tailieunhanh - Báo cáo khoa học: Subunit composition of the glycyl radical enzyme p-hydroxyphenylacetate decarboxylase

p-Hydroxyphenylacetate decarboxylase from Clostridium difficilecatalyses the decarboxylation ofp-hydroxyphenyl-acetate to yield the cytotoxic compoundp-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 contrast with the decarboxylase purified fromC. difficile, whichwas an almost inactive homo-dimeric protein (b2), the recombinant enzyme was a hetero-octameric (b4c4 ), cata-lytically competent complex, . | Eur. J. Biochem. 1-6 2004 FEBS 2004 doi Subunit composition of the glycyl radical enzyme p-hydroxyphenylacetate decarboxylase A small subunit HpdC is essential for catalytic activity Paula I. Andrei1 Antonio J. Pierik1 Stefan Zauner2 Luminita C. Andrei-Selmer3 and Thorsten Selmer1 1Laboratorium fur Mikrobiologie Fachbereich Biologie Philipps-Universitdt Marburg Germany 2Institut fur Zellbiologie und angewandte Botanik Fachbereich Biologie Philipps-Universitdt Marburg Germany 3Institut fur Klinische Immunologie und Transfusionsmedizin Justus-Liebig Universitat Giessen Germany p-Hydroxyphenylacetate decarboxylase from Clostridium difficile catalyses the decarboxylation of p-hydroxyphenyl-acetate 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 b2 the recombinant enzyme was a hetero-octameric p4y4 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. Keywords Clostridium difficile cresol glycyl radical enzymes S-adenosyl-methionine radical enzymes Tanne-rella forsythensis. Clostridium difficile is a spore forming strict anaerobic bacterium that causes gastrointestinal infections in humans ranging from asymptomatic colonization to severe diarrhoea pseudomembranous colitis toxic megacolon colon perforation and .

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