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 organism concerning its ability to ferment amino acids. The formation of p-cresol as the main fermentation product of tyrosine by C. difficile is unique among clostridial species. The enzyme responsible for p-cresol formation is p-hydroxyphenylacetate decarboxylase. The enzyme was purified from C. difficile strain DMSZ 1296(T) and initially characterized. The N-terminal amino-acid sequence was 100% identical to an open reading frame in the unfinished genome 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-lyase-like proteins from Escherichia coli and Archaeoglobus fulgidus. The enzyme decarboxylated p-hydroxyphenylacetate (K(m) = 2.8 mM) and 3,4-dihydroxyphenylacetate (K(m) = 0.5 mM). It was competitively inhibited by the substrate analogues p-hydroxyphenylacetylamide and p-hydroxymandelate with K(i) values of 0.7 mM and 0.48 mM, respectively. The protein was readily and irreversibly inactivated by molecular oxygen. Although the purified enzyme was active 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 decarboxylase as a novel glycyl radical enzyme and the substrate specificity of the enzyme, a catalytic mechanism involving ketyl radicals as intermediates is proposed.
Selmer
T
T
Laboratorium für Mikrobiologie, Fachbereich Biologie, Philipps-Universität, Marburg, Germany. selmer@mailer.uni-marburg.de
Andrei
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PI
D000123:Acetyltransferases / Q000737:chemistry / Q000235:genetics; D000595:Amino Acid Sequence; D019611:Archaeoglobus fulgidus / Q000201:enzymology; D001667:Binding, Competitive; D002262:Carboxy-Lyases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D002384:Catalysis; D002851:Chromatography, High Pressure Liquid; D016360:Clostridioides difficile / Q000201:enzymology* / Q000254:growth & development / Q000378:metabolism; D003067:Coenzymes / Q000737:chemistry / Q000378:metabolism; D003408:Cresols / Q000378:metabolism*; D019992:Databases as Topic; D004791:Enzyme Inhibitors / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology; D005609:Free Radicals / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D010100:Oxygen / Q000378:metabolism; D010648:Phenylacetates / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment; D020539:Sequence Analysis, Protein; D013379:Substrate Specificity
D003067:Coenzymes; D003408:Cresols; D004791:Enzyme Inhibitors; D005609:Free Radicals; D010648:Phenylacetates; C032538:4-cresol; D000123:Acetyltransferases; C022560:formate C-acetyltransferase; C425067:4-hydroxyphenylacetate decarboxylase; D002262:Carboxy-Lyases; D010100:Oxygen