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

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.

SUBMITTER: Hassan-Abdallah A

PROVIDER: S-EPMC1993822 | biostudies-literature | 2005 Sep

REPOSITORIES: biostudies-literature

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

Hassan-Abdallah Alshaimaa A Zhao Guohua G Eschenbrenner Michael M Chen Zhi-Wei ZW Mathews F Scott FS Jorns Marilyn Schuman MS

Protein expression and purification 20050413 1

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 ...[more]

PMID: 15922624

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Project description:UnlabelledSarcosine (N-methylglycine) is present in many environments inhabited by pseudomonads and is likely most often encountered as an intermediate in the metabolism of choline, carnitine, creatine, and glyphosate. While the enzymology of sarcosine metabolism has been relatively well studied in bacteria, the regulatory mechanisms governing catabolism have remained largely unknown. We previously determined that the sarcosine-catabolic (sox) operon of Pseudomonas aeruginosa is induced by the AraC family regulator GbdR in response to glycine betaine and dimethylglycine. However, induction of these genes was still observed in response to sarcosine in a gbdR deletion mutant, indicating that an independent sarcosine-responsive transcription factor also acted at this locus. Our goal in this study was to identify and characterize this regulator. Using a transposon-based genetic screen, we identified PA4184, or SouR (sarcosine oxidation and utilization regulator), as the sarcosine-responsive regulator of the sox operon, with tight induction specificity for sarcosine. The souR gene is required for appreciable growth on sarcosine as a carbon and nitrogen source. We also characterized the transcriptome response to sarcosine governed by SouR using microarray analyses and performed electrophoretic mobility shift assays to identify promoters directly regulated by the transcription factor. Finally, we characterized PA3630, or GfnR (glutathione-dependent formaldehyde neutralization regulator), as the regulator of the glutathione-dependent formaldehyde detoxification system in P. aeruginosa that is expressed in response to formaldehyde released during the catabolism of sarcosine. This study expands our understanding of sarcosine metabolic regulation in bacteria through the identification and characterization of the first known sarcosine-responsive transcriptional regulator.ImportanceThe Pseudomonas aeruginosa genome encodes many diverse metabolic pathways, yet the specific transcription regulators controlling their expression remain mostly unknown. Here, we used a genetic screen to identify the sarcosine-specific regulator of the sarcosine oxidase operon, which we have named SouR. SouR is the first bacterial regulator shown to respond to sarcosine, and it is required for growth on sarcosine. Sarcosine is found in its free form and is also an intermediate in the catabolic pathways of glycine betaine, carnitine, creatine, and glyphosate. The similarity of SouR to the regulators of carnitine and glycine betaine catabolism suggests evolutionary diversification within this regulatory family to allow response to structurally similar but physiologically distinct ligands.

Dataset Information

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

Publications

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

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