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PubMed2000

In vivo selection of protease cleavage sites by using chimeric Sindbis virus libraries.

Identifying protease cleavage sites contributes to our understanding of their specificity and biochemical properties and can help in designing specific inhibitors. One route to this end is the generation and screening of random libraries of cleavage sites. Both synthetic and phage-displayed libraries have been extensively used in vitro. We describe a novel system based on recombinant Sindbis virus which can be used to identify cleavage sites in vivo, thus eliminating the need for a purified enzyme and overcoming the problem of choosing the correct in vitro conditions. As a model we used the serine protease of the hepatitis C virus (HCV). We engineered the gene coding for this enzyme and two specific cleavage sites in the Sindbis virus structural gene and constructed libraries of viral genomes with a random sequence at either of the cleavage sites. The system was designed so that only viral genomes coding for sequences cleaved by the protease would produce viable viruses. With this system we selected viruses containing sequences mirroring those of the natural HCV protease substrates which were cleaved with comparable efficiencies.

Antibodies↗
PubMed1998

Putrescine-modified nerve growth factor: bioactivity, plasma pharmacokinetics, blood-brain/nerve barrier permeability, and nervous system biodistribution.

Previous investigations from our laboratory have demonstrated that the covalent modification of a variety of proteins, including antioxidant enzymes, with the naturally occurring polyamines--putrescine (PUT), spermidine, and spermine--dramatically increases their permeability coefficient-surface area product (PS) at the blood-brain and blood-nerve barriers after parenteral administration. In the present study, we have covalently modified nerve growth factor (NGF) with PUT by targeting carboxylic groups for their graded modification by controlling the ionization of these groups with pH. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis, western, and isoelectric focusing analyses demonstrated conversion of NGF to its polyamine-modified derivatives at different pH values. Although the immunoreactivity of PUT-NGF determined by ELISA and western analysis decreased with decreasing pH, the biological activity of PUT-NGF was not affected at any pH as determined by survival and neurite extension of dorsal root ganglia and PC12 cultures. Plasma pharmacokinetics after a single intravenous bolus administration revealed intact PUT-NGF through 10 min and 73-82% intact protein at 15 min. The PS value for PUT-NGF was maximized and the residual plasma volume (Vp) of the protein in the blood vessels minimized when the pH of the modification reaction was >6.4. The biodistribution of PUT-NGF at 15 min showed 22-33% intact protein in different brain regions, which represented 0.4-5.9 ng of PUT-NGF in different brain regions, a physiological dose that is capable of eliciting a bioresponse. The design of this polyamine-modified NGF derivative that has enhanced permeability at the blood-brain and blood-nerve barriers with retained bioactivity may obviate the necessity to create small-molecule mimics of NGF and may be applicable to neurotrophins, engineered multifunctional chimeric neurotrophins, antioxidant enzymes, and other therapeutic proteins with specific clinical application to neurological diseases.

Animals↗
PubMed1993

In vivo antitumor activity of 5-fluorocytosine on human colorectal carcinoma cells genetically modified to express cytosine deaminase.

A human colorectal carcinoma cell line, WiDr, was genetically engineered to express the nonmammalian enzyme, cytosine deaminase (CD). Expression of CD in WiDr cells (WiDr/CD) did not alter the growth rate of these cells when grown in vitro or as solid tumor xenografts in nude mice. However, expression of CD did increase the sensitivity of these cells to the nontoxic prodrug, 5-fluorocytosine (FCyt), decreasing the 50% inhibitory concentration for FCyt from 26,000 microM in parental WiDr cells to 27 microM in WiDr/CD cells. The increase in sensitivity to FCyt in WiDr/CD cells was the result of the CD-mediated conversion of FCyt to 5-fluorouracil (FUra) and subsequent FUra anabolites. The half-life of the prodrug, FCyt, was determined to be approximately 40 min in nude mice. A single i.p. injection of 500 mg FCyt/kg body weight resulted in a transient FCyt plasma level of approximately 4000 microM while osmotic minipumps or constant tail vein infusions of FCyt achieved continual FCyt plasma levels of 5 microM and 50 microM, respectively, with no overt signs of toxicity. Significant antitumor effects were observed in nude mice bearing tumors derived from WiDr/CD cells when these animals were given 500 mg FCyt/kg i.p. for 10 consecutive days. These antitumor effects were demonstrated by decreases in tumor growth rate, tumor size, tumor weight, and thymidine incorporation into tumor DNA. This antitumor effect was significant but less profound if FCyt was administered by constant tail vein infusion. WiDr and WiDr/CD cells were very sensitive to FUra in vitro (50% inhibitory concentration approximately 5 microM). However, no significant antitumor effects were observed in nude mice bearing tumors derived from either WiDr or WiDr/CD cells when these animals were treated with various doses of FUra. Taken collectively, these data indicate that nontoxic plasma levels of FCyt can be attained which can produce profound antitumor effects on tumors engineered to express CD and that these antitumor effects are significantly better than those that can be achieved using FUra. These positive data support the continued development of a gene therapy approach to colorectal carcinoma involving the selective expression of CD in colorectal tumors with subsequent administration of FCyt.

Animals↗
PubMed1985

Redesigning enzymes by site-directed mutagenesis.

The systematic alteration of protein structure has now become possible with genetic engineering. Recent developments in techniques for the chemical synthesis of DNA fragments and in recombinant DNA technology have enabled the facile modification of proteins by highly specific mutagenesis of their genes. Enzymes with novel properties may be produced in large quantities from the mutant genes. Kinetic analysis of the mutant enzymes can be combined with high-resolution structural data from protein X-ray crystallography to provide direct measurements on the relationships between structure and function. In particular, the strength and nature of enzyme-substrate interactions and their roles in catalysis and specificity may be studied. The tyrosyl-tRNA synthetase from Bacillus stearothermophilus is being systematically analysed by site-directed mutagenesis. A fine-structure analysis is revealing the subtle roles of hydrogen bonding in catalysis and specificity. Modification of the residues that hydrogen-bond with ATP and tyrosine shows how the energetics must be analysed in terms of an exchange reaction with solvent water. Based on this idea, and structural data, an enzyme of vastly improved enzyme-substrate affinity has been engineered. There thus appear to be real prospects of engineering proteins of new specificities, activities and structural properties. Direct information is also being gathered on the nature of enzyme catalysis. For example, the catalysis of formation of Tyr-AMP from Tyr and ATP does not appear to use the classical mechanisms of acid-base or covalent catalysis. Instead, there just appears to be a binding site that stabilizes the high-energy pentacoordinate intermediate in the reaction.

Adenosine Triphosphate↗
PubMed2002

Metabolic engineering through cofactor manipulation and its effects on metabolic flux redistribution in Escherichia coli.

Applications of genetic engineering or metabolic engineering have increased in both academic and industrial institutions. Most current metabolic engineering studies have focused on enzyme levels and on the effect of the amplification, addition, or deletion of a particular pathway. Although it is generally known that cofactors play a major role in the production of different fermentation products, their role has not been thoroughly and systematically studied. It is conceivable that in cofactor-dependent production systems, cofactor availability and the proportion of cofactor in the active form may play an important role in dictating the overall process yield. Hence, the manipulation of these cofactor levels may be crucial in order to further increase production. We have demonstrated that manipulation of cofactors can be achieved by external and genetic means and these manipulations have the potential to be used as an additional tool to achieve desired metabolic goals. We have shown experimentally that the NADH/NAD(+) ratio can be altered by using carbon sources with different oxidation states. We have shown further that the metabolite distribution can be influenced by a change in the NADH/NAD(+) ratio as mediated by the oxidation state of the carbon source used. We have also demonstrated that the total NAD(H/(+)) levels can be increased by the overexpression of the pncB gene. The increase in the total NAD(H/(+)) levels can be achieved even in a complex medium, which is commonly used by most industrial processes. Finally, we have shown that manipulation of the CoA pool/flux can be used to increase the productivity of a model product, isoamyl acetate.

Acetyl Coenzyme A↗
PubMed1996

Enzymatic properties of mutant enzymes at Trp49 and Tyr57 of RNase Rh from Rhizopus niveus.

In order to establish the role of Tyr57 and Trp49 in the enzymatic reaction of RNase Rh, several mutant enzymes at Tyr57 and Trp49 were prepared by protein engineering and their enzymatic properties were investigated. Among the four mutant enzymes at Trp49 (W49F, W49Y, W49A, and W49I), W49F showed 16% of the activity of the native enzyme, but the others (W49Y, W49A, and W49I) showed greatly decreased activity. The data showed that Trp49 is very important for the enzyme activity. Among 8 mutant enzymes at the 57th position, Y57F and Y57W showed similar enzymatic activity toward RNA to that of the wild-type enzyme, but the others (Y57G, Y57A, Y57V, Y57M, and Y57K) are more active toward RNA and less active toward XpGs. The reason for the apparent increase for RNA activity is discussed from the view point of substrate inhibition. It is noteworthy that W49F and Y57W became more pyrimidine base- and purine base-preferential, respectively.

Amino Acid Sequence↗
PubMed2002

Characterization of soluble and membrane-bound family 3 lytic transglycosylases from Pseudomonas aeruginosa.

Lytic transglycosylases cleave the beta,1-->4 glycosidic linkages between the N-acetylmuramoyl (MurNAc) and N-acetylglucosaminyl (GlcNAc) residues of peptidoglycan with the concomitant formation of 1,6-anhydro-N-acetylmuramyl reaction products. The genes encoding two hypothetical lytic transglycosylases were identified in the genome of Pseudomonas aeruginosa PAO1 by a BLAST search using membrane-bound lytic transglycosylase B (MltB) from Escherichia coli as the query. The two genes were amplified by PCR and cloned as fusion proteins with C-terminal hexa-His sequences. Expression studies of the two genes in E. coli in the presence of [(3)H]palmitate resulted in the labeling of only one of the two enzymes. This enzyme, named MltB, was overexpressed to form insoluble inclusion bodies. Its gene was engineered to produce a truncated form of the enzyme lacking its N-terminal 17 residues which includes Cys17, the putative site of lipidation. This MltB derivative (named sMltB) was shown to not label with [(3)H]palmitate, and it was overexpressed in soluble form. The second, nonlabeled enzyme was overexpressed in soluble form and hence was named soluble lytic transglycosylase B (SltB). Both sMltB and SltB were purified to apparent homogeneity by a combination of affinity (Ni(2+)-NTA), cation-exchange (Mono S), and gel permeation (Superdex 75) chromatographies. The reaction products released by the two enzymes from purified, insoluble peptidoglycan were characterized by a novel high-performance anion-exchange chromatography (HPAEC) assay. Both enzymes produced the same three major soluble products which were identified as anhydromuropeptides based on ESI-MS analysis (cross-linked anhydrodisaccharide-tetrasaccharide, m/z obs 1824.9; anhydrodisaccharide-pentapeptide, m/z obs 922.2; and anhydrodisaccharide-tripeptide, m/z obs 851.3. The Michaelis-Menten kinetic parameters were also determined for the two enzymes using the same insoluble peptidoglycan substrate by aminosugar compositional analysis of soluble reaction products. At pH 5.8 and in the presence of 0.1% Triton, SltB was found to be more catalytically efficient, as reflected by its k(cat)/K(M) value, than sMltB.

Amino Acid Sequence↗
PubMed2006

Prevention and repair of protein damage by the Maillard reaction in vivo.

The aging human extracellular matrix (ECM) and tissues rich in long-lived proteins undergo extensive changes with age that include increased stiffening, loss of elasticity, insolubilization, and decreased proteolytic digestibility. Most if not all these changes can be duplicated by the Maillard reaction in vitro, that is, the incubation of the proteins with reducing sugars and oxoaldehydes. These carbonyls eventually form advanced glycation end products (AGEs) and crosslinks that impair proteolytic digestibility and alter protein conformation. To date, close to 20 AGEs have been found in the human skin, of which ornithine is the single major result of damage to arginine residues, and glucosepane the single major crosslink. Although redox active metals and oxoaldehydes appear to play an important role in protein damage in experimental diabetes, their role in diabetic humans is still poorly understood. Evidence for the existence of deglycating enzymes has been found in vertebrates, bacteria, and fungi. However, only the vertebrate enzymes can deglycate larger, intracellular proteins via an ATP-dependent mechanism. Protein engineering will thus be needed to adapt Amadoriase enzymes toward deglycation of ECM proteins for purpose of probing the role of advanced glycation in animal models of diabetes and age-related diseases. The blocking of the reactivity of the glucosepane precursor using potent nucleophiles may be useful in preventing age-related changes in ECM proteins. However, there currently is no evidence in support of the proposed ability of so-called "AGE breakers" to cleave existing crosslinks of the Maillard reaction in vivo, and other mechanisms of action should be sought for this class of compounds.

Arginine↗
PubMed2003

Creating polyketide diversity through genetic engineering.

Modular polyketide synthases (PKS) are large multifunctional enzymes that synthesize complex polyketides, a therapeutically important class of natural products. The linear order and composition of catalytic sites that comprise the PKS represent a "code" that determines the identity of the polyketide product. By re-programming the PKS through genetic engineering, it is possible to alter the code in a predictable manner to create specific structural modifications of polyketides and to produce new libraries of these natural products.

Carbohydrate Sequence↗
PubMed1988

The use of genetically engineered tryptophan to identify the movement of a domain of B. stearothermophilus lactate dehydrogenase with the process which limits the steady-state turnover of the enzyme.

A general technique for monitoring the intramolecular motion of a protein is described. Genetic engineering is used to replace all the natural tryptophan residues with tyrosine. A single tryptophan residue is then inserted at a specific site within the protein where motion is then detected from the fluorescence characteristics of this fluorophore. This technique has been used in B. stearothermophilus lactate dehydrogenase mutant (W80Y, W150Y, W203Y, G106W) to correlate the slow closure of a surface loop of polypeptide (residues 98-110) with the maximum catalytic velocity of the enzyme.

Genetic Engineering↗
PubMed2000

Protein engineering of subtilisin.

The serine protease subtilisin is an important industrial enzyme as well as a model for understanding the enormous rate enhancements affected by enzymes. For these reasons along with the timely cloning of the gene, ease of expression and purification and availability of atomic resolution structures, subtilisin became a model system for protein engineering studies in the 1980s. Fifteen years later, mutations in well over 50% of the 275 amino acids of subtilisin have been reported in the scientific literature. Most subtilisin engineering has involved catalytic amino acids, substrate binding regions and stabilizing mutations. Stability has been the property of subtilisin which has been most amenable to enhancement, yet perhaps least understood. This review will give a brief overview of the subtilisin engineering field, critically review what has been learned about subtilisin stability from protein engineering experiments and conclude with some speculation about the prospects for future subtilisin engineering.

Calcium↗
PubMed1986

Natural variation of tyrosyl-tRNA synthetase and comparison with engineered mutants.

We report the cloning and sequence analysis of the gene for the tyrosyl-tRNA synthetase from Bacillus caldotenax and properties of the gene product. The amino acid sequence of the tyrosyl-tRNA synthetase was found to be 99% homologous with the corresponding enzyme from B. stearothermophilus, with only four amino acid differences. Two of these natural variations were found to involve active site residues of the enzyme and correspond to mutations that have been engineered previously in vitro. One, Thr-51----Ala-51, produced a more active enzyme, possessing a higher value of kcat/KM for ATP. Position 51 is a "hot spot" in the tyrosyl-tRNA synthetase, differing in enzymes derived from Escherichia coli, B. stearothermophilus, and B. caldotenax. The other, His-48----Asn-48, is found to be a neutral mutation but is in one of the rare regions that are conserved with other aminoacyl-tRNA synthetases. The equivalence of histidine and asparagine at position 48 extends the homology in this region to more enzymes. These residues, His-Ile-Gly-His, and now His-Ile-Gly-Asn, form part of the binding site for ATP in the transition state of the reaction. Although B. caldotenax is an obligate thermophile with an optimal growth temperature of 80 degrees C, as much as 20 degrees C above the growth optima of strains of Bacillus stearothermophilus, its tyrosyl-tRNA synthetase has an identical thermal stability in vitro to that from B. stearothermophilus.

Amino Acid Sequence↗
PubMed1998

Redesigning metabolic networks using mathematical programming

The design of new generation bioprocessing plants is increasingly dependent on the design of process-compatible microorganisms. The latter, whether through genetic or physiological manipulations, can be greatly assisted by metabolic engineering. An emerging powerful tool in metabolic engineering research is computer-assisted cell design using mathematical programming. In this work, the problem of optimizing cellular metabolic networks has been formulated as a Mixed Integer Nonlinear Programming (MINLP) model. The model can assist genetic engineers to identify which cellular enzymes should be modified, and the new levels of activity required to produce an optimal network. Results are presented from the tricarboxylic acid cycle in Dictyostelium discoideum. Copyright 1998 John Wiley & Sons, Inc.

Journal Article↗
PubMed2005

Structural and dynamic properties of cytochrome P450 BM-3 in pure water and in a dimethylsulfoxide/water mixture.

Solvent molecules play an important role for the structural and dynamical properties of proteins. A major focus of current protein engineering is the development of enzymes that are catalytically active in the presence of organic solvents. The monooxygenase P450 BM-3 is one of the best-studied enzymes and promising for industrial applications but with limited activity in the presence of organic solvents or cosolvents. To gain insights into the structural and dynamical properties of the heme domain of this enzyme in solution, molecular dynamics simulations in pure water and in a 14% DMSO/water mixture were performed. The results of the simulations show overall similar structural fluctuations in both solvent systems, with no indication of partial or global unfolding. In 14% DMSO, the regions comprising the helices E, F, and the EF loop (implicated in controlling the entry to the active site channel) undergo a large shift. Significant changes were also observed near the active site access channel at the residue R47. During the simulation, no DMSO molecule penetrated the active site. However, a significant accumulation of DMSO molecules close to the substrate-binding site and to the Flavin Mononucleotide (FMN) reductase domain interface was observed.

Bacillus megaterium↗
PubMed2003

Prediction of deleterious functional effects of amino acid mutations using a library of structure-based function descriptors.

An automated, active site-focused, computational method is described for use in predicting the effects of engineered amino acid mutations on enzyme catalytic activity. The method uses structure-based function descriptors (Fuzzy Functional Forms trade mark or FFFs trade mark ) to automatically identify enzyme functional sites in proteins. Three-dimensional sequence profiles are created from the surrounding active site structure. The computationally derived active site profile is used to analyze the effect of each amino acid change by defining three key features: proximity of the change to the active site, degree of amino acid conservation at the position in related proteins, and compatibility of the change with residues observed at that position in similar proteins. The features were analyzed using a data set of individual amino acid mutations occurring at 128 residue positions in 14 different enzymes. The results show that changes at key active site residues and at highly conserved positions are likely to have deleterious effects on the catalytic activity, and that non-conservative mutations at highly conserved residues are even more likely to be deleterious. Interestingly, the study revealed that amino acid substitutions at residues in close contact with the key active site residues are not more likely to have deleterious effects than mutations more distant from the active site. Utilization of the FFF-derived structural information yields a prediction method that is accurate in 79-83% of the test cases. The success of this method across all six EC classes suggests that it can be used generally to predict the effects of mutations and nsSNPs for enzymes. Future applications of the approach include automated, large-scale identification of deleterious nsSNPs in clinical populations and in large sets of disease-associated nsSNPs, and identification of deleterious nsSNPs in drug targets and drug metabolizing enzymes.

Algorithms↗
PubMed2002

Engineering of an intersubunit disulfide bridge in the iron-superoxide dismutase of Mycobacterium tuberculosis.

With the aim of enhancing interactions involved in dimer formation, an intersubunit disulfide bridge was engineered in the superoxide dismutase enzyme of Mycobacterium tuberculosis. Ser-123 was chosen for mutation to cysteine since it resides at the dimer interface where the serine side chain interacts with the same residue in the opposite subunit. Gel electrophoresis and X-ray crystallographic studies of the expressed mutant confirmed formation of the disulfide bond under nonreducing conditions. However, the mutant protein was found to be less stable than the wild type as judged by susceptibility to denaturation in the presence of guanidine hydrochloride. Decreased stability probably results from formation of a disulfide bridge with a suboptimal torsion angle and exclusion of solvent molecules from the dimer interface.

Cloning, Molecular↗