Functional analysis by site-directed mutagenesis of the NAD+-reducing hydrogenase from Ralstonia eutropha
Functional analysis by site-directed mutagenesis of the NAD+-reducing hydrogenase from Ralstonia eutropha
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DOI：
10.1128/jb.184.22.6280-6288.2002
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发表时间：
2002-11-01
影响因子：
3.2
通讯作者：
Friedrich, B
中科院分区：
文献类型：
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作者：
Burgdorf, T;De Lacey, AL;Friedrich, B
真养罗尔斯通氏菌胞质[NiFe]氢化酶（SH）在有氧条件下将氢的氧化与NAD（+）的还原偶联。在催化亚基HoxH，所有六个保守的基序周围的[NiFe]网站的存在。这些图案中的五个通过定点诱变来改变，以剖析氢活化的分子机制。根据表型特征，将27个突变体分为四个不同的类别。突变体的主要类别，类1，未能增长的氢和缺乏H-2氧化活性。在这些分离株之一（HoxH 164 A），H-2结合受损。第11类突变体显示出相对于低H-2-氧化活性的高D-2/H+交换速率。一个代表性的（HoxH H16 L）显示D-2/H+交换，但失去了电子受体还原活性。这两种活动在III类突变体中同样受到影响。形成IV类的突变体显示出特别有趣的表型。由于O-2敏感的SH蛋白，它们在氢上表现出O-2敏感的生长。
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.