RESUMO
α-Mannosidosis is an autosomal recessive lysosomal storage disorder caused by mutations in the MAN2B1 gene, encoding lysosomal α-mannosidase. The disorder is characterized by a range of clinical phenotypes of which the major manifestations are mental impairment, hearing impairment, skeletal changes, and immunodeficiency. Here, we report an α-mannosidosis mutation database, amamutdb.no, which has been constructed as a publicly accessible online resource for recording and analyzing MAN2B1 variants (http://amamutdb.no). Our aim has been to offer structured and relational information on MAN2B1 mutations and genotypes along with associated clinical phenotypes. Classifying missense mutations, as pathogenic or benign, is a challenge. Therefore, they have been given special attention as we have compiled all available data that relate to their biochemical, functional, and structural properties. The α-mannosidosis mutation database is comprehensive and relational in the sense that information can be retrieved and compiled across datasets; hence, it will facilitate diagnostics and increase our understanding of the clinical and molecular aspects of α-mannosidosis. We believe that the amamutdb.no structure and architecture will be applicable for the development of databases for any monogenic disorder.
Assuntos
Bases de Dados Genéticas , alfa-Manosidase/genética , alfa-Manosidose/genética , Alelos , Estudos de Associação Genética , Genótipo , Mutação , Fenótipo , Conformação Proteica , Software , Relação Estrutura-Atividade , alfa-Manosidase/química , alfa-Manosidase/metabolismo , alfa-Manosidose/diagnóstico , alfa-Manosidose/metabolismoRESUMO
The main function of lysosomal proteins is to degrade cellular macromolecules. We purified a novel lysosomal protein to homogeneity from bovine kidneys. By gene annotation, this protein is defined as a bovine phospholipase B-like protein 1 (bPLBD1) and, to better understand its biological function, we solved its structure at 1.9 Å resolution. We showed that bPLBD1 has uniform noncomplex-type N-glycosylation and that it localized to the lysosome. The first step in lysosomal protein transport, the initiation of mannose-6-phosphorylation by a N-acetylglucosamine-1-phosphotransferase, requires recognition of at least two distinct lysines on the protein surface. We identified candidate lysines by analyzing the structural and sequentially conserved N-glycosylation sites and lysines in bPLBD1 and in the homologous mouse PLBD2. Our model suggests that N408 is the primarily phosphorylated glycan, and K358 a key residue for N-acetylglucosamine-1-phosphotransferase recognition. Two other lysines, K334 and K342, provide the required second site for N-acetylglucosamine-1-phosphotransferase recognition. bPLBD1 is an N-terminal nucleophile (Ntn) hydrolase. By comparison with other Ntn-hydrolases, we conclude that the acyl moiety of PLBD1 substrate must be small to fit the putative binding pocket, whereas the space for the rest of the substrate is a large open cleft. Finally, as all the known substrates of Ntn-hydrolases have amide bonds, we suggest that bPLBD1 may be an amidase or peptidase instead of lipase, explaining the difficulty in finding a good substrate for any members of the PLBD family.
Assuntos
Amidoidrolases/química , Lisofosfolipase/química , Lisossomos/enzimologia , Amidoidrolases/isolamento & purificação , Amidoidrolases/metabolismo , Animais , Domínio Catalítico , Bovinos , Cristalografia por Raios X , Células HeLa , Humanos , Ligação de Hidrogênio , Rim/enzimologia , Lisofosfolipase/isolamento & purificação , Lisofosfolipase/metabolismo , Modelos Moleculares , Processamento de Proteína Pós-Traducional , Estrutura Quaternária de Proteína , Estrutura Secundária de Proteína , Análise de Sequência de ProteínaRESUMO
α-Mannosidosis is a lysosomal storage disorder caused by mutations in the MAN2B1 gene. The clinical presentation of α-mannosidosis is variable, but typically includes mental retardation, skeletal abnormalities and immune deficiency. In order to understand the molecular aetiology of α-mannosidosis, we describe here the subcellular localization and intracellular processing of 35 MAN2B1 variants, including 29 novel missense mutations. In addition, we have analysed the impact of the individual mutations on the three-dimensional structure of the human MAN2B1. We categorize the MAN2B1 missense mutations into four different groups based on their intracellular processing, transport and secretion in cell culture. Impaired transport to the lysosomes is a frequent cause of pathogenicity and correlates with a lack of protein processing (groups 1 and 3). Mutant MAN2B1 proteins that find their way to the lysosomes are processed, but less efficiently than the wild-types (groups 2 and 4). The described four categories of missense mutations likely represent different pathogenic mechanisms. We demonstrate that the severity of individual mutations cannot be determined based only on their position in the sequence. Pathogenic mutations cluster into amino acids which have an important role on the domain interface (arginines) or on the folding of the enzyme (prolines, glycines, cysteines). Tolerated mutations generally include surface mutations and changes without drastic alteration of residue volume. The expression system and structural details presented here provide opportunities for the development of pharmacological therapy by screening or design of small molecules that might assist MAN2B1 folding and hence, transport and activity.
Assuntos
Mutação/genética , alfa-Manosidose/enzimologia , alfa-Manosidose/genética , Substituição de Aminoácidos , Animais , Células CHO , Células COS , Chlorocebus aethiops , Cricetinae , Cricetulus , Regulação da Expressão Gênica , Células HeLa , Humanos , Espaço Intracelular/metabolismo , Manosidases/química , Manosidases/genética , Modelos Moleculares , Conformação Proteica , Transporte Proteico/genéticaRESUMO
The structure of phosphoribosyl anthranilate isomerase (TrpF) from the hyperthermophilic archaeon Pyrococcus furiosus (PfTrpF) has been determined at 1.75 Å resolution. The PfTrpF structure has a monomeric TIM-barrel fold which differs from the dimeric structures of two other known thermophilic TrpF proteins. A comparison of the PfTrpF structure with the two known bacterial thermophilic TrpF structures and the structure of a related mesophilic protein from Escherichia coli (EcTrpF) is presented. The thermophilic TrpF structures contain a higher proportion of ion pairs and charged residues compared with the mesophilic EcTrpF. These residues contribute to the closure of the central barrel and the stabilization of the barrel and the surrounding α-helices. In the monomeric PfTrpF conserved structural water molecules are mostly absent; instead, the structural waters are replaced by direct side-chain-main-chain interactions. As a consequence of these combined mechanisms, the P. furiosus enzyme is a thermodynamically stable and entropically optimized monomeric TIM-barrel enzyme which defines a good framework for further protein engineering for industrial applications.
Assuntos
Aldose-Cetose Isomerases/química , Pyrococcus furiosus/enzimologia , Sequência de Aminoácidos , Cristalografia por Raios X , Temperatura Alta , Íons/química , Modelos Moleculares , Dados de Sequência Molecular , Conformação Proteica , Multimerização Proteica , Estrutura Secundária de Proteína , Pyrococcus furiosus/química , Alinhamento de Sequência , Água/químicaRESUMO
Unfolded glycoproteins retained in the endoplasmic reticulum (ER) are degraded via the ER-associated degradation (ERAD) pathway. These proteins are subsequently transported to the cytosol and degraded by the proteasomal complex. Although the sequential events of ERAD are well described, its regulation remains poorly understood. The cytosolic mannosidase, Man2C1, plays an essential role in the catabolism of cytosolic free oligomannosides, which are released from the degraded proteins. We have investigated the impact of Man2C1 overexpression on protein glycosylation and the ERAD process. We demonstrated that overexpression of Man2C1 led to modifications of the cytosolic pool of free oligomannosides and resulted in accumulation of small Man(2-4)GlcNAc(1) glycans in the cytosol. We further correlated this accumulation with incomplete protein glycosylation and truncated lipid-linked glycosylation precursors, which yields an increase in N-glycoprotein en route to the ERAD. We propose a model in which high mannose levels in the cytosol interfere with glucose metabolism and compromise N-glycan synthesis in the ER. Our results show a clear link between the intracellular mannose-6-phosphate level and synthesis of the lipid-linked precursors for protein glycosylation. Disturbance in these pathways interferes with protein glycosylation and upregulated ERAD. Our findings support a new concept that regulation of Man2C1 expression is essential for maintaining efficient protein N-glycosylation.
Assuntos
Retículo Endoplasmático/metabolismo , Manosidases/biossíntese , Complexo de Endopeptidases do Proteassoma/metabolismo , Regulação para Cima , Glicosilação , Células HeLa , Humanos , Manosefosfatos/metabolismo , Manosidases/química , Oligossacarídeos/metabolismo , Transfecção , Uridina Difosfato Glucose/metabolismo , alfa-ManosidaseRESUMO
Psychrophilic alkaline phosphatase (AP) from the Antarctic strain TAB5 was subjected to directed evolution in order to identify the key residues steering the enzyme's cold-adapted activity and stability. A round of random mutagenesis and further recombination yielded three thermostable and six thermolabile variants of the TAB5 AP. All of the isolated variants were characterised by their residual activity after heat treatment, Michaelis-Menten kinetics, activation energy and microcalorimetric parameters of unfolding. In addition, they were modelled into the structure of the TAB5 AP. Mutations which affected the cold-adapted properties of the enzyme were all located close to the active site. The destabilised variants H135E and H135E/G149D had 2- and 3-fold higher kcat, respectively, than the wild-type enzyme. Wild-type AP has a complex heat-induced unfolding pattern while the mutated enzymes loose local unfolding transitions and have large shifts of the Tm values. Comparison of the wild-type and mutated TAB5 APs demonstrates that there is a delicate balance between the enzyme activity and stability and that it is possible to improve the activity and thermostability simultaneously as demonstrated in the case of the H135E/G149D variant compared to H135E.
Assuntos
Fosfatase Alcalina/química , Engenharia de Proteínas/métodos , Fosfatase Alcalina/genética , Sítios de Ligação , Varredura Diferencial de Calorimetria , Dimerização , Evolução Molecular Direcionada , Cinética , Mutagênese Sítio-Dirigida , Mutação , Conformação Proteica , Desnaturação Proteica , Dobramento de Proteína , Recombinação Genética , Temperatura , TermodinâmicaRESUMO
Alkaline phosphatases (APs) are non-specific phosphohydrolases that are widely used in molecular biology and diagnostics. We describe the structure of the cold active alkaline phosphatase from the Antarctic bacterium TAB5 (TAP). The fold and the active site geometry are conserved with the other AP structures, where the monomer has a large central beta-sheet enclosed by alpha-helices. The dimer interface of TAP is relatively small, and only a single loop from each monomer replaces the typical crown domain. The structure also has typical cold-adapted features; lack of disulfide bridges, low number of salt-bridges, and a loose dimer interface that completely lacks charged interactions. The dimer interface is more hydrophobic than that of the Escherichia coli AP and the interactions have tendency to pair with backbone atoms, which we propose to result from the cold adaptation of TAP. The structure contains two additional magnesium ions outside of the active site, which we believe to be involved in substrate binding as well as contributing to the local stability. The M4 site stabilises an interaction that anchors the substrate-coordinating R148. The M5 metal-binding site is in a region that stabilises metal coordination in the active site. In other APs the M5 binding area is supported by extensive salt-bridge stabilisation, as well as positively charged patches around the active site. We propose that these charges, and the TAP M5 binding, influence the release of the product phosphate and thus might influence the rate-determining step of the enzyme.
Assuntos
Fosfatase Alcalina/química , Bactérias/enzimologia , Metais/química , Fosfatase Alcalina/genética , Sítios de Ligação , Cristalização , Cristalografia por Raios X , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Estrutura Terciária de Proteína , Relação Estrutura-AtividadeRESUMO
The crystal structure of L-lactate oxidase (LOX) from Aerococcus viridans has been determined at 2.1 A resolution. LOX catalyzes the flavin mononucleotide (FMN) dependent oxidation of lactate to pyruvate and hydrogen peroxide. LOX belongs to the alpha-hydroxy-acid oxidase flavoenzyme family; members of which bind similar substrates and to some extent have conserved catalytic properties and structural motifs. LOX crystallized as two tightly packed tetramers in the asymmetric unit, each having fourfold symmetry. The present structure shows a conserved FMN coordination, but also reveals novel residues involved in substrate binding compared with other family members.
Assuntos
Oxigenases de Função Mista/química , Streptococcaceae/enzimologia , Sequência de Aminoácidos , Sítios de Ligação , Cristalização , Cristalografia por Raios X , Mononucleotídeo de Flavina/química , Dados de Sequência Molecular , Estrutura Quaternária de Proteína , Alinhamento de SequênciaRESUMO
Mutation analysis performed on six Italian families with alpha-mannosidosis type II allowed the identification of five new mutations in the MAN2B1 gene: c.157G>T, c.562C>T, c.599A>T, c.293dupA, c.2402G>A (p.E53X, p.R188X, p.H200L, p.Y99VfsX61, p.G801D). Protein residues G801 and H200 are conserved among the four mammalian alpha-mannosidases cloned to date: human, cattle, cat and mouse. In vitro expression studies demonstrated that both missense mutations expressed no residual alpha-mannosidase activity indicating that they are disease-causing mutations. Modelling into the three-dimensional structure revealed that the p.H200L could involve the catalytic mechanism, whereas p.G801D would affect the correct folding of the enzyme.
Assuntos
Mutação Puntual , alfa-Manosidase/genética , alfa-Manosidose/genética , Animais , Catálise , Gatos , Bovinos , Linhagem Celular , Códon sem Sentido , Consanguinidade , Análise Mutacional de DNA , Humanos , Itália , Rim , Lisossomos/enzimologia , Camundongos , Modelos Moleculares , Mutagênese Sítio-Dirigida , Mutação de Sentido Incorreto , Reação em Cadeia da Polimerase , Conformação Proteica , Dobramento de Proteína , Proteínas Recombinantes de Fusão/metabolismo , Especificidade da Espécie , alfa-Manosidase/química , alfa-Manosidase/deficiência , alfa-Manosidase/metabolismo , alfa-Manosidose/classificação , alfa-Manosidose/enzimologiaRESUMO
The aim of this work is to develop a prokaryotic system capable of expressing membrane-bound receptors in quantities suitable for biochemical and biophysical studies. Our strategy exploits the endogenous high-level expression of the membrane protein bacteriorhodopsin (BR) in the Archaeon Halobacterium salinarum. We attempted to express the human muscarinic acetylcholine (M(1)) and adrenergic (a2b) receptors by fusing the coding region of the m1 and a2b genes to nucleotide sequences known to direct bacterio-opsin (bop) gene transcription. The fusions included downstream modifications to produce non-native carboxyl-terminal amino acids useful for protein identification and purification. bop mRNA and BR accumulation were found to be tightly coupled and the carboxyl-terminal coding region modifications perturbed both. m1 and a2b mRNA levels were low, and accumulation was sensitive to both the extent of the bop gene fusion and the specific carboxyl-terminal coding sequence modifications included. Functional a2b adrenergic receptor expression was observed to be dependent on the downstream coding region. This work demonstrates that a critical determinant of expression resides in the downstream coding region of the wild-type bop gene and manipulation of the downstream coding region of heterologous genes may affect their potential for expression in H. salinarum.
Assuntos
Bacteriorodopsinas/genética , Halobacterium salinarum/genética , Receptores Adrenérgicos/genética , Receptores Muscarínicos/genética , Sequência de Aminoácidos , Fusão Gênica Artificial , Bacteriorodopsinas/análise , Bacteriorodopsinas/biossíntese , Sequência de Bases , Western Blotting , Expressão Gênica , Halobacterium salinarum/crescimento & desenvolvimento , Halobacterium salinarum/metabolismo , Dados de Sequência Molecular , Plasmídeos , RNA Mensageiro/análise , RNA Mensageiro/biossíntese , Receptores Adrenérgicos/biossíntese , Receptores Adrenérgicos alfa 2/genética , Receptores Muscarínicos/biossíntese , Proteínas Recombinantes de Fusão/genética , Fatores de TempoRESUMO
Lysosomal alpha-mannosidase (LAM: EC 3.2.1.24) belongs to the sequence-based glycoside hydrolase family 38 (GH38). Two other mammalian GH38 members, Golgi alpha-mannosidase II (GIIAM) and cytosolic alpha-mannosidase, are expressed in all tissues. In humans, cattle, cat and guinea pig, lack of lysosomal alpha-mannosidase activity causes the autosomal recessive disease alpha-mannosidosis. Here, we describe the three-dimensional structure of bovine lysosomal alpha-mannosidase (bLAM) at 2.7A resolution and confirm the solution state dimer by electron microscopy. We present the first structure of a mammalian GH38 enzyme that offers indications for the signal areas for mannose phosphorylation, suggests a previously undetected mechanism of low-pH activation and provides a template for further biochemical studies of the family 38 glycoside hydrolases as well as lysosomal transport. Furthermore, it provides a basis for understanding the human form of alpha-mannosidosis at the atomic level. The atomic coordinates and structure factors have been deposited in the Protein Data Bank (accession codes 1o7d and r1o7dsf).
Assuntos
Lisossomos/enzimologia , Manosidases/química , Animais , Sítios de Ligação , Bovinos , Cristalografia por Raios X , Bases de Dados como Assunto , Dimerização , Drosophila melanogaster , Ativação Enzimática , Glicosídeo Hidrolases/química , Glicosilação , Ligação de Hidrogênio , Concentração de Íons de Hidrogênio , Rim/enzimologia , Lisossomos/metabolismo , Manose/metabolismo , Microscopia Eletrônica , Modelos Moleculares , Mutação , Conformação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , alfa-ManosidaseRESUMO
Human LAMAN (lysosomal a-mannosidase) was synthesized as a 120 kDa precursor in transfected COS cells [African-green-monkey kidney cells], which was partly secreted as a single-chain form and partly sorted to the lysosomes being subsequently cleaved into three peptides of 70, 40 and 15 kDa respectively. Both the secreted and the lysosomal forms contained endo H (endoglucosidase H)-resistant glycans, suggesting a common pathway through the trans-Golgi network. A fraction of LAMAN was retained intracellularly as a single-chain endo H-sensitive form, probably in the ER (endoplasmic reticulum). The inherited lack of LAMAN causes the autosomal recessive storage disease a-mannosidosis. To understand the biochemical consequences of the disease-causing mutations, 11 missense mutations and two in-frame deletions were introduced into human LAMAN cDNA by in vitro mutagenesis and the resulting proteins were expressed in COS cells. Some selected mutants were also expressed in Chinese-hamster ovary cells. T355P (Thr355Pro), P356R, W714R, R750W and L809P LAMANs as well as both deletion mutants were misfolded and arrested in the ER as inactive single-chain forms. Six of the mutants were transported to the lysosomes, either with less than 5% of normal specific activity (H72L, D196E/N and R220H LAMANs) or with more than 30% of normal specific activity (E402K LAMAN). F320L LAMAN resulted in much lower activity in Chinese-hamster ovary cells when compared with COS cells. Modelling into the three-dimensional structure revealed that the mutants with highly reduced specific activities contained substitutions of amino acids involved in the catalysis, either co-ordinating Zn2+ (His72 and Asp196), stabilizing the active-site nucleophile (Arg220) or positioning the active-site residue Asp319 (Phe320).
Assuntos
Lisossomos/enzimologia , Transporte Proteico/fisiologia , alfa-Manosidase/metabolismo , alfa-Manosidose/enzimologia , Animais , Células CHO/química , Células CHO/metabolismo , Células COS/química , Células COS/metabolismo , Bovinos , Linhagem Celular , Chlorocebus aethiops , Cricetinae , Genótipo , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Manosidases , Modelos Moleculares , Mutagênese Sítio-Dirigida/genética , Fenótipo , Estrutura Quaternária de Proteína , Transporte Proteico/genética , Transfecção/métodos , alfa-Manosidase/química , alfa-Manosidase/genética , alfa-Manosidose/genéticaRESUMO
We used an unbiased genome-wide approach to identify exonic variants segregating with diabetes in a multigenerational Finnish family. At least eight members of this family presented with diabetes with age of diagnosis ranging from 18 to 51 years and a pattern suggesting autosomal dominant inheritance. We sequenced the exomes of four affected members of this family and performed follow-up genotyping of additional affected and unaffected family members. We uncovered a novel nonsynonymous variant (p.Trp314Arg) in the Wolfram syndrome 1 (WFS1) gene that segregates completely with the diabetic phenotype. Multipoint parametric linkage analysis with 13 members of this family identified a single linkage signal with maximum logarithm of odds score 3.01 at 4p16.2-p16.1, corresponding to a region harboring the WFS1 locus. Functional studies demonstrate a role for this variant in endoplasmic reticulum stress, which is consistent with the ß-cell failure phenotype seen in mutation carriers. This represents the first compelling report of a mutation in WFS1 associated with dominantly inherited nonsyndromic adult-onset diabetes.
Assuntos
Diabetes Mellitus/genética , Proteínas de Membrana/genética , Síndrome de Wolfram/genética , Adolescente , Adulto , Estresse do Retículo Endoplasmático , Exoma/genética , Feminino , Genes Dominantes , Ligação Genética , Humanos , Masculino , Pessoa de Meia-Idade , LinhagemRESUMO
Acid α-glucosidase (GAA) is a lysosomal enzyme and a pharmacological target for Pompe disease, an inherited lysosomal storage disorder (LSD). An emerging treatment for LSDs is the use of pharmacological chaperones, small molecules that enhance total cellular activity of the target lysosomal protein. We have systematically studied thirteen inhibitors, which provide good lead compounds for the development of GAA chaperones. We have verified binding on GAA at low and neutral pH, mapping the range of pH during transport to lysosomes. These ligands inhibit GAA competitively and reversibly, and a few of the compounds show higher molecular stabilisation capacity than would be expected from their binding affinity. These molecules also increase lysosomal localisation of GAA variants in cells. In order to understand the specific molecular mechanism of the interactions, we docked the compounds to a homology model of the human GAA. Three factors contribute to the tightness of binding. Firstly, well-positioned hydroxy groups are essential to orient the ligand and make the binding specific. Secondly, the open nature of the GAA active site allows both large and small ligands to bind. The third and most important binding determinant is the positive charge on the ligand, which is neutralised by Asp 518 or Asp 616 on GAA. Our study creates a firm basis for the design of drugs to treat Pompe disease, as it provides a comparable study of the ligand properties. Our analysis suggests a useful drug design framework for specific pharmacological chaperones for human GAA.
Assuntos
Desenho de Fármacos , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Doença de Depósito de Glicogênio Tipo II/tratamento farmacológico , Doença de Depósito de Glicogênio Tipo II/enzimologia , alfa-Glucosidases/metabolismo , Humanos , Lisossomos/efeitos dos fármacos , Lisossomos/enzimologia , Lisossomos/metabolismo , Modelos Moleculares , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Relação Estrutura-Atividade , alfa-Glucosidases/químicaRESUMO
Alkaline phosphatases (APs) are commercially applied enzymes that catalyze the hydrolysis of phosphate monoesters by a reaction involving three active site metal ions. We have previously identified H135 as the key residue for controlling activity of the psychrophilic TAB5 AP (TAP). In this article, we describe three X-ray crystallographic structures on TAP variants H135E and H135D in complex with a variety of metal ions. The structural analysis is supported by thermodynamic and kinetic data. The AP catalysis essentially requires octahedral coordination in the M3 site, but stability is adjusted with the conformational freedom of the metal ion. Comparison with the mesophilic Escherichia coli, AP shows differences in the charge transfer network in providing the chemically optimal metal combination for catalysis. Our results provide explanation why the TAB5 and E. coli APs respond in an opposite way to mutagenesis in their active sites. They provide a lesson on chemical fine tuning and the importance of the second coordination sphere in defining metal specificity in enzymes. Understanding the framework of AP catalysis is essential in the efforts to design even more powerful tools for modern biotechnology.
Assuntos
Fosfatase Alcalina/metabolismo , Metais/metabolismo , Zinco/metabolismo , Fosfatase Alcalina/química , Sítios de Ligação , Varredura Diferencial de Calorimetria , Domínio Catalítico , Cristalografia por Raios X , Estabilidade Enzimática , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Metais/química , Modelos Moleculares , Mutação , Dobramento de Proteína , Temperatura , Termodinâmica , Zinco/químicaRESUMO
We have determined the structures of the wild type and seven active site variants of yeast inorganic pyrophosphatase (PPase) in the presence of Mg2+ and phosphate, providing the first complete structural description of its catalytic cycle. PPases catalyze the hydrolysis of pyrophosphate and require four divalent metal cations for catalysis; magnesium provides the highest activity. The crystal form chosen contains two monomers in the asymmetric unit, corresponding to distinct catalytic intermediates. In the "closed" wild-type active site, one of the two product phosphates has already dissociated, while the D115E variant "open" conformation is of the hitherto unobserved two-phosphate and two-"bridging" water active site. The mutations affect metal binding and the hydrogen bonding network in the active site, allowing us to explain the effects of mutations. For instance, in Y93F, F93 binds in a cryptic hydrophobic pocket in the absence of substrate, preserving hydrogen bonding in the active site and leading to relatively small changes in solution properties. This is not true in the presence of substrate, when F93 is forced back into the active site. Such subtle changes underline how low the energy barriers are between thermodynamically favorable conformations of the enzyme. The structures also allow us to associate metal binding constants to specific sites. Finally, the wild type and the D152E variant contain a phosphate ion adjacent to the active site, showing for the first time how product is released through a channel of flexible cationic side chains.
Assuntos
Pirofosfatase Inorgânica/química , Sítios de Ligação/genética , Catálise , Proteínas Fúngicas , Ligação de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Pirofosfatase Inorgânica/genética , Magnésio/química , Mutação , FosfatosRESUMO
Functional and structural studies require gene overexpression and purification of soluble proteins. We wanted to express proteins from the psychrophilic bacterium Vibrio salmonicida in Escherichia coli, but encountered solubility problems. To improve the solubility of the proteins, we compared the effects of six N-terminal fusion proteins (Gb1, Z, thioredoxin, GST, MBP and NusA) and an N-terminal His6-tag. The selected test set included five proteins from the fish pathogen V. salmonicida and two related products from the mesophilic human pathogen Vibrio cholerae. We tested the expression in two different expression strains and at three different temperatures (16, 23 and 37 degrees C). His6-tag was the least effective tag, and these vector constructs were also difficult to transform. MBP and NusA performed best, expressing soluble proteins with all fusion partners in at least one of the cell types. In some cases MBP, GST and thioredoxin fusions resulted in products of incorrect size. The effect of temperature is complex: in most cases level of expression increased with temperature, whereas the effect on solubility was opposite. We found no clear connection between the preferred expression temperature of the protein and the temperature of the original host organism's natural habitat.
Assuntos
Aliivibrio salmonicida/fisiologia , Proteínas de Bactérias/genética , Escherichia coli/genética , Proteínas de Bactérias/química , Clonagem Molecular , Temperatura Baixa , Regulação Bacteriana da Expressão Gênica , Solubilidade , Homologia Estrutural de ProteínaRESUMO
A glycosyl hydrolase family 38 enzyme, neutral alpha-mannosidase, has been proposed to be involved in hydrolysis of cytosolic free oligosaccharides originating either from ER-misfolded glycoproteins or the N-glycosylation process. Although this enzyme has been isolated from the cytosol, it has also been linked to the ER by subcellular fractionations. We have studied the subcellular localization of neutral alpha-mannosidase by immunofluorescence microscopy and characterized the human recombinant enzyme with natural substrates to elucidate the biological function of this enzyme. Immunofluorescence microscopy showed neutral alpha-mannosidase to be absent from the ER, lysosomes, and autophagosomes, and being granularly distributed in the cytosol. In experiments with fluorescent recovery after photo bleaching, neutral alpha-mannosidase had slower than expected two-phased diffusion in the cytosol. This result together with the granular appearance in immunostaining suggests that portion of the neutral alpha-mannosidase pool is somehow complexed. The purified recombinant enzyme is a tetramer and has a neutral pH optimum for activity. It hydrolyzed Man(9)GlcNAc to Man(5)GlcNAc in the presence of Fe(2+), Co(2+), and Mn(2+), and uniquely to neutral alpha-mannosidases from other organisms, the human enzyme was more activated by Fe(2+) than Co(2+). Without activating cations the main reaction product was Man(8)GlcNAc, and Cu(2+) completely inhibited neutral alpha-mannosidase. Our findings from enzyme-substrate characterizations and subcellular localization studies support the suggested role for neutral alpha-mannosidase in hydrolysis of soluble cytosolic oligomannosides.
Assuntos
Citosol/enzimologia , Oligossacarídeos/metabolismo , alfa-Manosidase/metabolismo , Animais , Autofagia , Células CHO/ultraestrutura , Cricetinae , Cricetulus , Retículo Endoplasmático/metabolismo , Fibroblastos/citologia , Fibroblastos/metabolismo , Fibroblastos/ultraestrutura , Glicosilação , Humanos , Hidrólise , Imunização , Imunoglobulina G/imunologia , Lisossomos/metabolismo , Masculino , Microscopia Confocal , Microscopia de Fluorescência , Pichia/crescimento & desenvolvimento , Pichia/metabolismo , Coelhos , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Frações SubcelularesRESUMO
Yeast inorganic pyrophosphatase (Y-PPase) is a model system for studying phosphoryl-transfer reactions catalysed by multiple metal ions. To understand the process requires knowledge of the positions of the protons in the active site, which can be best achieved by neutron diffraction analysis. In order to reduce the hydrogen incoherent-scattering background and to improve the signal-to-noise ratio of the neutron reflections, deuterated protein was produced. Deuterated protein 96% enriched with deuterium was produced in high yield and crystals as large as 2 mm on one side were obtained. These crystals have unit-cell parameters a = 58.9, b = 103.9, c = 117.0 A, alpha = beta = gamma = 90 degrees at 273 K and diffract neutrons to resolutions of 2.5-3 A. The X-ray structure of the perdeuterated protein has also been refined at 273 K to 1.9 A resolution.