Protein engineering on subtilisin.
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Bacillus↗
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Results for “enzyme engineering”
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Engineering hyperactive variants of human deoxyribonuclease I by altering its functional mechanism.
Human deoxyribonuclease I (DNase I), an enzyme used to treat cystic fibrosis patients, has been engineered to more effectively degrade double-stranded DNA to lower molecular weight fragments by altering its functional mechanism from the native single-stranded nicking pathway to a much more efficient one which results in increased double-stranded scission. By introducing positively charged amino acids at DNase I positions that can interact favorably with the proximal negatively charged phosphate groups of the DNA, we have created a hyperactive variant with approximately 35-fold higher DNA-degrading activity relative to wild type. This enhancement can be attributed to both a decrease in Km and an increase in Vmax. Furthermore, unlike wild-type DNase I, the hyperactive variants are no longer inhibited by physiological saline. Replacement of the same positions with negatively charged amino acids greatly reduced DNA cleavage activity, consistent with a repulsive effect with the neighboring DNA phosphates. In addition, these variants displayed similar activities toward a small synthetic substrate, p-nitrophenyl phenylphosphonate, suggesting that the difference in DNA cleavage activity is due to the interaction of the engineered charged residues with the DNA phosphate backbone rather than any change in catalytic machinery. Finally, experiments involving the repair of DNase I digested DNA with T4 DNA ligase and the Klenow fragment of DNA polymerase I suggest that single-stranded gaps are introduced by the hyperactive variants. Thus, the increased functional activity of the hyperactive variants may be explained in part by a shift toward a processive DNA nicking mechanism, which leads to a higher frequency of double-stranded breaks.
Calcium↗
Supervised enzyme network inference from the integration of genomic data and chemical information.
MOTIVATION: The metabolic network is an important biological network which relates enzyme proteins and chemical compounds. A large number of metabolic pathways remain unknown nowadays, and many enzymes are missing even in known metabolic pathways. There is, therefore, an incentive to develop methods to reconstruct the unknown parts of the metabolic network and to identify genes coding for missing enzymes. RESULTS: This paper presents new methods to infer enzyme networks from the integration of multiple genomic data and chemical information, in the framework of supervised graph inference. The originality of the methods is the introduction of chemical compatibility as a constraint for refining the network predicted by the network inference engine. The chemical compatibility between two enzymes is obtained automatically from the information encoded by their Enzyme Commission (EC) numbers. The proposed methods are tested and compared on their ability to infer the enzyme network of the yeast Saccharomyces cerevisiae from four datasets for enzymes with assigned EC numbers: gene expression data, protein localization data, phylogenetic profiles and chemical compatibility information. It is shown that the prediction accuracy of the network reconstruction consistently improves owing to the introduction of chemical constraints, the use of a supervised approach and the weighted integration of multiple datasets. Finally, we conduct a comprehensive prediction of a global enzyme network consisting of all enzyme candidate proteins of the yeast to obtain new biological findings. AVAILABILITY: Softwares are available upon request.
Algorithms↗
Innovative fermentation strategies for the production of extremophilic enzymes.
A new type of microfiltration (MF) bioreactor, developed in our laboratory, was investigated for use in improving efficiency of the production of extremophilic enzymes. In spite of the difficulties in cultivating hyperthermophiles, we achieved, in 300 h fermentation, more than 38 g/l dry weight of Sulfolobus solfataricus using a MF technique, and we demonstrated that the activity of alcohol dehydrogenase (ADH), as the reporter enzyme, was not affected by cell density. However, hyperthermophile cultivation is difficult to scale up because of evaporation and the very low growth rate. Thus, to achieve high productivity we cultivated, in the MF bioreactor, recombinant mesophilic hosts engineered for the production of two thermophilic enzymes, namely, trehalosyldextrin-forming enzyme (SsTDFE) and trehalose-forming enzyme (SsTFE) from Sulfolobus solfataricus. The traditional Luria-Bertani broth used for recombinant Escherichia coli growth was replaced with a semidefined medium. The latter was used in both the batch and the MF experiments, and the ratio of complex components (e.g., yeast extract and tryptone) to a simple carbon source (glycerol) was decreased during the fed-batch phase to further decrease the medium cost in view of industrial applications. The bioprocess developed was able to improve productivity 500 fold for rSsTFE and 60 fold for rSsTDFE with respect to the wild type cultivated in MF mode. Comparisons with another recombinant enzyme, alpha-glucosidase (rSsalphagly), from Sulfolobus solfataricus produced in our MF bioreactor are reported.
Bioreactors↗
Genetic engineering of human interferons from lymphoblastoid cells: I. Cloning of interferon species expressed in Sendai-induced Namalva cells.
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Base Sequence↗
Structural Characterization and Engineering of a GH134 β-Mannanase from Aspergillus nidulans for Enhancement of Activity and Stability.
Mannans are abundant plant hemicelluloses, and endo-β-mannanases are important biocatalysts for their conversion into functional manno-oligosaccharides. Here, we report the structural and functional characterization of a glycoside hydrolase family 134 β-mannanase from Aspergillus nidulans (AnGH134) and a structure-guided engineering strategy to improve its performance on locust bean gum. The 1.75 Å crystal structure reveals the conserved lysozyme-like fold of GH134 enzymes and supports an inverting catalytic mechanism with Glu43 and Asp55 as the putative catalytic residues. Docking, mutational, and molecular dynamics analyses indicate that AnGH134 uses an extended substrate-binding groove and that groove-exit residues and the C-terminal region contribute to productive catalysis. Guided by these findings, N-terminal fusion of CBM10 enhanced catalytic efficiency and thermal stability, whereas C-terminal fusion was detrimental. These results provide a framework for engineering GH134 mannanases.
Aspergillus nidulans↗
Simultaneous enhancement of thermostability and catalytic activity of phospholipase A(1) by evolutionary molecular engineering.
The thermal stability and catalytic activity of phospholipase A(1) from Serratia sp. strain MK1 were improved by evolutionary molecular engineering. Two thermostable mutants were isolated after sequential rounds of error-prone PCR performed to introduce random mutations and filter-based screening of the resultant mutant library; we determined that these mutants had six (mutant TA3) and seven (mutant TA13) amino acid substitutions. Different types of substitutions were found in the two mutants, and these substitutions resulted in an increase in nonpolar residues (mutant TA3) or in differences between side chains for polar or charged residues (mutant TA13). The wild-type and mutant enzymes were purified, and the effect of temperature on the stability and catalytic activity of the enzymes was investigated. The melting temperatures of the TA3 and TA13 enzymes were increased by 7 and 11 degrees C, respectively, compared with the melting temperature of the wild-type enzyme. Thus, we found that evolutionary molecular engineering was an effective and efficient approach for increasing thermostability without compromising enzyme activity.
Catalysis↗
Identification of copper ligands in Aspergillus oryzae tyrosinase by site-directed mutagenesis.
Copper ligands of the recombinant tyrosinase from the fungus Aspergillus oryzae expressed in Saccharomyces cerevisiae or Escherichia coli were identified by site-directed mutagenesis. The recombinant protyrosinases expressed in S. cerevisiae were assayed for catalytic activities of mono-oxygenase and L-dopa oxidase at pH 5.5 after acid shock at pH 3.0. Replacements of His-63, His-84, His-93, His-290, His-294, His-332 or His-333 with asparagine resulted in mutant enzymes exhibiting no activities. The site-directed mutant Cys82Ala showed that Cys-82 was also an essential residue for the activity. We obtained homogeneous preparations of activated tyrosinases from mutated thioredoxin fusion gene products expressed in E. coli by acid shock. The copper contents of engineered mutants and wild-type enzyme expressed in E. coli were determined by atomic absorption spectrophotometry. The wild-type enzyme contained 2 g-atoms of copper/mol of the subunit. The His63Asn, His84Asn, His93Asn, His290Asn, His294Asn, His332Asn, His333Asn or Cys82Ala substitution decreased copper binding by approx. 50%, indicating that the mutants contain only approx. 1 g-atom of copper/mol of the subunit. The five mutants His63Asn, His93Asn, His290Asn, His294Asn and Cys82Ala contain only one copper ion, which is fully detectable by EPR. From the correlation of g( parallel) and (Cu)A( parallel), we deduced that the nitrogen or sulphur donors in the copper ligands should be in a square or a distorted tetrahedral geometric environment. In further atomic absorption spectrophotometry experiments, no copper atom was observed in the seven double mutants His63Asn/His290Asn, His63Asn/His294Asn, His63Asn/His332Asn, His63Asn/His333Asn, Cys82Ala/His290Asn, His84Asn/His333Asn and His93Asn/His290Asn. We propose a new structure of active sites of tyrosinase from A. oryzae: the most likely binding sites of tyrosinase for Cu(A) are His-63, His-84 and His-93, with the remaining conserved Cys-82 providing the fourth ligand. Cu(B) liganded by four histidine residues, His-290, His-294, His-332 and His-333, is identified as new binding motif of Cu(B).
Alanine↗
The xylose reductase (AKR2B5) structure: homology and divergence from other aldo-keto reductases and opportunities for protein engineering.
The structure of xylose reductase from Candida tenuis (AKR2B5) has been determined and refined to 2.2 A resolution, both in holo and apo forms. These structures allow the recognition of numerous hydrophilic residues responsible for dimerization, a novel feature for the superfamily of enzymes. The residues allowing for dual NADH/NADPH cosubstrate specificity are also identified. Since xylose reductase functions in conjunction with an NAD(+)-specific xylitol dehydrogenase in the xylose assimilation pathway, this is a key step in engineering an enzyme specific for only NADH which will permit cosubstrate recycling between the two enzymes in a high-flux pathway. The structure of xylose reductase, combined with others in the superfamily provides an opportunity to examine and compare structural divergence as a function of sequence homology. It also suggests that the dimeric aldo-keto reductases (AKRs) from families 2 and 7 evolved from a common dimeric ancestor.
Alcohol Oxidoreductases↗
Preparation of artificial bifunctional enzymes by gene fusion.
Within the cellular framework operate a number of sequentially acting enzyme systems of varying complexity and spatial organization. In some cases they are found as bi- or even multi-functional enzymes, where each single polypeptide chain carries two or more catalytic functions. To mimic these enzymes, a number of different bifunctional enzymes have been prepared by in-frame gene fusion. Thus, beta-galactosidase and galactokinase as well as beta-galactosidase and galactose dehydrogenase were genetically fused. Different physical and chemical properties of the hybrid proteins have been investigated, such as protein stability, subunit interactions, linker regions and substrate channeling in vitro and in vivo. Important fields of application for artificial bifunctional enzymes can be found in biochemical analysis, protein purification and metabolic engineering.
Amino Acid Sequence↗
V79 Chinese hamster cells genetically engineered for cytochrome P450 and their use in mutagenicity and metabolism studies.
V79 Chinese hamster cells are being genetically engineered for stable expression of cytochromes P450 using a SV40 early promoter containing plasmid as the eukaryotic expression vector. V79 cells lack endogenous cytochrome P450 activity. Therefore, genetically engineered V79 cell lines are defined for the cytochrome P450 isoform acquired upon cDNA mediated gene transfer. V79 cells have a longstanding tradition as indicator cells for various biological endpoints, e.g. mutation, chromosomal aberration, cytotoxicity. The genetically engineered V79 cells combine these useful biological endpoints with metabolic competence. In this sense, V79 cell lines genetically engineered for stable expression of cytochromes P450 present newly developed tools for studying and understanding metabolism related problems in toxicology and pharmacology.
Animals↗
Elimination of major side effects due to ROS of therapeuticals through biotransformation control of the 57 cytochromes P450 isoenzymes.
Drug biotransformations are controlled in the body by tissue-differentiated choice of a selection of the induced and constitutive 57 isoforms of the human cytochromes P450 (CYP). CYP are the mixed function oxidase enzyme family (EC 1.14.14.1: mono-oxygenases that contain iron-dependent protohaematin IX). Many of the major side effects of clinical therapeutical agents have been attributed to the generation of highly toxic "reactive oxygen species" (ROS). Such ROS generation can be circumvented however by the ability to exert control over which forms of CYP are present: and as to which are bioactive in tissues other than the drug-target ones. Up to 57 isoenzymes of human cytochromes P450 are likely to be present: generation of a variety of species of ROS can be anticipated therefore with outcomes that include cancer, premature ageing, heart (arterial) damage and osteoporosis (in association with oestrogen/testosterone imbalance). Moreover, therapeutic vaccines could be constructed to remove the most harmful forms of cytochromes P450. Designer CYP protein enzymes can now be readily constructed by genetic engineering techniques: these include site-directed mutagenesis to place the modified cyp gene in suitable enzyme expression location in particular tissues only. Furthermore, gene therapy could be arranged to augment (or replace activities lost) for example the production of insulin in the Islets of Langerhans of the pancreas by production of the appropriate form of human insulin in the liver.
Biotransformation↗
Characterization and manipulation of the acyl chain selectivity of fatty acid amide hydrolase.
Fatty acid amide hydrolase (FAAH) is a mammalian integral membrane enzyme that catabolizes several neuromodulatory fatty acid amides, including the endogenous cannabinoid anandamide and the sleep-inducing lipid oleamide. FAAH belongs to a large group of hydrolytic enzymes termed the amidase signature (AS) family that is defined by a conserved, linear AS sequence of approximately 130 amino acids. Members of the AS family display strikingly different substrate selectivities, yet the primary structural regions responsible for defining substrate recognition in these enzymes remain unknown. In this study, a series of unbranched p-nitroanilide (pNA) substrates ranging from 6 to 20 carbons in length was used to probe the acyl chain binding specificity of FAAH, revealing that this enzyme exhibits a strong preference for acyl chains 9 carbons in length or longer. A fluorophosphonate inhibitor of FAAH containing a photoactivatable benzophenone group was synthesized and used to locate a region of the enzyme implicated in substrate binding. Protease digestion and mass spectrometry analysis of FAAH-inhibitor conjugates identified the major site of cross-linking as residues 487-493. Site-directed mutagenesis revealed that a single residue in this region, I491, strongly influenced substrate specificity of FAAH. For example, an I491A mutant displayed a greatly reduced binding affinity for medium-chain pNA substrates (7-12 carbons) but maintained nearly wild-type binding and catalytic constants for longer chain substrates (14-20 carbons). Mutation of I491 to aromatic or more polar residues generated enzymes with relative hydrolytic efficiencies for medium- versus long-chain pNAs that varied up to 90-fold. Collectively, these studies indicate that I491 participates in hydrophobic binding interactions with medium-chain FAAH substrates. Additionally, the significant changes in substrate selectivity achieved by single amino acid changes suggest that FAAH possesses a rather malleable substrate binding domain and may serve, along with other AS enzymes, as a template for the engineering of amidases with novel and/or tailored specificities.
Alanine↗
Molecular studies on the toxifying effects by genetically engineered cytochromes P450.
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Animals↗
Cholinesterases in neural development: new findings and toxicologic implications.
Developing animals are more sensitive than adults to acute cholinergic toxicity from anticholinesterases, including organophosphorus pesticides, when administered in a laboratory setting. It is also possible that these agents adversely affect the process of neural development itself, leading to permanent deficits in the architecture of the central and peripheral nervous systems. Recent observations indicate that organophosphorus exposure can affect DNA synthesis and cell survival in neonatal rat brain. New evidence that acetylcholinesterase may have a direct role in neuronal differentiation provides additional grounds for interest in the developmental toxicity of anticholinesterases. For example, correlative anatomic studies show that transient bursts of acetylcholinesterase expression often coincide with periods of axonal outgrowth in maturing avian, rodent, and primate brain. Some selective cholinesterase inhibitors effectively suppress neurite outgrowth in model systems like differentiating neuroblastoma cells and explanted sensory ganglia. When enzyme expression is altered by genetic engineering, acetylcholinesterase levels on the outer surface of transfected neurons correlate with ability to extend neurites. Certain of these "morphogenic" effects may depend on protein-protein interactions rather than catalytic acetylcholinesterase activity. Nonetheless, it remains possible that some pesticides interfere with important developmental functions of the cholinesterase enzyme family.
Acetylcholinesterase↗
On the safety of Trichoderma reesei.
Trichoderma reesei has a long history of safe use in industrial-scale enzyme production. Applications of cellulases and xylanases produced by this fungus are found in food, animal feed, pharmaceutical, textile and pulp and paper industries. T. reesei is non-pathogenic for man and it has been shown not to produce fungal toxins or antibiotics under conditions used for enzyme production. During recent years genetic engineering techniques have also been used to improve the industrial production strains of T. reesei and, in addition, considerable experience of safe use of recombinant T. reesei strains in industrial scale has accumulated. Thus, T. reesei can be generally considered not only a safe production organism of its natural enzymes but also a safe host for other harmless gene products.
Allergens↗
Enhanced electron transfer and lauric acid hydroxylation by site-directed mutagenesis of CYP119.
CYP119, a cytochrome P450 from a thermophilic organism for which a crystal structure is available, is shown here to hydroxylate lauric acid in a reaction supported by putidaredoxin and putidaredoxin reductase. This fatty acid hydroxylation activity is increased 15-fold by T214V and D77R mutations. The T214V mutation increases the rate by facilitating substrate binding and enhancing the associated spin state change, whereas the D77R mutation improves binding of the heterologous redox partner putidaredoxin to CYP119 and the rate of electron transfer from it to the heme group. A sequence alignment with P450(cam) can, therefore, be used to identify a part of the binding site for putidaredoxin on an unrelated P450 enzyme. This information can be used to engineer by mutagenesis an improved complementarity of the protein-protein interface that results in improved electron transfer from putidaredoxin to the P450 enzyme. As a result, the catalytic activity of the thermo- and barostable CYP119 has been incorporated into a catalytic system that hydroxylates fatty acids.
Amino Acid Sequence↗
Use and limitations of modular metabolic control analysis in medicine and biotechnology.
By Metabolic Control Analysis (MCA), it has been shown that control on flux is in most cases shared by several enzymes rather than concentrated on one "rate-limiting step." This analysis also allows the quantification of the control exerted by groups (modules) of enzymes. The modules may correspond to spatial compartments or to functional units. A brief outline of the modular approach to MCA is given. The criteria by which the system can be modularized and the concept of monofunctional unit are explained. Various studies in which control analysis was applied to biotechnological and medical issues are reviewed. In particular, MCA has turned out to be helpful in the assessment of the severity of enzyme deficiencies. Another application is the search for target enzymes or enzyme groups where pharmaceuticals can suppress the metabolism of pathogenic microorganisms most. In biotechnology, modular and "traditional" control analyses are valuable tools for choosing the most promising targets for genetic manipulation so as to increase a biosynthetic flux. As control coefficients are linear approximations, the effect of enhancing the activities of enzymes to a larger extent is often overestimated. Further limitations such as the restriction to stationary states, uncertainties due to spatial heterogeneities and the impact of experimental error are discussed.
Animals↗