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PubMed2005

Natural products to drugs: daptomycin and related lipopeptide antibiotics.

Daptomycin (Cubicin) is a lipopeptide antibiotic approved in the USA in 2003 for the treatment of skin and skin structure infections caused by Gram-positive pathogens. It is a member of the 10-membered cyclic lipopeptide family of antibiotics that includes A54145, calcium-dependent antibiotic (CDA), amphomycin, friulimicin, laspartomycin, and others. This review highlights research on this class of antibiotics from 1953 to 2005, focusing on more recent studies with particular emphasis on the interplay between structural features and antibacterial activities; chemical modifications to improve activity; the genetic organization and biosynthesis of lipopeptides; and the genetic engineering of the daptomycin biosynthetic pathway to produce novel derivatives for further chemical modification to develop candidates for clinical evaluation.

Anti-Bacterial Agents↗
PubMed1994

Protein dimerization. Inside job.

In a sophisticated combination of genetic engineering and organic synthesis, a general method for dimerizing recombinant intracellular proteins has been devised; the usefulness of the method should now be testable.

Models, Chemical↗
PubMed1997

Development of a standard reference material for diesel mutagenicity in the Salmonella plate incorporation assay.

The present study documents the mutagenicity of a new National Institute of Standards and Technology (NIST) standard reference material (SRM) in the Salmonella plate incorporation assay. This study is in response to a previous recommendation by the World Health Organization to develop large batches of new SRMs for biological and chemical research. SRM 1975 is a dichloromethane (DCM) extract of 5.6 kg of filter-collected combustion particulate matter (SRM 2975) from operating forklifts with diesel engines. The mutagenicity and a summary of the related chemical analysis of mutagens in SRM 1975 is presented in this paper, and are available from the NIST. Mutagenicity test conditions were: Salmonella typhimurium TA98, TA100 (standard strains); TA98NR, TA100NR (nitroreductase (NR) gene deficient); and YG1021 and YG1026 (NR gene addition); 10 dose levels in the linear portion of the dose-response curve; duplicate plates per dose; and S9 at 6.4% or 1.1 mg of protein/plate. Four rounds of testing were conducted. Rounds were conducted at least 1 week apart. Slopes (revertants/microg) were calculated by the linear regression rejection model of Bernstein and by the Stead and Krewski models which analyze non-linear data. The GeneTox Manager software package developed at the EPA was used to record the data and calculate the slopes. Results demonstrated: (1) the ranking of slopes without S9 was: YG1021 > TA98 > TA98NR > YG1026 > TA100 > TA100NR in all three statistical models; (2) the mutagenic activity of SRM 1975 was significantly increased by the presence of the NR gene; (3) the slope values for the TA100 series were significantly less than for the TA98 series; (4) in general, the addition of the S9 significantly reduced mutagenic activity; (5) the mutagenic activity of the SRM 1975 was stable over time and variability was low (generally less than 20% in slope values over the 4 rounds); and (6) agreement of the slope values among the three models was excellent due to the linear nature of the data. These data will be useful in ranking other diesel and air samples for mutagenic activity, for quality assurance of data generated in different laboratories, for quality control within a laboratory, and as positive control values for future air and automotive emission studies.

Mutagenicity Tests↗
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↗
PubMed1988

Bioengineering issues related to in situ remediation of contaminated soils and groundwater.

This chapter presents some of the engineering challenges in biological degradation of organic contaminants in surface soils and the subsurface environment. Extraction of contaminants from the subsurface is generally costly, slow, and difficult. This has led to an interest in using in situ techniques for biodegradation of contaminants. For some contaminants that can be readily used as primary energy sources for bacteria with or without the presence of oxygen, bacterial removal is relatively simple and, in some situations, occurs naturally. In other cases, such as hydrocarbon spills, in situ treatment is still attractive, but has the added cost and difficulty of supplying required oxygen and nutrients for the fairly efficient aerobic oxidation. There are some indications that hydrocarbons may be biodegraded in the absence of oxygen; but, in these cases, the rates of degradation appear to be slow. More research in this area is desirable. The major challenge to both engineers and scientists lies in the decomposition of hazardous chemicals that appear to be transformed by the process of co-metabolism, or else are so low in concentration that they can only serve as secondary substrates. In either case, primary substrates are required to supply the major energy requirements for bacterial growth and/or for activation of enzymes necessary for the transformation. Engineering experience in the utilization of such processes for degradation of contaminants in the environment is very limited. Little is currently known about substrate interactions and how to optimize the bacterial transformations. Chemical requirements for achieving co-metabolism are high. In addition, knowledge is lacking about methods for getting the correct amounts of chemicals to the locations where needed and in the form needed. The solution of these important issues presents a significant challenge to the engineering and science communities, requiring both basic laboratory studies and field demonstrations in well-characterized environments.

Bacteria↗
PubMed2005

A second-site suppressor strategy for chemical genetic analysis of diverse protein kinases.

Chemical genetic analysis of protein kinases involves engineering kinases to be uniquely sensitive to inhibitors and ATP analogs that are not recognized by wild-type kinases. Despite the successful application of this approach to over two dozen kinases, several kinases do not tolerate the necessary modification to the ATP binding pocket, as they lose catalytic activity or cellular function upon mutation of the 'gatekeeper' residue that governs inhibitor and nucleotide substrate specificity. Here we describe the identification of second-site suppressor mutations to rescue the activity of 'intolerant' kinases. A bacterial genetic selection for second-site suppressors using an aminoglycoside kinase APH(3')-IIIa revealed several suppressor hotspots in the kinase domain. Informed by results from this selection, we focused on the beta sheet in the N-terminal subdomain and generated a structure-based sequence alignment of protein kinases in this region. From this alignment, we identified second-site suppressors for several divergent kinases including Cdc5, MEKK1, GRK2 and Pto. The ability to identify second-site suppressors to rescue the activity of intolerant kinases should facilitate chemical genetic analysis of the majority of protein kinases in the genome.

Amino Acid Substitution↗
PubMed2002

Production of meiosis-activating sterols from metabolically engineered yeast.

Meiosis-activating sterols (MAS), a class of potent regulators of reproductive processes, are difficult to obtain by chemical synthesis or isolation from natural sources. We demonstrate the development of metabolically engineered strains of Saccharomyces cerevisiae that accumulate MAS as the predominant sterol product. Homologous recombination was used to construct an erg24Delta erg25Delta hem1Delta mutant RXY4.3, which lacked sterol Delta14 reductase, C-4 oxidase, and delta-aminolevulinate synthase. The HEM1 deletion allowed sterol import and rendered RXY4.3 viable under aerobic conditions. This mutant accumulated 4,4-dimethyl-5alpha-cholesta-8,14,24-trien-3beta-ol (FF-MAS), and a similar erg25Delta hem1Delta mutant produced 4,4-dimethyl-5alpha-cholesta-8,24-dien-3beta-ol (T-MAS). Based on consistent yields of approximately 5 mug of FF-MAS per mL of culture, fermentation of genetically modified yeast compares favorably with other approaches to produce MAS.

Cholestadienols↗
PubMedDate not supplied

The assessment of irritation using clinical methods and questionnaires.

Sensory irritant responses to chemical exposures are measured by a variety of methods; however, studies can be influenced from biases associated with study design and subject responses. This article reviews the different methods used to quantitate irritation. These methods primarily focus on eye and nasal mucosal irritation. Although methods to evaluate mouth, throat mucosal, and dermal irritation are also relevant, they are seldom used in actual practice. Measurements for eye irritation include tear film stability, epithelial damage, foam formation, blinking frequency, tear flow, inflammation, and hyperemia. Methods for detecting nasal mucosa irritation include measuring swelling of the nasal mucosa, peak airflows through the nose, acoustic rhinometry, and rhinostereometry, which measures thickness of the anterior nasal turbinate. Questionnaires are useful for defining a set of symptoms in an attempt to characterize dose-response relationships from controlled exposure studies or field studies, to compare rates of events in field studies, or to screen for disease. However, it is important to consider carefully the study design, goal of utilization, and constraints surrounding their application. Whichever method is used in medical surveillance or to evaluate effectiveness of industrial hygiene or engineering controls in preventing irritation effects from chemical exposure, the sensitivity, specificity, and predictive value of the irritation measurements are important factors in interpreting the results. This article reviews these various issues and offers some advice.

Air Pollutants, Occupational↗
PubMed2005

Grp78, Grp94, and Grp170 interact with alpha1-antitrypsin mutants that are retained in the endoplasmic reticulum.

In alpha1-antitrypsin (alpha1-AT) deficiency, a mutant form of alpha1-AT polymerizes in the endoplasmic reticulum (ER) of liver cells resulting in chronic hepatitis and hepatocellular carcinoma by a gain of toxic function mechanism. Although some aspects of the cellular response to mutant alpha1-AT Z have been partially characterized, including the involvement of several proteasomal and nonproteasomal mechanisms for disposal, other parts of the cellular response pathways, particularly the chaperones with which it interacts and the signal transduction pathways that are activated, are still not completely elucidated. The alpha1-AT Z molecule is known to interact with calnexin, but, according to one study, it does not interact with Grp78. To carry out a systematic search for the chaperones with which alpha1-AT Z interacts in the ER, we used chemical cross-linking of several different genetically engineered cell systems. Mutant alpha1-AT Z was cross-linked with Grp78, Grp94, calnexin, Grp170, UDP-glucose glycoprotein:glucosyltransferase, and two unknown proteins of approximately 110-130 kDa. Sequential immunoprecipitation/immunoblot analysis and coimmunoprecipitation techniques demonstrated each of these interactions without chemical cross-linking. The same chaperones were found to interact with two nonpolymerogenic alpha1-AT mutants that are retained in the ER, indicating that these interactions are not specific for the alpha1-AT Z mutant. Moreover, sucrose density gradient centrifugation studies suggest that approximately 85% of alpha1-AT Z exists in heterogeneous soluble complexes with multiple chaperones and approximately 15% in extremely large polymers/aggregates devoid of chaperones. Agents that perturb the synthesis and/or activity of ER chaperones such as tunicamycin and calcium ionophore A23187, have different effects on the solubility and degradation of alpha1-AT Z as well as on its residual secretion.

Cell Culture Techniques↗
PubMed2003

Homogeneous biocatalysis in organic solvents and water-organic mixtures.

Biocatalysis in non-aqueous media has undergone tremendous development during the last decade, and numerous reactions have been introduced and optimized for synthetic applications. In contrast to aqueous enzymology, biotransformations in organic solvents offer unique industrially attractive advantages, such as: drastic changes in the enantioselectivity of the reaction, the reversal of the thermodynamic equilibrium of hydrolysis reactions, suppression of water-dependent side reactions, and resistance to bacterial contamination. Currently, the field is dominated by heterogeneous biocatalysis based primarily on lyophilized enzyme powders, cross-linked crystals, and enzymes immobilized on inert supports that are mainly applied in enantioselective synthesis. However, low reaction rates are an inherent problem of the heterogeneous biocatalysis, while the homogeneous systems have the advantage that the elimination of diffusional barriers of substrates and products between organic and water phases results in an increase in the reaction rate. Here the discussion is focused on the correlation between activity and structure of the intact enzymes dissolved in neat organic solvents, as well as modifications of natural enzymes, which make them soluble and catalytically active in non-aqueous environment. Factors that influence conformation and stability of the enzymes are also discussed. Current developments in non-aqueous biocatalysts that combine advantages of protein modification and immobilization, i.e., HIP plastics, enzyme chips, ionic liquids, are introduced. Finally, engineering enzymes for biotransformations in non-conventional media by directed evolution is summarized.

Catalysis↗
PubMed2006

A comparative study of metabolic engineering anti-metabolite tolerance in Escherichia coli.

A problem in strain engineering is that mutations that benefit the expression of a phenotype in one environment may impose a cost to biological fitness in a new environment. The overall objective of this study was to improve understanding of this phenomenon within the context of a classic anti-metabolite selection strategy. We have engineered Escherichia coli using three mutagenesis techniques (chemical mutagenesis, insertional mutagenesis, and plasmid-based overexpression) and assessed the relative costs and benefits to biological fitness of mutants selected for tolerance to five amino acid analogs whose target amino acids (glutamatic acid, aspartic acid, tryptophan, glycine, and serine) differ in metabolic connectivity and biosynthetic energy requirements. Our major findings include (i) the fold increase in anti-metabolite tolerance, independent of mutagenesis strategy, was much greater for aspartic acid beta-hydroxamate (AAH) compared to all other tested hydroxamates, (ii) increased tolerance to glutamic acid gamma-hydroxamate (GAH) was not achieved using any of the mutagenesis strategies, and (iii) characteristics of the anti-metabolite, rather than those of the corresponding metabolite, were more important in determining the ability to increase tolerance.

Drug Tolerance↗
PubMed2005

Production and purification of self-assembling peptides in Ralstonia eutropha.

Self-assembling peptides have emerged as an attractive scaffold material for tissue engineering, yet the expense associated with solid phase chemical synthesis has limited their broad use. In addition, the fidelity of chemical synthesis constrains the length of polypeptides that can be produced homogeneously by this method. Template-derived biosynthesis by recombinant DNA technology may overcome both of these problems. However, recovery of polypeptides from recombinant protein expression systems typically involves multi-step purification schemes. In this study, we report an integrated approach to recombinantly produce and purify self-assembling peptides from the recently developed expression host Ralstonia eutropha. The purification is based on the specific affinity of carbohydrate binding modules (CBMs) to cellulose. In a first step, we identified CBMs that express well in R. eutropha by assembling a fusion library of green fluorescent protein (GFP) and CBMs and determining the fluorescence of cell-free extracts. Three GFP::CBM fusions were found to express at levels similar to GFP alone, of which two CBMs were able to mediate cellulose binding of the GFP::CBM fusion. These two CBMs were then fused to multiple repeats of the self-assembling peptide RAD16-I::E (N-RADARADARADARADAE-C). The fusion protein CBM::E::(RAD16-I::E)4 was expressed in R. eutropha and purified using the CBM's affinity for cellulose. Subsequent proteolytic cleavage with endoproteinase GluC liberated RAD16-I::E peptide monomers with similar properties to the chemically synthesized counterpart RAD16-I.

Carrier Proteins↗
PubMed2001

Characterization of an oxygen-dependent inducible promoter, the nar promoter of Escherichia coli, to utilize in metabolic engineering.

The nar promoters, whose transcription is maximally induced under microaerobic conditions in the presence of nitrate ion, were characterized in fed-batch culture to determine whether they can be used for metabolic engineering, by which overall production of valuable chemicals can be increased. For this purpose, we tested whether the expression level of a reporter gene, the lacZ gene from the nar promoter, could be maintained constant throughout the induction period by manipulation of dissolved oxygen (DO) levels at a given nitrate ion concentration. First, E. coli was grown under aerobic conditions (DO 80%) to absorbance at 600 nm (OD(600)) of 35, then the nar promoter was induced by reduction of DO to different levels, combined with different frequencies and duration of alternating microaerobic and aerobic conditions throughout the entire induction period. For a wild-type nar promoter (pMW61) in a mutant host E. coli with a mutation in the narG gene on the chromosome of the host (RK5265), it was possible to maintain production of beta-galactosidase activity per cell (specific beta-galactosidase activity) at a constant rate at 5000, 10,000, 15,000, and 20,000 Miller units, using different combinations of nitrate ion concentrations (0.1%, 0.5%, and 1%) and DO levels. In addition, it was possible to maintain production of specific beta-galactosidase activity at a constant rate at about 10,000 Miller units in the absence of nitrate ion when a nitrate-independent nar promoter (pMW618) in the narL(-) mutant of the W3110 E. coli strain (W3110narL(-)) was used. Based on these results, we conclude that the nar promoter system provides a convenient expression system for metabolic engineering as well as for maximal production of recombinant proteins under conditions of fed-batch culture.

Chromosomes, Bacterial↗
PubMed2002

Functionally orthogonal ligand-receptor pairs for the selective regulation of gene expression generated by manipulation of charged residues at the ligand-receptor interface of ER alpha and ER beta.

The reengineering of protein-small molecule interfaces represents a powerful tool of chemical biology. For many applications it is necessary to engineer receptors so that they do not interact with their endogenous ligands but are highly responsive to designed ligand analogues, which in turn do not interact with endogenous proteins. The chemical design strategy used to reengineer protein-small molecule interfaces is particularly challenging for interfaces involving relatively plastic receptor binding sites and therefore presents a unique challenge in molecular design. In this study we explore the scope and limitations of a new strategy for manipulating polar/charged residues across the ligand receptor interface of estradiol (E2) and the estrogen receptor (ER). Carboxylate-functionalized E2 analogues can activate ER alpha(Glu353-->Ala) and ER beta(Glu305-->Ala) with very large selectivites, demonstrating that this design strategy is extendable to other members of the steroid hormone receptor family. Neutral E2 analogues were found to complement ER alpha(E353A) with similar potencies but with generally lower selectivities. This suggests that the high selectivity observed with ligand-receptor pairs generated by exchanging charged residues across ligand-receptor interfaces is only due in part to their complementary shapes and that appropriate introduction of charged functionality on the ligand can provide substantial enhancement of selectivity by decreasing the engineered ligands affinity for the endogenous receptor. Attempts to modify the cationic residues by complementing Arg394-->Ala or Arg394-->Glu were not successful.

Cells, Cultured↗
PubMed2024

Adeno-Associated Virus Engineering and Load Strategy for Tropism Modification, Immune Evasion and Enhanced Transgene Expression.

Gene therapy aims to add, replace or turn off genes to help treat disease. To date, the US Food and Drug Administration (FDA) has approved 14 gene therapy products. With the increasing interest in gene therapy, feasible gene delivery vectors are necessary for inserting new genes into cells. There are different kinds of gene delivery vectors including viral vectors like lentivirus, adenovirus, retrovirus, adeno-associated virus et al, and non-viral vectors like naked DNA, lipid vectors, polymer nanoparticles, exosomes et al, with viruses being the most commonly used. Among them, the most concerned vector is adeno-associated virus (AAV) because of its safety, natural ability to efficiently deliver gene into cells and sustained transgene expression in multiple tissues. In addition, the AAV genome can be engineered to generate recombinant AAV (rAAV) containing transgene sequences of interest and has been proven to be a safe gene vector. Recently, rAAV vectors have been approved for the treatment of various rare diseases. Despite these approvals, some major limitations of rAAV remain, namely nonspecific tissue targeting and host immune response. Additional problems include neutralizing antibodies that block transgene delivery, a finite transgene packaging capacity, high viral titer used for per dose and high cost. To deal with these challenges, several techniques have been developed. Based on differences in engineering methods, this review proposes three strategies: gene engineering-based capsid modification (capsid modification), capsid surface tethering through chemical conjugation (surface tethering), and other formulations loaded with AAV (virus load). In addition, the major advantages and limitations encountered in rAAV engineering strategies are summarized.

Dependovirus↗
PubMed2003

Beta-propellers: associated functions and their role in human diseases.

The beta-propeller fold appears as a very fascinating architecture based on four-stranded antiparallel and twisted beta-sheets, radially arranged around a central tunnel. Similar to the alpha/beta-barrel (TIM-barrel) fold, the beta-propeller has a wide range of different functions, and is gaining substantial attention. Some proteins containing beta-propeller domains have been implicated in the pathogenesis of a variety of diseases such as cancer, Alzheimer, Huntington, arthritis, familial hypercholesterolemia, retinitis pigmentosa, osteogenesis, hypertension, and microbial and viral infections. This article reviews some aspects of 3D structure, amino acids sequence regularities, and biological functions of the proteins containing beta-propeller domains. Major emphasis has been laid on beta-propellers whose functions are associated to human diseases. Recent research efforts reported in the fields of protein engineering, drug design, and protein structure-function relationship studies, concerning the beta-propeller architecture, have also been discussed.

Disease↗