RESUMO
BACKGROUND: To contain intra-hospital transmission of third-generation cephalosporin-resistant Enterobacterales (3GCR-E), contact isolation precautions are recommended. AIM: To quantify transmissions of 3GCR Escherichia coli and 3GCR Klebsiella pneumoniae within a hospital. METHODS: An automated outbreak detection system (AODS) was used to identify clusters (N≥2) of 3GCR Enterobacterales for the years 2016, 2018 and 2020. Clusters were defined by phenotypic agreement of microbiological results and spatial and temporal relationship. Core genome multi-locus sequence typing (cgMLST) was used to confirm whether the cluster isolates were transmitted between patients. FINDINGS: A total of 4343 3GCR E. coli and 1377 K. pneumoniae isolates were analysed. Among the 3GCR E. coli isolates, the AODS identified 304 isolates as cluster isolates, the median cluster size was two (range: 2-5). The cgMLST analysis revealed that a total of 23 (7.5%) 3GCR E. coli cluster isolates were transmission-associated, of which 20 isolates (87%) were detected in intensive care patients. Among the 3GCR K. pneumoniae isolates, the AODS identified 73 isolates as cluster isolates, the median cluster size was two (range: 2-4). CgMLST revealed that 35 (48%) 3GCR K. pneumoniae cluster isolates were transmission associated, of which 27 isolates (77%) were detected in intensive care patients. CONCLUSION: For 3GCR K. pneumoniae, cgMLST confirmed the AODS results more frequently than for 3GCR E. coli. Therefore, contact isolation precautions for 3GCR K. pneumoniae may be appropriate on intensive care units, but only in certain circumstances, such as outbreaks, for Enterobacterales with lower transmissibility, such as E. coli.
Assuntos
Escherichia coli , Infecções por Klebsiella , Humanos , Escherichia coli/genética , Tipagem de Sequências Multilocus , Klebsiella pneumoniae/genética , Controle de Infecções/métodos , Cefalosporinas/farmacologia , beta-Lactamases/genética , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Testes de Sensibilidade Microbiana , Infecções por Klebsiella/microbiologiaRESUMO
UNLABELLED: AIM of the study was to analyse the influence of a concomitant vitamin D deficiency on the results of (99m)Tc-MIBI studies in patients (pts) with primary hyperparathyroidism (pHPT). PATIENTS, METHODS: Between January 1998 and May 2004, 71 pts with pHPT had undergone operation after a (99m)Tc-MIBI study of whom 54 pts (76%) had normal values of 25-OH-vitamin D3 and 17 pts (24%) had vitamin D deficiency. Results of a dual-phase (99m)Tc-MIBI protocol with SPECT were compared with histopathology. RESULTS: In 54 pts with normal vitamin D values late SPECT images identified more lesions (n=51, sensitivity 91%) than early planar (n=45, sensitivity 82%) or late planar images (n=50, sensitivity 88%). In 17 pts with vitamin D deficiency late SPECT images identified more lesions (n=13, sensitivity 72%) than early planar (n=10, sensitivity 56%) or late planar images (n=10, sensitivity 56%) too. In pts with vitamin D deficiency the sensitivity of a (99m)Tc-MIBI SPECT study was lower than in those with normal vitamin D status (72% vs. 91%) and dependent on the value for PTH. However, the results did not reach statistical significance: early planar: p=0.1625; late planar: p=0.0039; (99m)Tc-MIBI SPECT: p=0.1180. CONCLUSION: The likelihood of a pathological (99m)Tc-MIBI study being obtained in pts with pHPT is dependent on the parathyroid hormone level. However, a negative influence of a low vitamin D level on the scintigraphic detection rate of a parathyroid adenoma could not be proven which may be due to the low number of pts with vitamin D deficiency.
Assuntos
Adenoma/diagnóstico por imagem , Hiperparatireoidismo/diagnóstico por imagem , Neoplasias das Paratireoides/diagnóstico por imagem , Tecnécio Tc 99m Sestamibi , Deficiência de Vitamina D/diagnóstico por imagem , Humanos , Hiperparatireoidismo/complicações , Hiperparatireoidismo/patologia , Tamanho do Órgão , Hormônio Paratireóideo/sangue , Cintilografia , Compostos Radiofarmacêuticos , Estudos Retrospectivos , Ultrassonografia , Deficiência de Vitamina D/complicações , Deficiência de Vitamina D/patologiaRESUMO
The activities of three Rab-specific factors with GDP/GTP exchange activity, Vps9p, Rabex-5 and DSS4, with their cognate GTPases, Ypt51p, Rab5 and Ypt1p, have been analysed quantitatively. In contrast to other exchange factors examined and to DSS4, Vps9p, and by analogy probably Rabex-5, have considerably lower affinity than GDP to the respective GTPases. In keeping with this, they are relatively weak exchangers, with a maximal rate constant for GDP release from the ternary complex between exchange factor, GTPase and GDP of ca 0.01 s(-1), which is several orders of magnitude lower than for other exchange factors examined. If interaction with these proteins is a mandatory aspect of the Rab cycle, this suggests that the overall rate of cycling might be controlled at this point of the cycle. Surprisingly, DSS4, which has the thermodynamic potential to displace GDP effectively from Ypt1p, also does this very slowly, again with a maximal rate constant of ca 0.01 s(-1). An additional, and based on present knowledge, unique, feature of the Ypt1p.DSS4 complex, is that the association of GTP (or GDP) is more than 10(3)-fold slower than to Ypt1p, thus leading to a long life-time of the binary complex between the two proteins, even at the high nucleotide concentrations that prevail in the cell. This leads to the conclusion that the protein-protein complex is likely to have an important biological significance in addition to its probable role in GTP/GDP exchange.
Assuntos
Proteínas de Transporte/metabolismo , Proteínas Fúngicas/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Proteínas de Transporte Vesicular , Proteínas rab de Ligação ao GTP/metabolismo , Transferência de Energia , Fluorescência , Guanosina Difosfato/análogos & derivados , Guanosina Difosfato/metabolismo , Guanosina Trifosfato/análogos & derivados , Guanosina Trifosfato/metabolismo , Cinética , Naftalenossulfonatos , Ligação Proteica , TermodinâmicaRESUMO
Point mutations in the human gene encoding coenzyme B12 (adenosylcobalamin)-dependent methylmalonyl-CoA mutase give rise to an inherited disorder of propionic acid metabolism termed mut methylmalonic aciduria. Almost all such mutations alter amino acids in the homodimeric human enzyme that are identical to residues in the catalytic alpha-subunit of the heterodimeric methylmalonyl-CoA mutase from the bacterium Propionibacterium shermanii, to which the mature human enzyme shows an overall 65% sequence identity. To explore how specific mutations might cause the observed clinical phenotype, 12 known mutations were mapped onto a three-dimensional homology model of the subunit of the human enzyme, generated using the program MODELLER on the basis of the recently published 2.0 A X-ray crystal structure of the P. shermanii methylmalonyl-CoA mutase. Eight mutations are found in the C-terminal B12-binding domain, of which 4 (G623R, G626C, G630E, G703R) are in direct contact with the corrin and are clustered around the histidine ligand (H627) provided by the protein to coordinate the cobalt atom of the B12 cofactor. Introduction of a side chain, particularly one that is charged, at any of these positions is expected to disrupt the flavodoxin-like fold and severely impair its binding of B12. Mutation at either of two other highly conserved glycine residues in this domain (G648D, G717V) also disrupts critical elements in the fold as would the introduction of an additional positive charge in the mutation H678R. Mutation of an arginine in a solvent-exposed loop to a hydrophobic residue (R694W) is also pathogenic. The remaining mutations have been mapped to the N-terminal region of the mutase, two of which introduce a buried, uncompensated charge, either near the subunit interface (A377E), or near the narrow channel through which acyl-CoA esters gain access to the active site (W105R). The extreme N-terminus of methylmalonyl-CoA mutase is predicted to make extensive contacts with the other subunit, and a mutant in this region (R93H) may prevent the correct assembly of the dimer.
Assuntos
Erros Inatos do Metabolismo dos Aminoácidos/genética , Ácido Metilmalônico/urina , Metilmalonil-CoA Mutase/química , Modelos Moleculares , Mutação Puntual , Homologia de Sequência , Sequência de Aminoácidos , Sítios de Ligação , Cobamidas/metabolismo , Simulação por Computador , Cristalização , Humanos , Metilmalonil-CoA Mutase/genética , Metilmalonil-CoA Mutase/metabolismo , Dados de Sequência Molecular , Propionibacterium/enzimologia , Dobramento de Proteína , Difração de Raios XRESUMO
Rab GTPases play a key role in the regulation of membrane traffic. Posttranslational geranylgeranylation is critical for their biological activity and is conferred by a Rab geranylgeranyl transferase (RabGGTase). To study the interactions between Rab proteins and RabGGTase, we used in vitro ligation methodology to generate a fluorescent semi-synthetic Rab7 protein. The obtained protein was functionally active and was used to demonstrate a micromolar affinity interaction of Rab7 with the RabGGTase in the absence of Rab escort protein (REP). This finding is consistent with an earlier proposed model according to which RabGGTase possesses two independent weak binding sites for REP and Rab proteins.
Assuntos
Alquil e Aril Transferases/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Alquil e Aril Transferases/química , Sítios de Ligação , Proteínas de Transporte/metabolismo , Clonagem Molecular , Transferência de Energia , Escherichia coli , Cinética , Reação em Cadeia da Polimerase , Processamento de Proteína Pós-Traducional , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Espectrometria de Fluorescência , Proteínas rab de Ligação ao GTP/química , proteínas de unión al GTP Rab7RESUMO
Adenosylcobalamin-dependent methylmalonyl-CoA mutase catalyzes the interconversion of methylmalonyl-CoA and succinyl-CoA via radical intermediates generated by substrate-induced homolysis of the coenzyme carbon-cobalt bond. From the structure of methylmalonyl-CoA mutase it is evident that the deeply buried active site is completely shielded from solvent with only a few polar contacts made between the protein and the substrate. Site-directed mutants of amino acid His244, a residue close to the inferred site of radical chemistry, were engineered to investigate its role in catalysis. Two mutants, His244Ala and His244Gln, were characterized using kinetic and spectroscopic techniques. These results confirmed that His244 is not an essential residue. However, compared with that of the wild type, k(cat) was lowered by 10(2)- and 10(3)-fold for the His244Gln and His244Ala mutants, respectively, while the K(m) for succinyl-CoA was essentially unchanged in both cases. The primary kinetic tritium isotope effect (k(H)/k(T)) for the His244Gln mutant was 1.5 +/- 0.3, and tritium partitioning was now found to be dependent on the substrate used to initiate the reaction, indicating that the rearrangement of the substrate radical to the product radical was extremely slow. The His244Ala mutant underwent inactivation under aerobic conditions at a rate between 1 and 10% of the initial rate of turnover. The crystal structure of the His244Ala mutant, determined at 2.6 A resolution, indicated that the mutant enzyme is unaltered except for a cavity in the active site which is occupied by an ordered water molecule. Molecular oxygen reaching this cavity may lead directly to inactivation. These results indicate that His244 assists directly in the unusual carbon skeleton rearrangement and that alterations in this residue substantially lower the protection of reactive radical intermediates during catalysis.
Assuntos
Cobamidas/química , Metilmalonil-CoA Mutase/química , Aerobiose/genética , Alanina/genética , Sítios de Ligação/genética , Cristalização , Cristalografia por Raios X , Deutério/química , Radicais Livres/química , Glutamina/genética , Histidina/genética , Cinética , Metilmalonil-CoA Mutase/genética , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Propionibacterium/enzimologia , Propionibacterium/genética , Espectrofotometria Ultravioleta , Análise Espectral , Trítio/químicaRESUMO
Methylmalonyl-CoA mutase from Propionibacterium shermanii is an adenosylcobalamin-dependent enzyme which catalyzes the reversible isomerization of methylmalonyl-CoA and succinyl-CoA. The rate of tritium loss from 5'-[3H]adenosylcobalamin during the enzymic reaction and the relative rates of tritium appearance in substrate and product were examined. Upon the addition of methylmalonyl-CoA to a solution of holoenzyme, tritium was completely released from the cofactor within about 500 ms. No tritium was found either bound to the enzyme or released into the water. The radioactivity was found in methylmalonyl-CoA and succinyl-CoA in a constant ratio of 1 to 3, which did not change during the first 300 ms of the reaction. Upon the addition of succinyl-CoA to a solution of holoenzyme, tritium was released at essentially the same rate, and the radioactivity was found in methylmalonyl-CoA and succinyl-CoA in the identical constant ratio of 1 to 3. The tritium isotope effect on the enzyme-catalyzed hydrogen transfer, measured using 14C-labeled methylmalonyl-CoA as substrate, was kH/kT = 4.9. This low value shows that hydrogen transfer is only partly rate limiting and that at least one subsequent slow step, such as product release, contributes substantially to the overall reaction velocity. The identical partitioning of tritium, regardless of the substrate used, shows that the rearrangement of the substrate radical into the product radical is not rate limiting. The very low tritium isotope effect and the fact that all the tritium is found bound either to the CoA esters or to the cofactor make it very unlikely that a protein radical is an intermediate in the methylmalonyl-CoA mutase-catalyzed rearrangement.
Assuntos
Cobamidas/metabolismo , Metilmalonil-CoA Mutase/metabolismo , Propionibacterium/enzimologia , Trítio/farmacologia , Acil Coenzima A/metabolismo , Deutério/metabolismo , Cinética , Estrutura Molecular , Ligação Proteica , Trítio/metabolismoRESUMO
GTPases of the Rab family are key components of vesicular transport in eukaryotic cells. Posttranslational attachment of geranylgeranyl moieties is essential for Rab function. Geranylgeranyltransferase type II (GGTase-II) catalyzes the modification of Rab proteins once they are in complex with their escort protein (REP). Upon completion of prenylation, REP and modified Rab leave the enzyme, enabling a new round of catalysis. We have studied the mechanism underlying substrate binding and product release in the geranylgeranylation of Rab proteins. Binding of the Rab7:REP-1 complex to GGTase-II was found to be strongly modulated by geranylgeranyl pyrophosphate (GGpp). The affinity of GGTase-II for the Rab7:REP-1 complex increases from ca. 120 nM to ca. 2 nM in the presence of GGpp. To study the effect of GGpp on interaction of the enzyme with its product, we generated semisynthetic doubly prenylated Rab7 bearing a fluorescent reporter group. Using this novel compound, we demonstrated that the affinity of doubly prenylated Rab7:REP-1 complex for GGTase-II was 2 and 18 nM in the absence and presence of GGpp, respectively. The difference in affinities originates mainly from a difference in the dissociation rates. Thus, binding of the new isoprenoid substrate molecule facilitates the product release by GGTase-II. The affinity of GGpp for the prenylated Rab7:REP-1:GGTase-II was K(d) = 22 nM, with one molecule of GGpp binding per molecule of prenylated ternary complex. We interpreted this finding as an indication that the geranylgeranyl moieties transferred to Rab protein do not occupy the GGpp binding site of the GGTase-II. In summary, these results demonstrate that GGpp acts as an allosteric activator that stabilizes the Rab7:REP-1:GGTase-II complex and triggers product release upon prenylation, preventing product inhibition of the enzyme.
Assuntos
Alquil e Aril Transferases/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Alquil e Aril Transferases/química , Regulação Alostérica , Animais , Sítios de Ligação , Corantes Fluorescentes/metabolismo , Guanosina Difosfato/análogos & derivados , Guanosina Difosfato/metabolismo , Cinética , Substâncias Macromoleculares , Fosfatos de Poli-Isoprenil/química , Fosfatos de Poli-Isoprenil/metabolismo , Ligação Proteica , Prenilação de Proteína , Ratos , Espectrometria de Fluorescência , Especificidade por Substrato , Titulometria , ortoaminobenzoatos/metabolismo , Proteínas rab de Ligação ao GTP/síntese química , Proteínas rab de Ligação ao GTP/química , Proteínas rab de Ligação ao GTP/metabolismo , proteínas de unión al GTP Rab7RESUMO
Rab geranylgeranyltransferase (RabGGTase) catalyzes the prenylation of Rab proteins. Despite possessing a single active site, RabGGTase is able to add geranylgeranyl moieties onto each of the two C-terminal cysteine residues of Rab. We have studied the kinetics of Rab double prenylation employing a combination of a novel high pressure liquid chromatography (HPLC)-based in vitro prenylation assay and fluorescence spectroscopy. Transfer of the first geranylgeranyl group proceeds with a k(1) = 0.16 s(-1), while the conversion from singly to double prenylated Rab is 4-fold slower (k(2) = 0.039 s(-1)). We found that following the first transfer reaction, the conjugated lipid is removed from the active site of RabGGTase but mono-prenylated Rab.REP complex remains bound to RabGGTase with a K(d) < 1 nm. In contrast to the doubly prenylated Rab7.REP dissociation of the mono-prenylated species from RabGGTase was only weakly stimulated by phosphoisoprenoid. Based on the obtained rate constants we calculated that at least 72% of mono-prenylated Rab molecules proceed to double prenylation without dissociating from RabGGTase. The obtained data provides an explanation of how RabGGTase discriminates between mono-prenylated intermediate and double prenylated reaction product. It also indicates that the phosphoisoprenoid acts both as a substrate and as a sensor governing the kinetics of protein.protein interactions in the double prenylation reaction.
Assuntos
Alquil e Aril Transferases/metabolismo , Prenilação de Proteína , Proteínas rab de Ligação ao GTP/metabolismo , Animais , Cromatografia Líquida de Alta Pressão/métodos , Compostos de Dansil/metabolismo , Transferência de Energia/fisiologia , Indicadores e Reagentes/metabolismo , Cinética , Complexos Multienzimáticos , Fosfatos de Poli-Isoprenil/química , Ligação Proteica , Espectrometria de Fluorescência/métodos , Espectrometria de Massas por Ionização por Electrospray , proteínas de unión al GTP Rab7RESUMO
Rab geranylgeranyltransferase (RabGGTase or GGTase-II) catalyzes the post-translational prenylation of Rab proteins. Rab proteins are recognized as substrates only when they are complexed to Rab Escort Protein (REP). The classical model of prenylation complex assembly assumes initial formation of the Rab.REP binary complex, which subsequently binds to RabGGTase loaded with the isoprenoid donor geranylgeranyl pyrophosphate (GGpp). We demonstrate here that REP-1 can also associate with RabGGTase in the absence of Rab protein and that this interaction is dramatically strengthened by the presence of phosphoisoprenoids such as GGpp. The GGpp-dependent interaction between RabGGTase and REP-1 was observed using affinity precipitations and gel filtration and was quantitated on the basis of fluorescence assays. In the presence of GGpp, REP-1 binds to RabGGTase with a K(d) value of approximately 10 nm, while in its absence the affinity between the two proteins is in the micromolar range. We further demonstrate that binding of Rab7 to the RabGGTase.GGpp.REP-1 complex occurs without prior dissociation of REP-1. Analysis of binding and prenylation rate constants indicate that the RabGGTase.GGpp.REP-1 complex can function as a kinetically competent intermediate of the prenylation reaction. We conclude that, depending on the prevailing concentrations, binding of REP-1 to RabGGTase in the presence of GGpp may serve as an alternative pathway for the assembly of the prenylation machinery in vivo. Implications of these findings for the role of REP-1 in the prenylation reaction are discussed.
Assuntos
Alquil e Aril Transferases/metabolismo , Fosfatos de Poli-Isoprenil/metabolismo , Prenilação de Proteína , Proteínas rab de Ligação ao GTP/metabolismo , Precipitação Química , Cromatografia em Gel , Cinética , Complexos Multienzimáticos/metabolismo , Ligação Proteica , proteínas de unión al GTP Rab7RESUMO
The adenosylcobalamin-dependent methylmalonyl-CoA mutase catalyzes the reversible rearrangement of methylmalonyl-CoA into succinyl-CoA by a free-radical mechanism. The recently solved X-ray crystal structure of methylmalonyl-CoA mutase from Propionibacterium shermanii has shown that tyrosine 89 is an active-site residue involved in substrate binding. The role of tyrosine 89, a conserved residue among methylmalonyl-CoA mutases, has been investigated by using site-directed mutagenesis to replace this residue with phenylalanine. The crystal structure of the Tyr89Phe mutant was determined to 2.2 A resolution and was found to be essentially superimposable on that of wild-type. Mutant and wild-type enzyme have very similar KM values, but kcat for the Tyr89Phe mutant is 580-fold lower than for wild-type. The rate of release of tritium from 5'-[3H]adenosylcobalamin during the enzymatic reaction and its rate of appearance in substrate and product were measured. The tritium released was found to partition unequally between methylmalonyl-CoA and succinyl-CoA, in a ratio of 40:60 when the reaction was initiated by addition of methylmalonyl-CoA and in a ratio of 10:90 when the reaction was initiated by addition of succinyl-CoA. The overall release of tritium was four times faster when succinyl-CoA was used as substrate. The tritium isotope effect on the enzyme catalyzed hydrogen transfer, measured with methylmalonyl-CoA as a substrate, was kH/kT = 30, which is within the expected range for a full primary kinetic tritium isotope effect. The different partitioning of tritium, dependent upon which substrate was used, and the normal value for the kinetic tritium isotope effect contrast markedly with the behavior of wild-type mutase. It appears that the loss of a single interaction involving the hydroxyl group of tyrosine 89 both affects the stability of radical intermediates and decreases the rate of interconversion of the substrate- and product-derived radicals.
Assuntos
Metilmalonil-CoA Mutase/química , Tirosina/metabolismo , Substituição de Aminoácidos/genética , Sítios de Ligação , Cobamidas/metabolismo , Cristalização , Cristalografia por Raios X , Deutério/metabolismo , Estabilidade Enzimática , Radicais Livres/metabolismo , Cinética , Metilmalonil-CoA Mutase/genética , Metilmalonil-CoA Mutase/isolamento & purificação , Mutagênese Sítio-Dirigida , Fenilalanina/genética , Propionibacterium/enzimologia , Trítio/metabolismo , Tirosina/genéticaRESUMO
Mammalian geranylgeranyltransferase type II (GGTase-II) is a 100-kDa heterodimer that catalyzes the transfer of two 20-carbon geranylgeranyl groups from geranylgeranyl pyrophosphate onto C-terminal cysteine residues of Rab GTPases. This modification is essential for the biological activity of Rab proteins. Geranylgeranylation can be performed in vitro using recombinant GGTase-II but so far large-scale production of the enzyme was challenging. We report here the design of a two plasmid expression system that will produce GGTase-II at levels as high as 15 mg/L in Escherichia coli. The protein was produced as a heterodimer with the alpha subunit bearing a cleavable tandem 6His-glutathione S-transferase (GST) tag that was used for two-step purification of the enzyme. Purified enzyme was functionally active as determined by in vitro prenylation and phosphoisoprenoid binding assay. Furthermore, the GST-tagged GGTase-II was used for preparative in vitro prenylation of the Rab7:REP-1 complex. Using this procedure, 10 mg of doubly prenylated Rab7:REP-1 complex were obtained.
Assuntos
Alquil e Aril Transferases/metabolismo , Escherichia coli , Proteínas rab de Ligação ao GTP/metabolismo , Alquil e Aril Transferases/genética , Alquil e Aril Transferases/isolamento & purificação , Sequência de Aminoácidos , Animais , Sequência de Bases , Cromatografia em Gel , Eletroforese em Gel de Poliacrilamida , Escherichia coli/genética , Vetores Genéticos/genética , Mamíferos , Dados de Sequência Molecular , Peso Molecular , Prenilação de Proteína , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , proteínas de unión al GTP Rab7RESUMO
Geranylgeranyltransferase type II (GGTase-II) modifies small monomeric GTPases of the Rab family by attaching geranylgeranyl moieties onto two cysteines of their C-terminus. We investigated to what extent GGTase-II discriminates between its native substrate geranylgeranyl pyrophosphate (GGpp) and other phosphoisoprenoids, including farnesyl pyrophosphate (Fpp). On the basis of a novel fluorescent assay, we demonstrated that GGpp binds to GGTase-II with an affinity of 8 +/- 4 nM, while Fpp is bound less strongly (K(d) = 60 +/- 8 nM). Analysis of the binding kinetics of four different phosphoisoprenoids indicated that in all cases association is rapid, with rate constants in the range of 0.15 nM(-1) s(-1). In contrast, the dissociation rates differed greatly, depending on the phosphoisoprenoid used, with weak binding substrates generally displaying an increased rate of dissociation. The affinity of GGpp and Fpp for GGTase-II was also determined in the presence of the Rab7-REP-1 complex. The affinity for GGpp was essentially unaffected by the presence of the complex; Fpp on the other hand bound less strongly to the GGTase-II under these conditions, resulting in a K(d) of 260 +/- 60 nM. In vitro prenylation experiments were used to establish that Fpp not only does bind to GGTase-II but also is transferred with an observed rate constant of 0.082 s(-1) which is very similar to that of GGpp. The implications of the low level of discrimination by GGTase-II for the in vivo specificity of the enzyme and the use of farnesyltransferase inhibitors in anti-cancer therapy are discussed.
Assuntos
Alquil e Aril Transferases/metabolismo , Fosfatos de Poli-Isoprenil/metabolismo , Prenilação de Proteína , Proteínas rab de Ligação ao GTP , Proteínas Adaptadoras de Transdução de Sinal , Marcadores de Afinidade/metabolismo , Alquil e Aril Transferases/química , Animais , Proteínas de Transporte/metabolismo , Corantes Fluorescentes/metabolismo , Humanos , Cinética , Fosfatos de Poli-Isoprenil/química , Ligação Proteica , Sesquiterpenos , Espectrometria de Fluorescência , Especificidade por Substrato , ortoaminobenzoatos/metabolismoRESUMO
Posttranslational prenylation of proteins is a widespread phenomenon and the majority of prenylated proteins are geranylgeranylated members of the Rab GTPase family. Geranylgeranylation is catalyzed by Rab geranylgeranyltransferase (RabGGTase) and is critical for the ability of Rab protein to mediate vesicular docking and fusion of various intracellular vesicles. RabGGTase consists of a catalytic alpha/beta heterodimer and an accessory protein termed Rab escort protein (REP-1) that delivers the newly prenylated Rab proteins to their target membrane. Mutations in the REP-1 gene in humans lead to an X-chromosome-linked defect known as choroideremia--a debilitating disease that inevitably culminates in complete blindness. Here we report in vitro assembly and purification of the stoichiometric ternary complex of RabGGTase with REP-1 stabilized by a hydrolysis-resistant phosphoisoprenoid analog--farnesyl phosphonyl(methyl)phoshonate. The complex formed crystals of extended plate morphology under low ionic-strength conditions. X-ray diffraction data were collected to 2.8 A resolution at the ESRF. The crystals belong to the monoclinic space group P2(1), with unit-cell parameters a = 68.7, b = 197.7, c = 86.1 A, beta = 113.4 degrees. Preliminary structural analysis revealed the presence of one molecule in the asymmetric unit.