Characterization of the Catalase-Peroxidase KatG from Burkholderia pseudomallei by Mass Spectrometry

• Published in: The Journal of biological chemistry 2003-09, Vol.278 (37), p.35687-35692
• Main Authors: Donald, Lynda J., Krokhin, Oleg V., Duckworth, Harry W., Wiseman, Benjamin, Deemagarn, Taweewat, Singh, Rahul, Switala, Jack, Carpena, Xavi, Fita, Ignacio, Loewen, Peter C.
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Bacterial Proteins; Binding Sites; Burkholderia pseudomallei - enzymology; Hydrolysis; Mass Spectrometry; Molecular Sequence Data; Peptide Fragments - chemistry; Peroxidases - chemistry; Peroxidases - metabolism; Protein Conformation; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M304053200

The electron density maps of the catalase-peroxidase from Burkholderia pseudomallei (BpKatG) presented two unusual covalent modifications. A covalent structure linked the active site Trp111 with Tyr238 and Tyr238 with Met264, and the heme was modified, likely by a perhydroxy group added to the vinyl group on ring I. Mass spectrometry analysis of tryptic digests of BpKatG revealed a cluster of ions at m/z 6585, consistent with the fusion of three peptides through Trp111, Tyr238, and Met264, and a cluster at m/z ∼4525, consistent with the fusion of two peptides linked through Trp111 and Tyr238. MS/MS analysis of the major ions at m/z 4524 and 4540 confirmed the expected sequence and suggested that the multiple ions in the cluster were the result of multiple oxidation events and transfer of CH3-S to the tyrosine. Neither cluster of ions at m/z 4525 or 6585 was present in the spectrum of a tryptic digest of the W111F variant of BpKatG. The spectrum of the tryptic digest of native BpKatG also contained a major ion for a peptide in which Met264 had been converted to homoserine, consistent with the covalent bond between Tyr238 and Met264 being susceptible to hydrolysis, including the loss of the CH3-S from the methionine. Analysis of the tryptic digests of hydroperoxidase I (KatG) from Escherichia coli provided direct evidence for the covalent linkage between Trp105 and Tyr226 and indirect evidence for a covalent linkage between Tyr226 and Met252. Tryptic peptide analysis and N-terminal sequencing revealed that the N-terminal residue of BpKatG is Ser22.