Dinucleotide Binding Research Articles

A Cryptochrome 2 mutation yields advanced sleep phase in humans.

Familial Advanced Sleep Phase (FASP) is a heritable human sleep phenotype characterized by very early sleep and wake times. We identified a missense mutation in the human Cryptochrome 2 (CRY2) gene that co-segregates with FASP in one family. The mutation leads to replacement of an alanine residue at position 260 with a threonine (A260T). In mice, the CRY2 mutation causes a shortened circadian period and reduced phase-shift to early-night light pulse associated with phase-advanced behavioral rhythms in the light-dark cycle. The A260T mutation is located in the phosphate loop of the flavin adenine dinucleotide (FAD) binding domain of CRY2. The mutation alters the conformation of CRY2, increasing its accessibility and affinity for FBXL3 (an E3 ubiquitin ligase), thus promoting its degradation. These results demonstrate that CRY2 stability controlled by FBXL3 plays a key role in the regulation of human sleep wake behavior.

Genome-wide identification and analysis of CKX genes in Poncirus trifoliata

ABSTRACTCytokinin oxidase/dehydrogenases (CKXs) in plants are coded by a small multigene family and play important roles in maintaining cytokinin homeostasis. In this study, four CKX genes (i.e. PsCKX1, PsCKX2, PsCKX5, and PsCKX7) were cloned from Poncirus trifoliata. All PsCKXs contained a highly conserved flavin adenine dinucleotide (FAD) binding domain and a cytokinin dehydrogenase 1, FAD/cytokinin binding domain. PsCKX1 and PsCKX2 shared 66.2% and 65.4% identity with AtCKX6 and AtCKX1, respectively, while PsCKX5 and PsCKX7 exhibited less than 45% identity with AtCKXs. The expression analysis under abiotic conditions (NaCl, ABA, 6-BA and drought) revealed that the four PsCKX genes could respond to at least one treatment, and the expression patterns were diverse in root and leaf. Overexpressing four PsCKX genes in tobacco led to diverse phenotypic variations in transgenic plant, including leaf shape, root architecture, and plant height. In addition, the data showed that PsCKX2 and PsCKX5 hold promise to obtain citrus dwarf rootstock with a stronger root system, since the overexpression of them resulted in dwarf plants with more lateral roots. Taken together, the work lays the basis for applications of PsCKX genes in future.

Characterization of Glycerol Dehydrogenase from Thermoanaerobacterium thermosaccharolyticum DSM 571 and GGG Motif Identification.

Glycerol dehydrogenases (GlyDHs) are essential for glycerol metabolism in vivo, catalyzing its reversible reduction to 1,3-dihydroxypropranone (DHA). The gldA gene encoding a putative GlyDH was cloned from Thermoanaerobacterium thermosaccharolyticum DSM 571 (TtGlyDH) and expressed in Escherichia coli. The presence of Mn(2+) enhanced its enzymatic activity by 79.5%. Three highly conserved residues (Asp(171), His(254), and His(271)) in TtGlyDH were associated with metal ion binding. Based on an investigation of glycerol oxidation and DHA reduction, TtGlyDH showed maximum activity towards glycerol at 60°C and pH 8.0 and towards DHA at 60°C and pH 6.0. DHA reduction was the dominant reaction, with a lower Km(DHA) of 1.08 ± 0.13 mM and Vmax of 0.0053 ± 0.0001 mM/s, compared with glycerol oxidation, with a Km(glycerol) of 30.29 ± 3.42 mM and Vmax of 0.042 ± 0.002 mM/s. TtGlyDH had an apparent activation energy of 312.94 kJ/mol. The recombinant TtGlyDH was thermostable, maintaining 65% of its activity after a 2-h incubation at 60°C. Molecular modeling and site-directed mutagenesis analyses demonstrated that TtGlyDH had an atypical dinucleotide binding motif (GGG motif) and a basic residue Arg(43), both related to dinucleotide binding.

Mutated form (G52E) of inactive diphtheria toxin CRM197: molecular simulations clearly display effect of the mutation to NAD binding

Mutated form (G52E) of diphtheria toxin (DT) CRM197 is an inactive and nontoxic enzyme. Here, we provided a molecular insight using comparative molecular dynamics (MD) simulations to clarify the influence of a single point mutation on overall protein and active-site loop. Post-processing MD analysis (i.e. stability, principal component analysis, hydrogen-bond occupancy, etc.) is carried out on both wild and mutated targets to investigate and to better understand the mechanistic differences of structural and dynamical properties on an atomic scale especially at nicotinamide adenine dinucleotide (NAD) binding site when a single mutation (G52E) happens at the DT. In addition, a docking simulation is performed for wild and mutated forms. The docking scoring analysis and docking poses results revealed that mutant form is not able to properly accommodate the NAD molecule.

MECP2 Is a Frequently Amplified Oncogene with a Novel Epigenetic Mechanism That Mimics the Role of Activated RAS in Malignancy.

An unbiased genome-scale screen for unmutated genes that drive cancer growth when overexpressed identified methyl cytosine-guanine dinucleotide (CpG) binding protein 2 (MECP2) as a novel oncogene. MECP2 resides in a region of the X-chromosome that is significantly amplified across 18% of cancers, and many cancer cell lines have amplified, overexpressed MECP2 and are dependent on MECP2 expression for growth. MECP2 copy-number gain and RAS family member alterations are mutually exclusive in several cancer types. The MECP2 splicing isoforms activate the major growth factor pathways targeted by activated RAS, the MAPK and PI3K pathways. MECP2 rescued the growth of a KRAS(G12C)-addicted cell line after KRAS downregulation, and activated KRAS rescues the growth of an MECP2-addicted cell line after MECP2 downregulation. MECP2 binding to the epigenetic modification 5-hydroxymethylcytosine is required for efficient transformation. These observations suggest that MECP2 is a commonly amplified oncogene with an unusual epigenetic mode of action. MECP2 is a commonly amplified oncogene in human malignancies with a unique epigenetic mechanism of action. Cancer Discov; 6(1); 45-58. ©2015 AACR.This article is highlighted in the In This Issue feature, p. 1.

Molecular Dynamics Assisted Mechanistic Study of Isoniazid-Resistance against Mycobacterium tuberculosis InhA.

Examination of InhA mutants I16T, I21V, I47T, S94A, and I95P showed that direct and water mediated H-bond interactions between NADH and binding site residues reduced drastically. It allowed conformational flexibility to NADH, particularly at the pyrophosphate region, leading to weakening of its binding at dinucleotide binding site. The highly scattered distribution of pyrophosphate dihedral angles and chi1 side chain dihedral angles of corresponding active site residues therein confirmed weak bonding between InhA and NADH. The average direct and water mediated bridged H-bond interactions between NADH and mutants were observed weaker as compared to wild type. Further, estimated NADH binding free energy in mutants supported the observed weakening of InhA-NADH interactions. Similarly, per residue contribution to NADH binding was also found little less as compared to corresponding residues in wild type. This investigation clearly depicted and supported the effect of mutations on NADH binding and can be accounted for isoniazid resistance as suggested by previous biochemical and mutagenic studies. Further, structural analysis of InhA provided the crucial points to enhance the NADH binding affinity towards InhA mutants in the presence of direct InhA inhibitors to combat isoniazid drug resistance. This combination could be a potential alternative for treatment of drug resistant tuberculosis.

STING Activation by Translocation from the ER Is Associated with Infection and Autoinflammatory Disease.

STING is an ER-associated membrane protein that is critical for innate immune sensing of pathogens. STING-mediated activation of the IFN-I pathway through the TBK1/IRF3 signaling axis involves both cyclic-dinucleotide binding and its translocation from the ER to vesicles. However, how these events are coordinated, and the exact mechanism of STING activation, remain poorly understood. Here, we found that the Shigella effector protein IpaJ potently inhibits STING signaling by blocking its translocation from the ER to ERGIC, even in the context of dinucleotide binding. Reconstitution using purified components revealed STING translocation as the rate-limiting event in maximal signal transduction. Furthermore, STING mutations associated with autoimmunity in humans were found to cause constitutive ER exit and to activate STING independent of cGAMP binding. Together, these data provide compelling evidence for an ER retention and ERGIC/Golgi-trafficking mechanism of STING regulation that is subverted by bacterial pathogens and is deregulated in human genetic disease.

Cyclin-dependent kinase five mediates activation of lung xanthine oxidoreductase in response to hypoxia.

BackgroundXanthine oxidoreductase (XOR) is involved in oxidative metabolism of purines and is a source of reactive oxygen species (ROS). As such, XOR has been implicated in oxidant-mediated injury in multiple cardiopulmonary diseases. XOR enzyme activity is regulated, in part, via a phosphorylation-dependent, post-translational mechanism, although the kinase(s) responsible for such hyperactivation are unknown.Methods and ResultsUsing an in silico approach, we identified a cyclin-dependent kinase 5 (CDK5) consensus motif adjacent to the XOR flavin adenine dinucleotide (FAD) binding domain. CDK5 is a proline-directed serine/threonine kinase historically linked to neural development and injury. We tested the hypothesis that CDK5 and its activators are mediators of hypoxia-induced hyperactivation of XOR in pulmonary microvascular endothelial cells (EC) and the intact murine lung. Using complementary molecular and pharmacologic approaches, we demonstrated that hypoxia significantly increased CDK5 activity in EC. This was coincident with increased expression of the CDK5 activators, cyclin-dependent kinase 5 activator 1 (CDK5r1 or p35/p25), and decreased expression of the CDK5 inhibitory peptide, p10. Expression of p35/p25 was necessary for XOR hyperactivation. Further, CDK5 physically associated with XOR and was necessary and sufficient for XOR phosphorylation and hyperactivation both in vitro and in vivo. XOR hyperactivation required the target threonine (T222) within the CDK5-consensus motif.Conclusions and SignificanceThese results indicate that p35/CDK5-mediated phosphorylation of T222 is required for hypoxia-induced XOR hyperactivation in the lung. Recognizing the contribution of XOR to oxidative injury in cardiopulmonary disease, these observations identify p35/CDK5 as novel regulators of XOR and potential modifiers of ROS-mediated injury.

Conservation of transcription factor binding specificities across 600 million years of bilateria evolution.

Divergent morphology of species has largely been ascribed to genetic differences in the tissue-specific expression of proteins, which could be achieved by divergence in cis-regulatory elements or by altering the binding specificity of transcription factors (TFs). The relative importance of the latter has been difficult to assess, as previous systematic analyses of TF binding specificity have been performed using different methods in different species. To address this, we determined the binding specificities of 242 Drosophila TFs, and compared them to human and mouse data. This analysis revealed that TF binding specificities are highly conserved between Drosophila and mammals, and that for orthologous TFs, the similarity extends even to the level of very subtle dinucleotide binding preferences. The few human TFs with divergent specificities function in cell types not found in fruit flies, suggesting that evolution of TF specificities contributes to emergence of novel types of differentiated cells.

Proteopedia: Rossmann fold: A beta-alpha-beta fold at dinucleotide binding sites.

The Rossmann fold is one of the most common and widely distributed super-secondary structures. It is composed of a series of alternating beta strand (β) and alpha helical (α) segments wherein the β-strands are hydrogen bonded forming a β-sheet. The initial beta-alpha-beta (βαβ) fold is the most conserved segment of Rossmann folds. As this segment is in contact with the ADP portion of dinucleotides such as FAD, NAD, and NADP it is also called as an "ADP-binding βαβ fold". The Proteopedia entry on the Rossmann fold (Available at: http://proteopedia.org/w/Rossmann_fold) was generated to illustrate several structural aspects of super families of FAD and NAD(P) binding proteins: (1) The coenzymes FAD and NAD(P) share the basic adenosine diphosphate (ADP) structure. (2) The βαβ fold motif that is common to both FAD and NAD(P) binding enzymes accommodates the common ADP component of these two coenzymes. (3) In both FAD and NAD(P) binding sites, the tight turn between the first β-strand and the α-helix is in contact with the two phosphate groups of ADP. (4) This hairpin curve includes the first two conserved glycines (Gly-x-Gly) that allow the sharp turn of the polypeptide backbone. (5) The two β-strands of the βαβ fold may constitute the core of a larger β-sheet that may include up to seven β-strands generally in parallel orientation. (6) The structures of segments between additional strands vary greatly and may be composed of a variety of structures such as multiple short helices or coils.

C-terminal binding proteins: central players in development and disease.

C-terminal binding proteins (CtBPs) were initially identified as binding partners for the E1A-transforming proteins. Although the invertebrate genome encodes one CtBP protein, two CtBPs (CtBP1 and CtBP2) are encoded by the vertebrate genome and perform both unique and duplicative functions. CtBP1 and CtBP2 are closely related and act as transcriptional corepressors when activated by nicotinamide adenine dinucleotide binding to their dehydrogenase domains. CtBPs exert transcriptional repression primarily via recruitment of a corepressor complex to DNA that consists of histone deacetylases (HDACs) and histone methyltransferases, although CtBPs can also repress transcription through HDAC-independent mechanisms. More recent studies have demonstrated a critical function for CtBPs in the transcriptional repression of pro-apoptotic genes such as Bax, Puma, Bik, and Noxa. Nonetheless, although recent efforts have characterized the essential involvement of CtBPs in promoting cellular survival, the dysregulation of CtBPs in both neurodegenerative disease and cancers remains to be fully elucidated.

Association of C677T transition of the human methylenetetrahydrofolate reductase (MTHFR) gene with male infertility.

The human methylenetetrahydrofolate reductase (MTHFR) gene encodes one of the key enzymes in folate metabolism. This gene is located on chromosome 1 (1p36.3), which has 12 exons. The aim of the present study was to investigate the possible association of the two (C677T and A1298C) polymorphisms of this gene with male infertility. In a case-control study, 250 blood samples were collected from IVF centres in Sari and Babol (Iran): 118 samples were from oligospermic men and 132 were from controls. Two single nucleotide polymorphisms of the MTHFR genotype were detected using polymerase chain reaction-restriction fragment length polymorphism. There was no association found between the A1298C variant and male infertility. However, carriers of the 677T allele (CT and TT genotypes) were at a higher risk of infertility than individuals with other genotypes (odds ratio 1.84; 95% confidence interval 1.11-3.04; P=0.0174). Structural analysis of human MTHFR flavoprotein showed that C677T transition played an important role in the change in affinity of the MTHFR-Flavin adenine dinucleotide binding site. Based on our results, we suggest that C677T transition in MTHFR may increase the risk of male infertility, and detection of the C677T polymorphism biomarker may be helpful in the screening of idiopathic male infertility.

An ancient oxidoreductase making differential use of its cofactors

Abstract Many transcription factors contribute to cellular homeostasis by integrating multiple signals. Signaling via the yeast Gal80 protein, a negative regulator of the prototypic transcription activator Gal4, is primarily regulated by galactose. ScGal80 from Saccharomyces cerevisiae has been reported to bind NAD(P). Here, we show that the ability to bind these ligands is conserved in KlGal80, a Gal80 homolog from the distantly related yeast Kluyveromyces lactis. However, the homologs apparently have diverged with respect to response to the dinucleotide. Strikingly, ScGal80 binds NAD(P) and NAD(P)H with more than 50-fold higher affinity than KlGal80. In contrast to ScGal80, where NAD is neutral, NAD and NADP have a negative effect in KlGal80 on its interaction with a KlGal4-peptide in vitro. Swapping a loop in the NAD(P) binding Rossmann-fold of ScGal80 into KlGal80 increases the affinity for NAD(P) and has a significant impact on KlGal4 regulation in vivo. Apparently, dinucleotide binding allows coupling of the metabolic state of the cell to regulation of the GAL/LAC genes. The particular sequences involved in binding determine how exactly the metabolic state is sensed and integrated by Gal80 to regulate Gal4.

Structure of the human P2Y12 receptor in complex with an antithrombotic drug

P2Y receptors (P2YRs), a family of purinergic G-protein-coupled receptors (GPCRs), are activated by extracellular nucleotides. There are a total of eight distinct functional P2YRs expressed in human, which are subdivided into P2Y1-like receptors and P2Y12-like receptors. Their ligands are generally charged molecules with relatively low bioavailability and stability in vivo, which limits our understanding of this receptor family. P2Y12R regulates platelet activation and thrombus formation, and several antithrombotic drugs targeting P2Y12R--including the prodrugs clopidogrel (Plavix) and prasugrel (Effient) that are metabolized and bind covalently, and the nucleoside analogue ticagrelor (Brilinta) that acts directly on the receptor--have been approved for the prevention of stroke and myocardial infarction. However, limitations of these drugs (for example, a very long half-life of clopidogrel action and a characteristic adverse effect profile of ticagrelor) suggest that there is an unfulfilled medical need for developing a new generation of P2Y12R inhibitors. Here we report the 2.6 Å resolution crystal structure of human P2Y12R in complex with a non-nucleotide reversible antagonist, AZD1283. The structure reveals a distinct straight conformation of helix V, which sets P2Y12R apart from all other known class A GPCR structures. With AZD1283 bound, the highly conserved disulphide bridge in GPCRs between helix III and extracellular loop 2 is not observed and appears to be dynamic. Along with the details of the AZD1283-binding site, analysis of the extracellular interface reveals an adjacent ligand-binding region and suggests that both pockets could be required for dinucleotide binding. The structure provides essential insights for the development of improved P2Y12R ligands and allosteric modulators as drug candidates.

N-Oxide Reduction of Quinoxaline-1,4-Dioxides Catalyzed by Porcine Aldehyde Oxidase SsAOX1

Quinoxaline-1,4-dioxides (QdNOs) are a class of quinoxaline derivatives that are widely used in humans or animals as drugs or feed additives. However, the metabolic mechanism, especially the involved enzymes, has not been reported in detail. In this study, the N-oxide reduction enzyme, porcine aldehyde oxidase SsAOX1 was identified and characterized. The SsAOX1 gene was cloned from pig liver through reverse-transcription polymerase chain reaction using degenerate primers, which encode a 147-kDa protein with typical aldehyde oxidase motifs, two [2Fe-2S] centers, a flavin adenine dinucleotide (FAD) binding domain, and a molybdenum cofactor domain. After heterologous expression in a prokaryote, purified SsAOX1 formed a functional homodimer under native conditions. Importantly, the SsAOX1 catalyzed the N-oxide reduction at the N1 position of three representative QdNOs (quinocetone, mequindox, and cyadox), which are commonly used as animal feed additives. SsAOX1 has the highest activity toward quinocetone, followed by mequindox and cyadox, with kcat/K(m) values of 1.94 ± 0.04, 1.27 ± 0.15, and 0.43 ± 0.09 minute(-1) μM(-1), respectively. However, SsAOX1 has the lowest substrate affinity for quinocetone, followed by the cyadox and mequindox, with K(m) values of 4.36 ± 0.56, 3.16 ± 0.48, and 2.96 ± 0.51 μM, respectively. In addition, using site-directed mutagenesis, we found that substitution of glycine 1019 with threonine endows SsAOX1 with N-oxide reductive activity at the N4 position. The goal of this study was to identify and characterize the N-oxide reduction enzyme for a class of veterinary drugs, QdNOs, which will aid in the elucidation of the metabolic pathways of QdNOs and will provide a theoretical basis for their administration and new veterinary drug design.

Nicotinamide Adenine Dinucleotide-induced Multimerization of the Co-repressor CtBP1 Relies on a Switching Tryptophan

The transcriptional co-repressor C-terminal binding protein (CtBP) interacts with a number of repressor proteins and chromatin modifying enzymes. How the biochemical properties including binding of dinucleotide, oligomerization, and dehydrogenase domains of CtBP1 direct the assembly of a functional co-repressor to influence gene expression is not well understood. In the current study we demonstrate that CtBP1 assembles into a tetramer in a NAD(H)-dependent manner, proceeding through a dimeric intermediate. We find that NAD-dependent oligomerization correlates with NAD(+) binding affinity and that the carboxyl terminus is required for assembly of a dimer of dimers. Mutant CtBP1 proteins that abrogate dinucleotide-binding retain wild type affinity for the PXDLS motif, but do not self-associate either in vitro or in vivo. CtBP1 proteins with mutations in the dehydrogenase domain still retain the ability to self-associate and bind target proteins. Both co-immunoprecipitation and mammalian two-hybrid experiments demonstrate that CtBP1 self-association occurs within the nucleus, and depends on dinucleotide binding. Repression of transcription does not depend on dinucleotide binding or an intact dehydrogenase domain, but rather depends on the amino-terminal domain that recruits PXDLS containing targets. We show that tryptophan 318 (Trp(318)) is a critical residue for tetramer assembly and likely functions as a switch for effective dimerization following NAD(+) binding. These results suggest that dinucleotide binding permits CtBP1 to form an intranuclear homodimer through a Trp(318) switch, creating a nucleation site for multimerization through the C-terminal domain for tetramerization to form an effective repression complex.

Conserved water-mediated recognition and dynamics of NAD+ (carboxamide group) to hIMPDH enzyme: water mimic approach toward the design of isoform-selective inhibitor

Inosine monophosphate dehydrogenase (IMPDH) enzyme involves in GMP biosynthesis pathway. Type I hIMPDH is expressed at lower levels in all cells, whereas type II is especially observed in acute myelogenous leukemia, chronic myelogenous leukemia cancer cells, and 10 ns simulation of the IMP–NAD+ complex structures (PDB ID. 1B3O and 1JCN) have revealed the presence of a few conserved hydrophilic centers near carboxamide group of NAD+. Three conserved water molecules (W1, W, and W1′) in di-nucleotide binding pocket of enzyme have played a significant role in the recognition of carboxamide group (of NAD+) to D274 and H93 residues. Based on H-bonding interaction of conserved hydrophilic (water molecular) centers within IMP–NAD+-enzyme complexes and their recognition to NAD+, some covalent modification at carboxamide group of di-nucleotide (NAD+) has been made by substituting the –CONH2group by –CONHNH2 (carboxyl hydrazide group) using water mimic inhibitor design protocol. The modeled structure of modified ligand may, though, be useful for the development of antileukemic agent or it could be act as better inhibitor for hIMPDH-II.

Elucidations of the Catalytic Cycle of NADH-Cytochrome b5 Reductase by X-ray Crystallography: New Insights into Regulation of Efficient Electron Transfer

NADH-Cytochrome b5 reductase (b5R), a flavoprotein consisting of NADH and flavin adenine dinucleotide (FAD) binding domains, catalyzes electron transfer from the two-electron carrier NADH to the one-electron carrier cytochrome b5 (Cb5). The crystal structures of both the fully reduced form and the oxidized form of porcine liver b5R were determined. In the reduced b5R structure determined at 1.68Å resolution, the relative configuration of the two domains was slightly shifted in comparison with that of the oxidized form. This shift resulted in an increase in the solvent-accessible surface area of FAD and created a new hydrogen-bonding interaction between the N5 atom of the isoalloxazine ring of FAD and the hydroxyl oxygen atom of Thr66, which is considered to be a key residue in the release of a proton from the N5 atom. The isoalloxazine ring of FAD in the reduced form is flat as in the oxidized form and stacked together with the nicotinamide ring of NAD+. Determination of the oxidized b5R structure, including the hydrogen atoms, determined at 0.78Å resolution revealed the details of a hydrogen-bonding network from the N5 atom of FAD to His49 via Thr66. Both of the reduced and oxidized b5R structures explain how backflow in this catalytic cycle is prevented and the transfer of electrons to one-electron acceptors such as Cb5 is accelerated. Furthermore, crystallographic analysis by the cryo-trapping method suggests that re-oxidation follows a two-step mechanism. These results provide structural insights into the catalytic cycle of b5R.

Crystallographic analysis and structure‐guided engineering of NADPH‐dependent Ralstonia sp. Alcohol dehydrogenase toward NADH cosubstrate specificity

The NADP⁺-dependent alcohol dehydrogenase from Ralstonia sp. (RasADH) belongs to the protein superfamily of short-chain dehydrogenases/reductases (SDRs). As an enzyme that accepts different types of substrates--including bulky-bulky as well as small-bulky secondary alcohols or ketones--with high stereoselectivity, it offers potential as a biocatalyst for industrial biotechnology. To understand substrate and cosubstrate specificities of RasADH we determined the crystal structure of the apo-enzyme as well as its NADP⁺-bound state with resolutions down to 2.8 Å. RasADH displays a homotetrameric quaternary structure that can be described as a dimer of homodimers while in each subunit a seven-stranded parallel β-sheet, flanked by three α-helices on each side, forms a Rossmann fold-type dinucleotide binding domain. Docking of the well-known substrate (S)-1-phenylethanol clearly revealed the structural determinants of stereospecificity. To favor practical RasADH application in the context of established cofactor recycling systems, for example, those involving an NADH-dependent amino acid dehydrogenase, we attempted to rationally change its cosubstrate specificity from NADP⁺ to NAD⁺ utilizing the structural information that NADP⁺ specificity is largely governed by the residues Asn15, Gly37, Arg38, and Arg39. Furthermore, an extensive sequence alignment with homologous dehydrogenases that have different cosubstrate specificities revealed a modified general SDR motif ASNG (instead of NNAG) at positions 86-89 of RasADH. Consequently, we constructed mutant enzymes with one (G37D), four (N15G/G37D/R38V/R39S), and six (N15G/G37D/R38V/R39S/A86N/S88A) amino acid exchanges. RasADH (N15G/G37D/R38V/R39S) was better able to accept NAD⁺ while showing much reduced catalytic efficiency with NADP⁺, leading to a change in NADH/NADPH specificity by a factor of ∼3.6 million.

SCF(FBXL3) ubiquitin ligase targets cryptochromes at their cofactor pocket.

The flavoprotein cryptochromes (CRYs) act as blue-light receptors in plants and insects, but perform light-independent functions at the core of the mammalian circadian clock. To drive clock oscillations, mammalian CRYs associate with the Period proteins (PERs) and together inhibit the transcription of their own genes. The SCFFbxl3 ubiquitin ligase complex controls this negative feedback loop by promoting CRY ubiquitylation and degradation. Yet, the molecular mechanisms of their interactions and the functional role of flavin adenine dinucleotide (FAD) binding in CRYs remain poorly understood. Here we report crystal structures of mammalian CRY2 in its apo, FAD-bound, and Fbxl3-Skp1-complexed forms. Distinct from other cryptochromes of known structures, mammalian CRY2 binds FAD dynamically with an open cofactor pocket. Strikingly, the F-box protein Fbxl3 captures CRY2 by simultaneously occupying its FAD-binding pocket with a conserved C-terminal tail and burying its PER-binding interface. This novel F-box protein-substrate bipartite interaction is susceptible to disruption by both FAD and PERs, suggesting a new avenue for pharmacological targeting of the complex and a multifaceted regulatory mechanism of CRY ubiquitylation.