RESUMO
The retinal light response in animals originates from the photoisomerization of an opsin-coupled 11-cis-retinaldehyde chromophore. This visual chromophore is enzymatically produced through the action of carotenoid cleavage dioxygenases. Vertebrates require two carotenoid cleavage dioxygenases, ß-carotene oxygenase 1 and retinal pigment epithelium 65 (RPE65), to form 11-cis-retinaldehyde from carotenoid substrates, whereas invertebrates such as insects use a single enzyme known as Neither Inactivation Nor Afterpotential B (NinaB). RPE65 and NinaB couple trans-cis isomerization with hydrolysis and oxygenation, respectively, but the mechanistic relationship of their isomerase activities remains unknown. Here we report the structure of NinaB, revealing details of its active site architecture and mode of membrane binding. Structure-guided mutagenesis studies identify a residue cluster deep within the NinaB substrate-binding cleft that controls its isomerization activity. Our data demonstrate that isomerization activity is mediated by distinct active site regions in NinaB and RPE65-an evolutionary convergence that deepens our understanding of visual system diversity.
Assuntos
Carotenoides , Carotenoides/metabolismo , Carotenoides/química , Animais , Domínio Catalítico , Retinaldeído/metabolismo , Retinaldeído/química , cis-trans-Isomerases/metabolismo , cis-trans-Isomerases/genética , cis-trans-Isomerases/química , Dioxigenases/metabolismo , Dioxigenases/química , Dioxigenases/genética , Humanos , Modelos Moleculares , Evolução MolecularRESUMO
RPE65 is the essential trans-cis isomerase of the classical retinoid (visual) cycle. Mutations in RPE65 give rise to severe retinal dystrophies, most of which are associated with loss of protein function and recessive inheritance. The only known exception is a c.1430G>A (D477G) mutation that gives rise to dominant retinitis pigmentosa with delayed onset and choroidal and macular involvement. Position 477 is distant from functionally critical regions of RPE65. Hence, the mechanism of D477G pathogenicity remains unclear, although protein misfolding and aggregation mechanisms have been suggested. We characterized a D477G knock-in mouse model which exhibited mild age-dependent changes in retinal structure and function. Immunoblot analysis of protein extracts from the eyes of these knock-in mice demonstrated the presence of ubiquitinated RPE65 and reduced RPE65 expression. We observed an accumulation of retinyl esters in the knock-in mice as well as a delay in rhodopsin regeneration kinetics and diminished electroretinography responses, indicative of RPE65 functional impairment induced by the D477G mutation in vivo. However, a cell line expressing D477G RPE65 revealed protein expression levels, cellular localization and retinoid isomerase activity comparable to cells expressing wild-type protein. Structural analysis of an RPE65 chimera suggested that the D477G mutation does not perturb protein folding or tertiary structure. Instead, the mutation generates an aggregation-prone surface that could induce cellular toxicity through abnormal complex formation as suggested by crystal packing analysis. These results indicate that a toxic gain-of-function induced by the D477G RPE65 substitution may play a role in the pathogenesis of this form of dominant retinitis pigmentosa.
Assuntos
Genes Dominantes , Retina/metabolismo , Retinose Pigmentar/genética , cis-trans-Isomerases/genética , Animais , Modelos Animais de Doenças , Regulação da Expressão Gênica/genética , Técnicas de Introdução de Genes , Humanos , Camundongos , Mutação , Agregação Patológica de Proteínas/genética , Dobramento de Proteína , Retina/patologia , Células Fotorreceptoras Retinianas Cones/metabolismo , Células Fotorreceptoras Retinianas Cones/patologia , Retinose Pigmentar/patologia , cis-trans-Isomerases/químicaRESUMO
One key to the success of Pseudomonas spp. is their ability to reside in hostile environments. Pseudomonas spp. possess a cis-trans isomerase (Cti) an enzyme that converts the cis-unsaturated fatty acids (FAs) of the membrane lipids to their trans-isomers to rigidify the membrane and thereby resist stresses. Whereas the posttranslational Cti regulation has been previously reported, transcriptional cti regulation remains to be studied in more details. Here, we have studied cti transcriptional regulation in the solvent-tolerant strain Pseudomonas putida F1. Two cti transcriptional start sites (cti-279 and cti-77) were identified with cti-279 transcript being dominant. Expression of cti was found to increase with temperature increase, addition of the organic solvent, octanol and in the stationary growth phase. We found that cti expression was repressed by the cyclic-AMP receptor protein (Crp) and repression required the cyclic-AMP ligand of Crp. Production of trans-unsaturated FAs was found to decrease after 24 h of growth. Although this decrease was accompanied by an increase in cyclopropane FA content, this was not at the expense of trans-unsaturated FAs demonstrating the absence of competition between Cti and Cfa in FA modification.
Assuntos
Proteínas de Bactérias/genética , Ácidos Graxos/química , Regulação Enzimológica da Expressão Gênica , Pseudomonas putida/genética , Microbiologia do Solo , cis-trans-Isomerases/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Estabilidade Enzimática , Ácidos Graxos/metabolismo , Isomerismo , Pseudomonas putida/química , Pseudomonas putida/enzimologia , Pseudomonas putida/metabolismo , Transcrição Gênica , cis-trans-Isomerases/química , cis-trans-Isomerases/metabolismoRESUMO
Pseudomonas aeruginosa PAO1 can utilize various aromatic hydrocarbons as a carbon source. Among the three genes involved in the gentisate pathway of P. aeruginosa, the gene product of PA2473 belongs to the ζ-class glutathione S-transferase and is predicted to be a maleylpyruvate isomerase. In this study, we determined the crystal structure of maleylpyruvate isomerase from Pseudomonas aeruginosa PAO1 (PaMPI) at a resolution of 1.8â¯Å. PaMPI functions as a dimer and shows the glutathione S-transferase fold. The structure comparison with other glutathione S-transferase structures enabled us to predict the glutathione cofactor binding site and suggests that PaMPI has differences in residues that make up the putative substrate binding site. Biochemical study of PaMPI showed that the protein has an MPI activity. Interestingly, unlike the reported glutathione S-transferases so far, the purified PaMPI showed isomerase activity without the addition of the reduced glutathione, although the protein showed much higher activity when the glutathione cofactor was added to the reaction mixture. Taken together, our studies reveal that the gene product of PA2473 functions as a maleylpyruvate isomerase and might be involved in the gentisate pathway.
Assuntos
Pseudomonas aeruginosa/enzimologia , cis-trans-Isomerases/química , Sítios de Ligação , Cristalografia por Raios X , Gentisatos/metabolismo , Glutationa/metabolismo , Humanos , Modelos Moleculares , Conformação Proteica , Multimerização Proteica , Infecções por Pseudomonas/microbiologia , Pseudomonas aeruginosa/química , Pseudomonas aeruginosa/metabolismo , Especificidade por Substrato , cis-trans-Isomerases/metabolismoRESUMO
RPE65, the retinal pigment epithelium (RPE) smooth endoplasmic reticulum (sER) membrane-associated retinoid isomerase, plays an indispensable role in sustaining visual function in vertebrates. An important aspect which has attracted considerable attention is the posttranslational modification by S-palmitoylation of RPE65. Some studies show that RPE65 is a palmitoylated protein, but others deny that conclusion. While it is considered to be mainly responsible for RPE65's membrane association, we still lack conclusive evidence about RPE65 palmitoylation. In this review, we provide an overview of the history and current understanding of RPE65 palmitoylation.
Assuntos
Proteínas do Olho/química , Lipídeos/química , Lipoilação , Processamento de Proteína Pós-Traducional , Epitélio Pigmentado da Retina/enzimologia , cis-trans-Isomerases/química , Animais , Retículo Endoplasmático , HumanosRESUMO
RPE65 is the isomerase catalyzing conversion of all-trans-retinyl ester (atRE) into 11-cis-retinol in the retinal visual cycle. Crystal structures of RPE65 and site-directed mutagenesis reveal aspects of its catalytic mechanism, especially retinyl moiety isomerization, but other aspects remain to be determined. To investigate potential interactions between RPE65 and lipid metabolism enzymes, HEK293-F cells were transfected with expression vectors for visual cycle proteins and co-transfected with either fatty acyl:CoA ligases (ACSLs) 1, 3, or 6 or the SLC27A family fatty acyl-CoA synthase FATP2/SLCA27A2 to test their effect on isomerase activity. These experiments showed that RPE65 activity was reduced by co-expression of ACSLs or FATP2. Surprisingly, however, in attempting to relieve the ACSL-mediated inhibition, we discovered that triacsin C, an inhibitor of ACSLs, also potently inhibited RPE65 isomerase activity in cellulo. We found triacsin C to be a competitive inhibitor of RPE65 (IC50 = 500 nm). We confirmed that triacsin C competes directly with atRE by incubating membranes prepared from chicken RPE65-transfected cells with liposomes containing 0-1 µM atRE. Other inhibitors of ACSLs had modest inhibitory effects compared with triascin C. In conclusion, we have identified an inhibitor of ACSLs as a potent inhibitor of RPE65 that competes with the atRE substrate of RPE65 for binding. Triacsin C, with an alkenyl chain resembling but not identical to either acyl or retinyl chains, may compete with binding of the acyl moiety of atRE via the alkenyl moiety. Its inhibitory effect, however, may reside in its nitrosohydrazone/triazene moiety.
Assuntos
Inibidores Enzimáticos/farmacologia , Triazenos/farmacologia , cis-trans-Isomerases/química , Sequência de Aminoácidos , Animais , Sítios de Ligação , Galinhas , Coenzima A Ligases/antagonistas & inibidores , Células HEK293 , Humanos , Dados de Sequência Molecular , Ligação Proteica , cis-trans-Isomerases/antagonistas & inibidores , cis-trans-Isomerases/genética , cis-trans-Isomerases/metabolismoRESUMO
Modulators of the visual cycle have been developed for treatment of various retinal disorders. These agents were designed to inhibit retinoid isomerase [retinal pigment epithelium-specific 65 kDa protein (RPE65)], the rate-limiting enzyme of the visual cycle, based on the idea that attenuation of visual pigment regeneration could reduce formation of toxic retinal conjugates. Of these agents, certain ones that contain primary amine groups can also reversibly form retinaldehyde Schiff base adducts, which contributes to their retinal protective activity. Direct inhibition of RPE65 as a therapeutic strategy is complicated by adverse effects resulting from slowed chromophore regeneration, whereas effective retinal sequestration can require high drug doses with potential off-target effects. We hypothesized that the RPE65-emixustat crystal structure could help guide the design of retinaldehyde-sequestering agents with varying degrees of RPE65 inhibitory activity. We found that addition of an isopropyl group to the central phenyl ring of emixustat and related compounds resulted in agents effectively lacking in vitro retinoid isomerase inhibitory activity, whereas substitution of the terminal 6-membered ring with branched moieties capable of stronger RPE65 interaction potentiated inhibition. The isopropyl derivative series produced discernible visual cycle suppression in vivo, albeit much less potently than compounds with a high affinity for the RPE65 active site. These agents were distributed into the retina and formed Schiff base adducts with retinaldehyde. Except for one compound [3-amino-1-(3-isopropyl-5-((2,6,6-trimethylcyclohex-1-en-1-yl)methoxy)phenyl)propan-1-ol (MB-007)], these agents conferred protection against retinal phototoxicity, suggesting that both direct RPE65 inhibition and retinal sequestration are mechanisms of potential therapeutic relevance.
Assuntos
Visão Ocular/efeitos dos fármacos , cis-trans-Isomerases/antagonistas & inibidores , Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Oxirredutases do Álcool/genética , Oxirredutases do Álcool/metabolismo , Animais , Sítios de Ligação , Bovinos , Dermatite Fototóxica/prevenção & controle , Feminino , Cinética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microssomos/enzimologia , Modelos Moleculares , Epitélio Pigmentado Ocular/efeitos dos fármacos , Regeneração/efeitos dos fármacos , Doenças Retinianas/prevenção & controle , Epitélio Pigmentado da Retina/efeitos dos fármacos , Bases de Schiff/química , cis-trans-Isomerases/química , cis-trans-Isomerases/genética , cis-trans-Isomerases/isolamento & purificação , cis-trans-Isomerases/metabolismoRESUMO
Visual function in vertebrates is dependent on the membrane-bound retinoid isomerase RPE65, an essential component of the retinoid cycle pathway that regenerates 11-cis-retinal for rod and cone opsins. The mechanism by which RPE65 catalyzes stereoselective retinoid isomerization has remained elusive because of uncertainty about how retinoids bind to its active site. Here we present crystal structures of RPE65 in complex with retinoid-mimetic compounds, one of which is in clinical trials for the treatment of age-related macular degeneration. The structures reveal the active site retinoid-binding cavity located near the membrane-interacting surface of the enzyme as well as an Fe-bound palmitate ligand positioned in an adjacent pocket. With the geometry of the RPE65-substrate complex clarified, we delineate a mechanism of catalysis that reconciles the extensive biochemical and structural research on this enzyme. These data provide molecular foundations for understanding a key process in vision and pharmacological inhibition of RPE65 with small molecules.
Assuntos
Epitélio Pigmentado da Retina/enzimologia , Retinoides/farmacologia , Visão Ocular/efeitos dos fármacos , cis-trans-Isomerases/antagonistas & inibidores , cis-trans-Isomerases/química , Animais , Sítios de Ligação , Biocatálise , Cristalografia por Raios X , Diterpenos/síntese química , Diterpenos/química , Diterpenos/farmacologia , Ligantes , Luz , Camundongos Endogâmicos C57BL , Simulação de Acoplamento Molecular , Estrutura Molecular , Palmitatos , Éteres Fenílicos/síntese química , Éteres Fenílicos/química , Éteres Fenílicos/farmacologia , Propanolaminas/síntese química , Propanolaminas/química , Propanolaminas/farmacologia , Ligação Proteica , Conformação Proteica , Epitélio Pigmentado da Retina/efeitos dos fármacos , Epitélio Pigmentado da Retina/efeitos da radiação , Retinoides/síntese química , Retinoides/química , Estereoisomerismo , Visão Ocular/fisiologia , Visão Ocular/efeitos da radiaçãoRESUMO
Plants synthesize carotenoids, which are essential for plant development and survival. These metabolites also serve as essential nutrients for human health. The biosynthetic pathway for all plant carotenoids occurs in chloroplasts and other plastids and requires 15-cis-ζ-carotene isomerase (Z-ISO). It was not known whether Z-ISO catalyzes isomerization alone or in combination with other enzymes. Here we show that Z-ISO is a bona fide enzyme and integral membrane protein. Z-ISO independently catalyzes the cis-trans isomerization of the 15-15' carbon-carbon double bond in 9,15,9'-cis-ζ-carotene to produce the substrate required by the subsequent biosynthetic-pathway enzyme. We discovered that isomerization depends upon a ferrous heme b cofactor that undergoes redox-regulated ligand switching between the heme iron and alternate Z-ISO amino acid residues. Heme b-dependent isomerization of a large hydrophobic compound in a membrane was previously undescribed. As an isomerase, Z-ISO represents a new prototype for heme b proteins and potentially uses a new chemical mechanism.
Assuntos
Proteínas de Arabidopsis/metabolismo , Heme/metabolismo , Ferro/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Plantas/metabolismo , Zea mays/química , cis-trans-Isomerases/metabolismo , zeta Caroteno/biossíntese , Arabidopsis/química , Arabidopsis/enzimologia , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Cloroplastos/genética , Cloroplastos/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Heme/química , Interações Hidrofóbicas e Hidrofílicas , Ferro/química , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Isomerismo , Proteínas de Membrana/química , Proteínas de Membrana/genética , Modelos Moleculares , Oxirredução , Proteínas de Plantas/química , Proteínas de Plantas/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Zea mays/enzimologia , Zea mays/genética , cis-trans-Isomerases/química , cis-trans-Isomerases/genéticaRESUMO
RPE65 is the retinoid isomerohydrolase that converts all-trans-retinyl ester to 11-cis-retinol, a key reaction in the retinoid visual cycle. We have previously reported that cone-dominant chicken RPE65 (cRPE65) shares 90% sequence identity with human RPE65 (hRPE65) but exhibits substantially higher isomerohydrolase activity than that of bovine RPE65 or hRPE65. In this study, we sought to identify key residues responsible for the higher enzymatic activity of cRPE65. Based on the amino acid sequence comparison of mammalian and other lower vertebrates' RPE65, including cone-dominant chicken, 8 residues of hRPE65 were separately replaced by their counterparts of cRPE65 using site-directed mutagenesis. The enzymatic activities of cRPE65, hRPE65, and its mutants were measured by in vitro isomerohydrolase activity assay, and the retinoid products were analyzed by HPLC. Among the mutants analyzed, two single point mutants, N170K and K297G, and a double mutant, N170K/K297G, of hRPE65 exhibited significantly higher catalytic activity than WT hRPE65. Further, when an amino-terminal fragment (Met(1)-Arg(33)) of the N170K/K297G double mutant of hRPE65 was replaced with the corresponding cRPE65 fragment, the isomerohydrolase activity was further increased to a level similar to that of cRPE65. This finding contributes to the understanding of the structural basis for isomerohydrolase activity. This highly efficient human isomerohydrolase mutant can be used to improve the efficacy of RPE65 gene therapy for retinal degeneration caused by RPE65 mutations.
Assuntos
Mutação de Sentido Incorreto , cis-trans-Isomerases , Substituição de Aminoácidos , Animais , Bovinos , Linhagem Celular , Galinhas , Doenças Genéticas Inatas/enzimologia , Doenças Genéticas Inatas/genética , Doenças Genéticas Inatas/terapia , Terapia Genética , Humanos , Mutagênese Sítio-Dirigida , Degeneração Retiniana/enzimologia , Degeneração Retiniana/genética , Degeneração Retiniana/terapia , Especificidade da Espécie , Relação Estrutura-Atividade , cis-trans-Isomerases/química , cis-trans-Isomerases/imunologia , cis-trans-Isomerases/metabolismoRESUMO
Over 70 different missense mutations, including a dominant mutation, in RPE65 retinoid isomerase are associated with distinct forms of retinal degeneration; however, the disease mechanisms for most of these mutations have not been studied. Although some mutations have been shown to abolish enzyme activity, the molecular mechanisms leading to the loss of enzymatic function and retinal degeneration remain poorly understood. Here we show that the 26 S proteasome non-ATPase regulatory subunit 13 (PSMD13), a newly identified negative regulator of RPE65, plays a critical role in regulating pathogenicity of three mutations (L22P, T101I, and L408P) by mediating rapid degradation of mutated RPE65s via a ubiquitination- and proteasome-dependent non-lysosomal pathway. These mutant RPE65s were misfolded and formed aggregates or high molecular complexes via disulfide bonds. Interaction of PSMD13 with mutant RPE65s promoted degradation of misfolded but not properly folded mutant RPE65s. Many mutations, including L22P, T101I, and L408P, were mapped on non-active sites. Although their activities were very low, these mutant RPE65s were catalytically active and could be significantly rescued at low temperature, whereas mutant RPE65s with a distinct active site mutation could not be rescued under the same conditions. Sodium 4-phenylbutyrate and glycerol displayed a significant synergistic effect on the low temperature rescue of the mutant RPE65s by promoting proper folding, reducing aggregation, and increasing membrane association. Our results suggest that a low temperature eye mask and sodium 4-phenylbutyrate, a United States Food and Drug Administration-approved oral medicine, may provide a promising "protein repair therapy" that can enhance the efficacy of gene therapy by reducing the cytotoxic effect of misfolded mutant RPE65s.
Assuntos
Domínio Catalítico , Doença/genética , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Mutação de Sentido Incorreto , cis-trans-Isomerases/genética , cis-trans-Isomerases/metabolismo , Animais , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Glicerol/farmacologia , Células HEK293 , Humanos , Camundongos , Modelos Moleculares , Proteínas Mutantes/química , Fenilbutiratos/farmacologia , Complexo de Endopeptidases do Proteassoma/metabolismo , Dobramento de Proteína/efeitos dos fármacos , Proteólise/efeitos dos fármacos , Epitélio Pigmentado da Retina/citologia , Temperatura , Ubiquitinação/efeitos dos fármacos , cis-trans-Isomerases/químicaRESUMO
RPE65 is a key metalloenzyme responsible for maintaining visual function in vertebrates. Despite extensive research on this membrane-bound retinoid isomerase, fundamental questions regarding its enzymology remain unanswered. Here, we report the crystal structure of RPE65 in a membrane-like environment. These crystals, obtained from enzymatically active, nondelipidated protein, displayed an unusual packing arrangement wherein RPE65 is embedded in a lipid-detergent sheet. Structural differences between delipidated and nondelipidated RPE65 uncovered key residues involved in substrate uptake and processing. Complementary iron K-edge X-ray absorption spectroscopy data established that RPE65 as isolated contained a divalent iron center and demonstrated the presence of a tightly bound ligand consistent with a coordinated carboxylate group. These results support the hypothesis that the Lewis acidity of iron could be used to promote ester dissociation and generation of a carbocation intermediate required for retinoid isomerization.
Assuntos
Ferro/química , Lipídeos/química , Fosfolipídeos/química , cis-trans-Isomerases/química , Sequência de Aminoácidos , Animais , Sítios de Ligação/genética , Catálise , Bovinos , Cristalografia por Raios X , Ferro/metabolismo , Microssomos/enzimologia , Modelos Moleculares , Dados de Sequência Molecular , Fosfolipídeos/metabolismo , Conformação Proteica , Estrutura Terciária de Proteína , Epitélio Pigmentado da Retina/enzimologia , Homologia de Sequência de Aminoácidos , Espectroscopia por Absorção de Raios X , cis-trans-Isomerases/genética , cis-trans-Isomerases/metabolismoRESUMO
Nicotine is an environmental toxicant in tobacco waste, imposing a serious hazard for human health. Some bacteria including Pseudomonas spp. strains are able to metabolize nicotine to non-toxic compounds. The pyrrolidine pathway of nicotine degradation in Pseudomonas putida S16 has recently been revealed. The maleate isomerase (Pp-Iso) catalyses the last step in nicotine degradation of P. putida S16, the cis-trans isomerization of maleate to fumarate. In this study, we determined the crystal structures of both wild type isomerase by itself and its C200A point mutant in complex with its substrate maleate, to resolutions of 2.95 Å and 2.10 Å respectively. Our structures reveal that Asn17 and Asn169 play critical roles in recognizing the maleate by site-directed mutants' analysis. Surprisingly, our structure shows that the maleate is completely wrapped inside the isomerase. Examination of the structure prompted us to hypothesize that the ß2-α2 loop and the ß6-α7 loop have a breathing motion that regulates substrate/solvent entry and product departure. Our results of molecular dynamics simulation and enzymatic activity assay are fully consistent with this hypothesis. The isomerase probably uses this breathing motion to prevent the solvent from entering the active site and prohibit unproductive side reactions from happening.
Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Simulação de Dinâmica Molecular , Pseudomonas putida/enzimologia , cis-trans-Isomerases/química , cis-trans-Isomerases/metabolismo , Biotransformação , Cristalografia por Raios X , Análise Mutacional de DNA , Fumaratos/metabolismo , Malatos/metabolismo , Ligação Proteica , Conformação Proteica , Pseudomonas putida/químicaRESUMO
The maleate isomerase (MI) catalysed isomerization of maleate to fumarate has been investigated using a wide range of computational modelling techniques, including small model DFT calculations, QM-cluster approach, quantum mechanical/molecular mechanical approach (QM/MM in the ONIOM formalism) and molecular dynamics simulations. Several fundamental questions regarding the mechanism were answered in detail, such as the activation and stabilization of the catalytic Cys in a rather hydrophobic active site. The two previously proposed mechanisms were considered, where either enediolate or succinyl-Cys intermediate forms. Small model calculations as well as an ONIOM-based approach suggest that an enediolate intermediate is too unstable. Furthermore, the formation of succinyl-Cys intermediate via the nucleophilic attack of Cys76(-) on the substrate C2 (as proposed experimentally) was found to be energetically unfeasible in both QM-cluster and ONIOM approaches. Instead, our results show that Cys194, upon activation via the substrate, acts as a nucleophile and Cys76 acts as an acid/base catalyst, forming a succinyl-Cys intermediate in a concerted fashion. Indeed, the calculated PA of Cys76 is always higher than that of Cys194 before or upon substrate binding in the active site. Furthermore, the mechanism proceeds via multiple steps by substrate rotation around C2-C3 with the assistance of the now negatively charged Cys76, leading to the formation of fumarate. Finally, our calculated barrier is in good agreement with experiment. These findings represent a novel mechanism in the racemase superfamily.
Assuntos
Proteínas de Bactérias/metabolismo , Cisteína/química , cis-trans-Isomerases/metabolismo , Proteínas de Bactérias/química , Catálise , Domínio Catalítico , Teoria Quântica , cis-trans-Isomerases/químicaRESUMO
Dunaliella bardawil is a marine unicellular green algal that produces large amounts of ß-carotene and is a model organism for studying the carotenoid synthesis pathway. However, there are still many mysteries about the enzymes of the D. bardawil lycopene synthesis pathway that have not been revealed. Here, we have identified a CruP-like lycopene isomerase, named DbLyISO, and successfully cloned its gene from D. bardawil. DbLyISO showed a high homology with CruPs. We constructed a 3D model of DbLyISO and performed molecular docking with lycopene, as well as molecular dynamics testing, to identify the functional characteristics of DbLyISO. Functional activity of DbLyISO was also performed by overexpressing gene in both E. coli and D. bardawil. Results revealed that DbLyISO acted at the C-5 and C-13 positions of lycopene, catalyzing its cis-trans isomerization to produce a more stable trans structure. These results provide new ideas for the development of a carotenoid series from engineered bacteria, algae, and plants.
Assuntos
Clorofíceas , Liases Intramoleculares , Licopeno , cis-trans-Isomerases , Proteínas de Algas/genética , Proteínas de Algas/metabolismo , Proteínas de Algas/química , Sequência de Aminoácidos , Carotenoides/metabolismo , Carotenoides/química , Clorofíceas/enzimologia , Clorofíceas/genética , Clorofíceas/química , Clorofíceas/metabolismo , Clorófitas/enzimologia , Clorófitas/genética , Clorófitas/química , Clorófitas/metabolismo , cis-trans-Isomerases/genética , cis-trans-Isomerases/metabolismo , cis-trans-Isomerases/química , Escherichia coli/genética , Escherichia coli/metabolismo , Licopeno/metabolismo , Licopeno/química , Simulação de Acoplamento Molecular , Alinhamento de SequênciaRESUMO
Orange petals of calendula (Calendula officinalis) accumulate red carotenoids with the cis-configuration at the C-5 or C-5' position (5-cis-carotenoids). We speculated that the orange-flowered calendula is a carotenoid isomerase (crtiso) loss-of-function mutant that impairs the cis-to-trans conversion of 5-cis-carotenoids. We compared the sequences and enzyme activities of CRTISO from orange- and yellow-flowered calendulas. Four types of CRTISO were expressed in calendula petals. The deduced amino acid sequence of one of these genes (CoCRTISO1) was different between orange- and yellow-flowered calendulas, whereas the sequences of the other three CRTISOs were identical between these plants. Analysis of the enzymatic activities of the CoCRTISO homologs showed that CoCRTISO1-Y, which was expressed in yellow petals, converted carotenoids from the cis-to-trans-configuration, whereas both CoCRTISO1-ORa and 1-ORb, which were expressed in orange petals, showed no activity with any of the cis-carotenoids we tested. Moreover, the CoCRTISO1 genotypes of the F2 progeny obtained by crossing orange and yellow lines linked closely to petal color. These data indicate that CoCRTISO1 is a key regulator of the accumulation of 5-cis-carotenoids in calendula petals. Site-directed mutagenesis showed that the deletion of Cys-His-His at positions 462-464 in CoCRTISO1-ORa and a Gly-to-Glu amino acid substitution at position 450 in CoCRTISO1-ORb abolished enzyme activity completely, indicating that these amino acid residues are important for the enzymatic activity of CRTISO.
Assuntos
Calendula/anatomia & histologia , Calendula/enzimologia , Carotenoides/metabolismo , Pigmentação , Folhas de Planta/anatomia & histologia , Folhas de Planta/enzimologia , cis-trans-Isomerases/metabolismo , Sequência de Aminoácidos , Calendula/genética , Calendula/metabolismo , Clonagem Molecular , Regulação da Expressão Gênica de Plantas , Genótipo , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Mutação , Folhas de Planta/genética , Folhas de Planta/metabolismo , Homologia de Sequência de Aminoácidos , cis-trans-Isomerases/química , cis-trans-Isomerases/genéticaRESUMO
Previously, we showed that mutating RPE65 residue Phe-103 preferentially produces 13-cis-retinol instead of 11-cis-retinol, supporting a carbocation/radical cation mechanism of retinol isomerization. We asked whether this modulation of specificity can occur with residues other than Phe-103 and what role it plays in substrate binding and isomerization. We modeled the substrate-binding cleft of RPE65 to identify residues lining its surface. Many are phenylalanines and tyrosines, including three Phe residues (Phe-61, Phe-312, and Phe-526) forming an arch-like arrangement astride the cleft and Tyr-338. Also, Phe-418 sits at the neck of the cleft, lending a bend to the volume enclosed by the cleft. All mutations of Phe-61, Phe-312, and Phe-418 result in severely impaired or inactive enzyme. However, mutation of Phe-526 and Tyr-338, like Phe-103, decreases 11-cis-retinol formation, whereas increasing the 13-cis isomer. Significantly, 2 of these 3 residues, Phe-103 and Tyr-338, are located on putatively mobile interstrand loops. We propose that residual densities located in the binding cleft of the RPE65 structure represents a post-cleavage snapshot consistent not only with a fatty acid product, as originally modeled, but also an 11-cis-retinol product. Substrate docking simulations permit 11-cis- or 13-cis-retinyl ester binding in this relatively closed cleft, with the latter favored in F103L, F526A, and Y338A mutant structures, but prohibit binding of all-trans-retinyl ester, suggesting that isomerization occurs early in the temporal sequence, with O-alkyl ester cleavage occurring later. These findings provide insight into the mechanism of isomerization central to the visual cycle.
Assuntos
Fenilalanina/química , Tirosina/química , Vitamina A/química , cis-trans-Isomerases/química , Substituição de Aminoácidos , Animais , Sítios de Ligação , Cristalografia por Raios X , Camundongos , Mutação de Sentido Incorreto , Fenilalanina/genética , Fenilalanina/metabolismo , Ligação Proteica , Tirosina/genética , Tirosina/metabolismo , Vitamina A/genética , Vitamina A/metabolismo , cis-trans-Isomerases/genética , cis-trans-Isomerases/metabolismoRESUMO
PURPOSE: To identify pathogenic mutations responsible for retinal dystrophy in three consanguineous Pakistani families. METHODS: A thorough ophthalmic examination including fundus examination and electroretinography was performed, and blood samples were collected from all participating members. Genomic DNA was extracted, and genome-wide linkage and/or exclusion analyses were completed with fluorescently labeled short tandem repeat microsatellite markers. Two-point Lod scores were calculated, and coding exons along with exon-intron boundaries of RPE65 gene were sequenced, bidirectionally. RESULTS: Ophthalmic examinations of the patients affected in all three families suggested retinal dystrophy with an early, most probably congenital, onset. Genome-wide linkage and/or exclusion analyses localized the critical interval in all three families to chromosome 1p31 harboring RPE65. Bidirectional sequencing of RPE65 identified a splice acceptor site variation in intron 2: c.95-1G>A, a single base substitution in exon 3: c.179T>C, and a single base deletion in exon 5: c.361delT in the three families, respectively. All three variations segregated with the disease phenotype in their respective families and were absent from ethnically matched control chromosomes. CONCLUSIONS: These results strongly suggest that causal mutations in RPE65 are responsible for retinal dystrophy in the affected individuals of these consanguineous Pakistani families.
Assuntos
Consanguinidade , Mutação/genética , Distrofias Retinianas/genética , cis-trans-Isomerases/genética , Sequência de Aminoácidos , Sequência de Bases , Cromossomos Humanos Par 1/genética , Sequência Conservada/genética , Análise Mutacional de DNA , Eletrorretinografia , Família , Feminino , Fundo de Olho , Predisposição Genética para Doença , Haplótipos/genética , Humanos , Escore Lod , Masculino , Dados de Sequência Molecular , Paquistão , Linhagem , Distrofias Retinianas/fisiopatologia , cis-trans-Isomerases/químicaRESUMO
Peptide bond cis/trans isomerases (PCTIases) catalyze an intrinsically slow rotational motion taking part in the conformational dynamics of a protein backbone in all of its folding states. In this way, PCTIases assist other proteins to shape their functionally active structure. They have been associated with viral, bacterial, and parasitic infection, signal transduction, cell differentiation, altered metabolic activity, apoptosis, and many other physiological and pathophysiological processes. The need to understand, characterize, and control biochemical steps which contribute to the folding of proteins is a problem being addressed in many laboratories today. This review discusses the biochemical basis that the peptidyl prolyl cis/trans isomerase (PPIase) family of PCTIases uses for the control of bioactivity. Special emphasis is given to recent developments in the field of biocatalytic features of PPIases, the mechanism of catalysis, and enzyme inhibition.
Assuntos
Peptídeos/química , cis-trans-Isomerases/metabolismo , Biocatálise , Inibidores Enzimáticos/farmacologia , Conformação Proteica , cis-trans-Isomerases/antagonistas & inibidores , cis-trans-Isomerases/químicaRESUMO
In order to broaden the available genetic variation of melon, we developed an ethyl methanesulfonate mutation library in an orange-flesh 'Charentais' type melon line that accumulates ß-carotene. One mutagenized M2 family segregated for a novel recessive trait, a yellow-orange fruit flesh ('yofI'). HPLC analysis revealed that 'yofI' accumulates pro-lycopene (tetra-cis-lycopene) as its major fruit pigment. The altered carotenoid composition of 'yofI' is associated with a significant change of the fruit aroma since cleavage of ß-carotene yields different apocarotenoids than the cleavage of pro-lycopene. Normally, pro-lycopene is further isomerized by CRTISO (carotenoid isomerase) to yield all-trans-lycopene, which is further cyclized to ß-carotene in melon fruit. Cloning and sequencing of 'yofI' CRTISO identified two mRNA sequences which lead to truncated forms of CRTISO. Sequencing of the genomic CRTISO identified an A-T transversion in 'yofI' which leads to a premature STOP codon. The early carotenoid pathway genes were up regulated in yofI fruit causing accumulation of other intermediates such as phytoene and ζ-carotene. Total carotenoid levels are only slightly increased in the mutant. Mutants accumulating pro-lycopene have been reported in both tomato and watermelon fruits, however, this is the first report of a non-lycopene accumulating fruit showing this phenomenon.