Palustrisredoxin reductase (PuR) is an essential component of the soluble cytochrome P450 electron-transfer chains in Rhodopseudomonas palustris
CGA009. PuR belongs to the oxygenase-coupled NADH-dependent ferredoxin reductase (ONFR) family of FAD-dependent electron-transfer enzymes [ferredoxin-NAD(P)H reductases; EC 18.104.22.168]. ONFRs not only support P450-dependent monooxygenation, but also the oxidation of hydrocarbons in the alkane hydroxylase and benzene/biphenyl dioxygenase systems (Senda et al.
). Electron transport is initiated by a single two-electron transfer from NAD(P)H to FAD in ONFR to produce fully reduced FADH2
, which provides one electron to the iron–sulfur cluster, in most cases a [2Fe–2S] cluster, of ferredoxins. These electrons are finally transferred to the terminal oxygenase. Cytochrome P450 (CYP) enzymes are involved in the oxidative metabolism of both endogenous and exogenous compounds, including therapeutic drugs and other environmental toxins and carcinogens (Medina & Gómez-Moreno, 2004
). These enzyme systems are of increasing interest for various applications, including the stereospecific synthesis of organic compounds and the degradation of pollutants (Sevrioukova et al.
; Zanno et al.
is a purple photosynthetic bacterium that belongs to the α-proteobacteria and is widely distributed in nature. It has versatile metabolic pathways (Larimer et al.
) and is capable of degrading numerous substituted aromatic acids and nitrogen-containing compounds, including amino acids and heterocyclic aromatic compounds. It also dehalogenates and degrades chlorinated benzoates and chlorinated fatty acids (McGrath & Harfoot, 1997
; Egland et al.
), which are found in industrial wastes. This organism has potential applications in waste recycling and biofuels because it can degrade and recycle diverse compounds including lignin monomers, fatty acids and dicarboxylic acids of the types derived from green plants, animal fats and seed oils. R. palustris
contains seven CYP proteins: two ferredoxins (RPA1731 and RPA1872) that are associated with CYP genes and one putative ferredoxin reductase (RPA3782) that is not clustered with any CYP genes. The RPA3782 protein (palustrisredoxin reductase; PuR) has been expressed and found to associate with the RPA1872 ferredoxin to catalyze electron transfer to the RPA1871 (CYP199A2) cytochrome P450 enzyme. Full details of the cloning of the PuR gene and activity studies will be published separately.
Two related crystal structures from the ONFR family have been solved to date: those of the ferredoxin-reductase component (BphA4) of biphenyl dioxygenase from Pseudomonas
sp. strain KKS102 (Senda et al.
) and putidaredoxin reductase from P. putida
(Sevrioukova et al.
). Unlike plant-type ferredoxin reductases, represented by spinach ferredoxin reductase, and thioredoxin reductases (TR), represented by adrenodoxin reductase (AdR), the topology of the two solved ONFR member structures is similar to the protein fold of the disulfide-reductase family (Sevrioukova et al.
). The mechanism of electron transport of BphA4 has been studied and a model of the complex of putidaredoxin reductase and putidaredoxin has been proposed (Kuznetsov et al.
). However, none of these studies has yielded the productive geometry of the flavin–nicotinamide interaction and the hydride-transfer mechanism in ONFR remains unclear. We hope that the structure of PuR will provide new information on these interactions. Here, we report the expression, purification, crystallization and dynamic light scattering of PuR. The results show that PuR forms a stable monomer and is suitable for crystallization.