Activation and Regulation of the 4-Hydroxyphenylacetate Decarboxylase System from C. difficile
Summary Humans must adopt to live in a microbial world. The number of microbes associated with the human body alone exceeds the total number of body cells by more than one order of magnitude. Besides, the overall genetic information harboured by the microbial consortium in the human gut exceeds by...
|Online Access:||PDF Full Text|
No Tags, Be the first to tag this record!
Humans must adopt to live in a microbial world. The number of microbes associated with the human body alone exceeds the total number of body cells by more than one order of magnitude. Besides, the overall genetic information harboured by the microbial consortium in the human gut exceeds by many times the human genomic information. Some of these informations exhibit detrimental effects on the human well-being. The endeavour to understand these reactions more precisely does not only inspire the scientific community, but was also the driving force for this work concerning the activation and regulation of the 4-hydroxyphenylacetate (HPA) decarboxylase system of Clostridium difficile.
The HPA decarboxylase system catalyses the final step in tyrosine-fermentation, liberating the toxic end product p-cresol. The postulated mechanism of this reaction includes a number of radical intermediates and is tightly regulated on the protein as well as on the DNA level. Besides the two protein partners (activating enzyme: HpdA, and decarboxylase: HpdBC) the system includes five redox-active iron-sulfur centres, is dependent on S-adenosylmethionine (SAM) as radical generator and requires an external electron-source.
During the time-course of this work it could be established that the activating enzyme contains three catalytically essential [4Fe-4S] clusters. The other members of this protein-family (SAM radical family) coordinate just one, previously already well characterised cluster. The catalytic need for the additional clusters could be evaluated by mutational analysis of the cluster binding sites and biochemical analysis. In the presence of SAM and an electron source the activating enzyme initiates the glycyl-radical formation in the main enzyme. Quantifying the 5’ deoxyadenosine liberated by this reaction, allowed the description of a futile cycle, which runs parallel to the catalytic process. It could also be validated that the activation process is dependent on an external electron-source and that storage of an electron is not possible.
In contrary, the decarboxylase, which also contains two [4Fe-4S] centres, is able to hold back electrons and uses them to dissipate the glycyl-radical over time. This process – first described as transient activation – makes the HPA decarboxylases unique among most other well described members of the glycyl-radical family, where the radical is stable over prolonged incubation times.
In this respect the recently solved crystal structure of CsdBC gave further evidence that the coordination of the [4Fe-4S] clusters is mediated by the small subunit (CsdC) and confirmed the hetero-octameric nature of the complex of the inactive precursor, which is essential for catalysis.
Not only for the fast activation of the system but also for the intrinsic inactivation mediated by the small subunit, the redox-properties of the [4Fe-4S] centres are of utmost importance in the regulation of the HPA-decarboxylase systems.|
|Physical Description:||102 Pages|