Pneumococcal phosphorylcholine esterase, Pce, contains a metal binuclear center that is essential for substrate binding and catalysis

The phosphorylcholine esterase from Streptococcus pneumoniae, Pce, catalyzes the hydrolysis of phosphorylcholine residues from teichoic and lipoteichoic acids attached to the bacterial envelope and comprises a globular N-terminal catalytic module containing a zinc binuclear center and an elongated C-terminal choline-binding module. The dependence of Pce activity on the metal/enzyme stoichiometry shows that the two equivalents of zinc are essential for the catalysis, and stabilize the catalytic module through a complex metal-ligand coordination network. The pH dependence of Pce activity toward the alternative substrate p-nitrophenylphosphorylcholine (NPPC) shows that k(cat) and k(cat)/K(m) depend on the protonation state of two protein residues that can be tentatively assigned to the ionization of the metal-bound water (hydrogen bonded to D89) and to H228. Maximum activity requires deprotonation of both groups, although the catalytic efficiency is optimum for the single deprotonated form. The drastic reduction of activity in the H90A mutant, which still binds two Zn2+ ions at neutral pH, indicates that Pce activity also depends on the geometry of the metallic cluster. The denaturation heat capacity profile of Pce exhibits two peaks with T(m) values of 39.6 degrees C (choline-binding module) and 60.8 degrees C (catalytic module). The H90A mutation reduces the high-temperature peak by about 10 degrees C. Pce is inhibited in the presence of 1 mM zinc, but this inhibition depends on pH, buffer, and substrate species. A reaction mechanism is proposed on the basis of kinetic data, the structural model of the Pce:NPPC complex, and the currently accepted mechanism for other Zn-metallophosphoesterases.     

NMR study of the preferred membrane orientation of polyisoprenols (dolichol) and the impact of their complex with polyisoprenyl recognition sequence peptides on membrane structure

Earlier NMR studies showed that the polyisoprenols (PIs) dolichol (C95), dolichylphosphate (C95-P) and undecaprenylphosphate (C55-P) could alter membrane structure by inducing in the lamellar phospholipid (PL) bilayer a nonlamellar or hexagonal (Hex II) structure. The destabilizing effect of C95 and C95-P on host fatty acyl chains was supported by small angle X-ray diffraction and freeze-fracture electron microscopy. Our present 1H- and 31P-NMR studies show that the addition of a polyisoprenol recognition sequence (PIRS) peptide to nonlamellar membranes induced by the PIs can reverse the hexagonal structure phase back to a lamellar structure. This finding shows that the PI:PIRS docking complex can modulate the polymorphic phase transitions in PL membranes, a finding that may help us better understand how glycosyl carrier-linked sugar chains may traverse membranes. Using an energy-minimized molecular modeling approach, we also determined that the long axis of C95 in phosphatidylcholine (PC) membranes is oriented approximately parallel to the interface of the lipid bilayer, and that the head and tail groups are positioned near the bilayer interior. In contrast, the phosphate head group of C95-P is anchored at the PC bilayer, and the angle between the long axis of C95-P and the bilayer interface is about 758, giving rise to a preferred conformation more perpendicular to the plane of the bilayer. Molecular modeling calculations further revealed that up to five PIRS peptides can bind cooperatively to a single PI molecule, and this tethered structure has the potential to form a membrane channel. If such a channel were to exist in biological membranes, it could be of functional importance in glycoconjugate translocation, a finding that has not been previously reported.     

Threading with chemostructural restrictions method for predicting fold and functionally significant residues: application to dipeptidylpeptidase IV (DPP-IV)

We present a new method for more accurate modeling of protein structure, called threading with chemostructural restrictions. This method addresses those cases in which a target sequence has only remote homologues of known structure for which sequence comparison methods cannot provide accurate alignments. Although remote homologues cannot provide an accurate model for the whole chain, they can be used in constructing practically useful models for the most conserved-and often the most interesting-part of the structure. For many proteins of interest, one can suggest certain chemostructural patterns for the native structure based on the available information on the structural superfamily of the protein, the type of activity, the sequence location of the functionally significant residues, and other factors. We use such patterns to restrict (1) a number of possible templates, and (2) a number of allowed chain conformations on a template. The latter restrictions are imposed in the form of additional template potentials (including terms acting as sequence anchors) that act on certain residues. This approach is tested on remote homologues of alpha/beta-hydrolases that have significant structural similarity in the positions of their catalytic triads. The study shows that, in spite of significant deviations between the model and the native structures, the surroundings of the catalytic triad (positions of C(alpha) atoms of 20-30 nearby residues) can be reproduced with accuracy of 2-3 A. We then apply the approach to predict the structure of dipeptidylpeptidase IV (DPP-IV). Using experimentally available data identifying the catalytic triad residues of DPP-IV (David et al., J Biol Chem 1993;268:17247-17252); we predict a model structure of the catalytic domain of DPP-IV based on the 3D fold of prolyl oligopeptidase (Fulop et al., Cell 1998;94:161-170) and use this structure for modeling the interaction of DPP-IV with inhibitor.     

Expression and binding properties of the two mannose-6-phosphate receptors differ during perinatal development in rat liver

Mammalian tissues express both cation-dependent (CD-MPR) and cation-independent (CI-MPR) mannose-6-phosphate receptors, which mediate the targeting of acid hydrolases to lysosomes. The coexistence of the two receptors in all cell types and tissues is still poorly understood. To determine whether these receptors might play a role in maturation, we studied their expression and binding properties in rat liver during perinatal development. CI-MPR expression decreases progressively from 18-day fetuses to adults, whereas the CD-MPR showed a transient decrease in newborn and at the 5th day after birth. Immunostaining of the tissues showed that both receptors localize to hepatocytes at all the ages and, additionally, the CD-MPR was reactive in megakaryocytes at early stages. Binding assays showed differences in the B(max) and K(D) values between the ages studied. These results demonstrate that both receptors change differentially during perinatal development, suggesting that they play distinct roles during organ maturation.     

Assessment of sweet potato [Ipomoea batatas (L.) Lam] for bioethanol production in southern Italy

The potential of sweet potato as an alternative crop for bioethanol production has been assessed. We evaluated the amount of soluble sugars, starch and cell wall polysaccharides in tubers of three sweet potato cultivars characterized by different pulp and peel colouration: “Yellow yam” with yellow flesh and brown peel, “White yam” with white flesh and white peel and “Orange yam” with orange flesh and brown peel. The results confirm the high concentration of carbohydrates in sweet potato tubers, especially “Yellow yam”, mainly in the form of starch (67%) and soluble sugars (26%). “Yellow yam”, which is the most widespread cultivar in Salento, appeared the best choice as biomass for bioethanol production. It is characterized by high productivity (20–40 tons/ha year). Results also suggest that “Yellow yam” cultivar has great potential as a bioethanol source in southern Italy with an estimated agroindustrial production yield higher than 2032 l/ha year.     

The influence of cosolvent concentration on enzymatic kinetic resolution of trans-2-phenyl-cyclopropane-1-carboxylic acid derivatives

A method to improve the enantioselectivity of lipase-catalyzed kinetic resolution (KR) of trans-2-phenyl-cyclopropane-1-carboxylic acid derivatives in water–acetone solution is presented. Two different approaches were compared: enzyme-catalyzed esterification and enzymatic hydrolysis of the target ester. A substantial influence of enzyme type, ethoxy group donor, and solvent on conversion and enantioselectivity of the enzymatic esterification was noted. While enzymatic esterification proceeds with poor enantioselectivity, the hydrolysis of target ester proceeds efficiently. Studies on the influence of cosolvent used for the enzymatic hydrolysis reaction showed that kinetic resolution can be performed in acetone and water buffer mixture predominantly containing organic solvent. Any change in organic solvent content resulted in a substantial decrease in enantioselectivity from almost E = 150 to less than 5.     

Carbohydrate metabolism in Bifidobacteria

Members of the genus Bifidobacterium can be found as components of the gastrointestinal microbiota, and are believed to play an important role in maintaining and promoting human health by eliciting a number of beneficial properties. Bifidobacteria can utilize a diverse range of dietary carbohydrates that escape degradation in the upper parts of the intestine, many of which are plant-derived oligo- and polysaccharides. The gene content of a bifidobacterial genome reflects this apparent metabolic adaptation to a complex carbohydrate-rich gastrointestinal tract environment as it encodes a large number of predicted carbohydrate-modifying enzymes. Different bifidobacterial strains may possess different carbohydrate utilizing abilities, as established by a number of studies reviewed here. Carbohydrate-degrading activities described for bifidobacteria and their relevance to the deliberate enhancement of number and/or activity of bifidobacteria in the gut are also discussed in this review.     

Structure,Dynamics, and Specificity of Endoglucanase D from Clostridium cellulovorans

The enzymatic degradation of cellulose is a critical step in the biological conversion of plant biomass into an abundant renewable energy source. An understanding of the structural and dynamic features that cellulases utilize to bind a single strand of crystalline cellulose and hydrolyze the β-1,4-glycosidic bonds of cellulose to produce fermentable sugars would greatly facilitate the engineering of improved cellulases for the large-scale conversion of plant biomass. Endoglucanase D (EngD) from Clostridium cellulovorans is a modular enzyme comprising an N-terminal catalytic domain and a C-terminal carbohydrate-binding module, which is attached via a flexible linker. Here, we present the 2.1-Å-resolution crystal structures of full-length EngD with and without cellotriose bound, solution small-angle X-ray scattering (SAXS) studies of the full-length enzyme, the characterization of the active cleft glucose binding subsites, and substrate specificity of EngD on soluble and insoluble polymeric carbohydrates. SAXS data support a model in which the linker is flexible, allowing EngD to adopt an extended conformation in solution. The cellotriose-bound EngD structure revealed an extended active-site cleft that contains seven glucose-binding subsites, but unlike the majority of structurally determined endocellulases, the active-site cleft of EngD is partially enclosed by Trp162 and Tyr232. EngD variants, which lack Trp162, showed a significant reduction in activity and an alteration in the distribution of cellohexaose degradation products, suggesting that Trp162 plays a direct role in substrate binding.     

Applicability of Arginyl-tRNA: Protein Arginyltransferase in Peptide Synthesis

A monoclonal antibody (MAb 1C3) to bovine β-casein was prepared, and a peptide (from 184 to 202 residues of β-casein) that could bind this monoclonal antibody was separated from a tryptic digest of β-casein. Other β-casein fragments, the peptide from 193 to 202 and the one from 1 to 189 residues, were also prepared for comparison. MAb 1C3 belongs to the IgG₁ subclass with a κ chain and its pI was 5.9. The binding affinity of β-casein and β-casein fragments to the monoclonal antibody was investigated by a competitive radioimmunoassay, and the order of affinity was β-casein peptide from 193 to 202 > β-casein > β-casein peptide from 184 to 202 > β-casein peptide from 1 to 189. On the basis of the results, the mechanism for the interaction between MAb 1C3 and β-casein fragments is discussed.     

Structure of a novel thermostable GH51 α-L-arabinofuranosidase from Thermotoga petrophila RKU-1

α‐L ‐arabinofuranosidases (EC 3.2.1.55) participate in the degradation of a variety of L ‐arabinose‐containing polysaccharides and interact synergistically with other hemicellulases in the production of oligosaccharides and bioconversion of lignocellulosic biomass into biofuels. In this work, the structure of a novel thermostable family 51 (GH51) α‐L ‐arabinofuranosidase from Thermotoga petrophila RKU‐1 (TpAraF) was determined at 3.1 Å resolution. The TpAraF tertiary structure consists of an (α/β)‐barrel catalytic core associated with a C‐terminal β‐sandwich domain, which is stabilized by hydrophobic contacts. In contrast to other structurally characterized GH51 AraFs, the accessory domain of TpAraF is intimately linked to the active site by a long β‐hairpin motif, which modifies the catalytic cavity in shape and volume. Sequence and structural analyses indicate that this motif is unique to Thermotoga AraFs. Small angle X‐ray scattering investigation showed that TpAraF assembles as a hexamer in solution and is preserved at the optimum catalytic temperature, 65°C, suggesting functional significance. Crystal packing analysis shows that the biological hexamer encompasses a dimer of trimers and the multiple oligomeric interfaces are predominantly fashioned by polar and electrostatic contacts.