Search PubMed⌕ Search

Biomedical subjects

A B Oppenheim

Publications and source records attributed to A B Oppenheim.

At least 19 recordsLinked to original sources
PubMed2001

High resolution structural analyses of mutant chitinase A complexes with substrates provide new insight into the mechanism of catalysis.

Chitinase A (ChiA) from the bacterium Serratia marcescens is a hydrolytic enzyme, which cleaves beta-1,4-glycosidic bonds of the natural biopolymer chitin to generate di-N-acetyl-chitobiose. The refined structure of ChiA at 1.55 A shows that residue Asp313, which is located near the catalytic proton donor residue Glu315, is found in two alternative conformations of equal occupancy. In addition, the structures of the cocrystallized mutant proteins D313A, E315Q, Y390F, and D391A with octa- or hexa-N-acetyl-glucosamine have been refined at high resolution and the interactions with the substrate have been characterized. The obtained results clearly show that the active site is a semiclosed tunnel. Upon binding, the enzyme bends and rotates the substrate in the vicinity of the scissile bond. Furthermore, the enzyme imposes a critical "chair" to "boat" conformational change on the sugar residue bound to the -1 subsite. According to our results, we suggest that residues Asp313 and Tyr390 along with Glu315 play a central role in the catalysis. We propose that after the protonation of the substrate glycosidic bond, Asp313 that interacts with Asp311 flips to its alternative position where it interacts with Glu315 thus forcing the substrate acetamido group of -1 sugar to rotate around the C2-N2 bond. As a result of these structural changes, the water molecule that is hydrogen-bonded to Tyr390 and the NH of the acetamido group is displaced to a position that allows the completion of hydrolysis. The presented results suggest a mechanism for ChiA that modifies the earlier proposed "substrate assisted" catalysis.

Amino Acid Substitution↗
PubMed2001

Compaction of single DNA molecules induced by binding of integration host factor (IHF).

We studied the interaction between the integration host factor (IHF), a major nucleoid-associated protein in bacteria, and single DNA molecules. Force-extension measurements of lambda DNA and an analysis of the Brownian motion of small beads tethered to a surface by single short DNA molecules, in equilibrium with an IHF solution, indicate that: (i) the DNA-IHF complex retains a random, although more compact, coiled configuration for zero or small values of the tension, (ii) IHF induces DNA compaction by binding to multiple DNA sites with low specificity, and (iii) with increasing tension on the DNA, the elastic properties of bare DNA are recovered. This behavior is consistent with the predictions of a statistical mechanical model describing how proteins bending DNA are driven off by an applied tension on the DNA molecule. Estimates of the amount of bound IHF in DNA-IHF complexes obtained from the model agree very well with independent measurements of this quantity obtained from the analysis of DNA-IHF crosslinking. Our findings support the long-held view that IHF and other histone-like proteins play an important role in shaping the long-scale structure of the bacterial nucleoid.

Bacterial Proteins↗
PubMed2001

An internal region of the RpoH heat shock transcription factor is critical for rapid degradation by the FtsH protease.

The proteolysis of regulatory proteins plays an important role in the control of gene expression. The Escherichia coli heat shock sigma factor RpoH (sigma(32)) is highly unstable. Its instability is determined by interactions with the DnaK chaperone machine, RNA polymerase and the ATP-dependent protease FtsH. Bradyrhizobium japonicum expresses three RpoH proteins of which RpoH(1) is highly stable. To determine which regions of E. coli RpoH determine protein lability, we generated a number of truncated versions and hybrid proteins. Truncation of N-terminal amino acids had no, and deletion of C-terminal amino acids only a minor effect on stability of RpoH. A major determinant of RpoH lability was mapped to a region of about 85 amino acids (residues 36-122) roughly comprising the sigma factor region 2. This is the first demonstration of an internal RpoH region being responsible for FtsH-mediated degradation.

ATP-Dependent Proteases↗
PubMed2000

Production and purification of SV40 major capsid protein (VP1) in Escherichia coli strains deficient for the GroELS chaperone machine.

Production of the major capsid protein of SV40, VP1, is of great interest for the study on capsid assembly in vitro. Production of soluble His6-VP1 in Escherichia coli strains deficient in the GroELS chaperone machine was substantially higher than in the wild-type strain. The His6-VP1 produced in a groEL mutant strain was readily purified. The protein was able to form higher-order structures as evidenced by analysis of the soluble fraction by gel filtration, by sedimentation in sucrose gradient, and by electron microscopy. We propose the use of groE mutants for the production of the major capsid protein of SV40 and perhaps also other papovaviruses.

Capsid↗
PubMed2000

Structures of chitobiase mutants complexed with the substrate Di-N-acetyl-d-glucosamine: the catalytic role of the conserved acidic pair, aspartate 539 and glutamate 540.

The catalytic domain of chitobiase (beta-N-1-4 acetylhexosaminidase) from Serratia marcescens, is an alpha/beta TIM-barrel. This enzyme belongs to family 20 of glycosyl hydrolases in which a conserved amino acid pair, aspartate-glutamate, is present (Asp539-Glu540). It was proposed that catalysis by this enzyme family is carried out by glutamate 540 acting as a proton donor and by the acetamido group of the substrate as a nucleophile. We investigated the role of Asp539 and Glu540 by site-directed mutagenesis, biochemical characterization and by structural analyses of chitobiase -substrate co-crystals. We found that both residues are essential for chitobiase activity. The mutations, however, led to subtle changes in the catalytic site. Our results support the model that Glu540 acts as the proton donor and that Asp539 acts in several different ways. Asp539 restrains the acetamido group of the substrate in a specific orientation by forming a hydrogen bond with N2 of the non-reduced (-1) sugar. In addition, this residue participates in substrate binding. It is also required for the correct positioning of Glu540 and may provide additional negative charge at the active site. Thus, these biochemical and structural studies provide a molecular explanation for the functional importance and conservation of these residues.

Acetylglucosamine↗
PubMed2000

Characterization of a conserved alpha-helical, coiled-coil motif at the C-terminal domain of the ATP-dependent FtsH (HflB) protease of Escherichia coli.

FtsH (HflB) is an ATP-dependent protease found in prokaryotic cells, mitochondria and chloroplasts. Here, we have identified, in the carboxy-terminal region of FtsH (HfIB), a short alpha helix predicted of forming a coiled-coil, leucine zipper, structure. This region appears to be structurally conserved. The presence of the coiled-coil motif in the Escherichia coli FtsH (HflB) was demonstrated by circular dichroism and cross-linking experiments. Mutational analysis showed that three highly conserved leucine residues are essential for FtsH (HfIB) activity in vivo and in vitro. Purified proteins mutated in the conserved leucine residues, were found to be defective in the degradation of E. coli sigma(32) and the bacteriophage lambda CII proteins. In addition, the mutant proteins were defective in the binding of CII The mutations did not interfere with the ATPase activity of FtsH (HflB). Finally, the mutant proteins were found to be more sensitive to trypsin degradation than the wild-type enzyme suggesting that the alpha helical region is an important structural element of FtsH (HflB).

ATP-Dependent Proteases↗
PubMed2000

A colicin-tolerant Escherichia coli mutant that confers hfl phenotype carries two mutations in the region coding for the C-terminal domain of FtsH (HflB).

An Escherichia coli mutant, ER437, which was originally isolated for colicin tolerance, was found to carry two amino acid changes in the C-terminal portion of FtsH (HflB). These mutations were demonstrated to reduce the ability of FtsH to degrade the phage lambda CII protein in vivo and in vitro, providing a rationalization for the mutant Hfl phenotype.

ATP-Dependent Proteases↗
PubMed2000

Differential degradation of Escherichia coli sigma32 and Bradyrhizobium japonicum RpoH factors by the FtsH protease.

The Escherichia coli heat shock sigma factor sigma32 (RpoH) is rapidly degraded under non-stress conditions. The integrity of the DnaK chaperone machinery and the ATP-dependent FtsH protease are required for sigma32 proteolysis in vivo. Bradyrhizobium japonicum expresses three sigma32-type transcription factors, RpoH1, RpoH2, and RpoH3, which are functional in E. coli. We compared the stability of these sigma factors with E. coli sigma32 stability. In E. coli C600 (wild-type), the half-lives of sigma32, RpoH1, RpoH2 and RpoH3 were 30 s, 7 min, 4 min and 4 min, respectively. The first three proteins were stabilized in ftsH mutant backgrounds, indicating that they are degraded by FtsH in the wild-type. Proteolysis of RpoH3 was FtsH-independent because this sigma factor was not stabilized in ftsH mutants. Interestingly, in a purified in vitro system, all four RpoH proteins were degraded by FtsH, indicating that in vivo protein degradation depends on additional cellular factors. Rationally designed point mutations of sigma32 and RpoH1 suggested that the highly conserved RpoH box does not play a major role in conferring stability to RpoH factors. Presumably, several regions distributed along the primary sequence of the sigma factor are important for FtsH-mediated proteolysis. Finally, we provide evidence that proteolysis of RpoH factors in vivo depends on the DnaK machinery, irrespective of the protease involved.

ATP-Dependent Proteases↗
PubMed2000

The thylakoid FtsH protease plays a role in the light-induced turnover of the photosystem II D1 protein.

The photosystem II reaction center D1 protein is known to turn over frequently. This protein is prone to irreversible damage caused by reactive oxygen species that are formed in the light; the damaged, nonfunctional D1 protein is degraded and replaced by a new copy. However, the proteases responsible for D1 protein degradation remain unknown. In this study, we investigate the possible role of the FtsH protease, an ATP-dependent zinc metalloprotease, during this process. The primary light-induced cleavage product of the D1 protein, a 23-kD fragment, was found to be degraded in isolated thylakoids in the dark during a process dependent on ATP hydrolysis and divalent metal ions, suggesting the involvement of FtsH. Purified FtsH degraded the 23-kD D1 fragment present in isolated photosystem II core complexes, as well as that in thylakoid membranes depleted of endogenous FtsH. In this study, we definitively identify the chloroplast protease acting on the D1 protein during its light-induced turnover. Unlike previously identified membrane-bound substrates for FtsH in bacteria and mitochondria, the 23-kD D1 fragment represents a novel class of FtsH substrate-functionally assembled proteins that have undergone irreversible photooxidative damage and cleavage.

ATP-Dependent Proteases↗
PubMed2000

Proteolysis of bacteriophage lambda CII by Escherichia coli FtsH (HflB).

FtsH (HflB) is a conserved, highly specific, ATP-dependent protease for which a number of substrates are known. The enzyme participates in the phage lambda lysis-lysogeny decision by degrading the lambda CII transcriptional activator and by its response to inhibition by the lambda CIII gene product. In order to gain further insight into the mechanism of the enzymatic activity of FtsH (HflB), we identified the peptides generated following proteolysis of the phage lambda CII protein. It was found that FtsH (HflB) acts as an endopeptidase degrading CII into small peptides with limited amino acid specificity at the cleavage site. beta-Casein, an unstructured substrate, is also degraded by FtsH (HflB), suggesting that protein structure may play a minor role in determining the products of proteolysis. The majority of the peptides produced were 13 to 20 residues long.

ATP-Dependent Proteases↗
PubMed1998

The SV40 capsid protein VP3 cooperates with the cellular transcription factor Sp1 in DNA-binding and in regulating viral promoter activity.

Chromatin structure and protein-protein interactions play an important role in eukaryotic gene function. Nucleosomal rearrangement at the simian virus 40 (SV40) regulatory region occurs at the late stages of the viral life cycle preceding viral assembly. The SV40 capsid proteins are required for this nucleosomal rearrangement suggesting that they participate in turning-off the viral promoters. In aiming to elucidate the role of the capsid proteins in gene regulation, we studied the interaction between VP3, an internal capsid protein, and the cellular transcription factor Sp1, a major regulator of both the early and late viral promoters. Our results showed that VP3 repressed transcription from the viral early promoter in vitro. We found significant cooperativity between Sp1 and VP3 in specific DNA-binding to the Sp1 binding site. In addition, protein-protein interactions between VP3 and Sp1 in the absence of DNA were observed. These findings have led us to conclude that the novel host-viral Sp1-VP3 complex down regulates viral transcription and further suggest that Sp1 participates in recruiting VP3 to the SV40 minichromosome in SV40 assembly.

Base Sequence↗
PubMed1998

Participation of IHF and a distant UP element in the stimulation of the phage lambda PL promoter.

We have previously identified a UP element in the phage lambda PL promoter, centred at position -90 from the transcription start site. Integration host factor (IHF), a heterodimeric DNA-binding and -bending protein, binds upstream of the lambda PL promoter in a region overlapping the UP element. Stimulation of transcription by IHF requires an intact alphaCTD and affects the initial binding of RNA polymerase to the promoter. We propose a model for the stimulation of PL by IHF in which IHF bends the DNA to bring the distal UP sequence in closer proximity to the promoter core sequences to allow the docking of the alphaCTD of RNA polymerase. Furthermore, IHF may also participate in protein-protein interactions with the alphaCTD. In support of this model, we found that alanine substitutions in alphaCTD at positions 265, 268, 270 and 275 reduced PL promoter activity. Mutations in the IHF DNA binding site, as well as IHF mutant proteins exhibiting a decreased ability to bend the DNA, were both defective in stimulating the PL promoter. In addition, some of the mutated IHF residues are clustered at a protein surface that interacts with the UP DNA sequence. These residues may also participate in protein-protein interactions with the alphaCTD.

Bacterial Proteins↗
PubMed1997

In vitro assembly of SV40 virions and pseudovirions: vector development for gene therapy.

SV40 is an attractive potential vector with high-efficiency gene transfer into a wide variety of human tissues, including the bone marrow, a critical target organ for the cure of many diseases. In the present study, the three SV40 capsid proteins, VP1, VP2, and VP3, were produced in Spodoptera frugiperda (Sf9) insect cells. Their co-production led to spontaneous assembly of SV40-like particles. Nuclear extracts containing the three proteins were allowed to interact with purified SV40 DNA, or with plasmid DNA produced and purified from Escherichia coli. The experiments demonstrated a physical association between the DNA and capsid proteins, protection from DNase I digestion, and the formation of infectious particles. The results indicate that intact, supercoiled DNA is being packaged and transmitted into the target cells. The transmitted DNA is biologically functional in gene expression and replication. The process, which utilizes naked DNA, is not dependent on the SV40 packaging signal ses. The procedure allows packaging of plasmids significantly larger than SV40 and permits the inclusion of potent regulatory signals, such as beta-globin locus control region (LCR) elements. These studies are the first step in the development of purified, in vitro-constructed pseudovirions for experimental and medical use.

Animals↗
PubMed1997

Impaired lysogenisation of the Escherichia coli rpoA341 mutant by bacteriophage lambda is due to the inability of CII to act as a transcriptional activator.

The C-terminus of the alpha subunit of Escherichia coli RNA polymerase is known to function in transcriptional activation at certain promoters. This region was previously shown to be necessary for full activation of the pE promoter by the phage lambda CII protein in vitro. In this work we investigated the inability of phage lambda to follow the lysogenic pathway in cells carrying the point mutation rpoA341 (a change of lysine 271 to glutamic acid). We found that neither overexpression of the cII gene nor stabilisation of the CII protein by the can1 mutation or by cIII gene overexpression was able to suppress the block in lysogenisation. In contrast, the lambda cin1 phage, which carries a CII-independent promoter for the expression of the cI gene, was able to efficiently lysogenise the rpoA341 mutant strain. Furthermore, the rpoA341 mutation prevented the activation of pE-lacZ and pI-lacZ transcriptional fusions by CII. Therefore we conclude that transcriptional activation by the cII gene product is abolished by the rpoA341 mutation, most probably due to impaired interaction between the CII activator and mutant RNA polymerase. The inability of RNA polymerase to respond to CII results in the impairment of lysogenisation of the rpoA341 mutant by phage lambda.

Bacteriophage lambda↗
PubMed1997

Isolation and characterization of the integration host factor genes of Pasteurella haemolytica.

Using a bacteriophage lambda complementation system in Escherichia coli, we cloned genes encoding subunits of the heterodimeric DNA binding/bending protein, integration host factor, from the bovine pathogen, Pasteurella haemolytica. Complementation of ihfA and ihfB mutations in E. coli demonstrated that the P. haemolytica gene products form functional heterologous heterodimers. The ihfA and ihfB genes encode polypeptides predicted to be 99 and 93 amino acids long, respectively, and are very similar to integration host factor subunits from other Gram-negative bacteria, although phylogenetic analysis indicated that the P. haemolytica sequences are distantly related to those from other bacteria. Most significant amino acid differences were restricted to the amino-terminal domains of the predicted peptides.

Bacterial Proteins↗
PubMed1997

Role of Escherichia coli cspA promoter sequences and adaptation of translational apparatus in the cold shock response.

A shift in growth temperature from 37 degrees C to 15 degrees C leads to a dramatic increase in the level of CspA, the major cold shock protein of Escherichia coli. To investigate the molecular basis of this induction, we considered the relevance of transcriptional and posttranscriptional controls by analyzing the steady-state levels of transcripts and the expression of reporter genes in cells carrying a set of cspA promoter fragments of variable length fused to lacZ or cat genes. We demonstrate that: (i) the core cspA promoter (from -40 to +16) responds to cold shock and a mutation at -36 increases the relative activity of the promoter at low temperature by threefold; (ii) the sequences upstream of -40 have a positive effect on expression at 37 degrees C, but no effect on the cold shock response; (iii) by virtue of their influence on mRNA stability, the downstream sequences (from +81 to +165) reduce expression at 37 degrees C and increase the intensity of the cold shock response; (iv) mutations in the GCACATCA and CCAAT motifs, present at +1/-4 and between the -10 and -35 elements, respectively, do not affect the cold shock response of the cspA promoter; (v) following cold shock, a modification of the protein synthetic machinery takes place that allows preferential translation of cspA mRNA relative to the non-cold shock cat and lacZ mRNAs. The quantitatively modest transcriptional activation shown by the core promoter of cspA following cold shock suggests that transcriptional activation can significantly contribute to cold shock induction only when coupled to posttranscriptional controls, such as alterations in mRNA stability and the translational apparatus.

Adaptation, Physiological↗
PubMed1997

Proteolysis of the phage lambda CII regulatory protein by FtsH (HflB) of Escherichia coli.

Rapid proteolysis plays an important role in regulation of gene expression. Proteolysis of the phage lambda CII transcriptional activator plays a key role in the lysis-lysogeny decision by phage lambda. Here we demonstrate that the E. coli ATP-dependent protease FtsH, the product of the host ftsH/hflB gene, is responsible for the rapid proteolysis of the CII protein. FtsH was found previously to degrade the heat-shock transcription factor sigma32. Proteolysis of sigma32 requires, in vivo, the presence of the DnaK-DnaJ-GrpE chaperone machine. Neither DnaK-DnaJ-GrpE nor GroEL-GroES chaperone machines are required for proteolysis of CII in vivo. Purified FtsH carries out specific ATP-dependent proteolysis of CII in vitro. The degradation of CII is at least 10-fold faster than that of sigma32. Electron microscopy revealed that purified FtsH forms ring-shaped structures with a diameter of 6-7 nm.

ATP-Dependent Proteases↗