1887

Abstract

Degenerate oligonucleotide primers based on internal peptide sequences obtained by HPLC from purified catalase were used to locate the and subsp. regions by PCR. Southern hybridization analysis with a probe derived from a 11 kb PCR-amplified fragment showed that a single copy of the putative catalase gene was present in the and subsp. chromosome. The nucleotide sequence of revealed a 1518 bp open reading frame for a protein with 505 amino acids and a predicted molecular mass of 58347 Da, whereas subsp. is 1368 nt long and encodes a polypeptide of 455 amino acids with a predicted molecular mass of 52584 Da. These catalases are highly homologous to typical monofunctional catalases from prokaryotes. The active-site residues, proximal and distal haem-binding ligands and NADPH-binding residues of the bovine liver catalase-type enzyme were highly conserved in KatA. cells carrying cloned had a catalase activity approximately 1000 times that of untransformed , but no detectable increase in catalase activity was observed with carrying cloned . Northern blotting showed the presence of a -specific transcript in subsp. , suggesting that the lack of catalase activity in this bacterium is due to a post-transcriptional alteration. Compared to the nucleotide sequence of , showed a single base-pair deletion and six mis-sense mutations, and these alterations were present in three other subsp. strains analysed. The deletion, located at 1338 bp from the initiation codon, originates a shift of the nucleotide reading frame and is responsible for the premature translation termination at 1368 bp, generating a KatB polypeptide 50 amino acid residues shorter than KatA. Moreover, four of the mis-sense mutations present in lead to non-conservative amino acid replacements, the most significant being that located at residue 317 (Pro in KatA→Ser in KatB) because the affected amino acid is involved in determining the proximal haem-binding site. Both the main alterations found in KatB (the deletion and the substitution in residue 317) seem to contribute to the lack of catalase activity in subsp. , as deduced from results obtained with chimeric catalase constructs.

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2000-02-01
2020-09-29
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