Metal Binding Is Critical for the Folding and Function of Laminin Binding Protein,Lmb of Streptococcus agalactiae

Lmb is a 34 kDa laminin binding surface adhesin of Streptococcus agalactiae. The structure of Lmb reported by us recently has shown that it consists of a metal binding crevice, in which a zinc ion is coordinated to three highly conserved histidines. To elucidate the structural and functional significance of the metal ion in Lmb, these histidines have been mutated to alanine and single, double and triple mutants were generated. These mutations resulted in insolubility of the protein and revealed altered secondary and tertiary structures, as evidenced by circular dichroism and fluorescence spectroscopy studies. The mutations also significantly decreased the binding affinity of Lmb to laminin, implicating the role played by the metal binding residues in maintaining the correct conformation of the protein for its binding to laminin. A highly disordered loop, proposed to be crucial for metal acquisition in homologous structures, was deleted in Lmb by mutation (ΔLmb) and its crystal structure was solved at 2.6 Å. The ΔLmb structure was identical to the native Lmb structure with a bound zinc ion and exhibited laminin binding activity similar to wild type protein, suggesting that the loop might not have an important role in metal acquisition or adhesion in Lmb. Targeted mutations of histidine residues confirmed the importance of the zinc binding crevice for the structure and function of the Lmb adhesin.     

RAPD analysis of genetic diversity among the isolates of Aspergillus flavus from different hosts and locations

Aflatoxin contamination is a major problem in maize, groundnut, chillies, cotton and tree nuts. These aflatoxins are low molecular weight toxic and carcinogenic secondary metabolites produced by Aspergillus flavus, A. parasiticus and A. nomius. In the present study, a total of 11 isolates of A. flavus isolated from groundnut, maize and chilli collected from different locations of Tamil Nadu, India were tested for their ability to produce aflatoxin B1 (AFB1) in vitro by indirect competitive enzyme-linked immunosorbent assay. The results show that the isolates vary in their level of toxin production. The amount of AFB1 produced by the toxigenic isolates of A. flavus ranged from 6.6 to 108.1?ng?ml^?1. Among the various isolates of A. flavus, the isolate VKR produced the highest amount (108.1?ng?ml^?1) of AFB1. The isolates viz. CBE1, CBE2, BSR1, BSR3 and BSR4 were found to be non-toxigenic. The genetic variability among these isolates was assessed by Random amplified polymorphic DNA (RAPD) analysis. DNA fragments of between 0.15 and 3.0?kb were obtained using 13 random primers, and each isolate differed in the size and number of PCR products indicating considerable polymorphism. Cluster analysis using Unweighted Pair Group Method with Arithmetic Mean clearly separated the isolates into four main clusters confirming the genetic diversity among the isolates of A. flavus. Both toxigenic and non-toxigenic isolates were intermingled in these four groups, indicating that no relationship exists between RAPD profile and the production of aflatoxin by A. flavus.     

The human xenobiotic-metabolizing enzyme arylamine N-acetyltransferase 1 (NAT1) is irreversibly inhibited by inorganic (Hg) and organic mercury (CH3Hg): Mechanism and kinetics

Human arylamine N-acetyltransferase 1 (NAT1) is a xenobiotic-metabolizing enzyme that biotransforms aromatic amine chemicals. We show here that biologically-relevant concentrations of inorganic (Hg²⁺) and organic (CH₃Hg⁺) mercury inhibit the biotransformation functions of NAT1. Both compounds react irreversibly with the active-site cysteine of NAT1 (half-maximal inhibitory concentration (IC₅₀) = 250 nM and k_inact = 1.4 × 10⁴ M⁻¹ s⁻¹ for Hg²⁺ and IC₅₀ = 1.4 μM and k_inact = 2 × 10² M⁻¹ s⁻¹ for CH₃Hg⁺). Exposure of lung epithelial cells led to the inhibition of cellular NAT1 (IC₅₀ = 3 and 20 μM for Hg²⁺ and CH₃Hg⁺, respectively). Our data suggest that exposure to mercury may affect the biotransformation of aromatic amines by NAT1.     

Conserved glycine residues in the P-loop of ATP synthases form a doorframe for nucleotide entrance

The phosphate binding loop (GXXXXGKT(S)) is conserved in several mononucleotide-binding proteins with similar three-dimensional structures. Although variations in other amino acids have been noted, the first glycine and glycine-lysine residues are highly conserved in all enzymes, whose role is yet to be understood. Alanine substitutions for critically positioned glycines—G234, G237, and G239—were generated for the catalytic A-subunit of A-ATP synthase from Pyrococcus horikoshii OT3, and their crystal structures were determined. They showed altered conformation for the phosphate binding loop, with G234A and G237A becoming flat and with G239A taking an intermediate conformation, resulting in the active-site region being closed to nucleotide entry. Furthermore, the essential amino acids S238 and K240, which normally interact with the nucleotide, become inaccessible. These mutant structures demonstrate the role of the strictly conserved glycine residues in guarding the active-site region for nucleotide entrance in archaea-type ATP synthases.     

Solution Structure,Determined by Nuclear Magnetic Resonance,of the b30-82 Domain of Subunit b of Escherichia coli F1Fo ATP Synthase

Subunit b, the peripheral stalk of bacterial F₁F_o ATP synthases, is composed of a membrane-spanning and a soluble part. The soluble part is divided into tether, dimerization, and δ-binding domains. The first solution structure of b30-82, including the tether region and part of the dimerization domain, has been solved by nuclear magnetic resonance, revealing an α-helix between residues 39 and 72. In the solution structure, b30-82 has a length of 48.07 Å. The surface charge distribution of b30-82 shows one side with a hydrophobic surface pattern, formed by alanine residues. Alanine residues 61, 68, 70, and 72 were replaced by single cysteines in the soluble part of subunit b, b22-156. The cysteines at positions 61, 68, and 72 showed disulfide formation. In contrast, no cross-link could be formed for the A70C mutant. The patterns of disulfide bonding, together with the circular dichroism spectroscopy data, are indicative of an adjacent arrangement of residues 61, 68, and 72 in both α-helices in b22-156.ATP synthesis by oxidative phosphorylation or photophosphorylation is a multistep membrane-located process that provides the bulk of cellular energy in eukaryotes and many prokaryotes. The majority of ATP synthesis is accomplished by the enzyme ATP synthase (EC 3.6.1.34), also called F₁F_o ATP synthase, which, in its simplest form, as in bacteria, is composed of eight different subunits (α₃, β₃, γ, δ, ɛ, a, b₂, and c_9-12). This multisubunit complex is divided into the F₁ headpiece, α₃:β₃, and a membrane-embedded ion-translocating part known as F_o, to which F₁ is attached by a central and a peripheral stalk (1, 5, 25). ATP is synthesized or hydrolyzed on the α₃:β₃ hexamer, and the energy provided for or released during that process is transmitted to the membrane-bound F_o sector, consisting of subunits a and c and part of subunit b (30, 31). The energy coupling between the two active domains occurs via the stalk part(s) (6). The central stalk is made of subunits γ and ɛ, and the peripheral stalk is formed by subunits δ and b. The peripheral stalk, which lies at the edge of the multisubunit assembly of the F₁F_o ATP synthase, acts as a stator to counter the tendency of the α₃:β₃ hexamer to follow the rotation of the central stalk and the attached c-ring, and to anchor the membrane-embedded a subunit (17, 36).In Escherichia coli, subunit b with its 156 residues extends with its soluble part (b_sol; b21-156) from the top of the F₁ sector down, into, and across the membrane, where it is associated with subunit a (2, 15, 32, 34). The 156-residue b subunit has been divided into four functional domains (28). They are, in order from the N to the C terminus; the membrane domain, the tether region, the dimerization domain, and the δ-binding domain. The structure of the synthesized 33-residue peptide comprising the N-terminal membrane-spanning region has been solved by ¹H NMR, showing an α-helical feature (14). The crystallographic structure of the major part of the dimerization domain, b62-122, revealed an α-helix with a length of 9.0 nm (12). Most recently, the NMR solution structure of the very C-terminal segment, b140-156, which interacts with the C terminus of subunit δ (δ91-177), has been determined by NMR spectroscopy (26). This molecule adopts a stable helix formation in solution with a flexible tail between amino acid residues 140 and 145. SAXS (26) and analytical ultracentrifuge studies have indicated that the soluble domain of subunit b (b21-156, b22-156) is dimeric in solution (12). So far, no high-resolution structure of the tether domain, including residues 25 to 52, or the N-terminal segment of the dimerization domain, which is formed by residues 53 to 122, is available (14).Here, we have turned our attention to the production and purification of residues 30 to 82 of subunit b (b30-82) from E. coli F₁F_o ATP synthase, which forms the remaining unsolved structural segment of subunit b. The structural features of this segment have been determined in solution using NMR spectroscopy. The introduction of a cysteine residue into b22-156 at four positions resulted in different intersubunit disulfide patterns, giving insight into the proximity of the residues.     

Real-time observation of strand exchange reaction with high spatiotemporal resolution

RecA binds to single-stranded (ss) DNA to form?a helical filament that catalyzes strand exchange with a homologous double-stranded (ds) DNA. The study of strand exchange in ensemble assays is limited by the diffusion limited homology search process, which masks the subsequent strand exchange reaction. We developed a single-molecule fluorescence assay with a few base-pair and millisecond resolution that can separate initial docking from the subsequent propagation of joint molecule formation. Our data suggest that propagation occurs in 3?bp increments with destabilization of the incoming dsDNA and concomitant pairing with the reference ssDNA. Unexpectedly, we discovered the formation of?a dynamic complex between RecA and the displaced DNA that remains bound transiently after joint molecule formation. This finding could have important implications for the irreversibility of strand exchange. Our model for strand exchange links structural models of RecA to its catalytic function.     

Analysis of structure, function, and evolutionary origin of the ob gene product--leptin

Leptin, the ob gene product, is a 167 amino acid polypeptide known to play a key role in regulating the fat stores of the body and is found in all eukaryotes, including mammals, aves, and also in invertebrates. To gain insight into the structure-function relation and origin of leptin, we have analyzed the amino acid sequence of leptin from 23 species by computing the frequency of occurrence of amino acids, their secondary structure, sequence homology, et cetera. Extensive conservation is observed within the leptin sequences of all the species, suggesting an evolutionary relatedness among them. It is interesting to note that human leptin shares a very high degree of homology with gorilla, chimpanzee, and orangutan indicative of a common function of leptin in them. Analysis of the codon bias in leptin from 11 species reveals that sminthopsis shows highest variation compared to human while less variation is observed in chimpanzee and orangutan, possibly reflecting the closeness in their evolution. Thus, understanding leptin's three-dimensional structure along with primary and secondary structure might enable us to understand the functional role played by this multifaceted adipocyte derived protein.     

Biological Treatment of a Pulp and Paper Industry Effluent by Pleurotus spp.

In this work, the ability of Pleurotus spp.:P. sajor-caju; P. platypus and P. citrinopileatus to treat pulp and paper mill effluent on a laboratory and pilot scale were studied. On the laboratory scale treatment, P. sajor-caju decolorized the effluent by 66.7% on day 6 of incubation. Inorganic chloride liberated by P. sajor-caju was 230.9% (814.0 mg/dl) and the COD was reduced by 61.3% (1302.0 mg/dl) on day 10 of treatment. In the pilot scale treatment maximum decolorization was obtained by P. sajor-caju (60.1%) on day 6 of the incubation. Inorganic chloride content was increased by 524.0 mg/dl (113.0%) and the COD was reduced by 1442.0 mg/dl (57.2%) by P. sajor-caju on day 7 of incubation. These results revealed that the treatment of pulp and paper mill effluent by P. sajor-caju proved as better candidate for the purpose than P. platypus and P. citrinopileatus.     

A New Medium for Bacillus thuringiensis Berliner

S ummary : A new solid medium for culturing Bacillus thuringiensis consists of groundnut cake, 10%; tamarind kernel powder, 1·5% and agar, 0·5%. The quantity of agar in the medium could be decreased from 1·5 to 0·5% by adding tamarind kernel powder. A spore yield (85% sporulation) of 3·2 g/l was obtained. Bacterial spores produced on the new solid medium were pathogenic to the larvae of the almond moth, Cadra cautella.