Identification of Two Catalases in Azotobacter vinelandii: a KatG Homologue and a Novel Bacterial Cytochrome c Catalase, CCC ^sub Av

Azotobacter vinelandii produces two detectable catalases during growth on minimal medium. The heat-labile catalase expressed during exponential growth phase was identified as a KatG homologue by liquid chromatography-tandem mass spectrometry (LC-MS/MS) using a mixed protein sample. The second catala...

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Bibliographic Details
Published inJournal of bacteriology Vol. 190; no. 3; p. 954
Main Authors Sandercock, James R, Page, William J
Format Journal Article
LanguageEnglish
Published Washington American Society for Microbiology 01.02.2008
Subjects
Bacteria
Bacteriology
Biochemistry
Chromatography
Enzymes
Mass spectrometry
Proteins
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Summary:Azotobacter vinelandii produces two detectable catalases during growth on minimal medium. The heat-labile catalase expressed during exponential growth phase was identified as a KatG homologue by liquid chromatography-tandem mass spectrometry (LC-MS/MS) using a mixed protein sample. The second catalase was heat resistant and had substantial residual activity after treatment at 90...C. This enzyme was purified by anion-exchange and size exclusion chromatography and was found to exhibit strong absorption at 407 nm, which is often indicative of associated heme moieties. The purified protein was fragmented by proteinase K and identified by LC-MS/MS. Some identity was shared with the MauG/bacterial cytochrome c peroxidase (BCCP) protein family, but the enzyme exhibited a strong catalase activity never before observed in this family. Because two putative c-type heme sites (CXXCH) were predicted in the peptide sequence and were demonstrated experimentally, the enzyme was designated a cytochrome c catalase (CCC...). However, the local organization of the CCC... heme motifs differed significantly from that of the BCCPs as the sites were confined to the C-terminal half of the catalase. A possible ... binding motif, previously described in the BCCPs, is also present in the CCC... peptide sequence. Some instability in the presence of EGTA was observed. Expression of the catalase was abolished in cccA mutants, resulting in a nearly 8,700-fold reduction in peroxide resistance in stationary phase. (ProQuest: ... denotes formulae/symbols omitted.)
ISSN:0021-9193
1098-5530