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.

• Klíčová slova
• Methylobacterium extorquens AM1, PQQ, PqqE, radical SAM enzyme,
• MeSH
• bakteriální proteiny chemie   metabolismus   MeSH
• endopeptidasy chemie   metabolismus   MeSH
• kofaktor PQQ biosyntéza   MeSH
• kyslík chemie   MeSH
• Methylobacterium extorquens enzymologie   MeSH
• molekulární sekvence - údaje MeSH
• S-adenosylmethionin chemie   MeSH
• sekvence aminokyselin MeSH
• sekvenční homologie aminokyselin MeSH
• spektroskopie Mossbauerova MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• bakteriální proteiny MeSH
• endopeptidasy MeSH
• kofaktor PQQ MeSH
• kyslík MeSH
• PqqE protein, Methylobacterium extorquens MeSH Prohlížeč
• S-adenosylmethionin MeSH