Recombinant expression of an l-amino acid oxidase from the fungus Hebeloma cylindrosporum in Pichia pastoris including fermentation.

l-amino acid oxidases (LAAOs) are flavoenzymes that catalyze the oxidative deamination of l-amino acids to the corresponding α-keto acids, ammonia, and hydrogen peroxide. Here, we show the overexpression, purification, and the characterization of LAAO4 from the fungus Hebeloma cylindrosporum in the yeast Pichia pastoris with a 9His-tag and compare this with the recently characterized 6His-hcLAAO4 expressed in E. coli. The expression of the enzyme with an ER-signal sequence in P. pastoris resulted in a glycosylated, secreted protein. The enzymatic activity without activation was higher after expression in P. pastoris compared to E. coli. Due to treatment with acidic pH, a striking increase of activity could be detected for both expression systems resulting in similar specific activities after acid activation. Regarding the substrate spectrum, temperature stability, Km, and vmax values, hcLAAO4 showed very few differences when produced in these two expression systems. A higher yield of hcLAAO4 could be obtained by fermentation.

L-Amino acid oxidase of the fungus Hebeloma cylindrosporum displays substrate preference towards glutamate.

Catabolism of amino acids is a central process in cellular nitrogen turnover, but only a few of the mechanisms involved have been described from basidiomycete fungi. This study identified one such mechanism, the l-amino acid oxidase (Lao1) enzyme of Hebeloma cylindrosporum, by 2D gel separation and MS. We determined genomic DNA sequences of lao1 and part of its upstream gene, a putative pyruvate decarboxylase (pdc2), and cloned the cDNA of lao1. The two genes were also identified and annotated from the genome of Laccaria bicolor. The lao1 and pdc2 gene structures were conserved between the two fungi. The intergenic region of L. bicolor possessed putative duplications not detected in H. cylindrosporum. Lao1 sequences possessed dinucleotide-binding motifs typical for flavoproteins. Lao1 was less than 23 % identical to Lao sequences described previously. Recombinant Lao1 of H. cylindrosporum was expressed in Escherichia coli, purified and refolded with SDS to gain catalytic activity. The enzyme possessed broad substrate specificity: 37 l-amino acids or derivatives served as effective substrates. The highest activities were recorded with l-glutamate, but positively charged and aromatic amino acids were also accepted. Michaelis constants for six amino acids varied from 0.5 to 6.7 mM. We have thus characterized a novel type of Lao-enzyme and its gene from the basidiomycete fungus H. cylindrosporum.

Characterization of the ER-located zinc transporter ZnT1 and identification of a vesicular zinc storage compartment in Hebeloma cylindrosporum.

Metal tolerance of filamentous fungi is a poorly understood mechanism. In order to unravel the molecular basis of zinc (Zn) tolerance in the ectomycorrhizal fungal model Hebeloma cylindrosporum, we carried out a functional screening of an H. cylindrosporum cDNA library in the zrc1Δ mutant strain of Saccharomyces cerevisiae to search for genes conferring Zn tolerance to yeast cells. This strategy allowed the isolation of HcZnT1, a gene belonging to the cation diffusion facilitator family, which induced tolerance to Zn, but not to other metals. HcZnT1 was constitutively expressed in Hebeloma cells, whatever the Zn status of the medium and the fungal cell type (mycelia, sporocarps, mycorrhizas). A HcZnT1:GFP fusion protein was expressed in yeast and the corresponding fluorescence was recorded on endoplasmic reticulum membranes. Taken together, these different findings suggest a dual role of HcZnT1 in Zn homeostasis of fungal cells, by supplying requested Zn ions for the functioning of the endoplasmic reticulum as well as by detoxifying the cytosol under Zn stress. Zn pools were also investigated by using the Zn-specific fluorophore zinquin in H. cylindrosporum cells. Zinquin labeling revealed compartmentalization in intracellular vesicles interspersed throughout the cytoplasm that do not correspond to vacuolar compartments. Altogether the present data represent the first steps into the understanding of Zn homeostasis and tolerance in Hebeloma.