Crucial contribution of the multiple copies of the initiator tRNA genes in the fidelity of tRNA(fMet) selection on the ribosomal P-site in Escherichia coli

The accuracy of the initiator tRNA (tRNA(fMet)) selection in the ribosomal P-site is central to the fidelity of protein synthesis. A highly conserved occurrence of three consecutive G-C base pairs in the anticodon stem of tRNA(fMet) contributes to its preferential selection in the P-site. In a genetic screen, using a plasmid borne copy of an inactive tRNA(fMet) mutant wherein the three G-C base pairs were changed, we isolated Escherichia coli strains that allow efficient initiation with the tRNA(fMet) mutant. Here, extensive characterization of two such strains revealed novel mutations in the metZWV promoter severely compromising tRNA(fMet) levels. Low cellular abundance of the chromosomally encoded tRNA(fMet) allows efficient initiation with the tRNA(fMet) mutant and an elongator tRNA(Gln), revealing that a high abundance of the cellular tRNA(fMet) is crucial for the fidelity of initiator tRNA selection on the ribosomal P-site in E. coli. We discuss possible implications of the changes in the cellular tRNA(fMet) abundance in proteome remodeling.

Identification of Critical Residues of the Mycobacterial Dephosphocoenzyme A Kinase by Site-Directed Mutagenesis

Dephosphocoenzyme A kinase performs the transfer of the c-phosphate of ATP to dephosphocoenzyme A, catalyzing the last step of coenzyme A biosynthesis. This enzyme belongs to the P-loop-containing NTP hydrolase superfamily, all members of which posses a three domain topology consisting of a CoA domain that binds the acceptor substrate, the nucleotide binding domain and the lid domain. Differences in the enzymatic organization and regulation between the human and mycobacterial counterparts, have pointed out the tubercular CoaE as a high confidence drug target (HAMAP database). Unfortunately the absence of a three-dimensional crystal structure of the enzyme, either alone or complexed with either of its substrates/regulators, leaves both the reaction mechanism unidentified and the chief players involved in substrate binding, stabilization and catalysis unknown. Based on homology modeling and sequence analysis, we chose residues in the three functional domains of the enzyme to assess their contributions to ligand binding and catalysis using site-directed mutagenesis. Systematically mutating the residues from the P-loop and the nucleotide-binding site identified Lys14 and Arg140 in ATP binding and the stabilization of the phosphoryl intermediate during the phosphotransfer reaction. Mutagenesis of Asp32 and Arg140 showed catalytic efficiencies less than 5-10% of the wild type, indicating the pivotal roles played by these residues in catalysis. Non-conservative substitution of the Leu114 residue identifies this leucine as the critical residue from the hydrophobic cleft involved in leading substrate, DCoA binding. We show that the mycobacterial enzyme requires the Mg2+ for its catalytic activity. The binding energetics of the interactions of the mutant enzymes with the substrates were characterized in terms of their enthalpic and entropic contributions by ITC, providing a complete picture of the effects of the mutations on activity. The properties of mutants defective in substrate recognition were consistent with the ordered sequential mechanism of substrate addition for CoaE.

Binding of active site directed ligands to phospholipase A2: Implications on the molecular constraints and catalytic mechanism

Molecular constraints for the localization of active site directed ligands (competitive inhibitors and substrates) in the active site of phospholipase A2 (PLA2) are characterized. Structure activity relationships with known inhibitors suggest that the head : group interactions dominate the selectivity as well as a substantial part of the affinity. The ab initio fitting of the amide ligands in the active site was carried out to characterize the head group interactions. Based on a systematic coordinate space search, formamide is docked with known experimental constraints such as coordination of the carbonyl group to Ca2+ and hydrogen bond between amide nitrogen and ND1 of His48. An optimal position for a bound water molecule is identified and its significance for the catalytic mechanism is postulated. Unlike the traditional ''pseudo-triad'' mechanism, the ''Ca-coordinatedoxyanion'' mechanism proposed here invokes activation of the catalytic water to form the oxyanion in the coordination sphere of calcium. As it attacks the carbonyl carbon of the ester, a near-tetrahedral intermediate is formed. As the second proton of the catalytic water is abstracted by the ester oxygen, its reorientation and simultaneous cleavage form hydrogen bond with ND1 of His48. In this mechanism of esterolysis, a catalytic role for the water co-ordinated to Ca2+ is recognised.

Interactions of L-serine at the active site of serine hydroxymethyltransferases: induction of thermal stability

Serine hydroxymethyltransferase (SHMT), EC 2.1.2.1, exhibits broad substrate and reaction specificity. In addition to cleaving many 3-hydroxyamino acids to glycine and an aldehyde, the enzyme also catalyzed the decarboxylation, transamination and racemization of several substrate analogues of amino acids. To elucidate the mechanism of interaction of substrates, especially L-serine with the enzyme, a comparative study of interaction of L-serine with the enzyme from sheep liver and Escherichia coli, was carried out. The heat stability of both the enzymes was enhanced in the presence of serine, although to different extents. Thermal denaturation monitored by spectral changes indicated an alteration in the apparent T, of sheep liver and E. coli SHMTs from 55 +/- 1 degrees C to 72 +/- 3 degrees C at 40 mM serine and from 67 +/- 1 degrees C to 72 +/- 1 degrees C at 20 mM serine, respectively. Using stopped flow spectrophotometry k values of (49 +/- 5)(.)10(-3) s(-1) and (69 +/- 7).10(-3) s(-1) for sheep liver and E. coli enzymes were determined at 50 mM serine. The binding of serine monitored by intrinsic fluorescence and sedimentation velocity measurements indicated that there was no generalized change in the structure of both proteins. However, visible CD measurements indicated a change in the asymmetric environment of pyridoxal 5'-phosphate at the active site upon binding of serine to both the enzymes. The formation of an external aldimine was accompanied by a change in the secondary structure of the enzymes monitored by far UV-CD spectra. Titration microcalorimetric studies in the presence of serine (8 mM) also demonstrated a single class of binding and the conformational changes accompanying the binding of serine to the enzyme resulted in a more compact structure leading to increased thermal stability of the enzyme.

Non-uniform slot injection (suction) or wall enthalpy into a steady nonsimilar compressible laminar boundary layer

Steady two-dimensional and axisymmetric compressible nonsimilar laminar boundary-layer flows with non-uniform slot injection (or suction) and non-uniform wall enthalpy have been studied from the starting point of the streamwise co-ordinate to the exact point of separation. The effect of different free stream Mach number has also been considered. The finite discontinuities arising at the leading and trailing edges of the slot for the uniform slot injection (suction) or wall enthalpy are removed by choosing appropriate non-uniform slot injection (suction) or wall enthalpy. The difficulties arising at the starting point of the streamwise co-ordinate, at the edges of the slot and at the point of separation are overcome by applying the method of quasilinear implicit finite difference scheme with an appropriate selection of finer step size along the streamwise direction. It is observed that the non-uniform slot injection moves the point of separation downstream but the non-uniform slot suction has the reverse effect. The increase of Mach number shifts the point of separation upstream due to the adverse pressure gradient. The increase of total enthalpy at the wall causes the separation to occur earlier while cooling delays it. The non-uniform total enthalpy at the wall (i.e., the cooling or heating of the wall in a slot) along the streamwise co-ordinate has very little effect on the skin friction and thus on the point of separation.

Identification of the active-site peptide of 2,3-dihydroxybenzoic acid decarboxylase from Aspergillus oryzae

The non-oxidative decarboxylation of aromatic acids is a poorly understood reaction. The transformation of 2,3-dihydroxybenzoic acid to catechol in the fungal metabolism of indole is a prototype of such a reaction. 2,3-Dihydroxybenzoic acid decarboxylase (EC 4.1.1.46) which catalyzes this reaction was purified to homogeneity from anthranilate induced cultures of Aspergillus oryzae using affinity chromatography. The enzyme did not require cofactors like NAD(+), PLP, TPP or metal ions for its activity. There was no spectral evidence for the presence of enzyme bound cofactors. The preparation, which was adjudged homogeneous by the criteria of SDS-PAGE, sedimentation analysis and N-terminal analysis, was characterized for its physicochemical and kinetic parameters. The enzyme was inactivated by group-specific modifiers like diethyl pyrocarbonate (DEPC) and N-ethylmaleimide (NEM). The kinetics of inactivation by DEPC suggested the presence of a single class of essential histidine residues, the second order rate constant of inactivation for which was 12.5 M(-1) min(-1). A single class of cysteine residues was modified by NEM with a second order rate constant of 33 M(-1) min(-1). Substrate analogues protected the enzyme against inactivation by both DEPC and NEM, suggesting the Location of the essential histidine and cysteine to be at the active site of the enzyme. The incorporation of radiolabelled NEM in a differential labelling experiment was 0.73 mol per mol subunit confirming the presence of a single essential cysteine per active-site. Differentially labelled enzyme was enzymatically cleaved and the peptide bearing the label was purified and sequenced. The active-site peptide LLGLAETCK and the N-terminal sequence MLGKIALEEAFALPRFEEKT did not bear any similarity to sequences reported in the Swiss-Prot Protein Sequence Databank, a reflection probably of the unique primary structure of this novel enzyme. The sequences reported in this study will appear in the Swiss-Prot Protein Sequence Databank under the accession number P80402.

Injection cooling of blunt bodies flying at high Mach numbers

A study of transpiration cooling of blunt bodies such as a hemicylinder is made by solving Navier-Stokes equations. An upwind, implicit time-marching code is developed for this purpose. The study is conducted for both perfect-gas and real-gas (chemical equilibrium) flows. Investigations are carried out for a special wall condition that is referred to as no heat flow into the wall condition. The effects of air injection on wall temperature are analyzed. Analyses are carried out for Mach numbers ranging between 6-10 and Reynolds numbers ranging between 10(6)-10(7). Studies are made for spatially constant as well as spatially varying mass injection rate distributions, White cold air injection reduces the wall temperature substantially, transpiration cooling is relatively less effective when the gas is in chemical equilibrium.

Probing the role of the fully conserved Cys126 in triosephosphate isomerase by site-specific mutagenesis - distal effects on dimer stability

Cys126 is a completely conserved residue in triosephosphate isomerase that is proximal to the active site but has been ascribed no specific role in catalysis. A previous study of the C126S and C126A mutants of yeast TIM reported substantial catalytic activity for the mutant enzymes, leading to the suggestion that this residue is implicated in folding and stability [Gonzalez-Mondragon E et al. (2004) Biochemistry43, 3255–3263]. We re-examined the role of Cys126 with the Plasmodium falciparum enzyme as a model. Five mutants, C126S, C126A, C126V, C126M, and C126T, were characterized. Crystal structures of the 3-phosphoglycolate-bound C126S mutant and the unliganded forms of the C126S and C126A mutants were determined at a resolution of 1.7–2.1 Å. Kinetic studies revealed an approximately five-fold drop in kcat for the C126S and C126A mutants, whereas an approximately 10-fold drop was observed for the other three mutants. At ambient temperature, the wild-type enzyme and all five mutants showed no concentration dependence of activity. At higher temperatures (> 40 °C), the mutants showed a significant concentration dependence, with a dramatic loss in activity below 15 μm. The mutants also had diminished thermal stability at low concentration, as monitored by far-UV CD. These results suggest that Cys126 contributes to the stability of the dimer interface through a network of interactions involving His95, Glu97, and Arg98, which form direct contacts across the dimer interface.

Probing the role of the fully conserved Cys126 in triosephosphate isomerase by site-specific mutagenesis - distal effects on dimer stability

DatabaseStructural data are available in the Protein Data Bank under the accession numbers 3PVF, 3PY2, and 3PWA. Structured digital abstract Tim binds to Tim by x-ray crystallography (View interaction).

The nature of charge ordering in rare earth manganates and its strong dependence on the size of the A-site cations

Charge ordering in rare earth manganates of the type Ln(0.5)A(0.5)MnO(3) (Ln = rare earth, A = alkaline earth) is highly sensitive to the average radius of the A-site cations, [r(A)]. Tn the small [r(A)] regime (e.g., Y0.5Ca0.5MnO3), charge ordering occurs in the paramagnetic state, the transformation to an antiferromagnetic state occurring at still lower temperatures. At moderate [r(A)] values (e.g., Nd0.5Sr0.5MnO3), a ferromagnetic metallic state transforms to a charge-ordered antiferromagnetic state with cooling. These two distinct types of charge ordering and associated properties are explained in terms of the variation of the exchange couplings J(FM) and J(AFM) with [r(A)] and the invariance of the single-ion Jahn-Teller energy with [r(A)]. A qualitative temperature-[r(A)] phase diagram, consistent with the experimental observations, has been constructed to describe the properties of the manganates in the different [r(A)] regimes. (C) 1997 Academic Press.

Structures of the catalytic site mutants D99A and H48Q and the calcium-loop mutant D49E of phospholipase A(2)

Crystal structures of the active-site mutants D99A and H48Q and the calcium-loop mutant D49E of bovine phospholipase A(2) have been determined at around 1.9 Angstrom resolution. The D99A mutant is isomorphous to the orthorhombic recombinant enzyme, space group P2(1)2(1)2(1), The H48Q and the calcium-loop mutant D49E are isomorphous to the trigonal recombinant enzyme, space group P3(1)21, The two active-site mutants show no major structural perturbations. The structural water is absent in D99A and, therefore, the hydrogen-bonding scheme is changed. In H48Q, the catalytic water is present and hydrogen bonded to Gln48 N, but the second water found in native His48 is absent. In the calcium-loop mutant D49E, the two water molecules forming the pentagonal bipyramid around calcium are absent and only one O atom of the Glu49 carboxylate group is coordinated to calcium, resulting in only four ligands.

Aerosol radiative forcing due to enhanced black carbon at an urban site in India

[1] During a comprehensive aerosol field campaign, simultaneous measurements were made of aerosol spectral optical depths, black carbon mass concentration (M-b), total (M-t) and size segregated aerosol mass concentrations over an urban continental location, Bangalore (13 degreesN, 77 degreesE, 960 m msl), in India. Large amounts of BC were observed; both in absolute terms and fraction of total mass (similar to11%) and submicron mass (similar to23%) implying a significantly low single scatter albedo. The aerosol visible optical depth (tau(p)) was in the range 0.24 to 0.45. Estimated surface forcing is as high as -23 W m(-2) and top of the atmosphere (TOA) forcing is +5 Wm(-2) during relatively cleaner periods (tau(p) similar to 0.24). The net atmospheric absorption translates to an atmospheric heating of similar to0.8 K day(-1) for cleaner periods and similar to1.5 K day(-1) for less cleaner periods (tau(p) similar to 0.45). Our observations raise several issues, which may have impacts to regional climate and monsoon.

Glasslike ordering and spatial inhomogeneity of magnetic structure in Ba3FeRu2O9: Role of Fe/Ru site disorder

Several doped 6H hexagonal ruthenates, having the general formula Ba3MRu2O9, have been studied over a significant period of time to understand the unusual magnetism of ruthenium metal. However, among them, the M = Fe compound appears different since it is observed that unlike others, the 3d Fe ions and 4d Ru ions can easily exchange their crystallographic positions, and as a result many possible magnetic interactions become realizable. The present study involving several experimental methods on this compound establishes that the magnetic structure of Ba3FeRu2O9 is indeed very different from all other 6H ruthenates. Local structural study reveals that the possible Fe/Ru site disorder further extends to create local chemical inhomogeneity, affecting the high-temperature magnetism of this material. There is a gradual decrease of Fe-57 Mossbauer spectral intensity with decreasing temperature (below 100 K), which reveals that there is a large spread in the magnetic ordering temperatures, corresponding to many spatially inhomogeneous regions. However, finally at about 25 K, the whole compound is found to take up a global glasslike magnetic ordering.

Structural model for the Cu-B site of dopamine beta-hydroxylase: Crystal structure of a copper(II) complex showing N3OS coordination with an axial sulfur ligation

A copper(II) complex containing a NSO-donor Schiff base and NN-donor 2,2'-bipyridine has been prepared and structurally characterized. The square pyramidal complex with an axial sulfur ligation is a structural model for the CUB site of dopamine-hydroxylase in its oxidized form. The copper(II) complex is catalytically active in the oxidation of ascorbic acid by dioxygen mediated by a copper(I) species which is proposed to have a four-coordinate structure with a N3S coordination geometry.