Journal article
Cloning, expression and crystallization of heterotetrameric sarcosine oxidase from Pseudomonas maltophilia
Protein expression and purification, v 43(1)
2005
PMID: 15922624
Featured in Collection : UN Sustainable Development Goals @ Drexel
Abstract
Heterotetrameric sarcosine oxidase (TSOX) is a complex bifunctional enzyme that catalyzes the oxidation of the methyl group in sarcosine (
N-methylglycine) and transfer of the oxidized methyl group into the one-carbon metabolic pool. In addition to four different subunits, TSOX contains three coenzymes (FAD, FMN, and NAD) and a binding site for tetrahydrofolate, the coenzyme acceptor of the oxidized methyl group from sarcosine. Based on preliminary success in crystallization of the natural enzyme, the genes encoding the subunits for TSOX from
Pseudomonas maltophilia (pTSOX) were cloned by functional screening of a genomic library. Recombinant enzyme exhibiting the same specific activity as natural pTSOX could not be isolated using a similar or identical purification procedure. This difficulty was overcome by affinity purification of recombinant pTSOX containing a C-terminal (His)
6 tag on the subunit (γ) encoded by
soxG, the gene located at the 3′ end of the pTSOX operon. Affinity-purified pTSOX could not be crystallized, a problem traced to microheterogeneity in the recombinant enzyme where about half of the FMN is present in a modified form that is not found in the natural enzyme and may be a biosynthetic intermediate. The modified flavin was eliminated by expression of the recombinant enzyme in the presence of sarcosine, the same reagent used to induce expression of the natural enzyme. Homogenous recombinant pTSOX was isolated from cells grown in the presence of sarcosine by chromatography on affinity and hydrophobic interaction matrices. High quality crystals that diffract to 1.85
Å resolution have been obtained.
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Details
- Title
- Cloning, expression and crystallization of heterotetrameric sarcosine oxidase from Pseudomonas maltophilia
- Creators
- Alshaimaa Hassan-Abdallah - Department of Biochemistry and Molecular Biology, Drexel University College of Medicine, Philadelphia, PA 19102, USAGuohua Zhao - Department of Biochemistry and Molecular Biology, Drexel University College of Medicine, Philadelphia, PA 19102, USAMichael Eschenbrenner - Department of Biochemistry and Molecular Biology, Drexel University College of Medicine, Philadelphia, PA 19102, USAZhi-wei Chen - Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, MO 63110, USAF. Scott Mathews - Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, MO 63110, USAMarilyn Schuman Jorns - Department of Biochemistry and Molecular Biology, Drexel University College of Medicine, Philadelphia, PA 19102, USA
- Publication Details
- Protein expression and purification, v 43(1)
- Publisher
- Elsevier
- Resource Type
- Journal article
- Language
- English
- Academic Unit
- Biochemistry and Molecular Biology; Civil, Architectural, and Environmental Engineering
- Web of Science ID
- WOS:000231367100005
- Scopus ID
- 2-s2.0-23244445190
- Other Identifier
- 991014877812504721
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- Collaboration types
- Domestic collaboration
- Web of Science research areas
- Biochemical Research Methods
- Biochemistry & Molecular Biology
- Biotechnology & Applied Microbiology