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The serine/threonine protein kinase casein kinase 1α (CK1α) functions as a negative regulator of Wnt signaling, phosphorylating β-catenin at serine 45 (P-S45) to initiate its eventual ubiquitin-mediated degradation. We previously showed that the repurposed, FDA-approved anthelminthic drug pyrvinium potently inhibits Wnt signaling and Moreover, we proposed that pyrvinium's Wnt inhibitory activity was the result of its function as an activator of CK1α. An understanding of the mechanism by which pyrvinium activates CK1α is important because pyrvinium was given an orphan drug designation by the FDA to treat familial adenomatous polyposis, a precancerous condition driven by constitutive Wnt signaling. In the current study, we show that pyrvinium stimulates the phosphorylation of S45 β-catenin, a known CK1α substrate, in a cell-based assay, and does so in a dose- and time-dependent manner. Alternative splicing of CK1α results in four forms of the protein with distinct biological properties. We evaluated these splice products and identified the CK1α splice variant, CK1αS, as the form that exhibits the most robust response to pyrvinium in cells. Kinetic studies indicate that pyrvinium also stimulates the kinase activity of purified, recombinant CK1αS , increasing its catalytic efficiency (/) toward substrates. These studies provide strong and clear mechanistic evidence that pyrvinium enhances CK1α kinase activity.
Cytochrome P450 enzymes (P450s) are broadly distributed among living organisms and play crucial roles in natural product biosynthesis, degradation of xenobiotics, steroid biosynthesis, and drug metabolism. P450s are considered as the most versatile biocatalysts in nature because of the vast variety of substrate structures and the types of reactions they catalyze. In particular, P450s can catalyze regio- and stereoselective oxidations of nonactivated C-H bonds in complex organic molecules under mild conditions, making P450s useful biocatalysts in the production of commodity pharmaceuticals, fine or bulk chemicals, bioremediation agents, flavors, and fragrances. Major efforts have been made in engineering improved P450 systems that overcome the inherent limitations of the native enzymes. In this review, we focus on recent progress of different strategies, including protein engineering, redox-partner engineering, substrate engineering, electron source engineering, and P450-mediated metabolic engineering, in efforts to more efficiently produce pharmaceuticals and other chemicals. We also discuss future opportunities for engineering and applications of the P450 systems.
© 2020 Li et al.
Human cytochrome P450 (P450) 11B2 catalyzes the formation of aldosterone, the major endogenous human mineralocorticoid. Aldosterone is important for the regulation of electrolyte homeostasis. Mutations and overexpression of P450 11B2 (also known as aldosterone synthase) can lead to hypertension, congestive heart failure, and diabetic nephropathy. The enzyme is therefore a target for drug development to manage these various disorders. P450 11B2 catalyzes aldosterone formation from 11-deoxycorticosterone through three distinct oxidation steps. It is currently unknown to which degree these reactions happen in sequence without the intermediate products dissociating from the enzyme ( processively) or whether these reactions happen solely distributively, in which the intermediate products must first dissociate and then rebind to the enzyme before subsequent oxidation. We present here a comprehensive investigation of processivity in P450 11B2-catalyzed reactions using steady-state, pre-steady-state, pulse-chase, equilibrium-binding titrations, and stopped-flow binding studies. We utilized the data obtained to develop a kinetic model for P450 11B2 and tested this model by enzyme kinetics simulations. We found that although aldosterone is produced processively, the enzyme preferentially utilizes a distributive mechanism that ends with the production of 18-OH corticosterone. This seemingly contradictory observation could be resolved by considering the ability of the intermediate product 18-OH corticosterone to exist as a lactol form, with the equilibrium favoring the ring-closed lactol configuration. In summary, our refined model for P450 11B2 catalysis indicates isomerization of the intermediate to a lactol can explain why P450 11B2 must produce aldosterone through a processive mechanism despite favoring a distributive mechanism.
© 2019 Reddish and Guengerich.
Cytochrome P450 (P450) enzymes are major catalysts involved in the oxidations of most drugs, steroids, carcinogens, fat-soluble vitamins, and natural products. The binding of substrates to some of the 57 human P450s and other mammalian P450s is more complex than a two-state system and has been proposed to involve mechanisms such as multiple ligand occupancy, induced-fit, and conformational-selection. Here, we used kinetic analysis of binding with multiple concentrations of substrates and computational modeling of these data to discern possible binding modes of several human P450s. We observed that P450 2D6 binds its ligand rolapitant in a mechanism involving conformational-selection. P450 4A11 bound the substrate lauric acid via conformational-selection, as did P450 2C8 with palmitic acid. Binding of the steroid progesterone to P450 21A2 was also best described by a conformational-selection model. Hexyl isonicotinate binding to P450 2E1 could be described by either a conformational-selection or an induced-fit model. Simulation of the binding of the ligands midazolam, bromocriptine, testosterone, and ketoconazole to P450 3A4 was consistent with an induced-fit or a conformational-selection model, but the concentration dependence of binding rates for varying both P450 3A4 and midazolam concentrations revealed discordance in the parameters, indicative of conformational-selection. Binding of the P450s 2C8, 2D6, 3A4, 4A11, and 21A2 was best described by conformational-selection, and P450 2E1 appeared to fit either mode. These findings highlight the complexity of human P450-substrate interactions and that conformational-selection is a dominant feature of many of these interactions.
© 2019 Guengerich et al.
O -Alkylguanine DNA-alkyltransferase (AGT), a DNA repair protein, can form crosslinks with DNA. The AGT-DNA crosslinks are known to be mutagenic when AGT is heterologously expressed in Escherichia coli, as well as in mammalian cells. To understand the biological consequences, reliable access to AGT-oligonucleotide crosslinks is needed. This article describes the synthesis and characterization of site-specific AGT-oligonucleotide crosslinks at the N2-position of deoxyguanosine and N6-position of deoxyadenosine. We developed a post-oligomerization strategy for the synthesis of propargyl-modified oligonucleotides. Copper-catalyzed azide-alkyne cycloaddition was used as a key step to obtain the iodoacetamide-linked oligonucleotides, which serve as good electrophiles for the crosslinking reaction with cysteine-145 of the active site of AGT. Trypsinization of AGT and hydrolysis of oligonucleotides, combined with analysis by liquid chromatography-tandem mass spectrometry, was utilized to confirm the nucleobase-adducted peptides. This method provides a useful strategy for the synthesis and characterization of site-specific DNA-protein crosslinks, which can be further used to understand proteolytic degradation-coupled DNA repair mechanisms. © 2019 by John Wiley & Sons, Inc.
© 2019 John Wiley & Sons, Inc.
IMPACT STATEMENT - The development of autograft extenders is a significant clinical need in bone tissue engineering. We report new settable poly(thioketal urethane)-based autograft extenders that have bone-like mechanical properties and handling properties comparable to calcium phosphate bone cements. These settable autograft extenders remodeled to form new bone in a biologically stringent intertransverse process model of bone formation that does not heal when treated with calcium phosphate bone void fillers or cements alone. This is the first study to report settable autograft extenders with bone-like strength and handling properties comparable to ceramic bone cements, which have the potential to improve treatment of bone fractures and other orthopedic conditions.
Complex II (SdhABCD) is a membrane-bound component of mitochondrial and bacterial electron transport chains, as well as of the TCA cycle. In this capacity, it catalyzes the reversible oxidation of succinate. SdhABCD contains the SDHA protein harboring a covalently bound FAD redox center and the iron-sulfur protein SDHB, containing three distinct iron-sulfur centers. When assembly of this complex is compromised, the flavoprotein SDHA may accumulate in the mitochondrial matrix or bacterial cytoplasm. Whether the unassembled SDHA has any catalytic activity, for example in succinate oxidation, fumarate reduction, reactive oxygen species (ROS) generation, or other off-pathway reactions, is not known. Therefore, here we investigated whether unassembled SdhA flavoprotein, its homolog fumarate reductase (FrdA), and the human SDHA protein have succinate oxidase or fumarate reductase activity and can produce ROS. Using recombinant expression in , we found that the free flavoproteins from these divergent biological sources have inherently low catalytic activity and generate little ROS. These results suggest that the iron-sulfur protein SDHB in complex II is necessary for robust catalytic activity. Our findings are consistent with those reported for single-subunit flavoprotein homologs that are not associated with iron-sulfur or heme partner proteins.
The lumen of the endoplasmic reticulum (ER) provides an oxidizing environment to aid in the formation of disulfide bonds, which is tightly regulated by both antioxidant proteins and small molecules. On the cytoplasmic side of the ER, cytochrome P450 (P450) proteins have been identified as a superfamily of enzymes that are important in the formation of endogenous chemicals as well as in the detoxication of xenobiotics. Our previous report described oxidative inhibition of P450 Family 4 enzymes via oxidation of the heme-thiolate cysteine to a sulfenic acid (-SOH) (Albertolle, M. E. (2017) 292, 11230-11242). Further proteomic analyses of murine kidney and liver microsomes led to the finding that a number of other drug-metabolizing enzymes located in the ER are also redox-regulated in this manner. We expanded our analysis of sulfenylated enzymes to human liver and kidney microsomes. Evaluation of the sulfenylation, catalytic activity, and spectral properties of P450s 1A2, 2C8, 2D6, and 3A4 led to the identification of two classes of redox sensitivity in P450 enzymes: heme-thiolate-sensitive and thiol-insensitive. These findings provide evidence for a mammalian P450 regulatory mechanism, which may also be relevant to other drug-metabolizing enzymes. (Data are available via ProteomeXchange with identifier PXD007913.).
© 2018 by The American Society for Biochemistry and Molecular Biology, Inc.
E3 ubiquitin (UB) ligases E4B and carboxyl terminus of Hsc70-interacting protein (CHIP) use a common U-box motif to transfer UB from E1 and E2 enzymes to their substrate proteins and regulate diverse cellular processes. To profile their ubiquitination targets in the cell, we used phage display to engineer E2-E4B and E2-CHIP pairs that were free of cross-reactivity with the native UB transfer cascades. We then used the engineered E2-E3 pairs to construct "orthogonal UB transfer (OUT)" cascades so that a mutant UB (xUB) could be exclusively used by the engineered E4B or CHIP to label their substrate proteins. Purification of xUB-conjugated proteins followed by proteomics analysis enabled the identification of hundreds of potential substrates of E4B and CHIP in human embryonic kidney 293 cells. Kinase MAPK3 (mitogen-activated protein kinase 3), methyltransferase PRMT1 (protein arginine -methyltransferase 1), and phosphatase PPP3CA (protein phosphatase 3 catalytic subunit alpha) were identified as the shared substrates of the two E3s. Phosphatase PGAM5 (phosphoglycerate mutase 5) and deubiquitinase OTUB1 (ovarian tumor domain containing ubiquitin aldehyde binding protein 1) were confirmed as E4B substrates, and β-catenin and CDK4 (cyclin-dependent kinase 4) were confirmed as CHIP substrates. On the basis of the CHIP-CDK4 circuit identified by OUT, we revealed that CHIP signals CDK4 degradation in response to endoplasmic reticulum stress.
Cytochrome P450 (P450, CYP) research provides many opportunities for the application of kinetic isotope effect (KIE) strategies. P450s collectively catalyze oxidations of more substrates than any other group of enzymes, and CH bond cleavage is a major feature in a large fraction of these reactions. The presence of a significant primary deuterium KIE is evidence that hydrogen abstraction is at least partially rate-limiting in the reactions, and this appears to be the case in many P450 reactions. The first report of a KIE in (P450-linked) drug metabolism appeared in 1961 (for morphine N-demethylation), and in a number of cases, it has been possible to modulate the in vivo metabolism or toxicity of chemicals by deuterium substitution. A number of efforts are in progress to utilize deuterium substitution to alter the metabolism of drugs in an advantageous manner.
© 2017 Elsevier Inc. All rights reserved.