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Characterization of the Catalase-Peroxidase KatG from Burkholderia pseudomallei by Mass Spectrometry
- Source :
- Digital.CSIC. Repositorio Institucional del CSIC, instname
- Publication Year :
- 2003
- Publisher :
- Elsevier BV, 2003.
-
Abstract
- 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, Tyr 238, 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 CH 3-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 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 Trp105 and Tyr226 and indirect evidence for a covalent linkage between Tyr226 and Met 252. Tryptic peptide analysis and N-terminal sequencing revealed that the N-terminal residue of BpKatG is Ser22.<br />This work was supported by Grants BIO099-0865 and BIO2002-04419 from Direccion General de Investigacion Ciencia y Technologia (to I. F.) and Grant OGP9600 from the Natural Sciences and Engineering Research Council of Canada (to P. C. L.)
- Subjects :
- Burkholderia pseudomallei
Protein Conformation
Stereochemistry
Molecular Sequence Data
Homoserine
Peptide
medicine.disease_cause
Mass spectrometry
Biochemistry
Mass Spectrometry
chemistry.chemical_compound
Bacterial Proteins
medicine
Amino Acid Sequence
Molecular Biology
Escherichia coli
Heme
Catalase-peroxidase
chemistry.chemical_classification
Binding Sites
biology
Hydrolysis
Active site
Cell Biology
Peptide Fragments
Peroxidases
chemistry
Covalent bond
Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
biology.protein
Subjects
Details
- ISSN :
- 00219258
- Volume :
- 278
- Database :
- OpenAIRE
- Journal :
- Journal of Biological Chemistry
- Accession number :
- edsair.doi.dedup.....16d4100a87cd8cb898c5f75fbb8ed6a0