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At least 217 records · Page 12Linked to original sources
PubMed1999

Protein engineering of a human enzyme that hydrolyzes V and G nerve agents: design, construction and characterization.

Because of deficiencies in the present treatments for organophosphorus anticholinesterase poisoning, we are attempting to develop a catalytic scavenger that can be administered as prophylactic protection. Currently known enzymes are inadequate for this purpose because they have weak binding and slow turnover, so we are trying to make an appropriate enzyme by protein engineering techniques. One butyrylcholinesterase mutant, G117H, has the desired type of activity but reacts much too slowly. This communication describes an attempt to determine the reason for the slow reaction so that a more efficient enzyme might be designed. The results indicate that the mutation at residue 117 has resulted in a distortion of the transition state of the reaction of organophosphorus compounds with the active site serine. This information will be used to develop other mutants that avoid transition state stabilization sites.

Animals↗
PubMedDate not supplied

Rational modification of enzyme catalysis by engineering surface charge.

Changing the surface charge of subtilisin by site-directed mutagenesis produces enzymes with significantly shifted pH-activity profiles, higher catalytic activities and altered specificities. Insight into water as a dielectric, the role of ions in electrostatic shielding and the field effects on catalysis is obtained and suggests rules for tailoring pH-activity profiles.

Buffers↗
PubMed2001

Genetic and protein engineering of diagnostic enzymes, cholesterol oxidase and xylitol oxidase.

For a long time, clinical diagnosis has been made mainly using chemical methods. Recently, several excellent substrate-specific enzymes have been developed and these enzymes are used as diagnostic catalysts. Using enzymes, it is possible to assay for a specific substance from specimens of serum or urine without the need for isolation of the substance which simplifies the process and shortens the assay time. Furthermore, the use of enzymatic assay methods for diagnosis has been facilitated by the developments in genetic engineering which made it possible to overproduce enzymes inexpensively. Here, we review the diagnostic enzymes, cholesterol oxidase and xylitol oxidase, which were successfully overproduced in our laboratory. In particular, the catalytic activity and pH and thermal stabilities of cholesterol oxidase were improved.

Journal Article↗
PubMed2002

Genetically-engineered bacteria expressing human enzymes and their use in the study of mutagens and mutagenesis.

Expression of human enzymes in bacterial cells has made possible the development of a new generation of mutagenicity assays. Metabolic activation takes place within the internal milieu of the target cell, so that no membrane barrier is interposed between the reactive metabolite and the DNA target. This technology has been extended to all major classes of enzymes of xenobiotic biotransformation, including P450. Expression of sequence variants of mutagen-bioactivation enzymes provides a new tool for studying their structure and function.

Bacteria↗
PubMed1987

Control of oligomeric enzyme thermostability by protein engineering.

The ability to control the resistance of an enzyme to inactivation due to exposure to elevated temperatures is essential for the understanding of thermophilic behavior and for developing rational approaches to enzyme stabilization. By means of site-directed mutagenesis, point mutations have been engineered in the dimeric enzyme yeast triosephosphate isomerase that improve its thermostability. Cumulative replacement of asparagine residues at the subunit interface by residues resistant to heat-induced deterioration and approximating the geometry of asparagine (Asn-14----Thr-14 and Asn-78----Ile-78) nearly doubled the half-life of the enzyme at 100 degrees C, pH 6. Moreover, in an attempt to model the deleterious effects of deamidation, we show that replacement of interfacial Asn-78 by an aspartic acid residue increases the rate constant of irreversible thermal inactivation, drastically decreases the reversible transition temperature, and reduces the stability against dilution-induced dissociation.

Carbohydrate Epimerases↗
PubMed2006

Activating an enzyme by an engineered coiled coil switch.

We designed a de novo protein based on a circular permutant of RNaseT1, in which the enzymatic activity can be manipulated by engineered peptide binding. The circular permutant of RNaseT1 was obtained by tethering the original C- and N-termini with a GPAG linker and cleaving the molecule between Glu82 and Asn83. This mutant lacked enzymatic activity, due to the destabilization of entire protein structure. We previously reported the construction of ABC-type heterotrimeric coiled coil peptides, in which the A- and B-type peptides cannot form the folded trimeric structure without the C-type peptide. The introduction of the A- and B-type coiled coil peptides to the C- and N-termini of the circular permutant of RNaseT1, respectively, and the subsequent addition of the C-type coiled coil peptide enabled the RNaseT1 domain to refold properly, thus, restoring the enzymatic activity. The formation of the trimeric coiled coil structure should bring the cleaved sites of RNaseT1 close enough to refold the RNaseT1 domain spontaneously.

Enzyme Activation↗
PubMed1989

Control of enzyme activity by an engineered disulfide bond.

A novel approach to the control of enzyme catalysis is presented in which a disulfide bond engineered into the active-site cleft of bacteriophage T4 lysozyme is capable of switching the activity on and off. Two cysteines (Thr21----Cys and Thr142----Cys) were introduced by oligonucleotide-directed mutagenesis into the active-site cleft. These cysteines spontaneously formed a disulfide bond under oxidative conditions in vitro, and the catalytic activity of the oxidized (cross-linked) T4 lysozyme was completely lost. On exposure to reducing agent, however, the disulfide bond was rapidly broken, and the reduced (non-cross-linked) lysozyme was restored to full activity. Thus an enzyme has been engineered such that redox potential can be used to control catalytic activity.

Binding Sites↗
PubMed2005

Structural and kinetic basis for substrate selectivity in Populus tremuloides sinapyl alcohol dehydrogenase.

We describe the three-dimensional structure of sinapyl alcohol dehydrogenase (SAD) from Populus tremuloides (aspen), a member of the NADP(H)-dependent dehydrogenase family that catalyzes the last reductive step in the formation of monolignols. The active site topology revealed by the crystal structure substantiates kinetic results indicating that SAD maintains highest specificity for the substrate sinapaldehyde. We also report substantial substrate inhibition kinetics for the SAD-catalyzed reduction of hydroxycinnamaldehydes. Although SAD and classical cinnamyl alcohol dehydrogenases (CADs) catalyze the same reaction and share some sequence identity, the active site topology of SAD is strikingly different from that predicted for classical CADs. Kinetic analyses of wild-type SAD and several active site mutants demonstrate the complexity of defining determinants of substrate specificity in these enzymes. These results, along with a phylogenetic analysis, support the inclusion of SAD in a plant alcohol dehydrogenase subfamily that includes cinnamaldehyde and benzaldehyde dehydrogenases. We used the SAD three-dimensional structure to model several of these SAD-like enzymes, and although their active site topologies largely mirror that of SAD, we describe a correlation between substrate specificity and amino acid substitution patterns in their active sites. The SAD structure thus provides a framework for understanding substrate specificity in this family of enzymes and for engineering new enzyme specificities.

Acrolein↗
PubMed1995

Indoleglycerol phosphate synthase-phosphoribosyl anthranilate isomerase: comparison of the bifunctional enzyme from Escherichia coli with engineered monofunctional domains.

Putative domain--domain interactions of the monomeric bifunctional enzyme indoleglycerol phosphate synthase:phosphoribosyl anthranilate isomerase from Escherichia coli were probed by separating the domains on the gene level and expressing them as monofunctional proteins. The engineered monofunctional enzymes were found to be stable, monomeric proteins with virtually full catalytic activity. In addition, binding of indolyglycerol phosphate to the active site of indoleglycerol phosphate synthase and binding of reduced 1-[(2-carboxyphenyl)amino]-1-deoxyribulose 5-phosphate, a competitive inhibitor of both indoleglycerol phosphate synthase and phosphoribosyl anthranilate isomerase, were almost identical in both the mono- and bifunctional enzymes. Furthermore, no association between the monofunctional enzymes was found, neither in vitro, by sedimentation and gel filtration experiments, nor in vivo, by coexpression of the domains in the same cell. Thus, no selective advantages of the bifunctional enzyme from Escherichia coli over the respective monofunctional enzymes were found on a functional level. However, the phosphoribosyl anthranilate isomerase domain appears to stabilize the indoleglycerol phosphate synthase domain of the bifunctional enzyme from Escherichia coli by interactions that seem to subtly influence the kinetics of ligand binding.

Aldose-Ketose Isomerases↗
PubMedDate not supplied

The use of biosensor technology for the engineering of antibodies and enzymes.

Recently developed scientific instrumentation featuring surface plasmon resonance detection allows the detection of biomolecular interactions in real time and without chemical modification of the binding partners. These biosensors are proving invaluable tools in protein engineering, particularly in research aimed at the isolation and improvement of protein binders and catalysts from macromolecular repertoires containing billions of individual members. This article reviews the use of biosensor technology for the isolation and characterization of engineered antibodies and enzymes.

Animals↗
PubMed2002

Enzyme technology and bioprocess engineering.

The impact of directed evolution and site-specific mutagenesis on the industrial utility of enzymatic catalysis through the modification of enzyme structure and function is clearly an important area of research in bioprocess engineering. High-throughput screening for novel or improved enzyme activities, both by more efficiently exploring nature's diversity and by creating new diversity in the test tube, allows new bioprocesses to be developed. Similarly, innovations in enzyme technology that address novel ways to apply enzymes in bioprocesses also have an impact on bioprocess engineering. Several recent developments have been made in this latter aspect of bioprocess engineering.

Biotechnology↗
PubMed1997

The dimerization of Pseudomonas putida cytochrome P450cam: practical consequences and engineering of a monomeric enzyme.

Cytochrome P450cam dimerizes via the formation of an intermolecular disulfide bond, complicating the storage and handling of the enzyme, particularly at higher concentrations. The dimeric enzyme is 14% less active than the monomer and forms at a slow but significant rate even at 4 degrees C [k = 1.09 x 10(-3) mM(-1) h(-1)]. To eliminate any ambiguity introduced by dimer formation and to simplify handling and storage of the enzyme, site-directed mutagenesis was used to identify C334 as the single cysteine residue responsible for the formation of the disulfide linkage and to engineer a monomeric enzyme by substituting an alanine in its place. The C334A mutant is identical with the wild-type P450cam monomer in terms of optical spectra, camphor binding and turnover activity, but shows no evidence of dimerization and aggregation even at millimolar concentrations. Preliminary 1H NMR investigations also indicate a significant improvement in the quality of spectra obtained with this mutant. (C334A)P450cam is therefore proposed as an alternative to the wild-type enzyme-a base mutant otherwise identical with the wild-type but with improved handling characteristics.

Camphor↗