Department of Synthetic Biology and Bioenergy, The J. Craig Venter Institute, Rockville, MD 20850, USA.
Microbiology (Reading). 2011 May;157(Pt 5):1363-1374. doi: 10.1099/mic.0.044834-0. Epub 2011 Feb 24.
HynSL from Alteromonas macleodii 'deep ecotype' (AltDE) is an oxygen-tolerant and thermostable [NiFe] hydrogenase. Its two structural genes (hynSL), encoding small and large hydrogenase subunits, are surrounded by eight genes (hynD, hupH and hypCABDFE) predicted to encode accessory proteins involved in maturation of the hydrogenase. A 13 kb fragment containing the ten structural and accessory genes along with three additional adjacent genes (orf2, cyt and orf1) was cloned into an IPTG-inducible expression vector and transferred into an Escherichia coli mutant strain lacking its native hydrogenases. Upon induction, HynSL from AltDE was expressed in E. coli and was active, as determined by an in vitro hydrogen evolution assay. Subsequent genetic analysis revealed that orf2, cyt, orf1 and hupH are not essential for assembling an active hydrogenase. However, hupH and orf2 can enhance the activity of the heterologously expressed hydrogenase. We used this genetic system to compare maturation mechanisms between AltDE HynSL and its Thiocapsa roseopersicina homologue. When the structural genes for the T. roseopersicina hydrogenase, hynSL, were expressed along with known T. roseopersicina accessory genes (hynD, hupK, hypC1C2 and hypDEF), no active hydrogenase was produced. Further, co-expression of AltDE accessory genes hypA and hypB with the entire set of the T. roseopersicina genes did not produce an active hydrogenase. However, co-expression of all AltDE accessory genes with the T. roseopersicina structural genes generated an active T. roseopersicina hydrogenase. This result demonstrates that the accessory genes from AltDE can complement their counterparts from T. roseopersicina and that the two hydrogenases share similar maturation mechanisms.
来自深海嗜冷杆菌“深生态型”（AltDE）的 HynSL 是一种耐氧和耐热的 [NiFe] 氢化酶。它的两个结构基因（hynSL），编码小和大亚基氢化酶亚单位，被八个基因（hynD、hupH 和 hypCABDFE）包围，这些基因预测编码参与氢化酶成熟的辅助蛋白。包含十个结构和辅助基因以及三个相邻基因（orf2、cyt 和 orf1）的 13 kb 片段被克隆到 IPTG 诱导表达载体中，并转移到缺乏天然氢化酶的大肠杆菌突变株中。在诱导下，AltDE 的 HynSL 在大肠杆菌中表达，并通过体外产氢测定确定其具有活性。随后的遗传分析表明，orf2、cyt、orf1 和 hupH 对于组装活性氢化酶不是必需的。然而，hupH 和 orf2 可以增强异源表达的氢化酶的活性。我们使用该遗传系统比较了 AltDE HynSL 和其同源物 Thiocapsa roseopersicina 的成熟机制。当 T. roseopersicina 氢化酶的结构基因 hynSL 与已知的 T. roseopersicina 辅助基因（hynD、hupK、hypC1C2 和 hypDEF）一起表达时，没有产生活性氢化酶。此外，AltDE 辅助基因 hypA 和 hypB 与整套 T. roseopersicina 基因的共表达也没有产生活性氢化酶。然而，所有 AltDE 辅助基因与 T. roseopersicina 结构基因的共表达产生了活性 T. roseopersicina 氢化酶。该结果表明，AltDE 的辅助基因可以补充其与 T. roseopersicina 的对应基因，并且两种氢化酶具有相似的成熟机制。
