Structures of mannose-6-phosphate isomerase from Salmonella typhimurium bound to metal atoms and substrate: implications for catalytic mechanism

Mannose-6-phosphate isomerase (MPI) catalyzes the inter-conversion of mannose 6-phosphate and fructose 6-phosphate. X-ray crystal structures of MPI from Salmonella typhimurium in the apo form (with no metal bound) and in the holo form (with bound Zn2+) and two other structures with yttrium bound at an inhibitory site and complexed with Zn2+ and fructose 6-phosphate (F6P) were determined in order to gain insights into the structure and the isomerization mechanism. Isomerization involves acid/base catalysis with proton transfer between the C1 and C2 atoms of the substrate. His99, Lys132, His131 and Asp270 are close to the substrate and are likely to be the residues involved in proton transfer. The interactions observed at the active site suggest that the ring-opening step is probably catalyzed by His99 and Asp270. An active-site loop consisting of residues 130-133 undergoes conformational changes upon substrate binding. Zn2+ binding induces structural order in the loop consisting of residues 50-54. The metal atom appears to play a role in substrate binding and is probably also important for maintaining the architecture of the active site. Isomerization probably follows the previously suggested cis-enediol mechanism.

Structural effects of a dimer interface mutation on catalytic activity of triosephosphate isomerase.The role of conserved residues and complementary mutations

The active site of triosephosphate isomerase (TIM, EC: 5.3.1.1), a dimeric enzyme, lies very close to the subunit interface. Attempts to engineer monomeric enzymes have yielded well-folded proteins with dramatically reduced activity. The role of dimer interface residues in the stability and activity of the Plasmodium falciparum enzyme, PfTIM, has been probed by analysis of mutational effects at residue 74. The PfTIM triple mutant W11F/W168F/Y74W (Y74W*) has been shown to dissociate at low protein concentrations, and exhibits considerably reduced stability in the presence of denaturants, urea and guanidinium chloride. The Y74W* mutant exhibits concentration-dependent activity, with an approximately 22-fold enhancement of kcat over a concentration range of 2.5–40 μm, suggesting that dimerization is obligatory for enzyme activity. The Y74W* mutant shows an approximately 20-fold reduction in activity compared to the control enzyme (PfTIM WT*, W11F/W168F). Careful inspection of the available crystal structures of the enzyme, together with 412 unique protein sequences, revealed the importance of conserved residues in the vicinity of the active site that serve to position the functional K12 residue. The network of key interactions spans the interacting subunits. The Y74W* mutation can perturb orientations of the active site residues, due to steric clashes with proximal aromatic residues in PfTIM. The available crystal structures of the enzyme from Giardia lamblia, which contains a Trp residue at the structurally equivalent position, establishes the need for complementary mutations and maintenance of weak interactions in order to accommodate the bulky side chain and preserve active site integrity.

Protein Synthesis in Mycobacterium tuberculosis H37Rv and the Effect of Streptomycin in Streptomycin-Susceptible and -Resistant Strains

An efficient in vitro amino acid-incorporating system from Mycobacterium tuberculosis H37Rv was standardized. Ribonucleic acid (RNA) isolated from phage-infected M. smegmatis cells served as natural messenger RNA and directed the incorporation of 14C-amino acids into protein. The effects of various antitubercular drugs and “known inhibitors” of protein synthesis on amino acid incorporation were studied. Antibiotics like chloramphenicol and tetracycline inhibited mycobacterial protein synthesis, though they failed to prevent the growth of the organism. This failure was shown to be due to the impermeability of mycobacteria to these drugs by use of “membrane-active” agents along with the antibiotics in growth inhibition studies. Several independent streptomycin-resistant mutants of M. tuberculosis H37Rv were isolated. Streptomycin inhibited the incorporation of 14C-amino acids into proteins by whole cells of a streptomycin-susceptible strain by more than 90%, whereas very little or no inhibition was observed in either high-level or low-level streptomycin-resistant strains.

Analysis of the amino acid sequences of plant Bowman-Birk inhibitors

Plant seeds contain a large number of protease inhibitors of animal, fungal, and bacterial origin. One of the well-studied families of these inhibitors is the Bowman-Birk family(BBI). The BBIs from dicotyledonous seeds are 8K, double-headed proteins. In contrast, the 8K inhibitors from monocotyledonous seeds are single headed. Monocots also have a 16K, double-headed inhibitor. We have determined the primary structure of a Bowman-Birk inhibitor from a dicot, horsegram, by sequential edman analysis of the intact protein and peptides derived from enzymatic and chemical cleavage. The 76-residue-long inhibitor is very similar to that ofMacrotyloma axillare. An analysis of this inhibitor along with 26 other Bowman-Birk inhibitor domains (MW 8K) available in the SWISSPROT databank revealed that the proteins from monocots and dicots belong to related but distinct families. Inhibitors from monocots show larger variation in sequence. Sequence comparison shows that a crucial disulphide which connects the amino and carboxy termini of the active site loop is lost in monocots. The loss of a reactive site in monocots seems to be correlated to this. However, it appears that this disulphide is not absolutely essential for retention of inhibitory function. Our analysis suggests that gene duplication leading to a 16K inhibitor in monocots has occurred, probably after the divergence of monocots and dicots, and also after the loss of second reactive site in monocots.

Modeling of Freezing Phenomena Induced by Chemical Reactions

Spatial variations in the concentration of a reactive solute in solution are often encountered in a catalyst particle, and this leads to variation in the freezing point of the solution. Depending on the operating temperature, this can result in freezing of the solvent oil a portion of catalyst, rendering that part of the active area ineffective Freezing call occur by formation of a sharp front or it mush that separates the solid and fluid phases. In this paper, we model the extent of reduction in the active area due to freezing. Assuming that the freezing point decreases linearly with solute concentration, conditions for freezing to occur have been derived. At steady state, the ineffective fraction of catalyst pellet is found to be the same irrespective of the mode of freezing. Progress of freezing is determined by both the heat of reaction and the latent heat of fusion Unlike in freezing of alloys where the latter plays a dominant role, the exothermicity of the reaction has a significant effect on freezing in the presence of chemical reactions. A dimensionless group analogous to the Stefan number could be defined to capture the combined effect of both of these.

Intensity Oscillation in the Corona as Observed during the Total Solar Eclipse of 29 March 2006

We obtained the images of the eastern part of the solar corona in the Fe xiv 530.3 nm (green) and Fe x 637.4 nm (red) coronal emission lines during the total solar eclipse of 29 March 2006 at Manavgat, Antalya, Turkey. The images were obtained using a 35 cm Meade telescope equipped with a Peltier-cooled 2k x 2k CCD and 0.3 nm pass-band interference filters at the rates of 2.95 s (exposure times of 100 ms) and 2.0 s (exposure times of 300 ms) in the Fe xiv and Fe x emission lines,respectively. The analysis of the data indicates intensity variations at some locations with period of strongest power around 27 s for the green line and 20 s for the red line. These results confirm earlier findings of variations in the continuum intensity with periods in the range of 5 to 56 s by Singh et al. (Solar Phys. 170, 235, 1997). The wavelet analysis has been used to identify significant intensity oscillations at all pixels within our field of view. Significant oscillations with high probability estimates were detected for some locations only. These locations seem to follow the boundary of an active region and in the neighborhood, rather than within the loops themselves. These intensity oscillations may be caused by fast magneto-sonic waves in the solar corona and partly account for heating of the plasma in the corona.

Facile enantiospecific synthesis of (+)-iso-cladospolide B

Enantiospecific synthesis of bio-active butenolide (+)-iso-cladospolide B from D-(-)-tartaric acid in a short synthetic sequence is presented. Pivotal reaction sequence includes cross metathesis of an alkene and Wittig olefination. (C) 2010 Elsevier Ltd. All rights reserved.

Acylation of lysolecithin in the intestinal mucosa of rats

1. The presence of an active acyl-CoA–lysolecithin (1-acylglycerophosphorylcholine) acyltransferase was demonstrated in rat intestinal mucosa. 2. ATP and CoA were necessary for the incorporation of free [1-14C]oleic acid into lecithin (phosphatidylcholine). 3. The reaction was about 20 times as fast with [1-14C]oleoyl-CoA as with free oleic acid, CoA and ATP. 4. With 1-acylglycerophosphorylcholine as the acceptor, both oleic acid and palmitic acid were incorporated into the β-position of lecithin; the incorporation of palmitic acid was 60% of that of oleic acid. 5. Of the various analogues of lysolecithin tested as acyl acceptors from [1-14C]oleoyl CoA, a lysolecithin with a long-chain fatty acid at the 1-position was most efficient. 6. The enzyme was mostly present in the brush-border-free particulate fraction of the intestinal mucosa. 7. Of the various tissues of rats tested for the activity, intestinal mucosa was found to be the most active, with testes, liver, kidneys and spleen following it in decreasing order.

Equivalent constitutive model-based design of wave-absorbing material system and controller

A design methodology for wave-absorbing active material system is reported. The design enforces equivalence between an assumed material model having wave-absorbing behavior and a set of target feedback controllers for an array of microelectro-mechanical transducers which are integral part of the active material system. The proposed methodology is applicable to problems involving the control of acoustic waves in passive-active material system with complex constitutive behavior at different length-scales. A stress relaxation type one-dimensional constitutive model involving viscous damping mechanism is considered, which shows asymmetric wave dispersion characteristics about the half-line. The acoustic power flow and asymptotic stability of such material system are studied. A single sensor non-collocated linear feedback control system in a one-dimensional finite waveguide, which is a representative volume element in an active material system, is considered. Equivalence between the exact dynamic equilibrium of these two systems is imposed. It results in the solution space of the design variables, namely the equivalent damping coefficient, the wavelength(s) to be controlled and the location of the sensor. The characteristics of the controller transfer functions and their pole-placement problem are studied. (c) 2005 Elsevier Ltd. All rights reserved.

Development of twist in an emerging magnetic flux tube by poloidal field accretion

Aims. Following an earlier proposal for the origin of twist in the magnetic fields of solar active regions, we model the penetration of a wrapped up background poloidal field into a toroidal magnetic flux tube rising through the solar convective zone.Methods. The rise of the straight, cylindrical flux tube is followed by numerically solving the induction equation in a comoving Lagrangian frame, while an external poloidal magnetic field is assumed to be radially advected onto the tube with a speed corresponding to the rise velocity.Results. One prediction of our model is the existence of a ring of reverse current helicity on the periphery of active regions. On the other hand, the amplitude of the resulting twist depends sensitively on the assumed structure ( diffuse vs. concentrated/intermittent) of the active region magnetic field right before its emergence, and on the assumed vertical profile of the poloidal field. Nevertheless, in the model with the most plausible choice of assumptions a mean twist comparable to the observations results.Conclusions. Our results indicate that the contribution of this mechanism to the twist can be quite significant, and under favourable circumstances it can potentially account for most of the current helicity observed in active regions.

Noise minimization in RC-active filters using generalized impedance converters

A procedure has been given for minimizing the total output noise of a Generalized Impedance Converter (GIC), subject to constraints dictated by signal handling capability of the Operational Amplifiers and ease of microcircuit fabrication. The noise reduction is achieved only by the adjustment of RC elements of the GIC, and the total output noise after optimization in the example cited is close to the theoretical lower limit. The output noise of a higher-order filter can be reduced by RC-optimizing the individual GIC's of the active realization. Experimental results on a 20–24 kHz channel bank band-pass filter demonstrate the effectiveness of the above procedure.

Crystal Structure of Staphylococcus aureus Metallopeptidase (Sapep) Reveals Large Domain Motions between the Manganese-bound and Apo-states

Proteases belonging to the M20 family are characterized by diverse substrate specificity and participate in several metabolic pathways. The Staphylococcus aureus metallopeptidase, Sapep, is a member of the aminoacylase-I/M20 protein family. This protein is a Mn2+-dependent dipeptidase. The crystal structure of this protein in the Mn2+-bound form and in the open, metal-free state suggests that large interdomain movements could potentially regulate the activity of this enzyme. We note that the extended inactive conformation is stabilized by a disulfide bond in the vicinity of the active site. Although these cysteines, Cys(155) and Cys(178), are not active site residues, the reduced form of this enzyme is substantially more active as a dipeptidase. These findings acquire further relevance given a recent observation that this enzyme is only active in methicillin-resistant S. aureus. The structural and biochemical features of this enzyme provide a template for the design of novel methicillin-resistant S. aureus-specific therapeutics.

The stereochemistry of a-aminoisobutyric acid containing peptides

a-Aminoisobutyric acid (Aib), * a nonprotein amino acid first described synthetically, I has been found in diverse sources, ranging from peptides of microbial origin2s3 to the Murchison mete~r i te.E~a rly studies of the chemistry of Aib were directed towards the synthesis of model peptides containing this "sterically hindered" amino There have been several reports on the synthesis of Aib containing analogs of biologically active peptides.

Development stage-specific expression of fibroin in the silk worm Bombyx mori is regulated translationally

The contents of fibroin H RNA as a function of development have been quantitated in the posterior silk glands of Bombyx mori larvae on different days of 4th and 5th instars. The fibroin RNA levels increased during the feeding stages of larvae and the RNA got completely degraded during the interim moult. The patterns of accumulation of fibroin RNA were similar in both the instars. Although there was considerable increase in the fibroin RNA content during the 5th larval instar, the relative abundance of fibroin RNA in the total RNA was fairly constant during the 4th and 5th instars. The increased content of fibroin RNA in 5th instar was the consequence of an overall increase in transcription accompanying the development progress, rather than specific increase only in fibroin transcription. The contents of fibroin protein in the 4th and 5th instars of development have also been quantitated making use of a sensitive radioimmune assay with a purified, antifibroin antibody. There were substantial differences between 4th and 5th instars in the absolute fibroin contents as well as the relative proportion of fibroin in the total proteins. These results implied that although the fibroin gene was transcribed at the same efficiency during the 4th and 5th instars, the translational efficiency was much lower during the 4th instar. The extent of polyadenylation of fibroin RNA was similar in both instars. However, there was a two-fold increase in the polysome association of fibroin RNA in the 5th instar. Over and above this, there was substantial increase during the 5th instar in the contents of those tRNAs. (e.g. Gly, Ala and Ser) which are abundantly represented in fibroin and therefore directly related to the expression of fibroin. The increased polysome association of fibroin mRNA and the adequate supply of cognate tRNAs in the 5th instar, together contributes to the translational regulation of fibroin in a developmental stage-specific manner. Based on these observations, we propose that translational regulation plays a major role in the development stage-specific synthesis of fibroin in Bombyx mori.

Effect of a QSO on the interstellar medium of its host galaxy

We consider the hydrodynamic evolution of gas in the interstellar medium of the host galaxy of a quasar due to Compton heating by the QSO radiation. We show that a Lagrangean formulation of the problem is necessary. It is found that the "hydrodynamic time scale" becomes important compared to the Compton heating time scale. We also relax the "single fluid" approximation by considering the existence of clouds and taking into account the mass loss from stars. The results predict star burst activity, and thus we explain the blue colors of the active galaxies.