Regulation of expression of the Lactococcus lactis histidine operon.
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In Lactococcus lactis, the his operon contains all the genes necessary for histidine biosynthesis. It is transcribed from a unique promoter, localized 300 bp upstream of the first gene. The region corresponding to the untranslated 5' end of the transcript, named the his leader region, displays the typical features of the T box transcriptional attenuation mechanism which is involved in the regulation of many amino acid biosynthetic operons and tRNA synthetase genes in gram-positive bacteria. Here we describe the regulation of transcription of the his operon by the level of histidine in the growth medium. In the absence of histidine, two transcripts are present. One covers the entire operon, while the other stops at a terminator situated about 250 bp downstream of the transcription start point. DNA sequences implicated in regulation of the his operon were identified by transcriptional fusion with luciferase genes and site-directed mutagenesis. In addition to the previously defined sequences necessary for effective T-box-mediated regulation, new essential regions were identified. Eighteen percent of the positions of the his leader region were found to differ in seven distantly related strains of L. lactis. Analysis of the variable positions supports the folding model of the central part of the his leader region. Lastly, in addition to the T-box-mediated regulation, the operon is regulated at the level of initiation of transcription, which is repressed in the presence of histidine. An operator site, necessary for full repression, overlaps the terminator involved in the T box attenuation mechanism. The functionality of the operator is altered on plasmids with low and high copy numbers, suggesting that supercoiling may play a role in the expression of the his operon. The extents of regulation at the levels of initiation and attenuation of transcription are 6- to 8-fold and 14-fold, respectively. Together, the two levels of control allow a 120-fold range of regulation of the L. lactis operon by histidine. (+info)
Molecular dynamics of human methemoglobin: the transmission of conformational information between subunits in an alpha beta dimer.
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Spectroscopic studies indicate an interaction of the distal histidine with the heme iron as well as the transmission of distal heme perturbations across the alpha1beta1 interface. Molecular dynamics simulations have been used to explain the molecular basis for these processes. Using a human methemoglobin alpha beta dimer, it has been shown that at 235 K after 61 ps, a rearrangement occurs in the alpha-chain corresponding to the formation of a bond with the distal histidine. This transition does not take place in the beta-chain during a 100-ps simulation and is reversed at 300 K. The absence of the distal histidine transition in the isolated chains and with the interface frozen indicate the involvement of the alphabeta interface. A detailed analysis of the simulation has been performed in terms of RMS fluctuations, domain cross-correlation maps, the disruption of helix hydrogen bonds, as well changes in electrostatic interactions and dihedral angles. This analysis shows that the rearrangements in the alpha-chain necessary to bring the histidine closer to the iron involve alterations primarily in the CD loop and at the interface. Communication to the beta-chain distal pocket is propagated by increased interactions of the alpha-chain B helix with the beta-chain G-GH-H segment and the flexibility in the EF loop. The G helices shown to be involved in propagation of perturbation across the alpha1beta1 interface extend into the alpha1beta2 interfaces, providing a mechansim whereby distal interactions can modulate the T<==>R transition in hemoglobin. (+info)
Conversion of cucumber linoleate 13-lipoxygenase to a 9-lipoxygenating species by site-directed mutagenesis.
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Multiple lipoxygenase sequence alignments and structural modeling of the enzyme/substrate interaction of the cucumber lipid body lipoxygenase suggested histidine 608 as the primary determinant of positional specificity. Replacement of this amino acid by a less-space-filling valine altered the positional specificity of this linoleate 13-lipoxygenase in favor of 9-lipoxygenation. These alterations may be explained by the fact that H608V mutation may demask the positively charged guanidino group of R758, which, in turn, may force an inverse head-to-tail orientation of the fatty acid substrate. The R758L+H608V double mutant exhibited a strongly reduced reaction rate and a random positional specificity. Trilinolein, which lacks free carboxylic groups, was oxygenated to the corresponding (13S)-hydro(pero)xy derivatives by both the wild-type enzyme and the linoleate 9-lipoxygenating H608V mutant. These data indicate the complete conversion of a linoleate 13-lipoxygenase to a 9-lipoxygenating species by a single point mutation. It is hypothesized that H608V exchange may alter the orientation of the substrate at the active site and/or its steric configuration in such a way that a stereospecific dioxygen insertion at C-9 may exclusively take place. (+info)
DNA binding protein dbpA binds Cdk5 and inhibits its activity.
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Progress in the cell cycle is governed by the activity of cyclin dependent kinases (Cdks). Unlike other Cdks, the Cdk5 catalytic subunit is found mostly in differentiated neurons. Interestingly, the only known protein that activates Cdk5 (i.e. p35) is expressed solely in the brain. It has been suggested that, besides its requirement in neuronal differentiation, Cdk5 activity is induced during myogenesis. However, it is not clear how this activity is regulated in the pathway that leads proliferative cells to differentiation. In order to find if there exists any Cdk5-interacting protein, the yeast two-hybrid system was used to screen a HeLa cDNA library. We have determined that a C-terminal 172 amino acid domain of the DNA binding protein, dbpA, binds to Cdk5. Biochemical analyses reveal that this fragment (dbpA(Cdelta)) strongly inhibits p35-activated Cdk5 kinase. The protein also interacts with Cdk4 and inhibits the Cdk4/cyclin D1 enzyme. Surprisingly, dbpA(Cdelta) does not bind Cdk2 in the two-hybrid assay nor does it inhibit Cdk2 activated by cyclin A. It could be that dbpA's ability to inhibit Cdk5 and Cdk4 reflects an apparent cross-talk between distinct signal transduction pathways controlled by dbpA on the one hand and Cdk5 or Cdk4 on the other. (+info)
Enzymatical properties of psychrophilic phosphatase I.
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Phosphatase I purified from a psychrophile (Shewanella sp.) [Tsuruta et al. (1998) J. Biochem. 123, 219-225] dephosphorylated O-phospho-L-tyrosine and phospho-tyrosyl residues in phosphorylated poly(Glu4,Tyr1) random polymer (polyEY) and phosphorylated myelin basic protein (MBP) but not phosphoseryl and/or phosphothreonyl residues in phosphorylated histone H1, casein and phosphorylase a, indicating that the enzyme showed protein-tyrosine-phosphatase (PTPase, EC 3.1.3.48)-like activity in vitro. The enzyme was remarkably inhibited by diethylpyrocarbonate (DEPC), monoiodoacetic acid (MIAA), and monoiodoacetamide (MIAM). Binding of 1 mol of DEPC to 1 mol of the enzyme caused complete inhibition of the enzyme; and 0.88 mol of 1-carboxymethylated histidine per mole of the enzyme was found when 90% of enzyme activity was lost by modification with 14C-MIAA. These results indicated that this psychrophilic enzyme was a PTPase-like enzyme with histidine as its catalytic residue. (+info)
Mixed reconstitution of mutated subunits of HIV-1 reverse transcriptase coexpressed in Escherichia coli - two tags tie it up.
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The active form of HIV-1 reverse transcriptase (RT) is a p66/p51 heterodimer, in which the p51 subunit is generated by C-terminal proteolytic cleavage of p66. A well-known problem of p66 recombinant expression is partial cleavage of a 15-kDa peptide from the C-terminus by host proteases that can not be completely suppressed. In order to analyse the contribution of specific residues to a particular function in one distinct subunit, an expression and purification system is required that selects for the combination of the two individual subunits with the desired substitutions. We reconstituted the p66/p51 heterodimer from subunits coexpressed in Escherichia coli as an N-terminal fusion protein of glutathione S-transferase (GST) with p51 and a C-terminally His-tagged p66, respectively. The two-plasmid coexpression system ensures convenience for gene manipulation while degradation is reduced to a minimum, as dimerization protects the protein from further proteolysis. The combination of glutathione-agarose, phenyl-superose and Ni/nitrilotriacetate affinity chromatography allows rapid and selective purification of the desired subunit combination. Truncated forms of p51 are efficiently removed. Mobility-shift assay revealed that the preparations are free of p66 homodimer. In a successful test of the novel expression system, mixed reconstituted RTs with p51 selectively mutated in a putative nucleic acid binding motif (the so called helix clamp) show reduced binding of dsDNA in mobility-shift assays. This indicates the p51 subunit has an active role in DNA binding (+info)
Alteration of the conserved residue tyrosine-158 to histidine renders human O6-alkylguanine-DNA alkyltransferase insensitive to the inhibitor O6-benzylguanine.
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The DNA repair protein O6-alkylguanine-DNA alkyltransferase (AGT) protects cells from alkylation damage. O6-Benzylguanine (BG) is a potent inactivator of human AGT (ED50 of 0.1 microM) that is currently undergoing clinical trials to enhance chemotherapy by alkylating agents. In a screen of AGT mutants randomly mutated at position glycine-160, we found that the double mutant Y158H/G160A protected Escherichia coli from killing by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) even in the presence of BG and that the AGT activity of this mutant was strongly resistant to BG (ED50 of 180 microM). Because the single mutant G160A was not resistant to BG, this suggested that the presence of the charged histidine residue at position 158 was responsible. This hypothesis was confirmed by the construction of the single mutation Y158H. The Y158H-mutant AGT was slightly less active than wild-type AGT for the repair of methylated DNA in vitro, but it protected E. coli from killing by MNNG even in the presence of BG and had an ED50 for the inactivation by BG of 620 microM. In contrast, mutant Y158F had an ED5o of 0.2 microM. Previous studies (M. Xu-Welliver et al., Cancer Res., 58: 1936-1945, 1998) have shown that mutant P140K is highly resistant to BG (ED50 of >1200 microM). Models of human AGT suggest that the side chain of the lysine inserted into this mutant is close to tyrosine-158 and that the positively charged lysine side-chain may interfere with BG binding. The double mutants P140K/Y158H and P140K/Y158F resembled P140K and Y158H in being highly resistant to BG, but the use of a sensitive assay for reaction of BG with AGT indicated that their abilities to react were in the order P140K/ Y158H < P140K < P140K/Y158F. These results confirm that the presence of a positively charged residue close to the active site of human AGT renders it highly resistant to BG without substantially affecting activity toward methylated DNA substrates. Such mutants may limit the value of BG therapy if they arise in malignant cells during chemotherapy, but the mutant sequences may be useful for gene therapy approaches in which BG-resistant human AGTs are used to prevent hematopoietic toxicity. At least 28 AGT sequences (from 25 species) have now been described. In 25 of these, the position equivalent to 158 in the human AGT is also a tyrosine, and in the other 3, it is a phenylalanine. The importance of an aromatic ring side chain at this position is emphasized by previous studies (S. Edara et al., Carcinogenesis, 16: 1637-1642, 1995), which show that the replacement by alanine renders human AGT inactive. Our results show that histidine can also substitute for tyrosine at this position. (+info)
Characterization of airway and vascular responses in murine lungs.
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1. We characterized the responses of murine airways and pulmonary vessels to a variety of endogenous mediators in the isolated perfused and ventilated mouse lung (IPL) and compared them with those in precision-cut lung slices. 2. Airways: The EC50 (microM) for contractions of airways in IPL/slices was methacholine (Mch), 6.1/1.5>serotonin, 0.7/2.0>U46619 (TP-receptor agonist), 0.1/0.06>endothelin-1, 0.1/0.05. In the IPL, maximum increase in airway resistance (RL) was 0.6, 0.4, 0.8 and 11 cmH2O s ml(-1), respectively. Adenosine (< or =1 mM), bombesin (< or =100 microM), histamine (< or =10 mM), LTC4 (< or =1 microM), PAF (0.25 microM) and substance P (< or =100 microM) had only weak effects (<5% of Mch) on RL. 3. Vessels: The EC50 (microM) for vasoconstriction in the IPL was LTC4, 0.06>U46619, 0.05+info)