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At least 649 records · Page 36Linked to original sources
PubMed1987

Engineering herbicide resistance in plants by expression of a detoxifying enzyme.

Phosphinothricin (PPT) is a potent inhibitor of glutamine synthetase in plants and is used as a non-selective herbicide. The bar gene which confers resistance in Streptomyces hygroscopicus to bialaphos, a tripeptide containing PPT, encodes a phosphinothricin acetyltransferase (PAT) (see accompanying paper). The bar gene was placed under control of the 35S promoter of the cauliflower mosaic virus and transferred to plant cells using Agrobacterium-mediated transformation. PAT was used as a selectable marker in protoplast co-cultivation. The chimeric bar gene was expressed in tobacco, potato and tomato plants. Transgenic plants showed complete resistance towards high doses of the commercial formulations of phosphinothricin and bialaphos. These data present a successful approach to obtain herbicide-resistant plants by detoxification of the herbicide.

Journal Article↗
PubMed2004

Recombinant AAV-mediated gene delivery to the central nervous system.

Various regions of the brain have been successfully transduced by recombinant adeno-associated virus (rAAV) vectors with no detected toxicity. When using the cytomegalovirus immediate early (CMV) promoter, a gradual decline in the number of transduced cells has been described. In contrast, the use of cellular promoters such as the neuron-specific enolase promoter or hybrid promoters such as the chicken beta-actin/CMV promoter resulted in sustained transgene expression. The cellular tropism of rAAV-mediated gene transfer in the central nervous system (CNS) varies depending on the serotype used. Serotype 2 vectors preferentially transduce neurons whereas rAAV5 and rAAV1 transduce both neurons and glial cells. Recombinant AAV4-mediated gene transfer was inefficient in neurons and glial cells of the striatum (the only structure tested so far) but efficient in ependymal cells. No inflammatory response has been described following rAAV2 administration to the brain. In contrast, antibodies to AAV2 capsid and transgene product were elicited but no reduction of transgene expression was observed and readministration of vector without loss of efficiency was possible from 3 months after the first injection. Based on the success of pioneer work performed with marker genes, various strategies for therapeutic gene delivery were designed. These include enzyme replacement in lysosomal storage diseases, Canavan disease and Parkinson's disease; delivery of neuroprotective factors in Parkinson's disease, Huntington disease, Alzheimer's disease, amyotrophic lateral sclerosis, ischemia and spinal cord injury; as well as modulation of neurotransmission in epilepsy and Parkinson's disease. Several of these strategies have demonstrated promising results in relevant animal models. However, their implementation in the clinics will probably require a tight regulation and a specific targeting of therapeutic gene expression which still demands further developments of the vectors.

Animals↗
PubMed1999

Expression of the inducible NO synthase in human monocytic U937 cells allows high output nitric oxide production.

Nitric oxide (NO) produced by inducible NO synthase (iNOS, NOS-2) is an important component of the macrophage-mediated immune defense toward numerous pathogens. Murine macrophages produce NO after cytokine activation, whereas, under similar conditions, human macrophages produce low levels or no NO at all. Although human macrophages can express iNOS mRNA and protein on activation, whether they possess the complete machinery necessary for NO synthesis remains controversial. To define the conditions necessary for human monocytes/macrophages to synthesize NO when expressing a functional iNOS, the human monocytic U937 cell line was engineered to synthesize this enzyme, following infection with a retroviral expression vector containing human hepatic iNOS (DFGiNOS). Northern blot and Western blot analysis confirmed the expression of iNOS in transfected U937 cells both at the RNA and protein levels. NOS enzymatic activity was demonstrated in cell lysates by the conversion of L-[3H]arginine into L-[3H]citrulline and the production of NO by intact cells was measured by nitrite and nitrate accumulation in culture supernatants. When expressing functional iNOS, U937 cells were capable of releasing high levels of NO. NO production was strictly dependent on supplementation of the culture medium with tetrahydrobiopterin (BH4) and was not modified by stimulation of the cells with different cytokines. These observations suggest that (1) human monocytic U937 cells contain all the cofactors necessary for NO synthesis, except BH4 and (2) the failure to detect NO in cytokine-stimulated untransfected U937 cells is not due to the presence of a NO-scavenging molecule within these cells nor to the destabilization of iNOS protein. DFGiNOS U937 cells represent a valuable human model to study the role of NO in immunity toward tumors and pathogens.

Animals↗
PubMed2006

Structure and enzymatic properties of a chimeric bacteriophage RB69 DNA polymerase and single-stranded DNA binding protein with increased processivity.

In vivo, replicative DNA polymerases are made more processive by their interactions with accessory proteins at the replication fork. Single-stranded DNA binding protein (SSB) is an essential protein that binds tightly and cooperatively to single-stranded DNA during replication to remove adventitious secondary structures and protect the exposed DNA from endogenous nucleases. Using information from high resolution structures and biochemical data, we have engineered a functional chimeric enzyme of the bacteriophage RB69 DNA polymerase and SSB with substantially increased processivity. Fusion of RB69 DNA polymerase with its cognate SSB via a short six amino acid linker increases affinity for primer-template DNA by sixfold and subsequently increases processivity by sevenfold while maintaining fidelity. The crystal structure of this fusion protein was solved by a combination of multiwavelength anomalous diffraction and molecular replacement to 3.2 A resolution and shows that RB69 SSB is positioned proximal to the N-terminal domain of RB69 DNA polymerase near the template strand channel. The structural and biochemical data suggest that SSB interactions with DNA polymerase are transient and flexible, consistent with models of a dynamic replisome during elongation.

Crystallization↗
PubMed2001

Specificity assay of serine proteinases by reverse-phase high-performance liquid chromatography analysis of competing oligopeptide substrate library.

In this paper we present an HPLC method developed for quick activity and specificity analysis of serine proteinases. The method applies a carefully designed peptide library in which the individual components differ only at the potential cleavage site for enzymes. The library has seven members representing seven different cleavage sites and it offers substrates for both trypsin and chymotrypsin-like enzymes. The individual peptide substrates compete for the proteinase during the enzymatic reaction. The reaction is monitored by RP-HPLC separation of the components. We describe the systematic design of the competitive peptide substrate library and the test of the system with eight different serine proteinases. The specificity profiles of the investigated enzymes as determined by the new method were essentially identical to the ones reported in the literature, verifying the ability of the system to characterize substrate specificity. The tests also demonstrated that the system could detect even subtle specificity differences of two isoforms of an enzyme. In addition to recording qualitative specificity profiles, data provided by the system can be analyzed quantitatively, yielding specificity constant values. This method can be a useful tool for quick analysis of uncharacterized gene products as well as new forms of enzymes generated by protein engineering.

Amino Acid Sequence↗
PubMed2000

X-ray structure of yeast Hal2p, a major target of lithium and sodium toxicity, and identification of framework interactions determining cation sensitivity.

The product of the yeast HAL2 gene (Hal2p) is an in vivo target of sodium and lithium toxicity and its overexpression improves salt tolerance in yeast and plants. Hal2p is a metabolic phosphatase which catalyses the hydrolysis of 3'-phosphoadenosine-5'-phosphate (PAP) to AMP. It is, the prototype of an evolutionarily conserved family of PAP phosphatases and the engineering of sodium insensitive enzymes of this group may contribute to the generation of salt-tolerant crops. We have solved the crystal structure of Hal2p in complex with magnesium, lithium and the two products of PAP hydrolysis, AMP and Pi, at 1.6 A resolution. A functional screening of random mutations of the HAL2 gene in growing yeast generated forms of the enzyme with reduced cation sensitivity. Analysis of these mutants defined a salt bridge (Glu238 ellipsis Arg152) and a hydrophobic bond (Va170 ellipsis Trp293) as important framework interactions determining cation sensitivity. Hal2p belongs to a larger superfamily of lithium-sensitive phosphatases which includes inositol monophosphatase. The hydrophobic interaction mutated in Hal2p is conserved in this superfamily and its disruption in human inositol monophosphatase also resulted in reduced cation sensitivity.

Cloning, Molecular↗
PubMed2002

The mathematics of metabolic control analysis revisited.

In this minireview, several different approaches to derivation of the theorems and relationships of Metabolic Control Analysis (MCA) are discussed and an alternative approach is presented. This new approach consists of solving the steady-state mass balances for the intracellular metabolites using linearized kinetics. The application of linearized kinetics reflects the fact that MCA is based on linearization of the system equations in a reference steady state. Our derivation is valid for metabolic networks of arbitrary complexity, including those containing conserved moieties and branches. The value of our approach is its simplicity: the derivation is straightforward and therefore easy to follow. It can serve as a compact introduction to the mathematical basis of MCA.

Biomedical Engineering↗
PubMed1999

Combinatorial chemistry and its applications in agriculture and food.

Combinatorial chemistry has become a major focus of research activity in the pharmaceutical industry for development new therapeutic compounds. The same techniques could be potentially applied to benefit agricultural and food research. This article reviews the various procedures used in combinatorial chemistry, outlines some of the strengths and limitations of the various methods, and proposes potential areas in agriculture and food that could be benefited by this technology. These areas include developing new antimicrobial agents, antioxidants, and other additives, creating antigen-binding molecules for the detection or removal of food pathogens or toxicants, engineering food proteins and enzymes for specific functions, and modifying biosynthetic pathways for the production of novel natural products.

Chemistry, Agricultural↗
PubMed1990

Biochemistry and biotechnology of amino acid dehydrogenases.

Over the last decade, amino acid dehydrogenases such as alanine dehydrogenase (Ala DH), leucine dehydrogenase (Leu DH), and phenylalanine dehydrogenase (Phe DH) have been applied to the enantiomer-specific synthesis and analysis of various amino acids. In perticular, amino acid dehydrogenases from thermophiles have received much attention because of their high stability. Their productivity was enhanced and the purification facilitated by the gene cloning. The advances in biotechnological applications of these enzymes are based on fundamental studies concerning characteristics of the enzymes and reaction mechanism as described in this chapter. Further elucidation of the structure and function of these enzymes based on genetic engineering and protein engineering may enable their properties to be improved for their future uses in biotechnology.

Amino Acid Oxidoreductases↗
PubMed2006

Enzymes in bast fibrous plant processing.

The program COST Action 847 Textile Quality and Biotechnology (2000-2005) has given an excellent chance to review the possibilities of the research, aiming at development of the industrial application of enzymes for bast fibrous plant degumming and primary processing. The recent advancements in enzymatic processing of bast fibrous plants (flax, hemp, jute, ramie and alike plants) and related textiles are given. The performance of enzymes in degumming, modification of bast fibres, roving, yarn, related fabrics as well as enzymatic bonding of lignocellulosic composites is provided.

Bacillus↗
PubMed1986

Site-specific DNA splicing: a general procedure for the creation of a restriction site at a predetermined position in a DNA sequence.

A method is described for creating any of a wide array of restriction sites at a predetermined position in a known DNA sequence. The method utilizes the exonuclease activity of BAL 31 and a specially designed bifunctional oligodeoxynucleotide linker. The desired restriction site is generated when the linker is ligated to those BAL 31-digested DNA fragments which end with the target sequence. The proper ligation product is then identified by a highly specific hybridization procedure. The method is versatile and specific and is especially useful in the isolation of functional elements of a gene.

Base Sequence↗
PubMed1987

Rapid site-specific mutagenesis in plasmids.

A quick and simple method for introducing site-specific mutations into plasmids is described. The procedure involves restriction-enzyme digestion of the plasmid to give a linear fragment. A second preparation of the same plasmid is digested with other restriction enzymes to remove the targeted mutational region to give a gapped fragment. The linear fragment and the gapped fragment are mixed, then denatured and annealed in the presence of a short, synthetic oligodeoxynucleotide corresponding to the targeted region and containing the desired mutation. The mix is then transformed directly into cells where host enzymes fill single-stranded gaps to make a complete double-stranded, mutant plasmid.

Base Sequence↗
PubMed1994

Designing pathways for environmental purposes.

Genetic modifications intended to improve the properties of enzymes or entire metabolic pathways involved in hazardous waste treatment have resulted in the discovery of promising approaches for expanding the utility of biotreatment. Construction of hybrid metabolic pathways as well as hybrid enzymes, site-directed mutagenesis, and gene amplification have all facilitated the desired outcome of more complete and rapid contaminant removal and considerably broadened substrate specificity.

Biodegradation, Environmental↗
PubMed2004

Accessibility of N-acyl-D-mannosamines to N-acetyl-D-neuraminic acid aldolase.

N-Acetyl-D-neuraminic acid (NeuNAc) aldolase is an important enzyme for the metabolic engineering of cell-surface NeuNAc using chemically modified D-mannosamines. To explore the optimal substrates for this application, eight N-acyl derivatives of D-mannosamine were prepared, and their accessibility to NeuNAc aldolase was quantitatively investigated. The N-propionyl-, N-butanoyl-, N-iso-butanoyl-, N-pivaloyl-, and N-phenylacetyl-D-mannosamines proved to be as good substrates as, or even better than, the natural N-acetyl-D-mannosamine, while the N-trifluoropropionyl and benzoyl derivatives were poor. It was proposed that the electronic effects might have a significant influence on the enzymatic aldol condensation reaction of D-mannosamine derivatives, with electron-deficient acyl groups having a negative impact. The results suggest that N-propionyl-, N-butanoyl-, N-iso-butanoyl-, and N-phenylacetyl-D-mannosamines may be employed to bioengineer NeuNAc on cells.

Carbohydrate Conformation↗
PubMed2026

Generative artificial intelligence for enzyme design and biocatalysis.

Sparked by innovations in generative artificial intelligence (AI), the field of protein design has undergone a paradigm shift with an explosion of new models for optimizing existing enzymes or creating them from scratch. After more than one decade of low success rates for computationally designed enzymes, generative AI models are now frequently used for designing proficient enzymes. Here, we provide a comprehensive overview and classification of generative AI models for enzyme design, highlighting models with experimental validation relevant to real-world settings and outlining their respective limitations. We argue that generative AI models now have the maturity to create and optimize enzymes for industrial applications. Wider adoption of generative AI models with experimental feedback loops can speed up the development of biocatalysts and serve as a community assessment to inform the next generation of models.

Biocatalysis↗
PubMed2002

Anionic substitutes for catalytic aspartic acids in phosphoribulokinase.

Mutagenic substitution of the invariant D42 and D169 residues in phosphoribulokinase (PRK) with amino acids that contain neutral side chains (e.g., alanine or asparagine) results in large decreases in catalytic efficiency (10(5)- and 10(4)-fold for replacement of D42 and D169, respectively). To further evaluate the importance of anionic side chains at residues 42 and 169, substitutions of glutamic acid (D42E, D169E) and cysteine (D42C and D169C in an otherwise cysteine-free protein) have been engineered. All purified mutant enzymes bind the fluorescent alternative substrate trinitrophenyl-ATP and the allosteric effector NADH similarly to wild-type PRK. For D42E and D42C, V(max) exhibits substantial decreases of 135- and 220-fold, respectively. Comparable substitutions for D169 result in smaller effects; D169E and D169C exhibit decreases in V(max) of 39- and 26-fold, respectively. Thus, regardless of the type of substitution, changes at D42 more profoundly affect catalytic rate than do comparable changes at D169. Precedent with enzymes in which cysteine replaces an acidic residue suggests that oxidation of the thiolate to a sulfinate can convert low-activity cysteine mutants into enzymes with improved activity. Periodate oxidation of cysteine-free PRK results in a slight decrease in activity. In contrast, comparable treatment of D42C and D169C proteins increases activity by 5- and 7-fold, respectively. Thus, for reasonably efficient catalysis, PRK requires anionic character in the side chains of residues 42 and 169. The enzyme can, however, tolerate substantial structural and chemical variability at these residues.

Adenosine Triphosphate↗