Biochemical and genetic characterization of benzylsuccinate synthase fromThauera aromatica: a new glycyl radical enzyme catalysing the first step in anaerobic toluene metabolism
| Autor: | Patric Hörth, Emile Schiltz, Wolfgang Haehnel, Christina Leutwein, Henk Schulz, Hermann Schägger, Johann Heider, Birgitta Leuthner |
|---|---|
| Rok vydání: | 1998 |
| Předmět: |
Gram-Negative Facultatively Anaerobic Rods
Thauera aromatica Operon Protein subunit Molecular Sequence Data Restriction Mapping Microbiology Cofactor Open Reading Frames Bacterial Proteins Amino Acid Sequence Anaerobiosis Carbon-Carbon Lyases Cloning Molecular Molecular Biology chemistry.chemical_classification Sequence Homology Amino Acid biology Molecular mass Sequence Analysis DNA Blotting Northern biology.organism_classification Enzymes Amino acid Molecular Weight Enzyme Biochemistry chemistry Genes Bacterial Benzylsuccinate synthase biology.protein Sequence Alignment Toluene |
| Zdroj: | Molecular Microbiology. 28:615-628 |
| ISSN: | 1365-2958 0950-382X |
| DOI: | 10.1046/j.1365-2958.1998.00826.x |
| Popis: | Toluene is anoxically degraded to CO2 by the denitrifying bacterium Thauera aromatica. The initial reaction in this pathway is the addition of fumarate to the methyl group of toluene, yielding benzylsuccinate as the first intermediate. We purified the enzyme catalysing this reaction, benzylsuccinate synthase (EC 4.1.99-), and studied its properties. The enzyme was highly oxygen sensitive and contained a redox-active flavin cofactor, but no iron centres. The native molecular mass was 220 kDa; four subunits of 94 (alpha), 90 (alpha'), 12 (beta) and 10 kDa (gamma) were detected on sodium dodecyl sulphate (SDS) gels. The N-terminal sequences of the alpha- and alpha'-subunits were identical, suggesting a C-terminal degradation of half of the alpha-subunits to give the alpha'-subunit. The composition of native enzyme therefore appears to be alpha2beta2gamma2. A 5 kb segment of DNA containing the genes for the three subunits of benzylsuccinate synthase was cloned and sequenced. The masses of the predicted gene products correlated exactly with those of the subunits, as determined by electrospray mass spectrometry. Analysis of the derived amino acid sequences revealed that the large subunit of the enzyme shares homology to glycyl radical enzymes, particularly near the predicted radical site. The highest similarity was observed with pyruvate formate lyases and related proteins. The radical-containing subunit of benzylsuccinate synthase is oxygenolytically cleaved at the site of the glycyl radical, producing the alpha'-subunit. The predicted cleavage site was verified using electrospray mass spectrometry. In addition, a gene coding for an activating protein catalysing glycyl radical formation was found. The four genes for benzylsuccinate synthase and the activating enzyme are organized as a single operon; their transcription is induced by toluene. Synthesis of the predicted gene products was achieved in Escherichia coli in a T7-promotor/polymerase system. |
| Databáze: | OpenAIRE |
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