PubMed Health⌕ Search

SEARCH · PubMed Health

Results for “enzyme engineering”

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 325 records · Page 18Linked to original sources
PubMed2005

X-ray structure of the R69D phosphatidylinositol-specific phospholipase C enzyme: insight into the role of calcium and surrounding amino acids in active site geometry and catalysis.

Phosphatidylinositol-specific phospholipase Cs (PLCs) are a family of phosphodiesterases that catalyze the cleavage of the P-O bond via transesterification using the internal hydroxyl group of the substrate as a nucleophile, generating the five-membered cyclic inositol phosphate as an intermediate or product. To better understand the role of calcium in the catalytic mechanism of PLCs, we have determined the X-ray crystal structure of an engineered PLC enzyme from Bacillus thuringiensis to 2.1 A resolution. The active site of this enzyme has been altered by substituting the catalytic arginine with an aspartate at position 69 (R69D). This single-amino acid substitution converted a metal-independent, low-molecular weight enzyme into a metal ion-dependent bacterial PLC with an active site architecture similar to that of the larger metal ion-dependent mammalian PLC. The Ca(2+) ion shows a distorted square planar geometry in the active site that allows for efficient substrate binding and transition state stabilization during catalysis. Additional changes in the positions of the catalytic general acid/general base (GA/GB) were also observed, indicating the interrelation of the intricate hydrogen bonding network involved in stabilizing the active site amino acids. The functional information provided by this X-ray structure now allows for a better understanding of the catalytic mechanism, including stereochemical effects and substrate interactions, which facilitates better inhibitor design and sheds light on the possibilities of understanding how protein evolution might have occurred across this enzyme family.

Amino Acid Sequence↗
PubMed1998

The evolution of biotransformation technologies.

Biotransformation is a broad and growing field of biotechnology and encompasses both enzymatic and microbial biocatalysis. Progress has been made in research on the key drivers of biotransformations, including the isolation and characterization of microbes and their enzymes from, and their utilization in, extreme environments, the manipulation, alteration, and augmentation of metabolic pathways, and the use of combinatorial biosynthesis and biocatalytic methodologies for new compound development.

Benzopyrans↗
PubMed1999

Toward the engineering of a super efficient enzyme.

The catalytic activity of a mutant of Photobacterium leiognathi Cu, Zn superoxide dismutase in which the Glu59 residue, conserved in most bacterial variants of the enzyme, has been replaced by glutamine was investigated by pulse radiolysis. At neutral pH the enzyme was found to have a kcat/KM of 1.0 +/- 0.1 x 10(10) M-1s-1 the highest value ever found for any superoxide dismutase. Brownian dynamics simulation suggests that such a high value is due to an enhanced substrate attraction by the modified electric field distribution. The mutant is also characterized by an active-site widely accessible for the solvent, since iodide is able to interact with the copper atom with an affinity constant twice as high as that found in the native enzyme. The large solvent accessible surface of the copper site together with a favorable distribution of the protein-generated electric field gives rise to the most efficient enzyme ever found with activity close to the diffusion limit.

Amino Acid Substitution↗
PubMed1991

Identification of electrostatic interactions that determine the phosphorylation site specificity of the cAMP-dependent protein kinase.

"Charged-to alanine" scanning mutagenesis of the catalytic subunit of the Saccharomyces cerevisiae cAMP-dependent protein kinase (C1) identified three glutamate residues, E171, E214, and E274, that are involved in the recognition of a peptide substrate, kemptide (Leu1Arg2Arg3Ala4Ser5Leu6Gly7). These glutamate residues are conserved or conservatively substituted with asparate in the serine/threonine protein kinases that have a requirement for basic residues on the N-terminal side of their phosphorylation sites. Alanine replacement mutants in C1 were subjected to kinetic analysis using alanine-substituted peptides as substrates. The additivity or nonadditivity of the effects of the alanine substitutions on the catalytic efficiency (kcat/Km) was analyzed. This allowed the identification of electrostatic interactions between the three glutamate residues in the enzyme and the two arginine residues present in the peptide substrate. The data suggest that E171 interacts with Arg2 in the substrate and that E214 and E274 both interact with Arg3. This may be a general method for identifying simple intermolecular interactions involving proteins when there is no three-dimensional structure available of the complex of interacting species. The identification of these interactions provides the potential for rational protein engineering of enzymes with alternative specificities.

Amino Acid Sequence↗
PubMed2006

Molecular modeling of glycosyltransferases.

Glycosyltransferases, the enzymes that build oligosaccharides and glycoconjugates, have received much interest in recent years owing to their biological functions and their potential uses in biotechnology. The analysis of the wealth of sequences that are now available in databases allowed the classification in different families characterized by conserved peptide motifs. Nevertheless, only a limited number of crystal structures is available and molecular modeling appears to be an inescapable tool for rationalization of binding data, engineering of enzyme properties, and design of inhibitors that would be of interest as therapeutic compounds. Because of sequence diversity and limited experimental data, molecular modeling of these enzymes is not straightforward and utilizes the most recent tools, such as fold recognition programs.

Amino Acid Sequence↗
PubMedDate not supplied

A large increase in enzyme-substrate affinity by protein engineering.

A single point mutation has been engineered in the tyrosyl-tRNA synthetase that improves its affinity (KM) for its substrate ATP by a factor of 100. In the crystal structure of the tyrosyl tRNA synthetase (of Bacillus stearothermophilus), the side-chain hydroxyl of Thr 51 appears to make a weak hydrogen bond with the AMP moiety of the substrate intermediate, tyrosyl adenylate. In the absence of substrate, however, the hydroxyl group should make a strong hydrogen bond with water which would favour dissociation of the enzyme-substrate complex. We have used oligodeoxynucleotide-directed mutagenesis to construct two point mutants at this site: one to remove the hydroxyl group (Thr 51 leads to Ala 51) and the other, in addition, to distort the local polypeptide backbone (Thr 51 leads to Pro 51). We report here that both mutants have increased activity (kcat/KM for ATP) but one mutant (Pro 51) shows a massive 25-fold increase due mainly to a lowered KM for ATP. This demonstrates dramatically the potential of in vitro mutagenesis for improving the affinity of an enzyme for its substrate.

Amino Acyl-tRNA Synthetases↗
PubMed1995

The protein deficient in Lowe syndrome is a phosphatidylinositol-4,5-bisphosphate 5-phosphatase.

Lowe syndrome, also known as oculocerebrorenal syndrome, is caused by mutations in the X chromosome-encoded OCRL gene. The OCRL protein is 51% identical to inositol polyphosphate 5-phosphatase II (5-phosphatase II) from human platelets over a span of 744 aa, suggesting that OCRL may be a similar enzyme. We engineered a construct of the OCRL cDNA that encodes amino acids homologous to the platelet 5-phosphatase for expression in baculovirus-infected Sf9 insect cells. This cDNA encodes aa 264-968 of the OCRL protein. The recombinant protein was found to catalyze the reactions also carried out by platelet 5-phosphatase II. Thus OCRL converts inositol 1,4,5-trisphosphate to inositol 1,4-bisphosphate, and it converts inositol 1,3,4,5-tetrakisphosphate to inositol 1,3,4-trisphosphate. Most important, the enzyme converts phosphatidylinositol 4,5-bisphosphate to phosphatidylinositol 4-phosphate. The relative ability of OCRL to catalyze the three reactions is different from that of 5-phosphatase II and from that of another 5-phosphatase isoenzyme from platelets, 5-phosphatase I. The recombinant OCRL protein hydrolyzes the phospholipid substrate 10- to 30-fold better than 5-phosphatase II, and 5-phosphatase I does not cleave the lipid at all. We also show that OCRL functions as a phosphatidylinositol 4,5-bisphosphate 5-phosphatase in OCRL-expressing Sf9 cells. These results suggest that OCRL is mainly a lipid phosphatase that may control cellular levels of a critical metabolite, phosphatidylinositol 4,5-bisphosphate. Deficiency of this enzyme apparently causes the protean manifestations of Lowe syndrome.

Amino Acid Sequence↗
PubMed2003

Antibodies as therapeutic agents: vive la renaissance!

Until recently, the concept of antibodies as in vivo therapeutics was still considered to be an exceedingly ambitious goal. However, in 2003, the situation has been completely transformed, with 14 FDA-approved monclonal antibodies (mAbs), 70 in late stage clinical (Phase II+) trials and > 1000 in preclinical development. The driving force behind this reversal in fortune has been advances in antibody engineering and the emergence of novel discovery techniques which overcame stability and immunogenicity issues that had blighted previous clinical trials of murine antibodies. For indications as diverse as inflammation, cancer and infectious disease, it is clear that unique properties of antibodies make them safe, effective and versatile therapeutics. These drugs can be used to neutralise pathogens, toxins and endogenous mediators of pathology. As cell targeting reagents, antibodies can be used to modulate cytoplasmic cascades or to 'tag' specific cells for complement- or effector-mediated lysis. Antibodies can also be modified to deliver toxic or modulatory payloads (small molecules, radionuclides and enzymes) and engineered to bind multiple epitopes (bispecifics) or even to have novel catalytic activity (abzymes). The modular structure of immunoglobulins and the availability of antibody fragment libraries also make it possible to produce variable-domain therapeutics (Fab, single-chain and domain antibodies). Although exhibiting less favourable kinetics in vivo, these fragments are simple to express and have an increased tissue penetration, making them especially useful as neutralising agents or in the delivery of payload. The number of approved antibodies is expected to increase arithmetically in the near term, as the platform is adopted as a valid alternative to small molecule discovery. This review provides an introduction to the antibody discovery process and discusses the past, present and future applications of therapeutic antibodies, with reference to several FDA-approved precedents.

Animals↗
PubMed2006

In vitro display technologies reveal novel biopharmaceutics.

Display technologies are fundamental to the isolation of specific high-affinity binding proteins for diagnostic and therapeutic applications in cancer, neurodegenerative, and infectious diseases as well as autoimmune and inflammatory disorders. Applications extend into the broad field of antibody (Ab) engineering, synthetic enzymes, proteomics, and cell-free protein synthesis. Recently, in vitro display technologies have come to prominence due to the isolation of high-affinity human antibodies by phage display, the development of novel scaffolds for ribosome display, and the discovery of novel protein-protein interactions. In vitro display represents an emerging and innovative technology for the rapid isolation and evolution of high-affinity peptides and proteins. So far, only one clinical drug candidate produced by in vitro display technology has been approved by the FDA for use in humans, but several are in clinical or preclinical testing. This review highlights recent advances in various engineered biopharmaceutical products isolated by in vitro display with a focus on the commercial developments.

Biopharmaceutics↗
PubMed1996

A functional chimeric modular polyketide synthase generated via domain replacement.

BACKGROUND: Modular polyketide synthases (PKSs), such as 6-deoxyerythronolide B synthase (DEBS), are large multifunctional enzymes that catalyze the biosynthesis of structurally complex and medically important natural products. Active sites within these assemblies are organized into 'modules', such that each module catalyzes the stereospecific addition of a new monomer onto a growing polyketide chain and also sets the reduction level of the beta-carbon atom of the resulting intermediate. The core of each module is made up of a 'reductive segment', which includes all, some, or none of a set of ketoreductase (KR), dehydratase, and enoylreductase domains, in addition to a large interdomain region which lacks overt function but may contribute to structural stability and inter-domain dynamics within modules. The highly conserved organization of reductive segments within modules suggests that they might be able to function in unnatural contexts to generate novel organic molecules. RESULTS: To investigate domain substitution as a method for altering PKS function, a chimeric enzyme was engineered. Using a bimodular derivative of DEBS (DEBS1+TE), the reductive segment of module 2, which includes a functional KR, was replaced with its homolog from module 3 of DEBS, which contains a (naturally occurring) nonfunctional KR. A recombinant strain expressing the chimeric gene produced the predicted ketolactone with a yield (35 %) comparable to that of a control strain in which the KR2 domain was retained but mutationally inactivated. CONCLUSIONS: These results demonstrate considerable structural tolerance within an important segment found in virtually every PKS module. The domain boundaries defined here could be exploited for the construction of numerous loss-of-function and possibly even gain-of-function mutants within this remarkable family of multifunctional enzymes.

Multienzyme Complexes↗
PubMed1999

Interdomain signaling in glutamine phosphoribosylpyrophosphate amidotransferase.

The glutamine phosphoribosylpyrophosphate (PRPP) amidotransferase-catalyzed synthesis of phosphoribosylamine from PRPP and glutamine is the sum of two half-reactions at separated catalytic sites in different domains. Binding of PRPP to a C-terminal phosphoribosyltransferase domain is required to activate the reaction at the N-terminal glutaminase domain. Interdomain signaling was monitored by intrinsic tryptophan fluorescence and by measurements of glutamine binding and glutamine site catalysis. Enzymes were engineered to contain a single tryptophan fluorescence reporter in key positions in the glutaminase domain. Trp(83) in the glutamine loop (residues 73-84) and Trp(482) in the C-terminal helix (residues 471-492) reported fluorescence changes in the glutaminase domain upon binding of PRPP and glutamine. The fluorescence changes were perturbed by Ile(335) and Tyr(74) mutations that disrupt interdomain signaling. Fluoresence titrations of PRPP and glutamine binding indicated that signaling defects increased the K(d) for glutamine but had little or no effect on PRPP binding. It was concluded that the contact between Ile(335) in the phosphoribosyltransferase domain and Tyr(74) in the glutamine site is a primary molecular interaction for interdomain signaling. Analysis of enzymes with mutations in the glutaminase domain C-terminal helix and a 404-420 peptide point to additional signaling interactions that activate the glutamine site when PRPP binds.

Amidophosphoribosyltransferase↗
PubMed2004

Engineering of a polymeric bacterial protein as a scaffold for the multiple display of peptides.

Protein assemblies with a high degree of repetitiveness and organization are known to induce strong immune responses. For that reason they have been postulated for the design of subunit vaccines by means of protein engineering. The enzyme lumazine synthase from Brucella spp. (BLS) is highly immunogenic, presumably owing to its homodecameric arrangement and remarkable thermodynamic stability. Structural analysis has shown that it is possible to insert foreign peptides at the ten amino terminus of BLS without disrupting its general folding. These peptides would be displayed to the immune system in a highly symmetric three-dimensional array. In the present work, BLS has been used as a protein carrier of foreign peptides. We have established a modular system to produce chimeric proteins decorated with ten copies of a desired peptide as long as 27 residues and have shown that their folding and stability is similar to that of the wild-type protein. The knowledge about the mechanisms of dissociation and unfolding of BLS allowed the engineering of polyvalent chimeras displaying different predefined peptides on the same molecular scaffold. Moreover, the reassembly of mixtures of chimeras at different steps of the unfolding process was used to control the stoichiometry and spatial arrangement for the simultaneous display of different peptides on BLS. This strategy would be useful for vaccine development and other biomedical applications.

Amino Acid Sequence↗
PubMed2005

Saturation mutagenesis of 2,4-DNT dioxygenase of Burkholderia sp. strain DNT for enhanced dinitrotoluene degradation.

2,4-Dinitrotoluene (2,4-DNT) and 2,6-DNT are priority pollutants, and 2,4-DNT dioxygenase of Burkholderia sp. strain DNT (DDO) catalyzes the initial oxidation of 2,4-DNT to form 4-methyl-5-nitrocatechol and nitrite but has significantly less activity on other dinitrotoluenes and nitrotoluenes (NT). Hence, oxidation of 2,3-DNT, 2,4-DNT, 2,5-DNT, 2,6-DNT, 2NT, and 4NT were enhanced here by performing saturation mutagenesis on codon I204 of the alpha subunit (DntAc) of DDO and by using a membrane agar plate assay to detect catechol formation. Rates of degradation were quantified both by the formation of nitrite and by the formation of the intermediates with high performance liquid chromatography. The degradation of both 2,3-DNT and 2,5-DNT were achieved for the first time (no detectable activity with the wild-type enzyme) using whole Escherichia coli TG1 cells expressing DDO variants DntAc I204L and I204Y (0.70 +/- 0.03 and 0.22 +/- 0.02 nmol/min/mg protein for 2,5-DNT transformation, respectively). DDO DntAc variant I204L also transformed both 2,6-DNT and 2,4-DNT 2-fold faster than wild-type DDO (0.8 +/- 0.6 nmol/min/mg protein and 4.7 +/- 0.5 nmol/min/mg protein, respectively). Moreover, the activities of DDO for 2NT and 4NT were also enhanced 3.5-fold and 8-fold, respectively. Further, DntAc variant I204Y was also discovered with comparable rate enhancements for the substrates 2,4-DNT, 2,6-DNT, and 2NT but not 4NT. Sequencing information obtained during this study indicated that the 2,4-DNT dioxygenases of Burkholderia sp. strain DNT and B. cepacia R34 are more closely related than originally reported. This is the first report of engineering an enzyme for enhanced degradation of nitroaromatic compounds and the first report of degrading 2,5-DNT.

Bacterial Proteins↗
PubMed2000

Practical application of mammalian cytochrome P450.

Heterologous expression systems play an important role in the analysis of structure-function relationships of mammalian P450s. In addition, these expression systems allow practical application of mammalian P450s. Genetically engineered fused enzymes between mammalian P450 and yeast NADPH-P450 reductase have possible applications in bioconversion processes. Combined use of techniques reported thus far could produce steroid hormones in the recombinant yeast cells harboring four P450 species, CYP11A1, CYP17A1, CYP21B1 and CYP11B1. In an Escherichia coli expression system, the technology of the construction of the mitochondrial P450 electron transport chain has been established. The recombinant E. coli cells expressing CYP27B1, adrenodoxin and NADPH-adrenodoxin reductase would be applicable to a bioconversion process to produce 1alpha,25-dihydroxyvitamin D3. We also demonstrated the usefulness of heterologous expression systems for human liver microsomal P450s for the prediction of drug metabolism in the human body. Microsomal fractions prepared from recombinant yeast, insect and mammalian cells are commercially available and play an important role in preclinical drug development. Application of mammalian P450 to bioremediation with genetic engineering has also been developed. Thus, mammalian P450s appear to have great potential for a wide range of practical applications.

Journal Article↗
PubMed1986

Genetically engineered calmodulins differentially activate target enzymes.

Three mutant calmodulin (CaM) genes together with the normal chicken CaM cDNA have been expressed in bacteria for the purpose of determining structure/function relationships in CaM. The mutant CaM genes were generated by in vitro recombination between a chicken CaM cDNA and a processed pseudogene that encodes a full-length CaM but with 19 amino acid substitutions as compared to authentic vertebrate CaM. The calmodulin-like (CaML) proteins derived from the pseudogene are called CaML19, CaML16, and CaML3 and contain 19, 16, and 3 amino acid substitutions, respectively. CaML3 is functionally identical to CaM by all criteria tested. The functional characteristics of CaML16 and CaML19 are also indistinguishable yet quite different from normal CaM. CaML19 and CaML16 will maximally activate myosin light chain kinase but will only half-maximally activate calcineurin and CaM-dependent multiprotein kinase. In addition, CaML16 and CaML19 do not activate phosphorylase kinase. The differential activation of these enzymes does not result from the loss of Ca2+-binding sites, since CaML16 binds four Ca2+ with affinity similar to CaM or CaM23. It is more likely that the functional characteristics of the mutant proteins result from an altered tertiary structure, since the Ca2+-dependent enhancement of tyrosine fluorescence and limited proteolysis pattern of CaML16 are different from that of CaM. The data demonstrate that the nature of the interaction of CaM with myosin light chain kinase is different from its interaction with calcineurin, CaM-dependent multiprotein kinase, and phosphorylase kinase and may involve different functional domains in CaM.

Amino Acid Sequence↗
PubMed1995

A multifunctional flow-injection biosensor for the simultaneous determination of ammonia, creatinine, and urea.

A strategy for the multifunctionalization of the FIA biosensor was developed. The described multifunctional FIA system offers a fast and simple method for the simultaneous determination of ammonia, creatinine, and urea. The hydrolysis of creatinine by creatinine deiminase (CRDI) or of urea by urease forms ammonia, which is amperometrically detected by an oxygen electrode, based on an enzyme conversion system, glutamate dehydrogenase (GLDH)/glutamate oxidase (GLOD). The split of the stream into three after sample injection and confluence before the GLDH reactor resulted in a three-channel system, into which were set three parallel columns, respectively, filled with immobilized CRDI, urease, and CPG. A triple-peak recording was obtained by putting two delay coils at the channels involving CRDI and urease. Thus the interfering of the endogenous ammonia on the creatinine and urea assay is simultaneously compensated. Furthermore, the problem of great difference in concentration between urea and the other two components is resolved by taking advantage of the differentiated dilution effect for each channel caused from the split-stream, flow-injection system. Linear calibration ranges for ammonia, creatinine, and urea were 0.1-5, 0.2-10, and 2-40 mM, respectively. One run was finished within 5 minutes, and the system was reproducibility good (3 to 5%). The results of the urine assay obtained by the present method will be described in the near future.

Amino Acid Oxidoreductases↗
PubMed1993

Rational protein engineering and industrial application: structure prediction by homology and rational design of protein-variants with improved 'washing performance'--the alkaline protease from Bacillus alcalophilus.

The successful attempt is presented to engineer an enzyme with respect to its technical application by the use of computer-aided protein design techniques. Based on a modeled 3-D structure a number of mutants of a subtilisin-like protease was designed with the aim to increase its washing performance. The model of the highly alkaline subtilisin protease OPTICLEAN from Bacillus alcalophilus was developed by the process of 'modeling by homology' starting with the structure of subtilisin Carlsberg 1CSE.BRK from the Brookhaven protein databank. Amino acid changes and deletions were performed with the graphic protein design program BRAGI. Force field calculations and molecular dynamic simulations were made with AMBER 3.0. The comparison of the model and the later solved X-ray structure of OPTICLEAN shows a high similarity between the two structures. On the other hand, interesting deviations between the two structures were observed in some external loop regions. The comparison shows that the deviations are due to difficulties in the prediction of correct main chain torsion angles of additional prolines and the selection of correct loops in deletion or insertion regions.

Amino Acid Sequence↗