Subunit composition of the glycyl radical enzyme p-hydroxyphenylacetate decarboxylase. A small subunit, HpdC, is essential for catalytic activity.
p-Hydroxyphenylacetate decarboxylase from Clostridium difficile catalyses the decarboxylation of p-hydroxyphenylacetate to yield the cytotoxic compound p-cresol. The three genes encoding two subunits of the glycyl-radical enzyme and the activating enzyme have been cloned and expressed in Escherichia coli. The recombinant enzymes were used to reconstitute a catalytically functional system in vitro. In contrast with the decarboxylase purified from C. difficile, which was an almost inactive homo-dimeric protein (beta(2)), the recombinant enzyme was a hetero-octameric (beta(4)gamma(4)), catalytically competent complex, which was activated using endogenous activating enzyme from C. difficile or recombinant activating enzyme to a specific activity of 7 U.mg(-1). Preliminary results suggest that phosphorylation of the small subunit is responsible for the change of the oligomeric state. These data point to an essential function of the small subunit of the decarboxylase and may indicate unique regulatory properties of the system.
Andrei
Paula I
PI
Laboratorium für Mikrobiologie, Fachbereich Biologie, Philipps-Universität, Karl-von-Frisch Strasse, D-35032 Marburg, Germany.
Pierik
Antonio J
AJ
Zauner
Stefan
S
Andrei-Selmer
Luminita C
LC
Selmer
Thorsten
T
D000595:Amino Acid Sequence; D001483:Base Sequence; D002262:Carboxy-Lyases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002384:Catalysis; D016360:Clostridioides difficile / Q000201:enzymology*; D004789:Enzyme Activation; D004926:Escherichia coli / Q000235:genetics; D005798:Genes, Bacterial; D005998:Glycine / Q000737:chemistry; D008969:Molecular Sequence Data; D021122:Protein Subunits / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism
D021122:Protein Subunits; D011994:Recombinant Proteins; C425067:4-hydroxyphenylacetate decarboxylase; D002262:Carboxy-Lyases; D005998:Glycine