title: PqqE from Methylobacterium extorquens AM1: a radical S-adenosyl-l-methionine enzyme with an unusual tolerance to oxygen.
abstract: Methylobacterium extorquens AM1 is an aerobic facultative methylotroph known to secrete pyrroloquinoline quinone (PQQ), a cofactor of a number of bacterial dehydrogenases, into the culture medium. To elucidate the molecular mechanism of PQQ biosynthesis, we are focusing on PqqE which is believed to be the enzyme catalysing the first reaction of the pathway. PqqE belongs to the radical S-adenosyl-l-methionine (SAM) superfamily, in which most, if not all, enzymes are very sensitive to dissolved oxygen and rapidly inactivated under aerobic conditions. We here report that PqqE from M. extorquens AM1 is markedly oxygen-tolerant; it was efficiently expressed in Escherichia coli cells grown aerobically and affinity-purified to near homogeneity. The purified and reconstituted PqqE contained multiple (likely three) iron-sulphur clusters and showed the reductive SAM cleavage activity that was ascribed to the consensus [4Fe-4S](2+) cluster bound at the N-terminus region. Mössbauer spectrometric analyses of the as-purified and reconstituted enzymes revealed the presence of [4Fe-4S](2+) and [2Fe-2S](2+) clusters as the major forms with the former being predominant in the reconstituted enzyme. PqqE from M.extorquens AM1 may serve as a convenient tool for studying the molecular mechanism of PQQ biosynthesis, avoiding the necessity of establishing strictly anaerobic conditions.
authors.affiliation: Department of Chemical Biology and Genetics, Centre of the Region Haná for Biotechnological and Agricultural Research;
authors.forename: Natsaran
authors.initials: N
authors.lastname: Saichana
authors.affiliation: Department of Chemical Biology and Genetics, Centre of the Region Haná for Biotechnological and Agricultural Research;
authors.forename: Katsuyuki
authors.initials: K
authors.lastname: Tanizawa
authors.affiliation: Regional Centre of Advanced Technologies and Materials, Faculty of Science, Palacký University, 783 71 Olomouc, Czech Republic;
authors.forename: Jiří
authors.initials: J
authors.lastname: Pechoušek
authors.affiliation: Regional Centre of Advanced Technologies and Materials, Faculty of Science, Palacký University, 783 71 Olomouc, Czech Republic;
authors.forename: Petr
authors.initials: P
authors.lastname: Novák
authors.affiliation: Department of Biological Chemistry, Faculty of Agriculture, Yamaguchi University, Yamaguchi 753-8515; and.
authors.forename: Toshiharu
authors.initials: T
authors.lastname: Yakushi
authors.affiliation: Department of Bioscience and Biotechnology, University of the Ryukyus, Okinawa 903-0213, Japan.
authors.forename: Hirohide
authors.initials: H
authors.lastname: Toyama
authors.affiliation: Department of Chemical Biology and Genetics, Centre of the Region Haná for Biotechnological and Agricultural Research; jitka.frebortova@upol.cz.
authors.forename: Jitka
authors.initials: J
authors.lastname: Frébortová
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism; D010450:Endopeptidases / Q000737:chemistry* / Q000378:metabolism; D020601:Methylobacterium extorquens / Q000201:enzymology*; D008969:Molecular Sequence Data; D010100:Oxygen / Q000737:chemistry*; D045542:PQQ Cofactor / Q000096:biosynthesis*; D012436:S-Adenosylmethionine / Q000737:chemistry*; D017386:Sequence Homology, Amino Acid; D015204:Spectroscopy, Mossbauer
chemical_list: D001426:Bacterial Proteins; D045542:PQQ Cofactor; D012436:S-Adenosylmethionine; D010450:Endopeptidases; C098959:PqqE protein, Methylobacterium extorquens; D010100:Oxygen
keywords: Methylobacterium extorquens AM1; PQQ; PqqE; radical SAM enzyme