A new xylanase from a Trichoderma harzianum strain.

A new xylanase (XYL2) was purified from solid-state cultures of Trichoderma harzianum strain C by ultrafiltration and gel filtration. SDS-PAGE of the xylanase showed an apparent homogeneity and molecular weight of 18 kDa. It had the highest activity at pH 5.0 and 45 degrees C and was stable at 50 degrees C and pH 5.0 up to 4 h xylanase. XYL2 had a low Km with insoluble oat spelt xylan as substrate. Compared to the amino acid composition of xylanases from Trichoderma spp, xylanase XYL2 presented a high content of glutamate/glutamine, phenylalanine and cysteine, and a low content of serine. Xylanase XYL2 improved the delignification and selectivity of unbleached hardwood kraft pulp.

Chemical synthesis of dendrotoxin-I: revision of the reported structure.

Dendrotoxin I (DTX-I) is a 60-residue peptide from the venom of the black mamba snake Dendroaspis polylepis, which binds to neuronal K+ channels. The structure reported previously for DTX-I was synthesized for the first time by a solution procedure. The synthetic product was confirmed to have the correct primary and disulfide structure determined by peptide mapping, sequence analysis and mass measurements. Comparison of synthetic DTX-I with the natural one by high-performance liquid chromatography and capillary zone electrophoresis, as well as by sequence analysis, revealed that the Asn residue at position 12 in the synthetic peptide was Asp in the natural product. Synthesis of DTX-I with Asp at position 12 gave a peptide identical with the natural product in all aspects. NMR analysis of synthetic [Asn12]- and [Asp12]-DTX-I also supported our findings that the Asn residue at position 12 in the DTX-I molecule should be revised as Asp. [Asn12]- and [Asp12]-DTX-I had very similar binding affinities when tested against radiolabeled dendrotoxin binding to rat brain synaptosomal membranes.

A new potassium channel toxin from the sea anemone Heteractis magnifica: isolation, cDNA cloning, and functional expression.

A new potassium channel toxin, HmK, has been isolated from the sea anemone Heteractis magnifica. It inhibits the binding of [125I]-alpha-dendrotoxin (a ligand for voltage-gated K channels) to rat brain synaptosomal membranes with a Ki of about 1 nM, blocks K+ currents through Kv 1.2 channels expressed in a mammalian cell line, and facilitates acetylcholine release at the avian neuromuscular junction. HmK comprises of 35 amino acids (Mr 4055) with the sequence R1TCKDLIPVS10ECTDIRCRTS20MKYRLNLCRK30TCGSC35. A full assignment of the disulfide linkages was made by using partial reduction with tri(2-carboxyethyl)phosphine (TCEP) at acid pH and rapid alkylation with iodoacetamide. The disulfide bridges were identified as Cys3-Cys35, Cys12-Cys28, and Cys17-Cys32. A cDNA clone encoding HmK was isolated using RT-PCR from the total RNA obtained from sea anemone tentacles, while the 5'- and 3'-flanking regions of the cDNA were amplified by RACE. The full-length cDNA was 563 bp long and contained a sequence encoding a signal peptide of 39 amino acids. The coding region for matured HmK toxin was cloned and expressed as a glutathione S-transferase (GST) fusion product in the cytoplasm of Escherichia coli. After affinity purification and cleavage, the recombinant toxin was shown to be identical to native HmK in its N-terminal sequence, chromatographic behavior, and binding to dendrotoxin binding sites on rat brain membranes.

On the convergent evolution of animal toxins. Conservation of a diad of functional residues in potassium channel-blocking toxins with unrelated structures.

BgK is a K+ channel-blocking toxin from the sea anemone Bunodosoma granulifera. It is a 37-residue protein that adopts a novel fold, as determined by NMR and modeling. An alanine-scanning-based analysis revealed the functional importance of five residues, which include a critical lysine and an aromatic residue separated by 6.6 +/- 1.0 A. The same diad is found in the three known homologous toxins from sea anemones. More strikingly, a similar functional diad is present in all K+ channel-blocking toxins from scorpions, although these toxins adopt a distinct scaffold. Moreover, the functional diads of potassium channel-blocking toxins from sea anemone and scorpions superimpose in the three-dimensional structures. Therefore, toxins that have unrelated structures but similar functions possess conserved key functional residues, organized in an identical topology, suggesting a convergent functional evolution for these small proteins.