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 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 approximately 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.
Donald
Lynda J
LJ
Department of Chemistry, University of Manitoba, Winnipeg, Manitoba R3T 2N2, Canada.
Krokhin
Oleg V
OV
Duckworth
Harry W
HW
Wiseman
Benjamin
B
Deemagarn
Taweewat
T
Singh
Rahul
R
Switala
Jack
J
Carpena
Xavi
X
Fita
Ignacio
I
Loewen
Peter C
PC
D000595:Amino Acid Sequence; D001426:Bacterial Proteins; D001665:Binding Sites; D016957:Burkholderia pseudomallei / Q000201:enzymology*; D006868:Hydrolysis; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry; D010544:Peroxidases / Q000737:chemistry* / Q000378:metabolism; D011487:Protein Conformation; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D001426:Bacterial Proteins; D010446:Peptide Fragments; D010544:Peroxidases; C076136:catalase HPI