Phytases catalyze the hydrolysis of phosphomonoester bonds of phytate (myo-inositol hexakisphosphate), thereby creating lower forms of myo-inositol phosphates and inorganic phosphate. In this study, cDNA expression libraries were constructed from four basidiomycete fungi (Peniophora lycii, Agrocybe pediades, a Ceriporia sp., and Trametes pubescens) and screened for phytase activity in yeast. One full-length phytaseencoding cDNA was isolated from each library, except for the Ceriporia sp. library where two different phytase-encoding cDNAs were found. All five phytases were expressed in Aspergillus oryzae, purified, and characterized. The phytases revealed temperature optima between 40 and 60°C and pH optima at 5.0 to 6.0, except for the P. lycii phytase, which has a pH optimum at 4.0 to 5.0. They exhibited specific activities in the range of 400 to 1,200 U ⅐ mg, of protein ؊1 and were capable of hydrolyzing phytate down to myo-inositol monophosphate. Surprisingly, 1 H nuclear magnetic resonance analysis of the hydrolysis of phytate by all five basidiomycete phytases showed a preference for initial attack at the 6-phosphate group of phytic acid, a characteristic that was believed so far not to be seen with fungal phytases. Accordingly, the basidiomycete phytases described here should be grouped as 6-phytases (EC 3.1.3.26).Phytases (myo-inositol hexakisphosphate phosphohydrolases) belong to the family of histidine acid phosphatases sharing the sequence consensus pattern(27; http://www.expasy.ch /cgi-bin/get-prodoc-entry?PDOC00538). They are capable of catalyzing the hydrolysis of phosphomonoester bonds of phytate (salts of myo-inositol hexakisphosphate or myo-inositol 1,2,3,4,5,6-hexakis dihydrogen phosphate), thereby creating lower forms of myo-inositol phosphates and inorganic phosphate (22,24). Phytases are grouped according to the specific position of the phosphate ester group on the phytate molecule at which hydrolysis is initiated, i.e., as 3-phytases (EC 3.1.3.8) or as 6-phytases (EC 3.1.3.26).Phytate is the primary source of inositol and the primary storage form of phosphate in plant seeds (23). Seeds, cereal grains, and legumes are important components of food and, in particular, of animal feed preparations. However, monogastric animals such as poultry and swine are incapable of utilizing the phosphorus bound in phytate due to low levels of phytase activity in the digestive tract. Furthermore, phytate acts as an antinutrient by chelating divalent cations and preventing the uptake of minerals, e.g., Zn (9). Thus, phytases are used as a cereal feed additive that enhances the phosphorus and mineral uptake in monogastric animals and reduces the level of phosphate output in their manure. Recently, there have been several reports on the cloning of fungal PhyA phytases from Aspergillus niger (16,20, 28), Aspergillus fumigatus (19), Aspergillus terreus, Myceliophthora thermophila (14), Emericella nidulans, Talaromyces thermophilus (18), and Thermomyces lanuginosus (2). Based on their characteristics and on their s...
Three new benzodiazepine alkaloids belonging to the circumdatin series have been isolated as minor constituents of culture extracts of a terrestrial strain of the fungus Aspergillus ochraceus. Their structures were solved by MS and NMR comparison with previously reported circumdatin analogues.
The prolactin receptor (PRLR) is activated by binding of prolactin in a 2:1 complex, but the activation mechanism is poorly understood. PRLR has a conserved WSXWS motif generic to cytokine class I receptors. We have determined the nuclear magnetic resonance solution structure of the membrane proximal domain of the human PRLR and find that the tryptophans of the motif adopt a T-stack conformation in the unbound state. By contrast, in the hormone bound state, a Trp/Arg-ladder is formed. The conformational change is hormone-dependent and influences the receptor-receptor dimerization site 3. In the constitutively active, breast cancer-related receptor mutant PRLR(I146L), we observed a stabilization of the dimeric state and a change in the dynamics of the motif. Here we demonstrate a structural link between the WSXWS motif, hormone binding, and receptor dimerization and propose it as a general mechanism for class 1 receptor activation.
Three novel isocoumarin (or isochromen) metabolites, dichlorodiaportin [3-(3, 3-dichloro-2-hydroxy-propyl)-8-hydroxy-6-methoxy-isochromen-1-one] (1), diaportinol [3-(2, 3-dihydroxy-propyl)-8-hydroxy-6-methoxy-isochromen-1-one] (2), and diaportinic acid [2-hydroxy-3-(8-hydroxy-6-methoxy-1-oxo-1H-isochromen-3-yl)-propanoic acid] (3), were isolated from the cultures of Penicillium nalgiovense along with citreoisocoumarin (4) and 6-methyl-citreoisocoumarin (5). Their structures were elucidated by spectroscopic methods including UV, MS, and NMR.
The crystal structure of the complex between an N-terminally truncated G129R human prolactin (PRL) variant and the extracellular domain of the human prolactin receptor (PRLR) was determined at 2.5 Å resolution by x-ray crystallography. This structure represents the first experimental structure reported for a PRL variant bound to its cognate receptor. The binding of PRL variants to the PRLR extracellular domain was furthermore characterized by the solution state techniques, hydrogen exchange mass spectrometry, and NMR spectroscopy. Compared with the binding interface derived from mutagenesis studies, the structural data imply that the definition of PRL binding site 1 should be extended to include residues situated in the N-terminal part of loop 1 and in the C terminus. Prolactin (PRL)4 is a protein hormone secreted by the anterior pituitary in vertebrates and possesses physiological functions of remarkable diversity, including effects on reproduction, lactation, and growth. PRL belongs to a family of homologous proteins comprising PRL, growth hormone (GH), and placental lactogen (PL). The biological effects associated with this cytokine family are mediated by two distinct classes of cell surface receptors, the PRL receptors (PRLR) and the GH receptors (GHR). The PRL/PL/GH biology is governed by a delicate balance between receptor cross-reactivity and selectivity; PRL and PL bind selectively to PRLR, whereas GH is capable of binding both PRLR and GHR.After being proposed more than a decade ago (1), hormoneinduced receptor dimerization became generally accepted as the model for cytokine receptor activation. For the PRL family members, the model describes the signaling molecular entity as a ternary complex between one hormone molecule and a receptor homodimer assembled in a strictly sequential and hormonedependent fashion; first the hormone ligand engages via binding site 1 (BS1) in high affinity binding to one receptor chain forming a 1:1 hormone-receptor complex. This complex constitutes the template for binding a second, identical receptor molecule via binding site 2 (BS2), resulting in the active 1:2 complex. However, the model has been challenged by an increasing body of experimental evidence, initially reported for the homologous human erythropoietin receptor (2) and later for GHR (3) and PRLR (4). These studies suggest that preformed, inactive dimers exist in the absence of hormone. Thus, receptor dimerization is a necessary but not sufficient event for receptor activation and, notably, not strictly ligand-dependent. For both human erythropoietin receptor (5) and GHR (3), mechanistic models have been proposed, where receptor activation involves relative rotations and movements of receptor subunits induced by hormone binding. Allosteric reorganization of the intracellular receptor domains brings associated JAK2 kinases into close proximity, allowing their activation by cross-phosphorylation. This initial activation step triggers a cascade of molecular events leading to the functional receptor response (6).The...
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