David Pignol

Lipase activation by nonionic detergents. The crystal structure of the porcine lipase-colipase-tetraethylene glycol monooctyl ether complex.

The crystal structure of the ternary porcine lipase-colipase-tetra ethylene glycol monooctyl ether (TGME) complex has been determined at 2.8 Å resolution. The crystals belong to the cubic space group F23 with a = 289.1 Å and display a strong pseudo-symmetry corresponding to a P23 lattice. Unexpectedly, the crystalline two-domain lipase is found in its open configuration. This indicates that in the presence of colipase, pure micelles of the nonionic detergent TGME are able to activate the enzyme; a process that includes the movement of an N-terminal domain loop (the flap). The effects of TGME and colipase have been confirmed by chemical modification of the active site serine residue using diisopropyl p-nitrophenylphosphate (E600). In addition, the presence of a TGME molecule tightly bound to the active site pocket shows that TGME acts as a substrate analog, thus possibly explaining the inhibitory effect of this nonionic detergent on emulsified substrate hydrolysis at submicellar concentrations. A comparison of the lipase-colipase interactions between our porcine complex and the human-porcine complex (van Tilbeurgh, H., Egloff, M.-P., Martinez, C., Rugani, N., Verger, R., and Cambillau, C. (1993) Nature 362, 814-820) indicates that except for one salt bridge interaction, they are conserved. Analysis of the superimposed complexes shows a 5.4° rotation on the relative position of the N-terminal domains excepting the flap that moves in a concerted fashion with the C-terminal domain. This flexibility may be important for the binding of the complex to the water-lipid interface.

Isolation and Characterization of Environmental Bacteria Capable of Extracellular Biosorption of Mercury

ABSTRACT Accumulation of toxic metals in the environment represents a public health and wildlife concern. Bacteria resistant to toxic metals constitute an attractive biomass for the development of systems to decontaminate soils, sediments, or waters. In particular, biosorption of metals within the bacterial cell wall or secreted extracellular polymeric substances (EPS) is an emerging process for the bioremediation of contaminated water. Here the isolation of bacteria from soil, effluents, and river sediments contaminated with toxic metals permitted the selection of seven bacterial isolates tolerant to mercury and associated with a mucoid phenotype indicative of the production of EPS. Inductively coupled plasma-optical emission spectroscopy and transmission electron microscopy in conjunction with X-ray energy dispersive spectrometry revealed that bacteria incubated in the presence of HgCl2 sequestered mercury extracellularly as spherical or amorphous deposits. Killed bacterial biomass incubated in the presence of HgCl2 also generated spherical extracellular mercury deposits, with a sequestration capacity (40 to 120 mg mercury per g [dry weight] of biomass) superior to that of live bacteria (1 to 2 mg mercury per g [dry weight] of biomass). The seven strains were shown to produce EPS, which were characterized by Fourier transform-infrared (FT-IR) spectroscopy and chemical analysis of neutral-carbohydrate, uronic acid, and protein contents. The results highlight the high potential of Hg-tolerant bacteria for applications in the bioremediation of mercury through biosorption onto the biomass surface or secreted EPS.

Neutron crystallographic evidence of lipase-colipase complex activation by a micelle

The concept of lipase interfacial activation stems from the finding that the catalytic activity of most lipases depends on the aggregation state of their substrates. It is thought that activation involves the unmasking and structuring of the enzymes active site through conformational changes requiring the presence of oil‐in‐water droplets. Here, we present the neutron structure of the activated lipase–colipase–micelle complex as determined using the D2O/H2O contrast variation low resolution diffraction method. In the ternary complex, the disk‐shaped micelle interacts extensively with the concave face of colipase and the distal tip of the C‐terminal domain of lipase. Since the micelle‐ and substrate‐binding sites concern different regions of the protein complex, we conclude that lipase activation is not interfacial but occurs in the aqueous phase and is mediated by colipase and a micelle.

CRYSTAL STRUCTURE OF HUMAN LITHOSTATHINE, THE PANCREATIC INHIBITOR OF STONE FORMATION

Human lithostathine (HLIT) is a pancreatic glycoprotein which inhibits the growth and nucleation of calcium carbonate crystals. The crystal structure of the monomeric 17 kDa HLIT, determined to a resolution of 1.55 angstroms, was refined to a crystallographic R‐factor of 18.6%. Structural comparison with the carbohydrate‐recognition domains of rat mannose‐binding protein and E‐selectin indicates that the C‐terminal domain of HLIT shares a common architecture with the C‐type lectins. Nevertheless, HLIT does not bind carbohydrate nor does it contain the characteristic calcium‐binding sites of the C‐type lectins. In consequence, HLIT represents the first structurally characterized member of this superfamily which is not a lectin. Analysis of the charge distribution and calculation of its dipole moment reveal that HLIT is a strongly polarized molecule. Eight acidic residues which are separated by regular 6 angstrom spacings form a unique and continuous patch on the molecular surface. This arrangement coincides with the distribution of calcium ions on certain planes of the calcium carbonate crystal; the dipole moment of HLIT may play a role in orienting the protein on the crystal surface prior to the more specific interactions of the acidic residues.

Mechanism of calcite crystal growth inhibition by the N-terminal undecapeptide of lithostathine.

Pancreatic juice is supersaturated with calcium carbonate. Calcite crystals therefore may occur, obstruct pancreatic ducts, and finally cause a lithiasis. Human lithostathine, a protein synthesized by the pancreas, inhibits the growth of calcite crystals by inducing a habit modification: the rhombohedral {10 1̄4} usual habit is transformed into a needle-like habit through the {112̄0} crystal form. A similar observation was made with the N-terminal undecapeptide (pE1R11) of lithostathine. We therefore aimed at discovering how peptides inhibit calcium salt crystal growth. We solved the complete x-ray structure of lithostathine, including the flexible N-terminal domain, at 1.3 Å. Docking studies of pE1R11 with the (101̄4) and (11 2̄0) faces through molecular dynamics simulation resulted in three successive steps. First, the undecapeptide progressively unfolded as it approached the calcite surface. Second, mobile lateral chains of amino acids made hydrogen bonds with the calcite surface. Last, electrostatic bonds between calcium ions and peptide bonds stabilized and anchored pE1R11 on the crystal surface. pE1R11-calcite interaction was stronger with the (11 2̄0) face than with the (10 1̄4) face, confirming earlier experimental observations. Energy contributions showed that the peptide backbone governed the binding more than did the lateral chains. The ability of peptides to inhibit crystal growth is therefore essentially based on backbone flexibility.

PpsR: a multifaceted regulator of photosynthesis gene expression in purple bacteria.

Purple bacteria control the level of expression and the composition of their photosystem according to light and redox conditions. This control involves several regulatory systems that have been now well characterized. Among them, the PpsR regulator plays a central role, because it directly or indirectly controls the synthesis of all of the different components of the photosystem. In this review, we report our knowledge of the PpsR protein, highlighting the diversity of its mode of action and focusing on the proteins identified in four model purple bacteria (Rhodobacter capsulatus, Rhodobacter sphaeroides, Rubrivivax gelatinosus, Bradyrhizobium ORS278). This regulator exhibits unique regulatory features in each bacterium: it can activate and/or repress the expression of photosynthesis genes, its activity can be modulated or not by the redox conditions, it can interact with other specific regulators and therefore be involved differently in light and/or redox regulatory circuits.

Bacteriophytochrome and regulation of the synthesis of the photosynthetic apparatus in Rhodopseudomonas palustris: pitfalls of using laboratory strains

The synthesis of the photosynthetic apparatus of different strains of Rhodopseudomonas palustris has been studied as a function of the oxygen concentration and far-red light. For strain CEA001, only a small amount of photosynthetic apparatus is synthesized in the dark for oxygen concentration higher than 8% whereas synthesis is strongly enhanced by far-red light illumination. This enhancement is due to the action of a bacteriophytochrome (ORF2127/ORF2128), which antagonizes the repressor PpsR. On the contrary, a large fraction of photosystem is synthesized in the dark and far-red illumination induces no enhancement in strain CGA009. This difference in phenotype of strain CGA009 is explained by a single point-mutation R428C in the helix-turn-helix DNA binding motif of PpsR, rendering it inactive. In addition, a frame-shift mutation had occurred in the gene encoding bacteriophytochrome (ORF2127/ORF2128), conducting to a truncated inactive sensor. We propose that these mutations occurred in culture. Bacteria have developed a sophisticated regulatory process to synthesize their photosynthetic apparatus when light is available. This process is a critical advantage for the bacteria under natural conditions since they optimize their development depending on the available energy resources. On the contrary, under laboratory growth conditions where there is no substrate limitation, there is no crucial need for such a regulation and deleterious mutations affecting this process are of no importance.

The lipase/colipase complex is activated by a micelle: neutron crystallographic evidence.

The catalytic activity of most lipases depends on the aggregation state of their substrates. It is supposed that lipase activation requires the unmasking and structuring of the enzymes active site through conformational changes involving the presence of oil-in-water droplets. This phenomenon has been called interfacial activation. Here, we report the crystal structure of the pancreatic activated lipase/colipase/micelle complex as determined using the D2O/H2O contrast variation low resolution neutron diffraction method. We find that a disk-shaped micelle interacts extensively with the concave face of colipase (CL) and the distal tip of the C-terminal domain of lipase away from the active site of the enzyme. Such interaction appears to help stabilizing the lipase-CL interaction. Consequently, we conclude that lipase activation is not interfacial but occurs in the aqueous phase and it is mediated by CL and a micelle.

CYP201A2, a cytochrome P450 from Rhodopseudomonas palustris, plays a key role in the biodegradation of tributyl phosphate

Tributyl phosphate (TBP) is a toxic organophosphorous compound widely used in nuclear fuel processing and chemical industries. Rhodopseudomonas palustris, one of the most metabolically versatile photosynthetic bacteria, is shown here to degrade TBP efficiently under photosynthetic conditions. This study shows that this O2- and NADPH/FMNH2-dependent process was also catalyzed when TBP was incubated with membrane-associated proteins extracted from this strain. The effects of several regulators of cytochrome P450 activity on the TBP consumption suggest a key role for a cytochrome P450 in this process. Disruption of the rpa0241 gene encoding a putative cytochrome P450 led to a 60% decrease of the TBP catabolism, whereas reintroducing the gene in the mutant restored the wild-type phenotype. The rpa0241 gene was expressed and purified in Escherichia coli. Characterization by UV-visible spectroscopy of the purified recombinant membrane-bound protein (CYP201A2) encoded by the rpa0241 gene revealed typical spectral characteristics of cytochrome P450 with a large spin state change of the heme iron associated with binding of TBP (Kdu2009≈u200965xa0μM). It is proposed that CYP201A2 catalyzes the initial step of the biodegradation process of TBP.

Crystal structure of bovine procarboxypeptidase A-S6 subunit III, a highly structured truncated zymogen E.

Subunit III, a defective serine endopeptidase lacking the typical N‐terminal hydrophobic dipeptide is secreted by the pancreas of ruminant species as part of the bovine ternary complex procarboxypeptidase A‐S6. Two monoclinic crystal forms were obtained and subsequently used to solve its X‐ray structure. The highest resolution model of subunit III was refined at 1.7 A resolution to a crystallographic R‐factor of 18.4%, with r.m.s. bond deviations from ideality of 0.012 A. About 80% of the model presents the characteristic architecture of trypsin‐like proteases. The remaining zones, however, have well‐defined, unique conformations. The regions from residues 70 to 80 and from 140 to 155 present maximum distances of 16 and 18 A relative to serine proteases and zymogens. Comparisons with the structures of porcine elastase 1 and chymotrypsinogen A indicate that the specific binding pocket of subunit III adopts a zymogen‐like conformation and thus provide a basis for its inactivity. In general, the structural analysis of subunit III strongly suggests that it corresponds to a truncated version of a new class of highly structured elastase‐like zymogen molecules. Based on the structures of subunit III and elastase 1, it is concluded that large concerted movements are necessary for the activation of zymogen E.