Maeda Toshinari, Sanchez-Torres Viviana, Wood Thomas K
Artie McFerrin Department of Chemical Engineering, Texas A & M University, 220 Jack E. Brown Building, College Station, TX 77843-3122, USA.
Appl Microbiol Biotechnol. 2007 Oct;76(5):1035-42. doi: 10.1007/s00253-007-1086-6. Epub 2007 Aug 1.
In the past, it has been difficult to discriminate between hydrogen synthesis and uptake for the three active hydrogenases in Escherichia coli (hydrogenase 1, 2, and 3); however, by combining isogenic deletion mutations from the Keio collection, we were able to see the role of hydrogenase 3. In a cell that lacks hydrogen uptake via hydrogenase 1 (hyaB) and via hydrogenase 2 (hybC), inactivation of hydrogenase 3 (hycE) decreased hydrogen uptake. Similarly, inactivation of the formate hydrogen lyase complex, which produces hydrogen from formate (fhlA) in the hyaB hybC background, also decreased hydrogen uptake; hence, hydrogenase 3 has significant hydrogen uptake activity. Moreover, hydrogen uptake could be restored in the hyaB hybC hycE and hyaB hybC fhlA mutants by expressing hycE and fhlA, respectively, from a plasmid. The hydrogen uptake results were corroborated using two independent methods (both filter plate assays and a gas-chromatography-based hydrogen uptake assay). A 30-fold increase in the forward reaction, hydrogen formation by hydrogenase 3, was also detected for the strain containing active hydrogenase 3 activity but no hydrogenase 1 or 2 activity relative to the strain lacking all three hydrogenases. These results indicate clearly that hydrogenase 3 is a reversible hydrogenase.
过去，很难区分大肠杆菌中三种活性氢化酶（氢化酶1、2和3）的氢合成与氢摄取功能；然而，通过结合来自Keio文库的同基因缺失突变，我们得以了解氢化酶3的作用。在一个缺乏通过氢化酶1（hyaB）和氢化酶2（hybC）摄取氢的细胞中，氢化酶3（hycE）的失活降低了氢摄取。同样，在hyaB hybC背景下，从甲酸盐产生氢的甲酸氢裂解酶复合物（fhlA）的失活也降低了氢摄取；因此，氢化酶3具有显著的氢摄取活性。此外，通过分别从质粒表达hycE和fhlA，可以在hyaB hybC hycE和hyaB hybC fhlA突变体中恢复氢摄取。使用两种独立的方法（滤板测定法和基于气相色谱的氢摄取测定法）证实了氢摄取结果。相对于缺乏所有三种氢化酶的菌株，对于含有活性氢化酶3活性但没有氢化酶1或2活性的菌株，还检测到氢化酶3正向反应（氢形成）增加了30倍。这些结果清楚地表明氢化酶3是一种可逆氢化酶。
