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The chemically difficult decarboxylation of 4-hydroxyphenylacetate to p-cresol is catalysed by the enzyme 4-hydroxyphenylacetate decarboxylase (Hpd). The enzyme has been purified and characterised as the prototype of a novel class of glycyl radical enzymes (GREs). Previous studies have shown that the Hpd system from Clostridium difficile shows distinct properties, which distinguishes this novel GRE system from the well-characterised pyruvate formate lyase (Pfl) and anaerobic ribonucleotide reductase (Nrd) system. In this work, the similar genes from Clostridium scatologenes (Csd) and from Tannerella forsythensis (Tfd) were cloned and expressed in Escherichia coli. The individual recombinant proteins were produced in Escherichia coli, purified and initially characterized.
The recombinant proteins of CsdBC (hetero-octamer) and TfdBC (hetero-tetramer) were composed of large and small subunits in a 1 to 1 molar ratio and contained 4 irons and 4 sulfurs per heterodimer as that of the HpdBC (hetero-octamer), which distinguishes these systems from Pfl and Nrd. While the Csd system exhibited 4-hydroxyphenylacetate decarboxylase activity like Hpd and was activated by either HpdA or CsdA, the Tfd system was inactive under any conditions tested, but the glycyl radical formation was observed by EPR.
When the glycyl radical subunits of the individual decarboxylases were genetically combined with the small subunits of the other systems, soluble recombinant proteins were formed and purified for some of these combinations. For all combinations yielding soluble enzymes, the molar ratio of the glycyl radical and small subunits was 1:1 and the presence of an iron-sulfur centre per hetero-dimer was evident, while the oligomeric state differed from the wild-type complexes, exhibiting a lower order of complexity. All the purified hybrid decarboxylases were inactive under current assay conditions.
The recombinant activating enzymes (CsdA and TfdA) were monomers according to size exclusion chromatography and contained 7-8 mol iron and 6-7 mol acid labile sulfur per mol of enzyme, indicating at least one additional metal centre, as compared to the Pfl and Nrd activators. The catalytically essential [4Fe-4S]+ cluster in CsdA and HpdA was detected by EPR, which was different to that of Pfl-AE or Nrd-AE, there was no significant signal changes after addition of SAM.
The activation of CsdBC or HpdBC by its cognate activating enzyme was transient, yielding maximum specific activities within 10 min followed by a slow inactivation with t1/2 ~ 30 min, which was accompanied by a quenching of the glycyl radical. This radical quenching process was monitored by using the oxygen-induced cleavage of decarboxylase on SDS-PAGE, and also by EPR. Combining these results, it was proposed that the second iron-sulfur centre (I-cluster, which located within a ~ 60 amino acids long insert between the SAM cluster and the first glycin-rich motif) could be responsible for the ‘inactivase’ activity of HpdA or CsdA. Alternatively, the unique metal centre in the GRE decarboxylase itself may provide the radical quenching properties of the systems.