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Protein Expr Purif. 2005 Sep;43(1):33-43. Epub 2005 Apr 13.

Cloning, expression and crystallization of heterotetrameric sarcosine oxidase from Pseudomonas maltophilia.

Author information

1
Department of Biochemistry and Molecular Biology, Drexel University College of Medicine, Philadelphia, PA 19102, USA.

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 (gamma) 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 A resolution have been obtained.

PMID:
15922624
PMCID:
PMC1993822
DOI:
10.1016/j.pep.2005.03.023
[Indexed for MEDLINE]
Free PMC Article

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