No abstract

Cytochrome P450 (CYP) 4 enzymes constitute a superfamily of heme-containing monooxygenases (1, 2) that participate in a variety of biological processes such as carbon source assimilation, biosynthesis and biodegradation, xenobiotic detoxification, and metabolism of medicines (1, 2). The most common activity of P450 enzymes is the insertion of an oxygen atom from dioxygen into chemically inert carbon-hydrogen bonds, but other reaction types including dealkylation, desaturation, heteroatom oxidation, epoxidation, phenol coupling, and reductive dehalogenation are also known (3-6). The various activities of P450 enzymes are of great interest due to their potential applications in, for example, synthesis of fine chemicals and drug metabolites under mild conditions with high specificity.P450 enzymatic activity requires two electrons that are usually derived from NAD(P)H and delivered to the P450s by electron transfer proteins which are broadly divided into two classes (7,8). Class I systems are diverse, usually consisting of an oxygenase-coupled NAD(P)H-dependent ferredoxin reductase (ONFR) and an iron-sulfur ferredoxin. Such systems are the predominant forms in prokaryotes but are also found in eukaryotic mitochondrial membranes. ONFRs typically contain an FAD cofactor. Ferredoxin cluster types include [2Fe-2S], [3Fe-4S], [4Fe-4S], and combinations of these (7, 9). Non-ferredoxin FMN proteins have also been identified (10). Class II P450 enzymes are most common in eukaryotes and utilize an NADPH-cytochrome P450 reductase (CPR) containing prosthetic groups FAD and FMN (11). Recently other more diverse electron transfer systems for P450 enzymes have been discovered and these have been defined into several new classes (7,8).An important difference between the two main classes is that, whereas a single CPR supports the activity of all 57 human P450s and yeast CPRs support the activity of numerous P450s heterologously expressed in the organism, most class I systems show redox partner specificity. Putidaredoxin (Pdx) is well known to have an effector role in CYP101A1 activity (12, 13). The activity of CYP199A2 from Rhodopseudomonas palustris CGA009 has been reconstituted with palustrisredoxin (Pux), a [2Fe-2S] ferredoxin genomically associated with CYP199A2, and an ONFR, palustrisredoxin reductase (PuR) (14). The high demethylation activity of this system is severely compromised in the hybrid PdR/Pdx/CYP199A2 system (14) due to weak ferredoxin/P450 binding (14,15). Numerous P450 enzymes with potentially interesting and desirable activities are

Cellular iron homeostasis is dominated by FBXL5mediated degradation of iron regulatory protein 2 (IRP2), which is dependent on both iron and oxygen. However, how the physical interaction between FBXL5 and IRP2 is regulated remains elusive. Here, we show that the C-terminal substrate-binding domain of FBXL5 harbors a [2Fe2S] cluster in the oxidized state. A cryoelectron microscopy (cryo-EM) structure of the IRP2-FBXL5-SKP1 complex reveals that the cluster organizes the FBXL5 C-terminal loop responsible for recruiting IRP2. Interestingly, IRP2 binding to FBXL5 hinges on the oxidized state of the [2Fe2S] cluster maintained by ambient oxygen, which could explain hypoxia-induced IRP2 stabilization. Steric incompatibility also allows FBXL5 to physically dislodge IRP2 from iron-responsive element RNA to facilitate its turnover. Taken together, our studies have identified an iron-sulfur cluster within FBXL5, which promotes IRP2 polyubiquitination and degradation in response to both iron and oxygen concentrations.

BTG2 is the prototypical member of the TOB family and is known to be involved in cell growth, differentiation and DNA repair. As a transcriptional co-regulator, BTG2 interacts with CCR4-associated factor 1 (CAF1) and POP2 (CALIF), which are key components of the general CCR4/NOT multi-subunit transcription complex, and which are reported to play distinct roles as nucleases involved in mRNA deadenylation. Here we report the crystal structures of human BTG2 and mouse TIS21 to 2.3 Å and 2.2 Å resolution, respectively. The structures reveal the putative CAF1 binding site. CAF1 deadenylase assays were performed with wild-type BTG2 and mutants that disrupt the interaction with CAF1. The results reveal the suppressive role of BTG2 in the regulation of CAF1 deadenylase activity. Our study provides insights into the formation of the BTG2-CAF1 complex and the potential role of BTG2 in the regulation of CAF1.