TAILIEUCHUNG - Báo cáo Y học: Propionate CoA-transferase from Clostridium propionicum Cloning of the gene and identi®cation of glutamate 324 at the active site

Propionate CoA-transferase fromClostridium propionicum hasbeenpuri®edand thegene encoding the enzymehasbeen cloned and sequenced. The enzyme was rapidly and irre-versibly inactivated by sodium borohydride or hydroxyl-amine in the presence of propionyl-CoA. The reduction of the thiol ester between a catalytic site glutamate and CoA with borohydride and the cleavage by hydroxylamine were used to introduce a site-speci®c label, which was followed by MALDI-TOF-MS. This allowed the identi®cation of glutamate 324 at the active site. . | Eur. J. Biochem. 269 372-380 2002 FEBS 2002 Propionate CoA-transferase from Clostridium propionicum Cloning of the gene and identification of glutamate 324 at the active site Thorsten Selmer Angela Willanzheimer and Marc Hetzel FB Biologie Philipps-Universitãt Marburg Germany Propionate CoA-transferase from Clostridium propionicum has been purified and the gene encoding the enzyme has been cloned and sequenced. The enzyme was rapidly and irreversibly inactivated by sodium borohydride or hydroxylamine in the presence of propionyl-CoA. The reduction of the thiol ester between a catalytic site glutamate and CoA with borohydride and the cleavage by hydroxylamine were used to introduce a site-specific label which was followed by MALDI-TOF-MS. This allowed the identification of glutamate 324 at the active site. Propionate CoA-transferase and similar proteins deduced from the genomes of Escherichia coli Staphylococcus aureus Bacillus halodurans and Aeropyrum pernix are proposed to form a novel subclass of CoA-transferases. Secondary structure element predictions were generated and compared to known crystal structures in the databases. A high degree of structural similarity was observed between the arrangement of secondary structure elements in these proteins and glutaconate CoA-transferase from Acidaminococcus fermentans. Keywords Clostridium propionicum alanine metabolism CoA-transferase active site thiol ester. Clostridium propionicum has been isolated as an alanine fermenting organism from the black mud of San Francisco bay 1 . The fermentation products were acetate ammonia carbon dioxide and propionate 2 . In contrast to other organisms which ferment alanine according to the so-called randomising pathway with succinate as a symmetric intermediate C. propionicum ferments alanine via the nonrandomising pathway with acrylyl-CoA as characteristic intermediate. This pathway seems to be restricted to a limited number of organisms including Megasphaera elsdenii 3 Bacteroides

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