Expression, Purification, and Characterization of Blasticidin S Deaminase (BSD) from Aspergillus terreus: The Role of Catalytic Zinc in Enzyme Structure

• Published in: Journal of biochemistry (Tokyo) Vol. 127; no. 6; pp. 955 - 963
• Main Authors: Kimura, Makoto, Sekido, Shigeko, Isogai, Yasuhiro, Yamaguchi, Isamu
• Format: Journal Article
• Language: English
• Published: England Oxford University Press 01.06.2000
• Subjects: Amino Acid Motifs; Amino Acid Sequence; Aminohydrolases - chemistry; Aminohydrolases - genetics; Aminohydrolases - isolation & purification; Aminohydrolases - metabolism; Aspergillus - enzymology; Carrier Proteins - chemistry; catalytic and structural zinc; Circular Dichroism; Conserved Sequence; Cytidine Deaminase - chemistry; cytosine nucleoside/nucleotide deaminases heat-modifiable protein; Electrophoresis, Polyacrylamide Gel; Enzyme Stability; Escherichia coli - genetics; Molecular Sequence Data; Mutagenesis, Site-Directed; Phenylmercury Compounds - chemistry; Protein Denaturation; Protein Folding; protein folding and stability; Protein Structure, Quaternary; Recombinant Proteins - chemistry; Recombinant Proteins - metabolism; Sequence Alignment; site-directed mutagenesis; Spectrometry, Fluorescence; Zinc - chemistry
• ISSN: 0021-924X; 1756-2651
• DOI: 10.1093/oxfordjournals.jbchem.a022711

We established an efficient overproduction-purification system for blasticidin S deaminase (BSD) using the cDNA cloned from Aspergillus terreus. The estimated molecular mass of the purified enzyme indicated BSD was a tetramer. This tetrameric form was very resistant to denaturation by SDS and showed heat-modifiable behavior on SDS-PAGE; i.e., BSD migrated much slower (as a single band of 36 kDa) in its active conformation than its completely denatured polypeptide (13 kDa) if heat treatment in 2% SDS was not performed before electrophoresis. As predicted from the presence of the catalytic zinc-coordinating sequence motif conserved in the cytosine nucleoside/nucleotide deaminase family, BSD also contained one zinc per deaminase subunit. However, the predicted catalytic function appeared not to be the only role of this zinc in the enzyme. First, titration of the zinc-chelating -SH groups with p-hydroxymercuriphenylsulfonate led to dissociation of the BSD tetramer into unstable monomers or aimers. Second, depletion of zinc on reconstitution of chemically denatured BSD (with either guanidine-HCl or acidic pH) resulted in improper folding of the polypeptide. These results suggest that zinc also plays a structural role in maintenance of the protein structure. When we introduced mutations at Glu-56 (the proposed active site) and Cys-91 (a proposed catalytic zinc-binding Cys) in BSD, none of the resulting mutants (E56D, E56Q, C91A, C91S, and C91H) showed any detectable activity, as judged with the spectrophotometric assay. Replacements of Cys-91 resulted in gross perturbation of the enzyme structure although the catalytically essential Glu-56 was not necessarily required for proper folding of the enzyme. These results further support our proposal that the catalytic zinc coordinated by the conserved sequence motif is also structural in BSD.