PRIMARY STRUCTURE AND CHARACTERIZATION OF THE VANADIUM CHLOROPEROXIDASE FROM THE FUNGUS CURVULARIA-INAEQUALIS

Using reverse transcription of messenger RNA followed by amplification using the polymerase chain reaction, three overlapping cDNA fragments encompassing the encoding sequence of the vanadium chloroperoxidase f rom the fungus Curvularia inaequalis were isolated and sequenced. The sequence was confirmed by DNA sequence analysis of genomic DNA. The de duced amino acid sequence predicts a protein of 609 residues with a ma ss of 67488 Da. Competitive reverse-transcription polymerase chain rea ction analysis indicates that vanadium chloroperoxidase expression tak es place in the secondary-growth phase initiated by nutrient depletion . Southern-blot analysis of genomic DNA indicates that there is only a single gene encoding the vanadium chloroperoxidase and that no isoenz ymes are present. The N-terminal amino acid residue was blocked and co uld not be determined by amino acid sequencing, although evidence is p resented showing that the N-terminal region starts very close to the f irst encoded methionine residue. Although the vanadium chloroperoxidas e is secreted, it was not possible to assign a leader peptide. The pro tein contains two putative N-glycosylation sites but experiments indic ate that the protein is non-glycosylated, Two cysteine residues are pr esent in the protein both as free thiols; no disulphide bridging was f ound. Metal analysis revealed that iron, copper, and calcium do not co nstitute part of the protein. Zinc was found at a ratio of 0.3 +/- 0.0 4 mol/mol protein. Boiling and subsequent SDS/PAGE of the protein samp le showed a typical degradation pattern of the enzyme. Amino acid sequ ence analysis of the resulting peptides showed that the cleavage took place at Asp-Pro bonds of which six are located throughout the protein . No sequence similarity with other known peroxidases was found except for one small region, sharing limited similarity with bacterial halop eroxidases and other alpha/beta-hydrolase-fold enzymes. In the case of the bacterial bromoperoxidases from this group, a methionine located in this region was suggested to have a role in catalysis. Methionine, however, was not involved in the catalysis of the vanadium chloroperox idase.