Expression, gene cloning, and characterization of five novel phytases from four basidiomycete fungi: Peniophora lycii, Agrocybe pediades, a Ceriporia sp., and Trametes pubescens.

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 phytase-encoding 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 degrees 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. 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).

Humicola insolens cellobiose dehydrogenase: cloning, redox chemistry, and "logic gate"-like dual functionality.

1Cellobiose dehydrogenase is a hemoflavoenzyme that catalyzes the sequential electron-transfer from an electron-donating substrate (e.g. cellobiose) to a flavin center, then to an electron-accepting substrate (e.g. quinone) either directly or via a heme center after an internal electron-transfer from the flavin to heme. We cloned the dehydrogenase from Humicola insolens, which encodes a protein of 761 amino acid residues containing an N-terminal heme domain and a C-terminal flavin domain, and studied how the catalyzed electron transfers are regulated. Based on the correlation between the rate and redox potential, we demonstrated that with a reduced flavin center, the enzyme, as a reductase, could export electron from its heme center by a "outer-sphere" mechanism. With the "resting" flavin center, however, the enzyme could have a peroxidase-like function and import electron to its heme center after a peroxidative activation. The dual functionality of its heme center makes the enzyme a molecular "logic gate", in which the electron flow through the heme center can be switched in direction by the redox state of the coupled flavin center.

The consensus concept for thermostability engineering of proteins.

Previously, sequence comparisons between a mesophilic enzyme and a more thermostable homologue were shown to be a feasible approach to successfully predict thermostabilizing amino acid substitutions. The 'consensus approach' described in the present paper shows that even a set of amino acid sequences of homologous, mesophilic enzymes contains sufficient information to allow rapid design of a thermostabilized, fully functional variant of this family of enzymes. A sequence alignment of homologous fungal phytases was used to calculate a consensus phytase amino acid sequence. Upon construction of the synthetic gene, recombinant expression and purification, the first phytase obtained, termed consensus phytase-1, displayed an unfolding temperature (T(m)) of 78.0 degrees C which is 15-22 degrees C higher than the T(m) values of all parent phytases used in its design. Refinement of the approach, combined with site-directed mutagenesis experiments, yielded optimized consensus phytases with T(m) values of up to 90.4 degrees C. These increases in T(m) are due to the combination of multiple amino acid exchanges which are distributed over the entire sequence of the protein and mainly affect surface-exposed residues; each individual substitution has a rather small thermostabilizing effect only. Remarkably, in spite of the pronounced increase in thermostability, catalytic activity at 37 degrees C is not compromised. Thus, the design of consensus proteins is a potentially powerful and novel alternative to directed evolution and to a series of rational approaches for thermostability engineering of enzymes and other proteins.

Tandem translation of Bacillus subtilis initiation factor IF2 in E. coli. Over-expression of infBB.su in E. coli and purification of alpha- and beta-forms of IF2B.su.

The protein synthesis initiation factor, IF2, in Bacillus subtilis has previously been characterized as being present in two forms, alpha and beta, of molecular mass 79 and 68 kDa, respectively, on the basis of their cross-reaction with anti-E. coli IF2 antibodies and by the DNA sequence of the gene for IF2, infBB.su. In this work we have cloned infBB.su in E. coli cells. Two proteins of molecular mass identical to the B. subtilis IF2 alpha and -beta were over-expressed and purified using a new three-step ion-exchange chromatography procedure. The N-terminal amino acid sequence of the two proteins was determined and the results confirmed that the two forms were IF2 alpha and -beta, both encoded by the infB gene. The N-terminal amino acid sequence determined for IF2 beta is Met94-Gln-Asn-Asn-Gln-Phe. The presence of methionine at position 94 shows that this form is, in fact, the result of a second translational initiation in infBB.su mRNA, since the codon at amino acid position 94 is GUG, which is the normal codon for valine, but also known to be an initiator codon. This is a new example of the unusual tandem translation in E. coli of an open mRNA reading frame.

OmpT proteolysis of E. coli initiation factor IF2. Elimination of a cleavage site by site-directed mutagenesis.

A serious problem during purification of the E. coli initiation factor IF2 is a significant loss of native IF2 due to partial degradation. We have previously shown that the major fragment, IF2 gamma (65 kDa), is the result of cleavage of IF2 alpha at the peptide bond between lysine 289 and arginine 290. In this paper we demonstrate that the cleavage is a result of proteolysis by outer membrane protease OmpT occurring immediately after cell lysis and in the S30 supernatant. By protein engineering we have constructed an IF2 mutant Lys289-greater than Met and shown that the IF2 gamma cleavage site in this mutant protein is insensitive to cleavage by OmpT. However the mutant protein is cleaved by OmpT between arginine 279 and alanine 280, which is a novel sequence specificity for this protease.

Tandem translation of E. coli initiation factor IF2 beta: purification and characterization in vitro of two active forms.

Two forms of E. coli initiation factor IF2, IF2 alpha and IF2 beta, have been known for several years. Both forms are products of the gene infB with translational initiation at codon 1 (AUG) and codon 158 (GUG) in the same reading frame. In this work we demonstrate that IF2 beta exists in two forms, IF2 beta and IF2 beta' with initiation codons 158 (GUG) and 165 (AUG) and molecular masses of 79.7 kDa and 78.8 kDa respectively. We have recently described a fast purification method for IF2 alpha, using an FPLC procedure consisting of ion-exchange liquid chromatography on Q Sepharose HP, Mono Q and Mono S. After the Mono Q step, an apparently homogeneous IF2 beta was observed when analyzed by SDS-PAGE. However the chromatography on Mono S results in the elution of two peaks containing IF2 beta. The N-terminal amino acid sequence of the two proteins identified the first peak to be IF2 beta and the second as a protein which we term IF2 beta' starting seven residues downstream at the AUG codon 165. The activity in vitro of the two purified forms of IF2 beta was tested by measuring the stimulation of binding of the initiator fMet-tRNA(fMet) to 70S ribosomes in the presence of GTP and poly(A,U,G) as messenger-RNA. In this assay no difference in activity is detected.

[Hallucinogenic psilocybine containing mushrooms. Toxins contained in Danish wild mushrooms]. / Hallucinogene psilocybinholdige svampe. Dansk vildtvoksende rusgift.

A number of the wild Danish mushrooms contain the hallucinogenic agent psilocybin which resembles LSD in many ways. The commonest of these are the "liberty cap" or "magic mushrooms" (Psilocybe semilanceata). On the basis of experience from USA and western Europa, increase in employment of this mushrooms as a hallucinogenic intoxicant may be anticipated in Denmark. The history, epidemiology, botany and pharmacology of the mushroom are reviewed. Clinical pictures and treatment are described for: 1) Acute poisoning with psilocybin-containing fungi, 2) Late sequelae of consumption of psilocybin-containing fungi and 3) Poisoning with more poisonous fungi on account of incorrect identification. General practitioners, duty roster doctors, doctors in casualty departments and in acute psychiatric departments should be aware of these problems. Intoxication with psilocybin may be confused with panic anxiety or euphoria in persons with mydriasis and other sympathomimetic symptoms. The possibility of more serious mushroom poisoning on account of incorrect identification should be borne in mind.