Characterization of the catalase-peroxidase KatG from Burkholderia pseudomallei by mass spectrometry

The electron density maps of the catalase-peroxidase from Burkholderia pseudomallei (BpKatG) presented two unusual covalent modifications. A covalent structure linked the active site Trp(111) with Tyr(238) and Tyr(238) with Met(264), 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 Trp(111), Tyr(238), and Met(264), and a cluster at m/z similar to 4525, consistent with the fusion of two peptides linked through Trp(111) and Tyr(238). 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 Met(264) had been converted to homoserine, consistent with the covalent bond between Tyr(238) and Met(264) 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 Trp(105) and Tyr(226) and indirect evidence for a covalent linkage between Tyr(226) and Met(252). Tryptic peptide analysis and N-terminal sequencing revealed that the N-terminal residue of BpKatG is Ser(22).