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Results for “enzyme engineering”

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PubMed1991

Protein engineering of Rubisco.

Modification of the kinetic parameters of enzymes by protein engineering requires extensive knowledge of the structural details of the enzyme and its complexes with different reaction intermediate analogues. Such structural studies are described here for Rubisco, ribulose-1,5-bisphosphate carboxylase/oxygenase, which catalyzes the initial reactions of two important but competing physiological events in green plants; carbon dioxide fixation and photorespiration. Observed functional changes in mutants of Rubisco are correlated with structural details as well as with the defined conformational changes that occur during catalysis. A possible functional role of the small subunit of Rubisco is described based on a comparison of the active-site geometry of a bacterial L2 molecule with that of higher plant L8S8 molecules. The ultimate aim of these studies is to engineer Rubisco mutants that are more efficient than the wild-type enzyme by decreasing the oxygenase/carboxylase ratio.

Kinetics↗

PubMed2002

Characterization of heterologous and native enzyme activity profiles in metabolically engineered Zymomonas mobilis strains during batch fermentation of glucose and xylose mixtures.

Zymomonas mobilis has been metabolically engineered to broaden its substrate utilization range to include D-xylose and L-arabinose. Both genomically integrated and plasmid-bearing Z. mobilis strains that are capable of fermenting the pentose D-xylose have been created by incorporating four genes: two genes encoding xylose utilization metabolic enzymes (xylA/xylB) and two genes encoding pentose phosphate pathway enzymes (talB/tktA). We have characterized the activities of the four newly introduced enzymes for xylose metabolism, along with those of three native glycolytic enzymes, in two different xylose-fermenting Z. mobilis strains. These strains were grown on glucose-xylose mixtures in computer-controlled fermentors. Samples were collected and analyzed to determine extracellular metabolite concentrations as well as the activities of several intracellular enzymes in the xylose and glucose uptake and catabolism pathways. These measurements provide new insights on the possible bottlenecks in the engineered metabolic pathways and suggest methods for further improving the efficiency of xylose fermentation.

Acetates↗

PubMed2005

Engineering of phenylacetaldehyde reductase for efficient substrate conversion in concentrated 2-propanol.

Phenylacetaldehyde reductase (PAR) is suitable for the conversion of various aryl ketones and 2-alkanones to corresponding chiral alcohols. 2-Propanol acts as a substrate solvent and hydrogen donor of coupled cofactor regeneration during the conversion of substrates catalyzed by PAR. To improve the conversion efficiency in high concentrations of substrate and 2-propanol, selection of a PAR mutant library and the subsequent rearrangement of mutations were attempted. With only a single selection round and following the manual combination of advantageous mutations, PAR was successfully adapted for the conversion of high concentrations of substrate with concentrated 2-propanol. This method will be widely applicable for the engineering of enzymes potentially valuable for industry.

1-Propanol↗

PubMed2025

Aminoacyl-tRNA Specificity of a Ligase Catalyzing Non-ribosomal Peptide Extension.

Peptide aminoacyl-transfer ribonucleic acid ligases (PEARLs) are amide-bond-forming enzymes that extend the main chain of peptides by using aminoacyl-tRNA (aa-tRNA) as a substrate. In this study, we investigated the substrate specificity of the PEARL BhaBCAla from Bacillus halodurans, which utilizes Ala-tRNAAla. By leveraging flexizyme, a ribozyme capable of charging diverse acids onto a desired tRNA, we generated an array of aa-tRNAs in which we varied both the amino acid and the tRNA to dissect the substrate scope of BhaBCAla. We demonstrate that BhaBCAla catalyzes peptide extension with noncognate proteinogenic and noncanonical amino acids, hydroxy acids, and mercaptocarboxylic acids when attached to tRNAAla. For most of these, the efficiency was considerably reduced compared to Ala, indicating that the enzyme recognizes the amino acid. By variation of the different parts of the tRNA, enzyme specificity was shown to also depend on the acceptor stem and the anticodon arm of the tRNA. These findings establish the molecular determinants of PEARL specificity and provide a foundation for engineering these enzymes for broader applications in peptide synthesis.

RNA, Transfer, Amino Acyl↗

PubMed1998

Synthesis of amidrazones using an engineered papain nitrile hydratase.

To demonstrate the usefulness of an engineered papain nitrile hydratase as a biocatalyst, a peptide amidrazone was prepared by incubation of the nitrile MeOCO-Phe-Alanitrile with the Gln19Glu papain mutant in the presence of salicylic hydrazide as a nucleophile. The amidrazone results from nucleophilic attack by salicylic hydrazide at the imino carbon of the thioimidate adduct formed between the enzyme and the peptide nitrile substrate. Compared to wild-type enzyme, the engineered nitrile hydratase causes a better than 4000-fold increase in the rate of amidrazone formation and yields a product of much higher purity. The advantages over other nitrile-hydrolyzing enzymes and current limitations of the papain nitrile hydratase are discussed.

Catalysis↗

PubMed2005

Rational design of thermostable lactate oxidase by analyzing quaternary structure and prevention of deamidation.

Our current knowledge of protein unfolding is overwhelmingly related to reversible denaturation. However, to engineer thermostable enzymes for industrial applications and medical diagnostics, it is necessary to consider irreversible denaturation processes and/or the entire quaternary structure. In this study we have used lactate oxidase (LOD), which is employed in lactic acid sensors, as a model example to design thermostable variants by rational design. Twelve mutant proteins were tested and one of them displayed a markedly greater thermostability than all the mutants we had previously obtained by random mutagenesis. This mutant was designed so as to strengthen the interaction between the subunits and stabilize the quaternary structure. Since LOD is difficult to crystallize, its three-dimensional structure remains unknown. This study shows that it is possible to carry out rational design to improve thermostability using a computer-aided quaternary structure model based on the known tertiary structure of a related protein. Critical factors required for increasing the thermal stability of proteins by rational design, where the 3-D structure is not available, are discussed.

Amides↗

PubMed2000

Src-Abl tyrosine kinase chimeras: replacement of the adenine binding pocket of c-Abl with v-Src to swap nucleotide and inhibitor specificities.

Engineered protein kinases with unnatural nucleotide specificity and inhibitor sensitivity have been developed to trace kinase substrate targets. We first engineered unnatural nucleotide specificity into v-Src by mutating one residue, isoleucine 338, to alanine. This position is highly conserved among all kinases in the sense that it is always occupied by either a large hydrophobic residue or threonine. Because of the conservation of this residue and the highly conserved fold of the kinase family, we have attempted to generalize the engineering of all kinases on the basis of our success with v-Src. Although many kinases can be similarly engineered using v-Src as a blueprint, we encountered one kinase, c-Abl, which when mutated, does not display the ability to accept unnatural ATP analogues. To overcome this failure of the engineered c-Abl (T315A) to accept unnatural nucleotides, we developed a new strategy for introducing unnatural nucleotide specificity into kinases. We generated a chimeric kinase in which regions of the kinase domain of c-Abl were swapped with the corresponding regions of v-Src (I338A). Specifically, we engineered two chimeras in which the N-terminal lobe of the SH1 domain of c-Abl was swapped with that of v-Src. These kinase chimeras were found to have the same unnatural nucleotide specificity as that of v-Src (I338A), while retaining the peptide specificity of c-Abl. Thus, these chimeric kinases are suitable for identifying the direct substrates of c-Abl. These engineered chimeric enzymes provide a new strategy for constructing kinases with tailor-made ligand binding properties.

Adenosine Triphosphate↗

PubMed1997

A cell-based immunobiosensor with engineered molecular recognition--Part II: Enzyme amplification systems.

Immune cells in vivo routinely perform highly selective immunosensing in blood and tissues as part of their normal immune surveillance functions. We have been investigating the potential of exploiting the immunosensing detection abilities of excitable immune cells (i.e. the mast cell) for the development of whole cell immunobiosensors. A key feature is that these immune cells can be selectively engineered to recognize specific antigens in vitro. In the presence of antigen, these cells undergo excitable activation responses which result in increased metabolism and the exocytosis of stored intracellular mediators. We have previously determined that mast cell metabolic responses can be thermally transduced in real time, thus indicating the possibility of whole cell thermoelectric immunobiosensing. In this work we investigated the use of enzyme amplification systems to enhance the direct transduction of immune cell responses to analyte. It was found that with appropriate enzymes, peak outputs occurred within approximately 5 min (4-20 times faster than without enzymes) and peak response magnitudes were up to nine-fold greater than without enzymes.

Amine Oxidase (Copper-Containing)↗

PubMed2006

From monomeric to homodimeric endonucleases and back: engineering novel specificity of LAGLIDADG enzymes.

Monomeric homing endonucleases of the LAGLIDADG family recognize DNA in a bipartite manner, reflecting the underlying structural assembly of two protein domains (A and B) related by pseudo 2-fold symmetry. This architecture allows for changes in DNA specificity via the distinct combination of these half-site domains. The key to engineering such hybrid proteins lies in the LAGLIDADG two-helix bundle that forms both the domain interface and the endonuclease active site. In this study, we utilize domain A of the monomeric I-DmoI to demonstrate the feasibility of generating functional homodimeric endonucleases that recognize palindromic DNA sequences derived from the original, non-palindromic target. Wild-type I-DmoI domain A is capable of forming a homodimer (H-DmoA) that binds tightly to, but does not cleave efficiently, its anticipated DNA target. Partial restoration of DNA cleavage ability was obtained by re-engineering the LAGLIDADG dimerization interface (H-DmoC). Upon fusing two copies of H-DmoC via a short peptide linker, a novel, site-specific DNA endonuclease was created (H-DmoC2). Like I-DmoI, H-DmoC2 is thermostable and cleaves the new target DNA to generate the predicted 4 nt 3'-OH overhangs but, unlike I-DmoI, H-DmoC2 retains stringent cleavage specificity when substituting Mn2+ for Mg2+ as co-factor. This novel endonuclease allows speculation regarding specificity of monomeric LAGLIDADG proteins, while it supports the evolutionary genesis of these proteins by a gene duplication event.

Amino Acid Motifs↗

PubMedDate not supplied

Kinetic properties of native and mutagenized isoforms of mitochondrial alcohol dehydrogenase III purified from Kluyveromyces lactis.

By computer modelling and protein engineering we have investigated changes in two amino acid residues located in the coenzyme pocket of the yeast Kluyveromyces lactis mitochondrial alcohol dehydrogenase III. These two residues, Gly 225 and Ala 274, were hypothesized to be involved in the enzyme discrimination between NAD(H) and NADP(H). Upon changing Gly 225 to Ala we produced an enzyme (mutant G225A) showing very little difference from the wild-type. On the contrary, change at position 274 of Phe instead of Ala (mutant A274F) caused a significant increase of K(m) values for NAD(P) and for NADPH and even a more marked decrease in catalytic activity. The k(cat)/K(m) rates for NADP(H) were also decreased in this mutant. Enzymes with the double changes at 225 and 274 (mutant G225A-A274F) showed, apart the substantial low K(m) value for NADPH and its high catalytic efficiency, kinetic parameters relative to coenzymes which were not additive over the single substitutions. Surprisingly, enzymes with changes at the two positions reduced efficiently acetaldehyde, displaying a K(m) value 10-fold lower and a catalytic efficiency sevenfold higher with respect to parent or singularly mutated enzymes. None of the engineered enzymes would convert formaldehyde, glutaraldehyde or aromatic aldehydes but all enzymes reduced propionaldehyde and butyraldehyde at relative reaction rates approximately half of that exhibited by acetaldehyde. Interestingly only mutant A274F was able to oxidize methanol almost as well as ethanol. In addition, this mutant was capable to convert secondary and cyclic alcohols, at a rate not detected in the other isoforms. These results are in general agreement with the prediction that increasing the size of amino acids in the proximity of the coenzyme pocket would hamper the accommodation of NADP but discord the increased affinity for NADPH as well as for alcoholic or aldehydic substrates with high steric hindrance.

Aldehyde Oxidoreductases↗

PubMed2001

Characterization of a new feedback-resistant 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase AroF of Escherichia coli.

Tyrosine feedback-inhibits the 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAHP) synthase isoenzyme AroF of Escherichia coli. Here we show that an Asn-8 to Lys-8 substitution in AroF leads to a tyrosine-insensitive DAHP synthase. This mutant enzyme exhibited similar activities (v=30-40 U mg(-1)) and substrate affinities (K(m)(erythrose-4-phosphate)=0.5 mM, positive cooperativity with respect to phospho(enol)pyruvate) as the wild-type AroF, but showed decreased thermostability. An engineered AroF enzyme lacking the seven N-terminal residues also was tyrosine-resistant. These results strongly suggest that the N-terminus of AroF is involved in the molecular interactions occurring in the feedback-inhibition mechanism.

3-Deoxy-7-Phosphoheptulonate Synthase↗

PubMed1997

Interaction of aflatoxin B1 with cytochrome P450 2A5 and its mutants: correlation with metabolic activation and toxicity.

Among members of the mouse cytochrome P450 2A family, P450 2A5 is the best catalyst of aflatoxin B1 (AFB1) oxidation to its 8,9-epoxide (Pelkonen, P., Lang, M., Wild, C. P., Negishi, M., and Juvonen, R. O. (1994) Eur. J. Pharmacol., Environ. Toxicol. Pharmacol. Sect. 292, 67-73). Here we studied the role of amino acid residues 209 and 365 of the P450 2A5 in the metabolism and toxicity of AFB1 using recombinant yeasts. The two sites have previously been shown to be essential in the interaction of coumarin and steroids with the P450 2A5. Reducing the size of the amino acid at position 209 or introducing a negatively charged residue at this site increased the 8,9-epoxidation of AFB1 compared to the wild type. In addition, replacing the hydrophobic amino acid at the 365 position with a positively charged lysine residue strongly decreased the metabolism of AFB1. These mutations changed the KM values generally less than the Vmax values. The changes in AFB1 metabolism contrast with the changes in coumarin 7-hydroxylation caused by these amino acid substitutions, since reducing the size of the 209 residue strongly reduced coumarin metabolism and increased the K(M) values. On the other hand, the results with AFB1 are similar to those obtained with steroid hydroxylation. This suggests that the size of the substrate is important when interacting with the residue 209 of the protein. The catalytic parameters of AFB1 correlated generally with its toxicity to the recombinant yeasts expressing the activating enzyme and with the binding of AFB1 to yeast DNA. Furthermore high affinity substrates and inhibitors (e.g., methoxsalen, metyrapone, coumarin 311, 7-methylcoumarin, coumarin, and pilocarpine) of P450 2A5 could efficiently block the toxicity of AFB1. It is suggested that the recombinant yeasts expressing engineered P450 enzymes are a useful model to understand the substrate protein interactions, to study the relationship of metabolic parameters to toxicity, and to test potential inhibitors of metabolism based toxicity.

Aflatoxin B1↗

PubMed2002

Perspectives on biotechnological applications of archaea.

Many archaea colonize extreme environments. They include hyperthermophiles, sulfur-metabolizing thermophiles, extreme halophiles and methanogens. Because extremophilic microorganisms have unusual properties, they are a potentially valuable resource in the development of novel biotechnological processes. Despite extensive research, however, there are few existing industrial applications of either archaeal biomass or archaeal enzymes. This review summarizes current knowledge about the biotechnological uses of archaea and archaeal enzymes with special attention to potential applications that are the subject of current experimental evaluation. Topics covered include cultivation methods, recent achievements in genomics, which are of key importance for the development of new biotechnological tools, and the application of wild-type biomasses, engineered microorganisms, enzymes and specific metabolites in particular bioprocesses of industrial interest.

Archaea↗

PubMed1990

Bioreactors in medicine.

The use of immobilized enzyme reactors in medicine is briefly reviewed in this presentation. Emphasis is placed on the recent and emerging technology involved in development and clinical applications of such devices.

Biomedical Engineering↗

PubMed2006

Histaminase PEGylation: preparation and characterization of a new bioconjugate for therapeutic application.

Copper amine oxidase catalyses the oxidative deamination of primary amino groups of several biogenic amines, one of which is histamine, the principal chemical mediator of the first phase of allergic reactions. Looking forward to a possible future therapeutic application of this enzyme in the field of histamine-mediated afflictions, we developed a simple method for the purification of a histaminase from grass pea shoots, a source particularly enriched with the enzyme. Furthermore, in order to improve its therapeutic potential, in particular to reduce the high impurity due to its heterologous source, we conjugated the protein with poly(ethylene glycol) and tested the molecular, immunogenic and pharmacokinetic properties of the native and modified forms. The PEGylated enzyme showed molecular and enzymatic properties similar to those of the unmodified one, but the PEGylation extended the permanence of the injected drug in the body and eliminated its high immunogenic behaviour. The considerable ease of native histaminase production as well as the improved properties after PEGylation, make this engineered plant enzyme a suitable drug candidate for alternative treatment of histamine-mediated affections.

Algorithms↗

PubMed2001

Kinetic effects of a single-amino acid mutation in a highly variable loop (residues 114-120) of class IV ADH.

Class IV alcohol dehydrogenase shows a deletion at position 117 with respect to class I enzymes, which typically have a Gly residue. In class I structures, Gly117 is part of a loop (residues 114-120) that is highly variable within the alcohol dehydrogenase family. A mutant human class IV enzyme was engineered in which a Gly residue was inserted at position 117 (G117ins). Its kinetic properties, regarding ethanol and primary aliphatic alcohols, secondary alcohols and pH profiles, were determined and compared with the results obtained in previous studies in which the size of the 114-120 loop was modified. For the enzymes considered, a smaller loop was associated with a lower catalytic efficiency towards short-chain alcohols (ethanol and propanol) and secondary alcohols, as well as with a higher K(m) for ethanol at pH 7.5 than at pH 10.0. The effect can be rationalized in terms of a more open, solvent-accessible active site in class IV alcohol dehydrogenase, which disfavors productive binding of ethanol and short-chain alcohols, specially at physiological pH.

Alcohol Dehydrogenase↗

PubMed2002

Cytochrome P450-based gene therapy for cancer treatment: from concept to the clinic.

In the last 16 years, more than a dozen gene-directed enzyme prodrug therapies for cancer treatment have been evaluated in preclinical studies. However, only few of them have evolved to the stage of clinical trial. This review assesses current knowledge in the area of cancer gene therapy, emphasizing cytochrome p450 (CYP)-based prodrug activation systems. This approach is intuitively highly suitable for the treatment of cancers, since several major anticancer drugs are activated by liver CYP enzymes. Important features of this strategy include: 1) use of human CYP genes to avoid immune complications that may hamper expression of therapeutic genes of non-human origin and thereby inhibit prodrug activation, 2). use of well established and clinically effective anticancer prodrugs, 3). strong bystander cytotoxic effect seen with all liver-activated CYP prodrugs, 4). the potential to inhibit liver CYP activity and expression to increase the bioavailability of prodrugs for CYP-transduced tumors, 5). possible extension to many CYP enzymes and their potential anticancer prodrug substrates, and 6). it can be used to arm therapeutic conditionally replicating viruses. Historically, this strategy utilized CYP 2B1 to activate oxazaphosphorines. It is now becoming clear that the repertoire of prodrugs is expandable and that CYP gene candidates are not limited to naturally occurring CYP genes, but may also encompass engineered CYP enzymes, improved by site directed mutagenesis or other approaches. Encouraging results from a recent phase I/II clinical trial that have implemented this strategy, as well as emerging problems related to gene delivery are discussed in this review.

Animals↗

PubMed2003

Modeling of reaction kinetics for reactor selection in the case of L-erythrulose synthesis.

To choose the most effective process design in enzyme process development it is important to find the most effective reactor mode of operation. This goal is achieved by modeling of the reaction kinetics as a tool of enzyme reaction engineering. With the example of the transketolase catalyzed L-erythrulose synthesis we demonstrate how the most effective reactor mode can be determined by kinetic simulations. This is of major importance if the biocatalyst deactivation is caused by one of the substrates as in this case by glycolaldehyde. The cascade of two membrane reactors in series with soluble enzyme is proposed as a solution for the enzyme deactivation by one of the substrates.

Journal Article↗