(R)-Indolelactyl-CoA dehydratase, the key enzyme of tryptophan reduction to indolepropionate in Clostridium sporogenes
In this thesis the pathway of the fermentation from tryptophan to indolepropionate in Clostridium sporogenes and the key enzyme indolelactyl-CoA dehydratase were investigated. The intermediates and the end product were detected via mass spectrometry. Tryptophan disproportionates oxidatively via 3-in...
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|Summary:||In this thesis the pathway of the fermentation from tryptophan to indolepropionate in Clostridium sporogenes and the key enzyme indolelactyl-CoA dehydratase were investigated. The intermediates and the end product were detected via mass spectrometry. Tryptophan disproportionates oxidatively via 3-indolepyruvate to 3-indoleacetate and reductively via (R)-3-indolelactate and (E)-3-indoleacrylate to 3-indolepropionate (IPA). IPA, which is formed in the human intestine, is transferred via the blood to the brain. In the human brain IPA scavenges reactive oxygen species (ROS) and thus protects from Alzheimer’s disease. The activities of the enzymes from the reductive branch of tryptophan fermentation (tryptophan transaminase, indolelactate dehydrogenase, indolelactate dehydratase and indoleacrylate reductase) were determined in the cell-free extract. Similarly, phenylalaine is reduced by C. sprogenes via (R)-phenyllactate and (E)-cinnamate to 3-phenylpropionate. Further this thesis focused on the key enzyme indolelactyl-CoA dehydratase, which catalyzes a very unusual syn-elimination of a non-activated proton at the β-position and an OH-group at the α-position of the thioester carbonyl by employing radical intermediates. Surprisingly the dehydratase purified from cultures growing on tryptophan shows a higher specific activity with indolelactyl-CoA than with phenyllactyl-CoA. The reverse was the case with the dehydratase purified from cultures growing on phenylalanine. Both native dehydratases consist of three subunits, which were identified via peptide MALDI-TOF fingerprinting and Nano LC-MS as the CoA-transferase (FldA) and the heterodimeric dehdratase (FldBC). The peptide sequences of the three subunits from the culture growing on tryptophan as well as that on phenylalanine are identical and derived from the same genes fldABC. The recombinant FldBC produced in E. coli shows even a higher specific activity than the native dehydratase FldABC. It has been observed that the molecular mass of the subunit FldB of the native dehydratase is around 2 kDa smaller than that calculated from the gene. Peptide fingerprinting showed that the N- and C-termini remained, but one internal 4.3 kDa peptide could not be detected. The predicted model of FldBC based on the structure of 2-hydroxyisocaproyl-CoA dehydratase shows that the C-terminal half of this peptide could have been cut off by posttranslational splicing. Probably tryptophan and phenylalanine induce slightly different splicing positions, which result in different specific activities of the dehydratase.|