Department of Chemical Engineering, Stanford University, Stanford, California, United States of America.
PLoS One. 2009 Oct 26;4(10):e7565. doi: 10.1371/journal.pone.0007565.
[FeFe] hydrogenases are metalloenzymes involved in the anaerobic metabolism of H(2). These proteins are distinguished by an active site cofactor known as the H-cluster. This unique [6Fe-6S] complex contains multiple non-protein moieties and requires several maturation enzymes for its assembly. The pathways and biochemical precursors for H-cluster biosynthesis have yet to be elucidated.
We report an in vitro maturation system in which, for the first time, chemical additives enhance [FeFe] hydrogenase activation, thus signifying in situ H-cluster biosynthesis. The maturation system is comprised of purified hydrogenase apoprotein; a dialyzed Escherichia coli cell lysate containing heterologous HydE, HydF, and HydG maturases; and exogenous small molecules. Following anaerobic incubation of the Chlamydomonas reinhardtii HydA1 apohydrogenase with S-adenosyl methionine (SAM), cysteine, tyrosine, iron, sulfide, and the non-purified maturases, hydrogenase activity increased 5-fold relative to incubations without the exogenous substrates. No conditions were identified in which addition of guanosine triphosphate (GTP) improved hydrogenase maturation.
The in vitro system allows for direct investigation of [FeFe] hydrogenase activation. This work also provides a foundation for studying the biosynthetic mechanisms of H-cluster biosynthesis using solely purified enzymes and chemical additives.
[FeFe]氢化酶是参与 H(2)厌氧代谢的金属酶。这些蛋白质的特征是一个称为 H 簇的活性位点辅因子。这个独特的[6Fe-6S]复合物包含多个非蛋白部分，并且需要几个成熟酶来组装。H 簇生物合成的途径和生化前体尚未阐明。
我们报告了一种体外成熟系统，在该系统中，首次使用化学添加剂增强[FeFe]氢化酶的激活，从而表明原位 H 簇生物合成。成熟系统由纯化的氢化酶脱辅基蛋白组成；含有异源 HydE、HydF 和 HydG 成熟酶的透析大肠杆菌细胞裂解物；和外源小分子。在厌氧孵育含有 S-腺苷甲硫氨酸（SAM）、半胱氨酸、酪氨酸、铁、硫化物和非纯化成熟酶的莱茵衣藻 HydA1 脱辅基氢化酶后，与没有外源底物的孵育相比，氢化酶活性增加了 5 倍。没有确定添加三磷酸鸟苷（GTP）可以改善氢化酶成熟的条件。
体外系统允许直接研究[FeFe]氢化酶的激活。这项工作还为使用纯酶和化学添加剂研究 H 簇生物合成的生物合成机制提供了基础。
