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Biomedical subjects

E Daub

Publications and source records attributed to E Daub.

17 recordsLinked to original sources
PubMed2005

Investigation of bioisosteric effects on the interaction of substrates/ inhibitors with the methionyl-tRNA synthetase from Escherichia coli.

Aminoacyl-tRNA synthetases catalyze the stepwise coupling of specific amino acid substrates to their cognate tRNAs. The first intermediate formed in this process is the aminoacyl-adenylate, which then subsequently reacts with the 3'-terminus of the cognate tRNA to transfer the amino acid to the tRNA. This overall reaction is critical for protein biosynthesis and is quintessential to the viability of all organisms. Therefore, the selective inhibition of bacterial amino acid-tRNA synthetases is the focus of intense current interest for the development of novel antibacterial agents. In order to elucidate some of the critical factors involved in recognition and binding of potential inhibitors to these bacterial systems, the current report has focused on the methionyl-tRNA synthetase from Escherichia coli. This enzyme has been studied with two sets of bioisosteric replacements in the methionine and methionyl-adenylate structures. Replacements of the carboxyl group of methionine with the phosphinic and phosphonic acid moieties were used to probe the effects of including potential transition state analogs on enzyme inhibition. The contributions of the aminoacyl-adenylate structure and the effect that fluorination has on inhibitory activity were investigated utilizing 5'-O-[(L-methionyl)-sulfamoyl]adenosine and 5'-O-[(S-trifluoromethyl-L-homocysteinyl)-sulfamoyl]adenosine. The K(i) values for these compounds were determined to be 0.4 mM, 1.2 mM, 0.25 nM and 2.4 nM respectively. A discussion of this data in relation to structural information provided by the recent determination of the three-dimensional structures of the E. coli enzyme with several of these compounds is presented.

Adenosine↗
PubMed2001

Elucidation of solvent exposure, side-chain reactivity, and steric demands of the trifluoromethionine residue in a recombinant protein.

When incorporated into proteins, fluorinated amino acids have been utilized as 19F NMR probes of protein structure and protein-ligand interactions, and as subtle structural replacements for their parent amino acids which is not possible using the standard 20-amino acid repertoire. Recent investigations have shown the ability of various fluorinated methionines, such as difluoromethionine (DFM) and trifluoromethionine (TFM), to be bioincorporated into recombinant proteins and to be extremely useful as 19F NMR biophysical probes. Interestingly, in the case of the bacteriophage lambda lysozyme (LaL) which contains only three Met residues (at positions 1, 14, and 107), four 19F NMR resonances are observed when TFM is incorporated into LaL. To elucidate the underlying structural reasons for this anomalous observation and to more fully explore the effect of TFM on protein structure, site-directed mutagenesis was used to assign each 19F NMR resonance. Incorporation of TFM into the M14L mutant resulted in the collapse of the two 19F resonances associated with TFM at position 107 into a single resonance, suggesting that when position 14 in wild-type protein contains TFM, a subtle but different environment exists for the methionine at position 107. In addition, 19F and [1H-13C]-HMQC NMR experiments have been utilized with paramagnetic line broadening and K2PtCl4 reactivity experiments to obtain information about the probable spatial position of each Met in the protein. These results are compared with the recently determined crystal structure of LaL and allow for a more detailed structural explanation for the effect of fluorination on protein structure.

Bacteriophage lambda↗
PubMed1998

Overproduction and characterization of a dimeric non-zinc glyoxalase I from Escherichia coli: evidence for optimal activation by nickel ions.

The ubiquitous glyoxalase system converts toxic alpha-keto aldehydes into their corresponding nontoxic 2-hydroxycarboxylic acids, utilizing glutathione (GSH) as a cofactor. The first enzyme in this system, glyoxalase I (GlxI), catalyzes the isomerization of the hemithioacetal formed nonenzymatically between GSH and cytotoxic alpha-keto aldehydes. To study the Escherichia coli GlxI enzyme, the DNA encoding this protein, gloA, was isolated and incorporated into the plasmid pTTQ18. Nucleotide sequencing of the gloA gene predicted a polypeptide of 135 amino acids and Mr of 14 919. The gloA gene has been overexpressed in E. coli and shown to encode for GlxI. An effective two-step purification protocol was developed, yielding 150-200 mg of homogeneous protein per liter of culture. Electrospray mass spectrometry confirmed the monomeric weight of the purified protein, while gel filtration analysis indicated GlxI to be a homodimer of 30 kDa. Zinc, the natural metal ion found in the Homo sapiens and Saccharomyces cerevisiae GlxI, had no effect on the activity of E. coli GlxI. In contrast, the addition of NiCl2 to the growth medium or to purified E. coli apo-GlxI greatly enhanced the enzymatic activity. Inductively coupled plasma and atomic absorption analyses indicated binding of only one nickel ion per dimeric enzyme, suggesting only one functional active site in this homodimeric enzyme. In addition, the apoprotein regained maximal activity with one molar equivalence of nickel chloride, indicative of tight metal binding. The effects of pH on the kinetics of the nickel-activated enzyme were also studied. This is the first example of a non-zinc activated GlxI whose maximal activation is seen with Ni2+.

Amino Acid Sequence↗
PubMed1998

Identification of glyoxalase I sequences in Brassica oleracea and Sporobolus stapfianus: evidence for gene duplication events.

Glyoxalase I (GlxI) is the first of two enzymes involved in the cellular detoxification of methylglyoxal. A recent search of the National Center for Biotechnology Information (NCBI) databases with the protein sequence of Salmonella typhimurium GlxI identified two new hypothetical proteins with unassigned function. These two sequences, from Brassica oleracea and Sporobolus stapfianus, have significant sequence similarity to known GlxI sequences, suggesting that these two open reading frames encode for GlxI in these plants. Interestingly, analysis of these two new sequences indicates that they code for a protein composed of two fused monomers, a situation previously found solely in the yeast GlxI enzymes.

Amino Acid Sequence↗
PubMed1997

Incorporation of trifluoromethionine into a phage lysozyme: implications and a new marker for use in protein 19F NMR.

Much interest is currently focused on understanding the detailed contribution that particular amino acid residues make in protein structure and function. Although the use of site-directed mutagenesis has greatly contributed to this goal, the approach is limited to the standard repertoire of twenty amino acids. Fluorinated amino acids have been utilized successfully to probe protein structure and dynamics as well as point to the importance of specific residues to biological function. In our continuing investigations on the importance of the amino acid methionine in biological systems, the successful incorporation of L-S-(trifluoromethyl)homocysteine (L-trifluoromethionine; L-TFM) into bacteriophage lambda lysozyme (LaL), an enzyme containing three methionine residues, is reported. The L isomer of TFM was synthesized in an overall yield of 33% from N-acetyl-D,L-homocysteine thiolactone and trifluoromethyl iodide. An expression plasmid giving strong overproduction of LaL was prepared and transformed into an Escherichia coli strain auxotrophic for methionine permitting the expression of LaL in the presence of L-TFM. The analogue would not support growth of the auxotroph and was found to be inhibitory to cell growth. However, cells that were initially grown in a Met-rich media followed by protein induction under careful control of the respective concentrations of L-Met and L-TFM in the media, were able to overexpress TFM-labeled LaL (TFM-LaL) at both high (70%) and low (31%) levels of TFM incorporation. TFM-LaL at both levels of incorporation exhibited analogous activity to the wild type enzyme and were inhibited by chitooligosaccharides indicating that incorporation of the analogue did not hinder enzyme function. Interestingly, the 19F solution NMR spectra of the TFM-labeled enzymes consisted of four sharp resonances spanning a chemical shift range of 0.9 ppm, with three of the resonances showing very modest shielding changes on binding of chitopentaose. The 19F NMR analysis of TFM-LaL at both high and low levels of incorporation suggested that one of the methionine positions gives rise to two separate resonances. The intensities of these two resonances were influenced by the extent of incorporation which was interpreted as an indication that subtle conformational changes in protein structure are induced by incorporated TFM. The similarities and differences between Met and TFM were analyzed using ab initio molecular orbital calculations. The methodology presented offers promise as a new approach to the study of protein-ligand interactions as well as for future investigations into the functional importance of methionine in proteins.

Bacteriophage lambda↗
PubMed1997

Isolation and sequencing of a gene coding for glyoxalase I activity from Salmonella typhimurium and comparison with other glyoxalase I sequences.

The glyoxalase I gene (gloA) from Salmonella typhimurium has been isolated in Escherichia coli on a multi-copy pBR322-derived plasmid, selecting for resistance to 3 mM methylglyoxal on Luria-Bertani agar. The region of the plasmid which confers the methylglyoxal resistance in E. coli was sequenced. The deduced protein sequence was compared to the known sequences of the Homo sapiens and Pseudomonas putida glyoxalase I (GlxI) enzymes, and regions of strong homology were used to probe the National Center for Biotechnology Information protein database. This search identified several previously known glyoxalase I sequences and other open reading frames with unassigned function. The clustal alignments of the sequences are presented, indicating possible Zn2+ ligands and active site regions. In addition, the S. typhimurium sequence aligns with both the N-terminal half and the C-terminal half of the proposed GlxI sequences from Saccharomyces cerevisiae and Schizosaccharomyces pombe, suggesting that the structures of the yeast enzymes are those of fused dimers.

Amino Acid Sequence↗
PubMed1995

Investigations of the interactions of saccharides with the lysozyme from bacteriophage lambda.

The bacteriophage lambda R gene has been isolated into an Escherichia coli expression system and the R gene product, a lysozyme, has been overexpressed and purified to homogeneity using an efficient purification procedure. A turbidimetric assay utilizing chloroform-treated E. coli cells has been optimized to assess the bacteriolytic activity of the purified enzyme. Using this assay, oligomers of beta (1 --> 4) N-acetyl-D-glucosamine at high concentrations were shown to inhibit lysozyme but were not cleaved by the enzyme. Differential scanning calorimetry revealed that the thermal denaturation of lysozyme was found to increase in the presence of (GlcNAc)3 and (GlcNAc)5. The lysozyme was also expressed in an E. coli strain auxotrophic for methionine, allowing for the incorporation of [methyl-13C]methionine into the enzyme. An alteration of the [1H-13C]HMQC NMR spectra of the labelled enzyme was observed in the presence of (GlcNAc)5. Commercially available nitrophenyl glycosides did not act as substrates for lambda lysozyme. The results indicate that lambda lysozyme has specific interactions with oligosaccharides of N-acetylglucosamine, but is incapable of hydrolyzing these sugars. The relevance of the structure of peptidoglycan to the activity of lambda lysozyme is discussed.

Bacteriophage lambda↗
PubMed1990

Causes of pediatric eye injuries. A population-based study.

Eye injuries are an important cause of ocular morbidity in children. We conducted a population-based study of eye injuries requiring hospital admission for children younger than 16 years in the state of Maryland during the 1982 calendar year. The population-based estimate of the incidence of ocular trauma in Maryland children was 15.2 per 100,000 per year (95% confidence interval, 12.8 to 17.7). Male patients outnumbered female patients as victims of eye injuries by a ratio of approximately 4:1; eye injuries in 11- to 15-year-old children occurred at more than twice the rate than for younger children. The most common cause of pediatric ocular trauma was accidental blows and falls (37%). Sports and recreational activities accounted for 27% of all eye injuries, 39% of all nonpenetrating injuries, and 40% of all injuries in 11- to 15-year-old children. A comparison of the causes of eye injuries at statewide specialty trauma centers and community-based hospitals indicated that sports-related injuries were treated at general hospitals more than 10 times more frequently than at the trauma centers. Other important causes of eye injuries were burns (9%), car crashes (11%), and nonpowder firearm accidents (4%). We conclude that the majority of pediatric eye injuries are preventable, and that the implementation of well-established safety precautions would greatly reduce this source of visual disability in our nation's children.

Accidental Falls↗
PubMed1989

The interaction of E. coli integration host factor and lambda cos DNA: multiple complex formation and protein-induced bending.

The interaction of E. coli's integration Host Factor (IHF) with fragments of lambda DNA containing the cos site has been studied by gel-mobility retardation and electron microscopy. The cos fragment used in the mobility assays is 398 bp and spans a region from 48,298 to 194 on the lambda chromosome. Several different complexes of IHF with this fragment can be distinguished by their differential mobility on polyacrylamide gels. Relative band intensities indicate that the formation of a complex between IHF and this DNA fragment has an equilibrium binding constant of the same magnitude as DNA fragments containing lambda's attP site. Gel-mobility retardation and electron microscopy have been employed to show that IHF sharply bends DNA near cos and to map the bending site. The protein-induced bend is near an intrinsic bend due to DNA sequence. The position of the bend suggests that IHF's role in lambda DNA packaging may be the enhancement of terminase binding/cos cutting by manipulating DNA structure.

Bacterial Proteins↗
PubMed1988

Isolation, cloning, and sequencing of the Salmonella typhimurium ddlA gene with purification and characterization of its product, D-alanine:D-alanine ligase (ADP forming).

A gene coding for D-alanine:D-alanine (D-Ala-D-Ala) ligase (ADP forming) (EC 6.3.2.4) activity has been isolated from a lambda library of Salmonella typhimurium DNA. Insertion mutations in the gene indicate that the gene is not essential for growth of the bacterium. The encoded enzyme was purified from an overproducing strain of S. typhimurium. D-Ala-D-Ala ligase is a protein of 39,271 molecular weight and has a kcat of 644 min-1 at pH 7.2. A 2.4-kilobase SalI-SphI fragment containing the gene was sequenced, and the ddlA gene consists of 1092 nucleotides. The gene sequence was compared to the sequence of the ddl gene of Escherichia coli [Robinson, A. C., Kenan, D. J., Sweeney, J., & Donachie, W. D. (1986) J. Bacteriol. 167, 809-817]. Because of differences between the S. typhimurium gene and the E. coli ddl gene, the S. typhimurium gene has been named ddlA.

Amino Acid Sequence↗
PubMed1987

The overproduction of DNA terminase of coliphage lambda.

An artificial operon containing the genes coding for the two subunits of lambda DNA terminase, Nul and A, has been constructed. Derivatives of plasmid pBR322 served as the cloning vehicles. The transcription is driven by the pL promoter of phage lambda, and translation of the terminase genes was made efficient by the replacement of the wild-type ribosome-binding sites for those of lambda genes cII and/or D. The operon also carries the oL operator, and this enables regulation of its expression by a thermosensitive repressor. The synthesis of genes Nul and A products is extremely efficient upon derepression. Within 40 min after induction of the operon, the two subunits comprise about 20% of the total cellular protein mass. Crude extracts prepared from these overproducing strains are at least 100 times more active than extracts prepared from induced lambda lysogens in both promotion of lambda DNA packaging and cosmid cleaving. The ability to produce highly concentrated terminase would enormously facilitate the study of its structure and mechanism of action. These extracts are also extremely useful in techniques such as lambda DNA packaging, cosmid mapping and cosmid linearization to improve efficiency of integration into mouse eggs.

Bacteriophage lambda↗
PubMed1986

Biosynthetic alr alanine racemase from Salmonella typhimurium: DNA and protein sequence determination.

The nucleotide sequence of the alr gene encoding the biosynthetic alanine racemase in Salmonella typhimurium is reported. The sequence was determined by the dideoxy chain termination method of Sanger mostly from recombinants derived from shotgun and specific subcloning of a 2.6-kilobase region containing the alr gene. The final bridging of nonoverlapping contiguous sequences was accomplished with the use of synthetic site-specific primers. The alr gene was found to be 1077 base pairs in length encoding a protein of 359 amino acid residues. Comparison of alr with the dadB gene encoding the catabolic alanine racemase in S. typhimurium revealed almost identical size (1077 vs. 1068 base pairs) and 52% sequence identity. The respective gene products displayed 43% homology, which includes a decapeptide bearing the pyridoxal 5'-phosphate binding site.

Alanine Racemase↗
PubMed1984

Catabolic alanine racemase from Salmonella typhimurium: DNA sequence, enzyme purification, and characterization.

The alanine racemase encoded by the Salmonella typhimurium dadB gene was purified to 90% homogeneity from an overproducing strain. At 37 degrees C the enzyme has a specific activity of 1400 units/mg (V max, L- to D-alanine). Active enzyme molecules are monomers of Mr 39 000 with one molecule of pyridoxal 5'-phosphate bound per subunit. The Km's for L- and D-alanine are 8.2 and 2.1 mM, respectively. Measurement of turnover numbers yielded the expected Keq value of 1.0. Determination of 22 of the 25 N-terminal amino acid residues of the purified polypeptide allowed localization of cloned DNA encoding the structural gene. Sequencing of subcloned DNA revealed that the dadB gene encodes a polypeptide of 356 amino acids whose calculated molecular weight (apoenzyme) was 39 044.

Alanine Racemase↗
PubMed1983

Alterations in MAST suit pressure with changes in ambient temperature.

A study was undertaken to test the hypothesis that change in ambient air temperature has an effect on MAST suit pressure according to the ideal gas law. Two different MAST suits were tested on Resusci-Annie dummies. The MAST suits were applied in a cold room at 4.4 degrees C and warmed to 44 degrees C. Positive linear correlations were found in nine trials, but the two suits differed in their rate of increase in pressure. Three trials using humans were conducted showing increased pressure with temperature but at a lesser rate than with dummies. A correlation of 0.5 to 1.0 mm Hg increase in MAST suit pressure for each 1.0 degrees C increase in ambient temperature was found. Implications are discussed for the use of the MAST suit in environmental conditions where the temperature changes.

Gravity Suits↗
PubMed1975

Effects of pH and thiols on the kinetics of yeast glyoxalase I. An evaluation of the random pathway mechanism.

The disproportionation of alpha-ketoaldehydes, catalyzed by yeast glyoxalase I, has been reported to involve a random pathway mechanism where one branch utilizes the hemimercaptal of glutathione and the alpha-ketoaldehyde in a one-substrate pathway, and the other branch utilizes first glutathione and then the alpha-ketoaldehyde in an ordered two-substrate pathway. The relative importance of the two pathways has been evaluated at 5 degrees in the pH range 3-7, using methylglyoxal and phenylglyoxal as representative aliphatic and aromatic alpha-ketoaldehydes, by comparing initial rates of hemimercaptal formation in the absence of enzyme with initial rates of product formation in the presence of high enzyme concentrations. If the enzyme is not added last, the initial rates of product formation are the same as the initial rates of adduct formation even under conditions where it could be shown that dehydration of the hydrated alpha-ketoaldehyde is not entirely rate determining. If the enzyme is added after hemimercaptal formation, there is a "burst" of product formation equivalent to the amount of hemimercaptal, followed by a slower reaction, consistent with the one-substrate pathway. Additional support for this pathway was obtained from a study of the effects of added thiol reagents on the "burst" kinetics. The broad specificity of yeast glyoxalase I for both aliphatic and aromatic alpha-ketoaldehydes, reflected in Vmax values which are insensitive to the nature of the alpha-ketoaldehyde drops abruptly if the side chain of the alpha-ketoaldehyde is sterically crowded. The hemimercaptal of tert-butylglyoxal has a Vmax 300-fold smaller than Vmax for methylglyoxal; 2,4,6-trimethylphenylglyoxal is essentially inactive as a substrate even though the closely related compound 2,4-dimethylphenylglyoxal is a normal substrate. Analysis of the Vmax and Km (or Ki) values of these alpha-ketoaldehydes suggests that sterically crowded side chains affect both enzyme-substrate formation and the catalytic reaction.

Binding Sites↗