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At least 1,657 records · Page 92Linked to original sources
PubMed2003

Development of a novel bacterial artificial chromosome cloning system for functional studies.

Bacterial artificial chromosome (BAC) cloning systems currently in use generate high quality genomic libraries for gene mapping, identification, and sequencing. However, the most commonly used BAC cloning systems do not facilitate functional studies in eukaryotic cells. To overcome this limitation, we have developed pEBAC190G, a new BAC vector that combines the features of the first generation PAC/BAC vectors with eukaryotic elements that facilitate the transfection, episomal maintenance, and functional analysis of large genomic fragments in eukaryotic cells. A number of different cloning strategies may be used to retrofit genomic fragments from existing libraries into the new vector. The system was tested by the retrofitting of a 170kb NotI genomic fragment from the RPCI-11 BAC library into the NotI site of pEBAC190G. Clones from any eukaryotic genomic library harboured in this vector can be transferred from bacteria directly to eukaryotic cells for functional analysis.

Base Sequence↗
PubMed2002

Chorismate lyase: kinetics and engineering for stability.

By removing the enolpyruvyl group from chorismate, chorismate lyase (CL) produces p-hydroxybenzoate (p-HB) for the ubiquinone biosynthetic pathway. We have analyzed CL by several spectroscopic and chemical techniques and measured its kinetic (kcat=1.7 s(-1), K(m)=29 microM) and product inhibition parameters (K(p)=2.1 microM for p-HB). Protein aggregation, a serious problem with wild type CL, proved to be primarily due to the presence of two surface-active cysteines, whose chemical modification or mutation (to serines) gave greatly improved solution behavior and minor effects on enzyme activity. CL is strongly inhibited by its product p-HB; for this reason activity and inhibition measurements were analyzed by both initial rate and progress curve methods. The results are consistent, but in this case where the stable enzyme-product complex rapidly becomes the predominant form of the enzyme, progress curve methods are more efficient. We also report inhibition measurements with several substrate and product analogs that give information on ligand binding interactions of the active site. The biological function of the unusual product retention remains uncertain, but may involve a mechanism of directed delivery to the membrane-bound enzyme that follows CL in the ubiquinone pathway.

Anthranilate Synthase↗
PubMed1999

Protein redesign.

It has been demonstrated that, for a number of proteins, it is possible to dramatically alter the connectivities between elements of secondary structure. Remarkably large loop insertions are tolerated and many redesigns have generated proteins that successfully fold to stable, active structures. Some redesigns have been entirely the choice of the investigators, whereas others have incorporated a randomization and selection step to identify optimal sequences. These studies have provided basic guidelines for the rational manipulation of protein structure and stability, they have allowed the dissection of folding pathways and they have generated proteins with the potential for practical therapeutic applications.

Bacterial Proteins↗
PubMed1985

Disorders of cobalamin metabolism.

Recent developments in our knowledge of the biochemistry and metabolism of cobalamin have given us some insight into clinical disorders. N2O, which easily induces cobalamin deficiency, both in vivo and in vitro, has greatly contributed to the investigation of the cobalamin deficient state, especially in relation to folate and amino acid metabolism. Demonstration of the cobalamin analog in human serum and a new enzyme which requires cobalamin as a coenzyme has led to recent increased interest in this field. The disorders of cobalamin metabolism will be summarized briefly as well as those areas currently of particular interest.

5-Methyltetrahydrofolate-Homocysteine S-Methyltran↗
PubMed1996

Development of improved tRNAs for in vitro biosynthesis of proteins containing unnatural amino acids.

BACKGROUND: Chemically aminoacylated suppressor tRNAs have previously been used in vitro to generate mutant proteins in which unnatural amino acids are incorporated site-specifically. Although the existing methodology often provides adequate quantities of mutant proteins, the suppression efficiencies of some unnatural amino acids are not high enough to yield useful amounts of protein. In an effort to make this useful mutagenesis strategy more general, we report here the results of a search to find alternative tRNAs as a way of increasing suppression efficiencies. RESULTS: Three suppressor tRNAs have been generated by runoff transcription and their ability to deliver unnatural amino acids site-specifically into proteins has been assessed in an E. coli-derived in vitro transcription/translation system. Analysis of their ability to insert both polar and nonpolar residues in response to an amber codon in two proteins suggests that an E. coli tRNAAsn-derived suppressor offers a significant improvement in suppression efficiency over other previously used tRNAs. CONCLUSIONS: Use of an E. coli tRNAAsn-derived suppressor may provide substantially higher yields of proteins containing unnatural amino acids, in addition to offering a broader tolerance for polar amino acids. A comparison of suppressor tRNAs derived from tRNAAsn, tRNAGln or tRNAAsp with that derived from tRNAPhe supports emerging evidence that the identity of an amino acid may be important in message recognition.

Amino Acids↗
PubMed1983

Secondary tritium isotope effects as probes of the enzymic and nonenzymic conversion of chorismate to prephenate.

To obtain information about the degree of concert of both the nonenzymic and the enzyme-catalyzed rearrangement of chorismate to prephenate, we have determined the secondary tritium isotope effects at the bond-making position (C-9) and the bond-breaking position (C-5) of chorismate. The isotope effects were determined by the competitive method, using either [5-3H,7-14C )chorismate or [9-3H,7-14C]chorismate as the substrate. In the nonenzymic reaction (pH 7.5, 60 degrees C), KH/kT is 1.149 +/- 0.012 for bond breaking (C-9) and 0.992 +/- 0.012 for bond making (C-5). This indicates an asymmetric transition state in which the new bond is hardly, if at all, formed, while the bond between C-5 and oxygen is substantially broken. In the enzymic reaction (pH 7.5, 30 degrees C), the values of kH/kT in both positions are unity within experimental error. It is most likely that the isotope effects are suppressed in the enzymic process and that the rate-limiting transition state occurs before the rearrangement itself. The kinetically significant transition state presumably involves either the binding step of the small equilibrium proportion of the axial conformer of the substrate or an isomerization of enzyme-bound chorismate from the more stable conformer in which the carboxyvinyloxy group is equatorial to that in which this group is axial. Rearrangement would then proceed relatively rapidly from the higher energy axial conformer.

3-Phosphoshikimate 1-Carboxyvinyltransferase↗
PubMed1984

Chorismate mutase/prephenate dehydrogenase from Escherichia coli K12: purification, characterization, and identification of a reactive cysteine.

The bifunctional enzyme involved in tyrosine biosynthesis, chorismate mutase/prephenate dehydrogenase, has been isolated from extracts of a regulatory mutant of Escherichia coli K12. The pure enzyme is a homodimer of total molecular weight 78 000 and displays Michaelis-Menten kinetics for both activities. Fingerprinting and amino acid sequencing of tryptic and thermolytic peptides of the S-[14C]carboxymethylated enzyme allowed the identification of three unique cysteine-containing sequences per subunit. Chemical modification of the native enzyme with 5,5'-dithiobis(2-nitrobenzoate) or iodoacetamide showed that one sulfhydryl group per subunit was particularly reactive, and the integrity of this group was essential for both enzymic activities. This work supports previous proposals for a close spatial relationship between the active sites.

Amino Acid Sequence↗
PubMed2001

New insights into the metal center of 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase.

Metal binding properties for a series of metal-substituted forms of 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase, DAHPS(Tyr), have been followed by UV-vis and EPR spectroscopy. The results show that there are two metal species present at pH = 7.0 and these are coordinated in a distorted metal binding site with a mixed nitrogen and oxygen donor atom coordination set. There is no spectroscopic evidence for strong M-S interactions in this system at any pH. Metal saturation occurs at a substoichiometric ratio of 0.8-0.85 metal/monomer, and the binding trends mirror previously published enzyme activity profiles. There is a conformational change for CuDAHPS under basic conditions, and equivalent protein handling for apoDAHPS leads to apparent loss of metal binding ability. Addition of the substrate PEP does not alter the UV-vis spectra, but there are small changes in the EPR spectra of CuDAHPS(Tyr). Further addition of the substrate analogue A5P has no effect on either spectra. Taken together, these results serve to link previous studies on enzyme activity with the recently determined X-ray crystal structure for DAHPS(Phe) and represent the first detailed spectroscopic characterization of the metal binding properties of DAHPS(Tyr).

3-Deoxy-7-Phosphoheptulonate Synthase↗
PubMed2006

Redesigning the PheA domain of gramicidin synthetase leads to a new understanding of the enzyme's mechanism and selectivity.

The PheA domain of gramicidin synthetase A, a non-ribosomal peptide synthetase, selectively binds phenylalanine along with ATP and Mg2+ and catalyzes the formation of an aminoacyl adenylate. In this study, we have used a novel protein redesign algorithm, K*, to predict mutations in PheA that should exhibit improved binding for tyrosine. Interestingly, the introduction of two predicted mutations to PheA did not significantly improve KD, as measured by equilibrium fluorescence quenching. However, the mutations improved the specificity of the enzyme for tyrosine (as measured by kcat/KM), primarily driven by a 56-fold improvement in KM, although the improvement did not make tyrosine the preferred substrate over phenylalanine. Using stopped-flow fluorometry, we examined binding of different amino acid substrates to the wild-type and mutant enzymes in the pre-steady state in order to understand the improvement in KM. Through these investigations, it became evident that substrate binding to the wild-type enzyme is more complex than previously described. These experiments show that the wild-type enzyme binds phenylalanine in a kinetically selective manner; no other amino acids tested appeared to bind the enzyme in the early time frame examined (500 ms). Furthermore, experiments with PheA, phenylalanine, and ATP reveal a two-step binding process, suggesting that the PheA-ATP-phenylalanine complex may undergo a conformational change toward a catalytically relevant intermediate on the pathway to adenylation; experiments with PheA, phenylalanine, and other nucleotides exhibit only a one-step binding process. The improvement in KM for the mutant enzyme toward tyrosine, as predicted by K*, may indicate that redesigning the side-chain binding pocket allows the substrate backbone to adopt productive conformations for catalysis but that further improvements may be afforded by modeling an enzyme:ATP:substrate complex, which is capable of undergoing conformational change.

Chorismate Mutase↗
PubMed1999

Regulation of phenylalanine biosynthesis. Studies on the mechanism of phenylalanine binding and feedback inhibition in the Escherichia coli P-protein.

Isothermal titration calorimetry (ITC) and site-directed mutagenesis were used to study the interaction of Phe with (a) the Escherichia coli P-protein, a bifunctional chorismate mutase/prephenate dehydratase that is feedback inhibited by Phe, (b) PDT32, a 32 kDa P-protein fragment (residues 101-386) containing the prephenate dehydratase and regulatory domains, and (c) R12, a C-terminal 12 kDa P-protein fragment (residues 286-386) containing the regulatory domain. DeltaH(total) values for PDT32, which included the heats of Phe binding, conformational change, and dimerization, established that in developing a mechanism for end product feedback inhibition, the P-protein has evolved a ligand recognition domain that exhibits Phe-binding enthalpies comparable to those reported for other full-fledged amino acid receptor proteins. Sequence alignments of R12 with other Phe-binding enzymes identified two highly conserved regions, GALV (residues 309-312) and ESRP (residues 329-332). Site-directed mutagenesis and ITC established that changes in the GALV and ESRP regions affected Phe binding and feedback inhibition to different extents. Mutagenesis further showed that C374 was essential for feedback inhibition, but not for Phe binding, while W338 was involved in Phe binding, but not in the Phe-induced conformational change required for feedback inhibition.

Bacterial Proteins↗
PubMedDate not supplied

In vivo 15N-enrichment of metabolites in suspension cultured cells and its application to metabolomics.

The incorporation of stable isotopes in suspension cultured cells is very simple and useful as a preliminary experimental method in the experimental scene of plant metabolomics to elucidate the metabolic profiles of mutants and transformants. Stable isotope methods would afford a dynamic explanation of turnover speed that would concern the metabolic flux. Utilization of suspension cultured cells allows genes to be easily induced or suppressed, culture conditions to be controlled, and samples to be easily prepared. Stable isotope tracing allows an index of metabolic flux to be obtained. Here we present an experiment feeding (15)N-labeled inorganic salts to Arabidopsis (cell line T87) and Coptis cultured cells. Results of a comparison of (15)N labeling ratios of amino acids derived from T87 cells cultured under light with those cultured in the dark corresponded to transcriptional expressions revealed by microarray experiments published previously, demonstrating the validity of this procedure. Furthermore, (15)N labeling ratios of Coptis cultured cells revealed arginine and lysine metabolism inhibition, which should result in inhibition of polyamine biosynthesis and cell division. This very simple experiment allowed us to uncover metabolic dynamic features of the plant cell. Therefore this method is very useful for forming working hypotheses and experimental design.

Amino Acids↗
PubMed2002

Longitudinal (1)H relaxation optimization in TROSY NMR spectroscopy.

A general method to enhance the sensitivity of the multidimensional NMR experiments performed at high-polarizing magnetic field via the significant reduction of the longitudinal proton relaxation times is described. The method is based on the use of two vast pools of "thermal bath" 1H spins residing on hydrogens covalently attached to carbon and oxygen atoms in 13C,15N labeled and fully protonated or fractionally deuterated proteins to uniformly enhance longitudinal relaxation of the 1HN spins and concomitantly the sensitivity of multipulse NMR experiments. The proposed longitudinal relaxation optimization is implemented in the 2D [15N,1H]-LTROSY, 2D [15N,1H]-LHSQC and 3D LTROSY-HNCA experiments yielding the factor 2-2.5 increase of the maximal signal-to-noise ratio per unit time at 600 MHz. At 900 MHz, the predicted decrease of the 1HN longitudinal relaxation times can be as large as one order of magnitude, making the proposed method an important tool for protein NMR at high magnetic fields.

Bacillus subtilis↗
PubMed2003

Carboxylate-substituted radicals from phenylselenide derivatives. Designs on models for coenzyme B12-dependent enzyme-catalyzed rearrangements.

Laser flash photolysis (266 nm) of alpha- and beta-phenylselenyl esters, carboxylic acids, and carboxylates in aqueous acetonitrile media gave the corresponding radicals by homolytic cleavage of the phenylselenyl groups. In the beta-substituted systems, acid and carboxylate radicals reacted in intramolecular reporter reactions with approximately equal rate constants. For the alpha-substituted systems, an ester- and carboxylic acid-substituted radical reacted in an intramolecular reporter reaction with the same rate constants, but the analogous alpha-carboxylate radical, a radical anion, reacted an order of magnitude less rapidly and with an activation energy that is 3 kcal/mol greater than that found for analogues. A kinetic titration of the equilibrating alpha-acid and alpha-carboxylate radicals gave pKa = 4.6. The results indicate that alpha-ester and alpha-carboxylic acid radicals are unlikely to be appropriate models for alpha-carboxylate radicals, the intermediates formed in a large subset of coenzyme B12-dependent enzyme-catalyzed reactions.

Carboxylic Acids↗
PubMed1998

Methylmalonic acidaemia with bilateral globus pallidus involvement: a neuropathological study.

A 16-month-old boy was hospitalized because of a 1-day history of severe ketoacidosis with lethargy, hypotonia, vomiting, and important dyspnoea. Organic acid assay by gas chromatography-mass spectrometry confirmed the diagnosis of methylmalonic acidaemia (MMA). On the sixteenth day, he developed an acute extrapyramidal disorder. The CT scan of the brain disclosed bilaterally symmetric lucency of basal ganglia. He died at 17 months of age. Post-mortem neuropathological examination, showed severe necrosis with spongiosis, cystic cavitation and numerous lipid-laden macrophages of the globi pallidi, and mild spongiosis of subthalamic nuclei, mammillary bodies, portion of internal capsule adjacent to globus pallidus, superior cerebellar peduncles and tegmentum of brainstem. Pallidal infarction, a focal ischaemic lesion, demonstrates that ischaemia/energy depletion may be important in the etiology of the neuropathology of MMA.

Cobamides↗