino group of the substrate. Comparison of structures obtained under different conditions reveals different conformations of a flexible loop, which must move to allow substrate binding.

Amino Acid Sequence↗
PubMed2005

Evolution of highly active enzymes by homology-independent recombination.

The theta-class GST enzymes hGSTT1-1 (human GSTTheta-1-1) and rGSTT2-2 (rat GSTTheta-2-2) share 54.3% amino acid identity and exhibit different substrate specificities. Homology-independent techniques [incremental truncation for the creation of hybrid enzymes (ITCHY) and SCRATCHY] and low-homology techniques (recombination-dependent exponential amplification PCR) were used to create libraries of chimeric enzymes containing crossovers (C/Os) at positions not accessible by DNA family shuffling. High-throughput flow cytometric screening using the fluorogenic rGSTT2-2-specific substrate 7-amino-4-chloromethyl coumarin led to the isolation of active variants with either one or two C/Os. One of these enzymes, SCR23 (83% identity to hGSTT1-1), was encoded by a gene that exchanged helices 4 and 5 of hGSTT1-1 with the corresponding sequence from rGSTT2-2. Compared with either parent, this variant was found to have an improved k(cat) with the selection substrate and also exhibited activity for the conjugation of glutathione to ethacrynic acid, a compound that is not recognized by either parental enzyme. These results highlight the power of combinatorial homology-independent and low-homology recombination methods for the generation of unique, highly active enzymes and also suggest a possible means of enzyme "humanization."

Animals↗
PubMedDate not supplied

On the evolution of an oligocephalic enzyme. glutamine-chorismate-amidotransferase-free anthranilate phosphoribosyltransferases from mutant strains of Salmonella typhimurium.

(1) A procedure has been described for the purification of two glutamine-chorismate-amidotransferase-free anthranilate phosphoribosyltransferases from mutant strains TAX6trpR782 and trpAB1653trpR782 of Salmonella typhimurium. (2) The native enzymes tend to aggregate forming polymers of molecular weights 333,000 in the case of TAXtrpR782 and 220,000 and larger than 1X10(6) in the case of trpAB1653trpR782. In the presence of sodium dodecyl sulfate the polymer of trpAB1653trpR782 dissociates into a single component with molecular weight of 72,000. (3) In contrast to anthranilate phosphoribosyltransferase of the wild type component II, the glutamine-chorismate-amidotransferase-free proteins do not complex with component I. They do however show catalytical similarities with the wild type with respect to anthranilate phosphoribosyltransferase activity.

Anthranilate Phosphoribosyltransferase↗
PubMed2001

Combinatorial and Evolution-Based Methods in the Creation of Enantioselective Catalysts.

Combinatorial methods in the development of enantioselective homogeneous catalysts constitute a new branch of catalysis research. The goal is to prepare libraries of potential asymmetric catalysts, rather than choosing the traditional one-catalyst-at-a-time approach. Several conceptional advancements have been reported in the parallel preparation of chiral ligands. Currently the most meaningful systems constitute modularly constructed ligands on solid supports, which allow high degrees of structural diversity and thus the maximum probability of finding enantioselective catalysts or even new types of ligands for asymmetric catalysis. Search strategies have been developed which amongst other things, lead to catalysts not likely to have been discovered by traditional methods. Genuine application of such strategies involve thousands of catalysts and require high-throughput screening systems capable of assaying enantioselectivity. The first high-throughput ee-screening systems were in fact developed for use in the directed evolution of enantioselective enzymes, a process based on "evolution in the test tube" in which the appropriate methods of random mutagenesis, gene expression, and ee assays are combined. Since no screening system is likely to be universal, different approaches are necessary. Thus far these include assays based on UV/Vis, fluorescence, circular dichroism, mass spectrometry, and even modified gas chromatography as well as special forms of capillary electrophoresis. One of the most efficient systems involves the concept of the mass-spectrometric detection of deuterium-labeled pseudo-enantiomers and pseudo-prochiral compounds with which about 1000 exact ee determinations can be achieved per day, although the assay is restricted to kinetic resolution and/or reactions of prochiral compounds bearing enantiotopic groups. Super-high-throughput screening for enantioselectivity is possible in many cases by making use of chirally modified capillary array electrophoresis in a parallel step. Accordingly, 7000 to 30 000 ee determinations can be carried out per day. These and other analytical developments are expected to stimulate further research in the combinatorial search for asymmetric homogeneous catalysts and in the directed evolution of enantioselective enzymes for use in organic chemistry.

Journal Article↗
PubMed2000

Enantioselective enzymes for organic synthesis created by directed evolution.

A new concept for the creation of enzymes displaying improved enantioselectivity in a given reaction is described; it is based on "evolution in the test tube". Accordingly, proper molecular biological methods for random mutagenesis, gene expression, and high-throughput screening systems for the rapid assay of enantioselectivity are combined. Several rounds of mutagenesis and screening are generally necessary in order to create mutant enzymes that show high degrees of enantioselectivity, as in the case of the lipase-catalyzed hydrolytic kinetic resolution of a chiral ester in which the original enantioselectivity of 2 % ee (E = 1) increases to > 90% ee (E = 25).

Catalysis↗
PubMedDate not supplied

Alteration of substrate chain-length specificity of type II synthase for polyhydroxyalkanoate biosynthesis by in vitro evolution: in vivo and in vitro enzyme assays.

In our previous study, in vitro evolution of type II polyhydroxyalkanoate (PHA) synthase (PhaC1Ps) from Pseudomonas sp. 61-3 yielded eleven mutant enzymes capable of synthesizing homopolymer of (R)-3-hydroxybutyrate [P(3HB)] in recombinant Escherichia coli JM109. These recombinant strains were capable of accumulating up to approximately 400-fold more P(3HB) than strains expressing the wild-type enzyme. These mutations enhanced the ability of the enzyme to specifically incorporate the 3HB-coenzyme A (3HB-CoA) substrate or improved catalytic efficiency toward the various monomer substrates of C4 to C12 (R)-3-hydroxyacyl-CoAs which can intrinsically be channeled by PhaC1Ps into P(3HB-co-3HA) copolymerization. In this study, beneficial amino acid substitutions of PhaC1Ps were analyzed based on the accumulation level and the monomer composition of P(3HB-co-3HA) copolymers generated by E. coli LS5218 [fadR601 atoC(Con)] harboring the monomer supplying enzyme genes. Substitutions of Ser by Thr(Cys) at position 325 were found to lead to an increase in the total amount of P(3HB-co-3HA) accumulated, whereas 3HB fractions in the P(3HB-co-3HA) copolymer were enriched by substitutions of Gln by Lys(Arg, Met) at position 481. This strongly suggests that amino acid substitutions at positions 325 and 481 are responsible for synthase activity and/or substrate chain-length specificity of PhaC1Ps. These in vivo results were supported by the in vitro results obtained from synthase activity assays using representative single and double mutants and synthetic substrates, (R,S)-3HB-CoA and (R,S)-3-hydroxydecanoyl-CoA. Notably, the position 481 was found to be a determinant for substrate chain-length specificity of PhaC1Ps.

Acyltransferases↗
PubMed2005

Simultaneous optimization of enzyme activity and quaternary structure by directed evolution.

Natural evolution has produced efficient enzymes of enormous structural diversity. We imitated this natural process in the laboratory to augment the efficiency of an engineered chorismate mutase with low activity and an unusual hexameric topology. By applying two rounds of DNA shuffling and genetic selection, we obtained a 400-fold more efficient enzyme, containing three non-active-site mutations. Detailed biophysical characterization of the evolved variant suggests that it exists predominantly as a trimer in solution, but is otherwise similarly stable as the parent hexamer. The dramatic structural and functional effects achieved by a small number of seemingly innocuous substitutions highlights the utility of directed evolution for modifying protein-protein interactions to produce novel quaternary states with optimized activities.

Chorismate Mutase↗
PubMed1999

Divergent evolution of fucosyltransferase genes from vertebrates, invertebrates, and bacteria.

On the basis of function and sequence similarities, the vertebrate fucosyltransferases can be classified into three groups: alpha-2-, alpha-3-, and alpha-6-fucosyltransferases. Thirty new putative fucosyltransferase genes from invertebrates and bacteria and six conserved peptide motifs have been identified in DNA and protein databanks. Two of these motifs are specific of alpha-3-fucosyltransferases, one is specific of alpha-2-fucosyltransferases, another is specific of alpha-6-fucosyltransferases, and two are shared by both alpha-2- and alpha-6-fucosyltranserases. Based on these data, literature data, and the phylogenetic analysis of the conserved peptide motifs, a model for the evolution offucosyltransferase genes by successive duplications, followed by divergent evolution is proposed, with either two different ancestors, one for the alpha-2/6-fucosyltransferases and one for the alpha-3-fucosyltransferases or a single common ancestor for the two families. The expected properties of such an hypothetical ancestor suggest that the plant or insect alpha-3-fucosyltransferases using chitobiose as acceptor might be the present forms of this ancestor, since fucosyltransferases using chitobiose as acceptor are expected to be of earlier appearance in evolution than enzymes using N -acetyllactosamine. However, an example of convergent evolution of fucosyltransferase genes is suggested for the appearance of the Leaepitopes found in plants and primates.

Amino Acid Sequence↗
PubMed1999

An endoglucanase, EglA, from the hyperthermophilic archaeon Pyrococcus furiosus hydrolyzes beta-1,4 bonds in mixed-linkage (1-->3),(1-->4)-beta-D-glucans and cellulose.

The eglA gene, encoding a thermostable endoglucanase from the hyperthermophilic archaeon Pyrococcus furiosus, was cloned and expressed in Escherichia coli. The nucleotide sequence of the gene predicts a 319-amino-acid protein with a calculated molecular mass of 35.9 kDa. The endoglucanase has a 19-amino-acid signal peptide but not cellulose-binding domain. The P. furiosus endoglucanase has significant amino acid sequence similarities, including the conserved catalytic nucleophile and proton donor, with endoglucanases from glucosyl hydrolase family 12. The purified recombinant enzyme hydrolyzed beta-1,4 but not beta-1,3 glucosidic linkages and had the highest specific activity on cellopentaose (degree of polymerization [DP] = 5) and cellohexaose (DP = 6) oligosaccharides. To a lesser extent, EglA also hydrolyzed shorter cellodextrins (DP < 5) as well as the amorphous portions of polysaccharides which contain only beta-1,4 bonds such as carboxymethyl cellulose, microcrystalline cellulose, Whatman paper, and cotton linter. The highest specific activity toward polysaccharides occurred with mixed-linkage beta-glucans such as barley beta-glucan and lichenan. Kinetics studies with cellooliogsaccharides and p-nitrophenyl-cellooligosaccharides indicated that the enzyme had three glucose binding subsites (-I, -II, and -III) for the nonreducing end and two glucose binding subsites (+I and +II) for the reducing end from the scissile glycosidic linkage. The enzyme had temperature and pH optima of 100 degreesC and 6.0, respectively; a half-life of 40 h at 95 degreesC; and a denaturing temperature of 112 degreesC as determined by differential scanning calorimetry. The discovery of a thermostable enzyme with this substrate specificity has implications for both the evolution of enzymes involved in polysaccharide hydrolysis and the occurrence of growth substrates in hydrothermal vent environments.

Amino Acid Sequence↗
PubMed2004

The use of forced protein evolution to investigate and improve stability of family 10 xylanases. The production of Ca2+-independent stable xylanases.

Metal ions such as calcium often play a key role in protein thermostability. The inclusion of metal ions in industrial processes is, however, problematic. Thus, the evolution of enzymes that display enhanced stability, which is not reliant on divalent metals, is an important biotechnological goal. Here we have used forced protein evolution to interrogate whether the stabilizing effect of calcium in an industrially relevant enzyme can be replaced with amino acid substitutions. Our study has focused on the GH10 xylanase CjXyn10A from Cellvibrio japonicus, which contains an extended calcium binding loop that confers proteinase resistance and thermostability. Three rounds of error-prone PCR and selection identified a treble mutant, D262N/A80T/R347C, which in the absence of calcium is more thermostable than wild type CjXyn10A bound to the divalent metal. D262N influences the properties of the calcium binding site, A80T fills a cavity in the enzyme, increasing the number of hydrogen bonds and van der Waals interactions, and the R347C mutation introduces a disulfide bond that decreases the free energy of the unfolded enzyme. A derivative of CjXyn10A (CfCjXyn10A) in which the calcium binding loop has been replaced with a much shorter loop from Cellulomonas fimi CfXyn10A was also subjected to forced protein evolution to select for thermostablizing mutations. Two amino acid substitutions within the introduced loop and the A80T mutation increased the thermostability of the enzyme. This study demonstrates how forced protein evolution can be used to introduce enhanced stability into industrially relevant enzymes while removing calcium as a major stability determinant.

Binding Sites↗