Development of a cell-free system reveals an oxygen-labile step in the maturation of [NiFe]-hydrogenase 2 of Escherichia coli.
By combining extracts from strains lacking genes encoding either the maturation enzymes or the large subunits of hydrogenases 1, 2 and 3 we could reconstitute in vitro under strictly anaerobic conditions 10-15% of the hydrogenase activity present in wild type Escherichia coli extracts. Purified, unprocessed Strep-tagged variants of the hydrogenase 2 large subunit, HybC, isolated from either a ΔhybD (encoding the hydrogenase 2-specific protease) mutant or a strain deficient in HypF could also be matured to active, processed enzyme using this system. These studies reveal that minimally one step early on the hydrogenase maturation pathway is oxygen-labile.
Soboh
Basem
B
Institute for Mikrobiologie, Martin-Luther University Halle-Wittenberg, Halle (Saale), Germany.
Krüger
Sara
S
Kuhns
Martin
M
Pinske
Constanze
C
Lehmann
Anne
A
Sawers
R Gary
RG
D019878:Carboxyl and Carbamoyl Transferases / Q000235:genetics; D010450:Endopeptidases / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006864:Hydrogenase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010100:Oxygen / Q000378:metabolism*
D029968:Escherichia coli Proteins; C491587:HypC protein, E coli; D006864:Hydrogenase; C481721:hydrogenase 2, E coli; D019878:Carboxyl and Carbamoyl Transferases; C504838:hypF protein, E coli; D010450:Endopeptidases; C119558:hydrogenase maturating endopeptidase HYBD; D010100:Oxygen