Functional analysis by site-directed mutagenesis of the NAD+-reducing hydrogenase from Ralstonia eutropha

被引:38
作者
Burgdorf, T
De Lacey, AL
Friedrich, B
机构
[1] Humboldt Univ, Inst Biol, D-10115 Berlin, Germany
[2] CSIC, Inst Catalisis, Madrid 28049, Spain
关键词
D O I
10.1128/JB.184.22.6280-6288.2002
中图分类号
Q93 [微生物学];
学科分类号
071005 ; 100705 ;
摘要
The tetrameric cytoplasmic [NiFe] hydrogenase (SH) of Ralstonia eutropha couples the oxidation of hydrogen to the reduction of NAD(+) under aerobic conditions. In the catalytic subunit HoxH, all six conserved motifs surrounding the [NiFe] site are present. Five of these motifs were altered by site-directed mutagenesis in order to dissect the molecular mechanism of hydrogen activation. Based on phenotypic characterizations, 27 mutants were grouped into four different classes. Mutants of the major class, class 1, failed to grow on hydrogen and were devoid of H-2-oxidizing activity. In one of these isolates (HoxH 164A), H-2 binding was impaired. Class 11 mutants revealed a high D-2/H+ exchange rate relative to a low H-2-oxidizing activity. A representative (HoxH H16L) displayed D-2/H+ exchange but had lost electron acceptor-reducing activity. Both activities were equally affected in class III mutants. Mutants forming class IV showed a particularly interesting phenotype. They displayed O-2-sensitive growth on hydrogen due to an O-2-sensitive SH protein.
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收藏
页码:6280 / 6288
页数:9
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