p-Hydroxyphenylacetate decarboxylase from Clostridium difficile - A novel glycyl radical enzyme catalysing the formation of p-cresol

The human pathogenic bacterium Clostridium difficile is a versatile organis m concerning its ability to ferment amino acids. The formation of p-cresol as the main fermentation product of tyrosine by C. difficile is unique amon g clostridial species. The enzyme responsible for p-cresol formation is p-h ydroxyphenylacetate decarboxylase. The enzyme was purified from C. difficil e strain DMSZ 1296(T) and initially characterized. The N-terminal amino-aci d sequence was 100% identical to an open reading frame in the unfinished ge nome of C. difficile strain 630. The ORF encoded a protein of the same size as the purified decarboxylase and was very similar to pyruvate formate-lya se-like proteins from Escherichia coli and Archaeoglobus fulgidus. The enzy me decarboxylated p-hydroxyphenylacetate (K-m = 2.8 mM) and 3,4-dihydroxyph enylacetate (K-m = 0.5 mM). It was competitively inhibited by the substrate analogues p-hydroxyphenylacetylamide and p-hydroxymandelate with K-i value s of 0.7 mM and 0.48 mM, respectively. The protein was readily and irrevers ibly inactivated by molecular oxygen. Although the purified enzyme was acti ve in the presence of sodium sulfide, there are some indications for an as yet unidentified low molecular mass cofactor that is required for catalytic activity in vivo. Based on the identification of p-hydroxyphenylacetate de carboxylase as a novel glycyl radical enzyme and the substrate specificity of the enzyme, a catalytic mechanism involving ketyl radicals as intermedia tes is proposed.