Cloning and sequencing of chloroperoxidase cDNA

Area of Science:

• Biochemistry
• Molecular Biology
• Mycology

Background:

• Chloroperoxidase is a heme-containing enzyme found in Caldariomyces fumago.
• Understanding its structure is crucial for elucidating its catalytic mechanism.

Purpose of the Study:

• To determine the complete primary amino acid sequence of Caldariomyces fumago chloroperoxidase.
• To identify key structural features and potential functional sites within the enzyme.

Main Methods:

• Preparation of an oligod-d(T) 12-18 primed cDNA library from Caldariomyces fumago mRNA.
• Sequencing of a full-length cDNA insert cloned into the pBR322 plasmid.
• Amino acid sequencing to determine approximately 73% of the peptide sequence.

Main Results:

• The complete primary sequence of chloroperoxidase, comprising 300 amino acids (32,974 daltons), was derived.
• DNA sequence data correlated with known peptide sequences.
• A putative 21-amino acid signal peptide was identified.
• Three potential glycosylation sites (Asn-X-Thr/Ser) and three cysteine residues were found.
• Cys87 showed minimal homology to the active site of cytochrome P450cam, suggesting a role in heme binding.

Conclusions:

• The derived DNA sequence provides the complete primary structure of Caldariomyces fumago chloroperoxidase.
• Cys87 is proposed to function as a thiolate ligand for the heme iron.
• The gene utilizes the rare arginine codon AGG more frequently than typically observed in E. coli or yeast.