Identification of two catalases in Azotobacter vinelandii: a KatG homologue and a novel bacterial cytochrome c catalase, CCCAv.
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 degrees 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(Av)). However, the local organization of the CCC(Av) heme motifs differed significantly from that of the BCCPs as the sites were confined to the C-terminal half of the catalase. A possible Ca2+ binding motif, previously described in the BCCPs, is also present in the CCC(Av) 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.
Department of Biological Sciences, University of Alberta, Edmonton, Alberta, Canada T6G2E9.
James R
JR
Sandercock
William J
WJ
Page
D000595:Amino Acid Sequence; D016948:Azotobacter vinelandii / Q000187:drug effects / Q000201:enzymology* / Q000254:growth & development / Q000502:physiology*; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D002374:Catalase / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D002853:Chromatography, Liquid; D045304:Cytochromes c / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D004795:Enzyme Stability; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D006358:Hot Temperature; D006861:Hydrogen Peroxide / Q000494:pharmacology; D008969:Molecular Sequence Data; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D012808:Sigma Factor / Q000378:metabolism; D053719:Tandem Mass Spectrometry
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D012808:Sigma Factor; C069567:sigma factor KatF protein, Bacteria; D045304:Cytochromes c; D006861:Hydrogen Peroxide; D002374:Catalase; C051253:katG protein, E coli