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.

The phosphorylation state of the C-terminal domain (CTD) of the RNA polymerase II plays crucial roles in transcription and mRNA processing. Previous studies showed that the plant CTD phosphatase-like 1 (CPL1) dephosphorylates Ser-5-specific CTD and regulates abiotic stress response in Arabidopsis. Here, we report the identification of a K-homology domain-containing protein named SHINY1 (SHI1) that interacts with CPL1 to modulate gene expression. The shi1 mutant was isolated from a forward genetic screening for mutants showing elevated expression of the luciferase reporter gene driven by a salt-inducible promoter. The shi1 mutant is more sensitive to cold treatment during vegetative growth and insensitive to abscisic acid in seed germination, resembling the phenotypes of shi4 that is allelic to the cpl1 mutant. Both SHI1 and SHI4/CPL1 are nuclear-localized proteins. SHI1 interacts with SHI4/CPL1 in vitro and in vivo. Loss-of-function mutations in shi1 and shi4 resulted in similar changes in the expression of some stress-inducible genes. Moreover, both shi1 and shi4 mutants display higher mRNA capping efficiency and altered polyadenylation site selection for some of the stress-inducible genes, when compared with wild type. We propose that the SHI1-SHI4/CPL1 complex inhibits transcription by preventing mRNA capping and transition from transcription initiation to elongation.

Protein prenylation is believed to be catalyzed by three heterodimeric enzymes: FTase, GGTase1, GGTase2. Here, we report the identification of a previously unknown human prenyltransferase complex consisting of an orphan prenyltransferase α subunit, PTAR1, and the catalytic β subunit of GGTase2, RabGGTB. This enzyme, which we named GGTase3, geranylgeranylates FBXL2 to allow its localization at cell membranes, where this ubiquitin ligase mediates the polyubiquitylation of membrane-anchored proteins. In cells, FBXL2 is specifically recognized by GGTase3 despite having a typical C-terminal CaaX prenylation-motif that is predicted to be recognized by GGtase1. Our crystal structure analysis of the full-length GGTase3-FBXL2-SKP1 complex reveals an extensive multivalent interface specifically formed between the leucine-rich repeat domain of FBXL2 and PTAR1, which unmasks the structural basis of the substrate-enzyme specificity. By uncovering a missing prenyltransferase and its unique mode of substrate recognition, our findings call for a revision of the “prenylation code”.

The cytochrome P450 CYP101D2 from Novosphingobium aromaticivorans DSM12444 is closely related to CYP101D1 from the same bacterium and to P450cam (CYP101A1) from Pseudomonas putida. All three are capable of oxidizing camphor stereoselectively to 5-exo-hydroxycamphor. The crystal structure of CYP101D2 revealed that the likely ferredoxin-binding site on the proximal face is largely positively charged, similar to that of CYP101D1. However, both the native and camphor-soaked forms of CYP101D2 had open conformations with an access channel. In the active site of the camphor-soaked form, the camphor carbonyl interacted with the haem-iron-bound water. Two other potential camphor-binding sites were also identified from electron densities in the camphor-soaked structure: one located in the access channel, flanked by the B/C and F/G loops and the I helix, and the other in a cavity on the surface of the enzyme near the F helix side of the F/G loop. The observed open structures may be conformers of the CYP101D2 enzyme that enable the substrate to enter the buried active site via a conformational selection mechanism. The second and third binding sites may be intermediate locations of substrate entry and translocation into the active site, and provide insight into a multi-step substrate-binding mechanism.

Nisin is a widely used antibacterial lantibiotic polypeptide produced by Lactococcus lactis. NisP belongs to the subtilase family and functions in the last step of nisin maturation as the leader-peptide peptidase. Deletion of the nisP gene in LAC71 results in the production of a non-active precursor peptide with the leader peptide unremoved. Here, the 1.1 Å resolution crystal structure of NisP is reported. The structure shows similarity to other subtilases, which can bind varying numbers of Ca atoms. However, no calcium was found in this NisP structure, and the predicted calcium-chelating residues were placed so as to not allow NisP to bind a calcium ion in this conformation. Interestingly, a short peptide corresponding to its own 635-647 sequence was found to bind to the active site of NisP. Biochemical assays and native mass-spectrometric analysis confirmed that NisP possesses an auto-cleavage site between residues Arg647 and Ser648. Further, it was shown that NisP mutated at the auto-cleavage site (R647P/S648P) had full catalytic activity for nisin leader-peptide cleavage, although the C-terminal region of NisP was no longer cleaved. Expressing this mutant in L. lactis LAC71 did not affect the production of nisin but did decrease the proliferation rate of the bacteria, suggesting the biological significance of the C-terminal auto-cleavage of NisP.