The oxygen-tolerant and NAD+-dependent formate dehydrogenase from Rhodobacter capsulatus is able to catalyze the reduction of CO2 to formate
The oxygen-tolerant and NAD+-dependent formate dehydrogenase from Rhodobacter capsulatus is able to catalyze the reduction of CO2 to formate
复制标题
DOI：
10.1111/febs.12528
复制
发表时间：
2013-12-01
期刊：
影响因子：
5.4
通讯作者：
Leimkuehler, Silke
中科院分区：
文献类型：
--
作者：
Hartmann, Tobias;Leimkuehler, Silke
荚膜红杆菌甲酸脱氢酶(RcFDH)是一种耐氧蛋白质，其亚基组成为()(2)，定位于细胞质中。它属于金属和NAD(+)依赖的FDHS，配位有一个钼辅助因子，四个[Fe4S4]簇和一个[Fe2S2]簇与-亚基结合，一个[Fe4S4]簇和一个FMN结合在-亚基上，一个[Fe2S2]簇结合在-亚基上。RcFDH在Escherichiacoli中异源表达，并对其进行了鉴定。辅因子分析表明，双钼杂多酚鸟嘌呤二核苷酸辅因子结合在活性部位含有半胱氨酸配体的FDSA亚基上。以甲酸盐为底物，测得转化率为2189min(-1)。CO_2还原反应的k(CAT)为89min(-1)。对甲酸盐氧化的偏好显示了该酶的二氧化碳还原的能量障碍。此外，含有FMN和[Fe4S4]的亚基与含有[Fe2S2]的亚基一起形成黄递酶单位，具有NAD(+)还原和NADH氧化的活性。除了结构基因fdsG、fdsB和fdsa外，囊状芽孢杆菌的fds操纵子还含有fdsC和fdsD基因。表达研究表明，只有当FDSC和FdsD同时存在时，RcFDH才是活性的。这两种蛋白都参与了双钼杂多酚鸟嘌呤二核苷酸的修饰和插入RcFDH。
The formate dehydrogenase from Rhodobactercapsulatus (RcFDH) is an oxygen-tolerant protein with an ()(2) subunit composition that is localized in the cytoplasm. It belongs to the group of metal and NAD(+)-dependent FDHs with the coordination of a molybdenum cofactor, four [Fe4S4] clusters and one [Fe2S2] cluster associated with the -subunit, one [Fe4S4] cluster and one FMN bound to the -subunit, and one [Fe2S2] cluster bound to the -subunit. RcFDH was heterologously expressed in Escherichiacoli and characterized. Cofactor analysis showed that the bis-molybdopterin guanine dinucleotide cofactor is bound to the FdsA subunit containing a cysteine ligand at the active site. A turnover rate of 2189min(-1) with formate as substrate was determined. The back reaction for the reduction of CO2 was catalyzed with a k(cat) of 89min(-1). The preference for formate oxidation shows an energy barrier for CO2 reduction of the enzyme. Furthermore, the FMN-containing and [Fe4S4]-containing -subunit together with the [Fe2S2]-containing -subunit forms a diaphorase unit with activities for both NAD(+) reduction and NADH oxidation. In addition to the structural genes fdsG, fdsB, and fdsA, the fds operon in R.capsulatus contains the fdsC and fdsD genes. Expression studies showed that RcFDH is only active when both FdsC and FdsD are present. Both proteins are proposed to be involved in bis-molybdopterin guanine dinucleotide modification and insertion into RcFDH.