Per Olof Nyman

Characterization of a laccase gene from the white-rot fungus Trametes versicolor and structural features of basidiomycete laccases

A gene coding for the multi-copper phenol oxidase laccase has been isolated from the white-rot basidiomycete Trametes versicolor. The gene, which is preceded by a TATA box and a pyrimidine-rich region, is predicted to contain ten introns. The mature translation product, preceded by a 22-residue signal peptide, should consist of 498 residues. Comparisons with Edman degradation data of peptides from T. versicolor laccase strongly suggest that two disulfide bridges are formed by Cys-85/Cys-487 and Cys-117/Cys-205, respectively. The encoded protein contains five Cys, and the sequence surrounding the remaining Cys-452 is consistent with its involvement in the ligation of type-1 copper. Alignment of sequences indicates that T. versicolor laccase displays a Phe at the position corresponding to a residue (Met in ascorbate oxidase and azurin) considered important for the reduction potential of type-1 copper proteins.

The amino acid sequence of Stellacyanin from the lacquer tree.

Summary The complete amino acid sequence of stellacyanin, a blue copper-containing glycoprotein, is presented. The protein has been enzymatically cleaved by trypsin, chymotrypsin, and by the protease from the Staphylococcus aureus strain U8. Stellacyanin consists of a single polypeptide chain of 107 amino acid residues. The protein contains one SH-group and one disulfide bond. The carbohydrate moieties, which are not yet characterized, are attached to the protein at three different positions, all having the characteristic sequence Asn-X-Thr.

Insulin secretion is highly sensitive to desorption of plasma membrane cholesterol

Cholesterol‐rich clusters of SNARE (soluble NSF attachment protein receptor) proteins have been implicated as being important for exocytosis. Here we demonstrate the significance of cholesterol for normal biphasic insulin secretion in mouse β cells by removal of cholesterol from the plasma membrane using methyl‐β‐cyclodextrin (MBCD). Maximal inhibition of insulin secretion in static incubations was achieved using 0.1 mM MBCD. In in situ pancreatic perfusion measurements, both first and second phase insulin secretions were reduced by ~50% (P<0.05). This was accompanied by a reduced number of docked large dense core vesicles (LDCVs) (~40%;P<0.01) and a reduced exocytotic response (>50%; P<0.01). Fur‐ther, subcellular fractionations demonstrated move‐ment of the synaptosomal protein of 25 kDa (SNAP‐25) from the plasma membrane to the cytosol after MBCD treatment. The inhibitory actions of MBCD were coun‐teracted by subsequent addition of cholesterol. We hypothesize that desorption of cholesterol leads to the disturbance of a basic exocytotic mechanism partly due to migration of SNAP‐25, and we conclude that insulin secretion is highly sensitive to changes in plasma mem‐brane cholesterol.—Vikman, J., Jimenez‐Feltstrom, J., Nyman, P., Thelin, J., Eliasson, L. Insulin secretion is highly sensitive to desorption of plasma membrane cholesterol. FASEBJ. 23, 58‐67 (2009)

A CLUSTER OF GENES ENCODING MAJOR ISOZYMES OF LIGNIN PEROXIDASE AND MANGANESE PEROXIDASE FROM THE WHITE-ROT FUNGUS TRAMETES VERSICOLOR

A gene cluster from the white-rot basidiomycete Trametes (Coriolus) versicolor (Tv) PRL 572 containing three structural genes, LPGIII, LPGIV and MPGI, was characterized. The genes are arranged in the same transcriptional direction, within a 10-kb region, and found to encode quantitatively dominant isozymes of lignin peroxidase (LP) and manganese peroxidase (MP). The second gene in sequence, LPGIV, predicts a 346-amino-acid (aa) mature polypeptide (36.9 kDa, pI 4.31) which is identical with the partial aa sequence information available on the LP12 isozyme (43.1 kDa, pI 3.27). The first gene, LPGIII, encodes a 341-aa polypeptide (36.1 kDa, pI 3.93) which has not been identified at the protein level. However, the similarity of LPGIV would suggest that the predicted product is an LP-type enzyme. LPGIII and LPGIV are homologous to the tandemly arranged genes LPGII and LPGI, respectively, recently described by Jönsson and Nyman [Biochim. Biophys. Acta 1218 (1994) 408-412]. The homologous genes, LPGIII/LPGII and LPGIV/LPGI, are 99% and 96% identical in sequence, respectively, and are predicted to encode identical polypeptides, since base substitutions in the predicted exons are all synonymous. The third gene, MPGI, is different in intron-exon organization and predicted to be disrupted by five rather than six introns, as are the LP genes. The deduced polypeptide, 339 aa in size (35.9 kDa, pI 4.07), is identical with the partial aa sequence information available for isozyme MP2 (44.5 kDa, pI 3.09). The MPGI- and LPGIV-encoded polypeptides are 70% identical in sequence which suggests that MP and LP from Tv may be regarded as members of the same family within the plant peroxidase superfamily. Most importantly, this study identifies a gene encoding the MP2 isozyme, and further shows that genes encoding MP and LP can be closely linked on the chromosome and may be coordinately transcribed.

The complete triphosphate moiety of non-hydrolyzable substrate analogues is required for a conformational shift of the flexible C-terminus in E. coli dUTP pyrophosphatase

The molecular mechanism of substrate analogue interaction with Escherichia coli dUTPase was investigated, using the non‐hydrolyzable 2′‐deoxyuridine 5′‐(α,β‐imido)triphosphate (α,β‐imido‐dUTP). Binding of this analogue induces a difference in the far UV circular dichroism (CD) spectrum arguing for a significant change in protein conformation. The spectral shift is strictly Mg2+‐dependent, does not appear with dUDP instead of α,β‐imido‐dUTP and is not elicited if the flexible C‐terminal arm is deleted from the protein by limited tryptic digestion. Involvement of the C‐terminal arm in α,β‐imido‐dUTP binding is consistent with the finding that this analogue protects against tryptic hydrolysis at Arg‐141. Near UV CD of ligand‐enzyme complexes reveals a characteristic difference in the microenvironments of enzyme‐bound dUDP and α,β‐imido‐dUTP, a difference not observable in C‐terminally truncated dUTPase. The results suggest that (i) closing of the active site during the catalytic cycle, through the movement of the C‐terminal arm, requires the presence of the complete triphosphate moiety of the substrate in complex with Mg2+, and (ii) after catalytic cleavage the active site pops open to facilitate product release.

Differential regulation of mnp2, a new manganese peroxidase-encoding gene from the ligninolytic fungus Trametes versicolor PRL 572

ABSTRACT A peroxidase-encoding gene, mnp2, and its corresponding cDNA were characterized from the white-rot basidiomycete Trametes versicolor PRL 572. We used quantitative reverse transcriptase-mediated PCR to identify mnp2 transcripts in nutrient-limited stationary cultures. Although mnp2 lacks upstream metal response elements (MREs), addition of MnSO4 to cultures increased mnp2 transcript levels 250-fold. In contrast, transcript levels of an MRE-containing gene of T. versicolor, mnp1, increased only eightfold under the same conditions. Thus, the manganese peroxidase genes in T. versicolor are differentially regulated, and upstream MREs are not necessarily involved. Our results support the hypothesis that fungal and plant peroxidases arose through an ancient duplication and folding of two structural domains, since we found the mnp1 and mnp2 polypeptides to have internal homology.

dUTPase from the retrovirus equine infectious anemia virus: specificity, turnover and inhibition

The kinetic properties of dUTPase from equine infectious anemia virus (EIAV) were investigated. K M (1.1 ± 0.1 μM) and k cat (25 s−1) were found to be independent of pH in the neutral pH range. Above pH 8.0, K M increases slightly. Below pH 6.0, the enzyme is rapidly deactivated. Detergent was found to enhance activity, leaving K M and k cat unaffected. Compared to the Escherichia coli dUTPase, the EIAV enzyme is equally potent in hydrolyzing dUTP, but less specific. Inhibition of the viral enzyme by the nucleotides dTTP, dUMP and a synthetic analogue, 2′‐deoxyuridine 5′‐(α,β‐imido)triphosphate, is stronger by one order of magnitude.

A novel type of peroxidase gene from the white-rot fungus Trametes versicolor

The wood-decaying fungus Trametes versicolor secretes a large number of peroxidase isozymes, presumed to partake in the degradation of lignin. From enzymic studies, two types of peroxidases have been distinguished: lignin peroxidases and manganese peroxidases. We here report the finding of a T. versicolor peroxidase gene, PG V, which displays several features not observed in previously studied peroxidase genes from white-rot fungi, such as a high number of introns (12). Eight of the 12 introns have positions equivalent to introns of peroxidase genes from another white-rot fungus, Phanerochaete chrysosporium. The gene structure of PG V appears to be primarily related to known lignin peroxidase genes, while the encoded mature 339-residue protein has several characteristics in common with manganese peroxidases. Analyses further indicate that PG V encodes a Ser instead of an Asn at a position regarded as invariant within the enzyme superfamily, with the side chain involved in hydrogen bonding with the distal His.

Identification of tyrosine as a functional residue in the active site of Escherichia coli dUTPase

dUTP nucleotidohydrolase (dUTPase, EC 3.6.1.23) from E. coli contains a total of six tyrosine residues per trimer. About half of them were found to be susceptible to acetylation with N-acetylimidazole or to nitration with tetranitromethane with concomitant loss of activity. Deacetylation with N-hydroxylamine leads to full reactivation. Inhibitory products of dUTP hydrolysis, i.e., dUMP and inorganic pyrophosphate together with the cofactor Mg2+ protect significantly against inactivation and chemical modification. In the Cu(2+)-dUTPase complex, charge transfer from Cu2+ to the tyrosinate anion was perturbed by the presence of the substrate dUTP. These results, together with the occurrence of one tyrosine residue in a strictly conserved sequence motif suggest the critical importance of this residue for the function of the enzyme.

Trametes versicolor ligninase: Isozyme sequence homology and substrate specificity

The substrate specificity of three ligninase isozymes from the white‐rot fungus Trametes versicolor has been investigated using stereochemically defined synthetic dimeric models for lignin. The isozymes have been found to attack non‐phenolic β‐O‐4 as well as β‐1 lignin model compounds. This finding confirms the classification of the isozymes from T. versicolor as ligninases. The amino‐terminal residues of the three isozymes from T. versicolor have been determined using Edman degradation. Minor differences found between the sequences suggest the existence of several structural genes for ligninase in T. versicolor. Comparisons have been made with the sequences of three previously reported ligninases from Phanerochaete chrysosporium, another lignin‐degrading fungus. One of the sequences from P. chrysosporium is distinctly more similar to the T. versicolor isozymes than to the other two sequences from P. chrysosporium.