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PubMed1992

[Evaluation of the usefulness of enzyme tests for the diagnosis of the effects of various organic solvents in the conditions of occupational exposure].

The aim of the study was to evaluate the suitability of enzymatic tests for estimating the effects of organic solvents upon the organisms of daily exposed workers. The analysis was undertaken in 572 workers exposed to such solvents as toluene, xylene, cresols and benzine. Periodical examination was carried out for the purposes of the study. The level of excreting organic solvents' metabolites in urine and serum enzymes' activity were determined. Also, the activity of alanine and aspartate aminotransferases and of plasmatic cholinesterase was determined. The results of the study support the necessity of applying the enzymatic tests as a basic method of evaluating the toxic activity of organic solvents.

Air Pollutants, Occupational↗
PubMed2001

Telomerase: diagnostics, cancer therapeutics and tissue engineering.

The enzyme telomerase has a key role in controlling the lifespan of human cells. It is absent from most somatic tissues but is reactivated in more than 85% of cancers, making the enzyme ideal as a marker of cancer cells and as a therapeutic target. In the context of normal human cells, the enzyme can extend cellular lifespan without causing cancer-associated changes or altering phenotypic properties. This capability could solve a major obstacle in the use of normal human cells for tissue engineering, that is, the induction of cellular senescence.

Journal Article↗
PubMed1999

[Purification and properties of human lysozyme in engineered bacterium E. coli].

To obtain the SDS-PAGE-pure human lysozyme, the crude enzyme of engineered bacterium E. coli was purified by chromatography on cation ion exchange of Express-Ion S. The optimum reaction temperature and pH of this lysozyme were 45 degrees C and 6.5, respectively. The isoelectric point is pH 8.91, and Km of the enzyme for Micrococcus lysodeikticus is 0.0311 mg/mL. The thermal stability of the engineered enzyme is more sensible than hen egg white lysozyme and human milk lysozyme. The sequence of 5 amino acids in N-end is same as designed, except an Met at the first. The affects of some metal ion on this enzyme were shown. Cu2+ of 0.01 mmol/L could completely inactivate the enzyme.

Amino Acid Sequence↗
PubMed2000

Extended-range glucose sensor employing engineered glucose dehydrogenases.

An enzyme glucose sensor with an expanded dynamic range was constructed using a novel strategy. This strategy was based on a new concept of utilizing protein-engineered enzymes with a different Michaelis constant, which allows for the expanded dynamic range. We used the engineered Escherichia coli pyrroloquinoline quinone glucose dehydrogenase (PQQGDH) of which His775 was substituted for Asp which showed an increased Km value (25-fold). We first constructed the composite colorimetric analytical system employing the wild-type PQQGDH and His775Asp and evaluated its dynamic range. The composite colorimetric analytical system was constructed and showed a wide dynamic range of 0.5-30 mM with less than +/-5% error. The composite colorimetric analytical system, an extended-range colorimetric analytical system, enabled the determination of the concentration of glucose over a 30-fold range that could not have been achieved using the single colorimetric analytical system. Furthermore, we have demonstrated the composite amperometric glucose sensor employing the combination of His775Asn and His775Asp. The extended-range glucose sensor acquired not only the expanded dynamic range (3-70 mM) that covered both dynamic ranges of the single enzyme sensors but also the narrower substrate specificity of glucose due to the inherited property of engineered enzymes.

Biosensing Techniques↗
PubMed2000

Engineering a living cell to desired metabolite concentrations and fluxes: pathways with multifunctional enzymes.

With molecular genetics enabling modulation of the concentrations of cellular enzymes, metabolic engineering becomes limited by the question of which modulations of the enzyme concentrations are required to bring about a desired pattern of cellular metabolism. In an earlier paper (Kholodenko et al. (1998). Biotechnol. Bioeng. 59, 239-247) we derived a method to determine the required modulations. This method, however, cannot be immediately applied to cellular pathways with enzymes catalyzing more than one step in metabolism (multifunctional enzymes). In the present paper we show to which extent the presence of multifunctional enzymes limits biotechological ambitions, which one might otherwise pursue in vain. In particular, it is impossible to change the concentration of a single intermediate and leave the rest of metabolism unperturbed if that intermediate interacts directly with a multifunctional enzyme. The analytical machinery of Metabolic Control Analysis is used to relate the desired and ensuing changes in the metabolic pattern. An explicit solution to this problem of engineering metabolism is then given in the form of a single matrix equation.

Animals↗
PubMed1998

Knockout animals in toxicology: assessment of toxin bioactivation pathways using mice deficient in xenobiotic metabolizing enzymes.

Genetically engineered animal models represent a substantial improvement in in vivo assessment of toxic pathways. Several transgenic mouse lines have been designed to detect specific toxic markers in response to xenobiotic exposure. They are suitable for in vivo large scale screening of potentially toxic effects of drugs and other xenobiotics, and are used as bioassay models for carcinogenicity testing. This contribution will focus on a different strategy, using transgenic knockout mouse lines, to investigate with more accuracy some metabolic pathways leading to the bioactivation or the bioinactivation of xenobiotics. Through direct knockout of cytochrome P450 (CYP) genes or the knockout of the transcriptional activator of CYP genes AHR (aryl hydrocarbon receptor), the involvement of hepatic metabolic enzymes in xenobiotic bioactivation will be exemplified.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMedDate not supplied

Conversion of allosteric inhibition to activation in phosphofructokinase by protein engineering.

Many enzymes are subject to allosteric control, often with inhibitors and activators binding to the same effector site. Phosphofructokinase in Escherichia coli is such an enzyme, being inhibited by phosphoenolpyruvate (PEP) and activated by ADP and GDP. How do individual interactions with effectors affect the balance between activation and inhibition, especially when both ligands share aspects of the same binding site? We find that mutation of a single residue in the effector site, Glu----Ala 187, leads to PEP being an activator rather than an inhibitor. With low concentrations of the substrate fructose-6-phosphate, the mutant enzyme is more than one hundred times more active than wild-type enzyme at millimolar concentrations of PEP. The classical Monod-Wyman-Changeux two-state model is too simple to account for the properties of the mutant enzyme.

Adenosine Diphosphate↗
PubMed2001

Degradation of explosives by nitrate ester reductases.

Explosive-contaminated land poses a hazard both to the environment and to human health. Microbial enzymes, either in their native or heterologous hosts, are a powerful and low-cost tool for eliminating this environmental hazard. As many explosives have only been present in the environment for 10 years, and with similar molecules not known in Nature, the origin of enzymes specialized for the breakdown of explosives is of particular interest. Screening of environmental isolates resulted in the discovery of flavoproteins capable of denitrating the explosives pentaerythritol tetranitrate (PETN) and glycerol trinitrate. These nitrate ester reductases are related in sequence and structure to Old Yellow Enzyme from Saccharomyces carlsbergenisis. All the members of this family have alpha/beta barrel structures and FMN as a prosthetic group, and reduce various electrophilic substrates. The nitrate ester reductases are, however, unusual in that they display activity towards the highly recalcitrant, aromatic explosive 2,4,6-trinitrotoluene, via a reductive pathway resulting in nitrogen liberation. We have embarked on a detailed study of the structure and mechanism of PETN reductase from a strain of Enterobacter cloacae. Work is focused currently on relating structure and function within this growing family of enzymes, with a view to engineering novel enzymes exhibiting useful characteristics.

Biodegradation, Environmental↗
PubMed1998

Hybrid enzymes: manipulating enzyme design.

Hybrid enzymes are engineered to contain elements of two or more enzymes. Hybrid-enzyme approaches, by taking advantage of the vast array of enzymatic properties that nature has evolved, as well as the strategies that nature has used to evolve them, are becoming an increasingly important avenue for obtaining novel enzymes with desired activities and properties.

Enzymes↗
PubMed2002

Complexities in ETS-domain transcription factor function and regulation: lessons from the TCF (ternary complex factor) subfamily. The Colworth Medal Lecture.

The ETS-domain transcription factor family can be divided into a series of subfamilies. Elk-1 represents the founding member of the ternary complex factor (TCF) subfamily. By focusing on the TCF subfamily, we can demonstrate the complexities that exist in the function and regulation of ETS-domain transcription factors. This article focuses on Elk-1 in detail and summarizes the functions of other TCFs. The key themes covered include the domain structure of the TCFs, the mechanisms of complex formation with serum response factor, regulation of TCFs by mitogen-activated protein kinase cascades, and transcriptional regulatory properties of the TCFs. Finally, the emerging role of the TCFs in vivo is discussed. A picture is developing indicating that, while these proteins exhibit significant sequence and functional conservation, key differences in their structure and regulation are being identified which may relate to unique functions of these proteins in vivo.

Amino Acid Sequence↗
PubMed2002

Gellan gum biosynthesis in Sphingomonas paucimobilis ATCC 31461: genes, enzymes and exopolysaccharide production engineering.

The commercial gelling agent, gellan, is an extracellular polysaccharide (EPS) produced by Sphingomonas paucimobilis ATCC 31461. In recent years, significant progress in understanding the relationship between gellan structure and properties and elucidation of the biosynthesis and engineering of this recent product of biotechnology has been made. This review focuses on recent advances in this field. Emphasis is given to identification and characterization of genes and enzymes involved, or predicted to be involved, in the gellan biosynthetic pathway, at the level of synthesis of sugar-activated precursors, of the repeat unit assembly and of gellan polymerization and export. Identification of several genes, biochemical characterization of the encoded enzymes and elucidation of crucial steps of the gellan pathway indicate that possibilities now exist for exerting control over gellan production at any of the three levels of its biosynthesis. However, a better knowledge of the poorly understood steps and of the bottlenecks and regulation of the pathway, the characterization of the composition, structure and functional properties of gellan-like polymers produced either by the industrial strain under different culture conditions or by mutants are still required for eventual success of the metabolic engineering of gellan production.

Biological Transport↗
PubMed2006

Engineering human cytochrome P450 enzymes into catalytically self-sufficient chimeras using molecular Lego.

The membrane-bound human cytochrome P450s have essential roles in the metabolism of endogenous compounds and drugs. Presented here are the results on the construction and characterization of three fusion proteins containing the N-terminally modified human cytochrome P450s CYP2C9, CY2C19 and CYP3A4 fused to the soluble NADPH-dependent oxidoreductase domain of CYP102A1 from Bacillus megaterium. The constructs, CYP2C9/BMR, CYP2C19/BMR and CYP3A4/BMR are well expressed in Escherichia coli as holo proteins. The chimeras can be purified in the absence of detergent and the purified enzymes are both active and correctly folded in the absence of detergent, as demonstrated by circular dichroism and functional studies. Additionally, in comparison with the parent P450 enzyme, these chimeras have greatly improved solubility properties. The chimeras are catalytically self-sufficient and present turnover rates similar to those reported for the native enzymes in reconstituted systems, unlike previously reported mammalian cytochrome P450 fusion proteins. Furthermore the specific activities of these chimeras are not dependent on the enzyme concentration present in the reaction buffer and they do not require the addition of accessory proteins, detergents or phospholipids to be fully active. The solubility, catalytic self-sufficiency and wild-type like activities of these chimeras would greatly simplify the studies of cytochrome P450 mediated drug metabolism in solution.

Amino Acid Motifs↗
PubMed2003

Molecular evolution and genetic engineering of C4 photosynthetic enzymes.

The majority of terrestrial plants, including many important crops such as rice, wheat, soybean, and potato, are classified as C(3) plants that assimilate atmospheric CO(2) directly through the C(3) photosynthetic pathway. C(4) plants, such as maize and sugarcane, evolved from C(3) plants, acquiring the C(4) photosynthetic pathway in addition to the C(3) pathway to achieve high photosynthetic performance and high water- and nitrogen-use efficiencies. Consequently, the transfer of C(4) traits to C(3) plants is one strategy being adopted for improving the photosynthetic performance of C(3) plants. The recent application of recombinant DNA technology has made considerable progress in the molecular engineering of photosynthetic genes in the past ten years. It has deepened understanding of the evolutionary scenario of the C(4) photosynthetic genes. The strategy, based on the evolutionary scenario, has enabled enzymes involved in the C(4) pathway to be expressed at high levels and in desired locations in the leaves of C(3) plants. Although overproduction of a single C(4) enzyme can alter the carbon metabolism of C(3) plants, it does not show any positive effects on photosynthesis. Transgenic C(3) plants overproducing multiple enzymes are now being produced for improving the photosynthetic performance of C(3) plants.

Evolution, Molecular↗
PubMed2006

PHA synthase engineering toward superbiocatalysts for custom-made biopolymers.

Poly-3-hydroxyalkanoates [P(3HA)s] are biologically produced polyesters that have attracted much attention as biodegradable polymers that can be produced from biorenewable resources. These polymers have many attractive properties for use as bulk commodity plastics, fishing lines, and medical uses that are dependent on the repeating unit structures. Despite the readily apparent benefits of using P(3HA)s as replacements for petrochemical-derived plastics, the use and distribution of P(3HA)s have been limited by their cost of production. This problem is currently being addressed by the engineering of enzymes involved in the production of P(3HA)s. Polyhydroxyalkanoate (PHA) synthase (PhaC) enzymes, which catalyze the polymerization of 3-hydroxyacyl-CoA monomers to P(3HA)s, were subjected to various forms of protein engineering to improve the enzyme activity or substrate specificity. This review covers the recent history of PHA synthase engineering and also summarizes studies that have utilized engineered PHA synthases.

Acyltransferases↗
PubMed1998

Protein engineering the surface of enzymes.

The protein surface is the interface through which a protein senses the external world. Its composition of charged, polar and hydrophobic residues is crucial for the stability and activity of the protein. The charge state of seven of the twenty naturally occurring amino acids is pH dependent. A total of 95% of all titratable residues are located on the surface of soluble proteins. In evolutionary related families of proteins such residues are particularly prone to substitutions, insertions and deletions. We present here an analysis of the residue composition of 4038 proteins, selected from 125 protein families with < 25% identity between core members of each family. Whereas only 16.8% of the residues were truly buried, 40.7% were > 30% exposed on the surface and the remainder were < 30% exposed. The individual residue types show distinct differences. The data presented provides an important new approach to protein engineering of protein surfaces. Guidelines for the optimization of solvent exposure for a given residue are given. The cutinase family of enzymes has been investigated. The stability of native cutinase has been studied as a function of pH, and has been compared with the cutinase activity towards tributyrin. Whereas the onset of enzymatic activity is linked with the deprotonation of the active site HIS188, destabilization of the 3D structure as determined by differential scanning calorimetry is coupled with the loss of activity at very basic pH values. A modeling investigation of the pH dependence of the electrostatic potentials reveals that the activity range is accompanied by the development of a highly significant negative potential in the active site cleft. The 3D structures of three mutants of the Fusarium solani pisi cutinase have been solved to high resolution using X-ray diffraction analysis. Preliminary X-ray data are presented.

Binding Sites↗
PubMed1999

Engineering of improved microbes and enzymes for bioremediation.

Bioremediation with microorganisms is an attractive alternative to conventional techniques, such as incineration and chemical treatment, for disposing of pollutants. Recent progress in molecular biology, microbiology, and genetics is providing the driving force towards engineering improved microbes and enzymes for bioremediation. A number of genetic engineering approaches have been developed in the past several years that have proven useful in introducing/evolving the desired properties for different biodegradative pathways or enzymes. The initial excitement generated in this area should continue to pave the way for rational or irrational design of microbes or enzymes with novel remedial properties.

Amino Acid Sequence↗
PubMed2006

Modulation of therapeutic antibody effector functions by glycosylation engineering: influence of Golgi enzyme localization domain and co-expression of heterologous beta1, 4-N-acetylglucosaminyltransferase III and Golgi alpha-mannosidase II.

The effector functions elicited by IgG antibodies strongly depend on the carbohydrate moiety linked to the Fc region of the protein. Therefore several approaches have been developed to rationally manipulate these glycans and improve the biological functions of the antibody. Overexpression of recombinant beta1,4-N-acetylglucosaminyltransferase III (GnT-III) in production cell lines leads to antibodies enriched in bisected oligosaccharides. Moreover, GnT-III overexpression leads to increases in non-fucosylated and hybrid oligosaccharides. Such antibody glycovariants have increased antibody-dependent cellular cytotoxicity (ADCC). To explore a further variable besides overexpression of GnT-III, we exchanged the localization domain of GnT-III with that of other Golgi-resident enzymes. Our results indicate that chimeric GnT-III can compete even more efficiently against the endogenous core alpha1,6-fucosyltransferase (alpha1,6-FucT) and Golgi alpha-mannosidase II (ManII) leading to higher proportions of bisected non-fucosylated hybrid glycans ("Glyco-1" antibody). The co-expression of GnT-III and ManII led to a similar degree of non-fucosylation as that obtained for Glyco-1, but the majority of the oligosaccharides linked to this antibody ("Glyco-2") are of the complex type. These glycovariants feature strongly increased ADCC activity compared to the unmodified antibody, while Glyco-1 (hybrid-rich) features reduced complement-dependent cytotoxicity (CDC) compared to Glyco-2 or unmodified antibody. We show that apart from GnT-III overexpression, engineering of GnT-III localization is a versatile tool to modulate the biological activities of antibodies relevant for their therapeutic application.

Animals↗