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

被引:37
|
作者
Donald, LJ
Krokhin, OV
Duckworth, HW
Wiseman, B
Deemagarn, T
Singh, R
Switala, J
Carpena, X
Fita, I
Loewen, PC [1 ]
机构
[1] Univ Manitoba, Dept Microbiol, Winnipeg, MB R3T 2N2, Canada
[2] Univ Manitoba, Dept Chem, Winnipeg, MB R3T 2N2, Canada
[3] Univ Manitoba, Dept Phys, Winnipeg, MB R3T 2N2, Canada
[4] CSIC, Inst Biol Mol Barcelona, ES-08034 Barcelona, Spain
关键词
D O I
10.1074/jbc.M304053200
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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).
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收藏
页码:35687 / 35692
页数:6
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