RESUMEN
Adenosylcobalamin (AdoCbl) or coenzyme B12-dependent enzymes catalyze intramolecular group-transfer reactions and ribonucleotide reduction in a wide variety of organisms from bacteria to animals. They use a super-reactive primary-carbon radical formed by the homolysis of the coenzyme's Co-C bond for catalysis and thus belong to the larger class of "radical enzymes." For understanding the general mechanisms of radical enzymes, it is of great importance to establish the general mechanism of AdoCbl-dependent catalysis using enzymes that catalyze the simplest reactions-such as diol dehydratase, glycerol dehydratase and ethanolamine ammonia-lyase. These enzymes are often called "eliminases." We have studied AdoCbl and eliminases for more than a half century. Progress has always been driven by the development of new experimental methodologies. In this chapter, we describe our investigations on these enzymes, including their metabolic roles, gene cloning, preparation, characterization, activity assays, and mechanistic studies, that have been conducted using a wide range of biochemical and structural methodologies we have developed.
Asunto(s)
Etanolamina Amoníaco-Liasa , Animales , Cobamidas/química , Cobamidas/metabolismo , Etanolamina Amoníaco-Liasa/química , Etanolamina Amoníaco-Liasa/metabolismo , Glicerol , Hidroliasas , Fosfotreonina/análogos & derivadosRESUMEN
Post-transcriptional upregulation is an effective way to increase the expression of transgenes and thus maximize the yields of target chemicals from metabolically engineered organisms. Refractory elements in the 3' untranslated region (UTR) that increase mRNA half-life might be available. In Saccharomyces cerevisiae, several terminator regions have shown activity in increasing the production of proteins by upstream coding genes; among these terminators the DIT1 terminator has the highest activity. Here, we found in Saccharomyces cerevisiae that two resident trans-acting RNA-binding proteins (Nab6p and Pap1p) enhance the activity of the DIT1 terminator through the cis element GUUCG/U within the 3'-UTR. These two RNA-binding proteins could upregulate a battery of cell-wall-related genes. Mutagenesis of the DIT1 terminator improved its activity by a maximum of 500% of that of the standard PGK1 terminator. Further understanding and improvement of this system will facilitate inexpensive and stable production of complicated organism-derived drugs worldwide.
Asunto(s)
Regiones no Traducidas 3' , Expresión Génica , Transferasas de Hidroximetilo y Formilo/genética , Proteínas de Unión al ARN/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Regiones Terminadoras Genéticas , Transferasas de Hidroximetilo y Formilo/metabolismo , Proteínas de Unión al ARN/genética , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
BACKGROUND: Deoxyuridine triphosphate nucleotidohydrolase (dUTPase) hydrolyzes dUTP to dUMP and pyrophosphate to maintain the cellular thymine-uracil ratio. dUTPase is also a target for cancer chemotherapy. However, the mechanism defining its substrate affinity remains unclear. Sequence comparisons of various dUTPases revealed that Arabidopsis thaliana dUTPase has a unique tryptophan at position 93, which potentially contributes to its degree of substrate affinity. To better understand the roles of tryptophan 93, A. thaliana dUTPase was studied. RESULTS: Enzyme assays showed that A. thaliana dUTPase belongs to a high-affinity group of isozymes, which also includes the enzymes from Escherichia coli and Mycobacterium tuberculosis. Enzymes from Homo sapiens and Saccharomyces cerevisiae are grouped as low-affinity dUTPases. The structure of the homo-trimeric A. thaliana dUTPase showed three active sites, each with a different set of ligand interactions between the amino acids and water molecules. On an α-helix, tryptophan 93 appears to keep serine 89 in place via a water molecule and to specifically direct the ligand. Upon being oriented in the active site, the C-terminal residues close the active site to promote the reaction. CONCLUSIONS: In the high-affinity group, the prefixed direction of the serine residues was oriented by a positively charged residue located four amino acids away, while low-affinity enzymes possess small hydrophobic residues at the corresponding sites.
Asunto(s)
Proteínas de Arabidopsis/química , Dominio Catalítico , Pirofosfatasas/química , Triptófano/química , Secuencia de Aminoácidos , Arabidopsis/enzimología , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Sitios de Unión/genética , Unión Competitiva , Cristalografía por Rayos X , Interacciones Hidrofóbicas e Hidrofílicas , Ligandos , Modelos Moleculares , Datos de Secuencia Molecular , Unión Proteica , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Pirofosfatasas/genética , Pirofosfatasas/metabolismo , Homología de Secuencia de Aminoácido , Especificidad por Sustrato , Triptófano/genética , Triptófano/metabolismoRESUMEN
Combinatorial screening used together with a broad library of gene expression cassettes is expected to produce a powerful tool for the optimization of the simultaneous expression of multiple enzymes. Recently, we proposed a highly tunable protein expression system that utilized multiple genome-integrated target genes to fine-tune enzyme expression in yeast cells. This tunable system included a library of expression cassettes each composed of three gene-expression control elements that in different combinations produced a wide range of protein expression levels. In this study, four gene expression cassettes with graded protein expression levels were applied to the expression of three cellulases: cellobiohydrolase 1, cellobiohydrolase 2, and endoglucanase 2. After combinatorial screening for transgenic yeasts simultaneously secreting these three cellulases, we obtained strains with higher cellulase expressions than a strain harboring three cellulase-expression constructs within one high-performance gene expression cassette. These results show that our method will be of broad use throughout the field of metabolic engineering.
Asunto(s)
Celulasa/biosíntesis , Escherichia coli/metabolismo , Proteínas Fúngicas/biosíntesis , Expresión Génica , Phanerochaete/genética , Saccharomyces cerevisiae/metabolismo , Talaromyces/genética , Celulasa/genética , Escherichia coli/genética , Proteínas Fúngicas/genética , Phanerochaete/enzimología , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genética , Saccharomyces cerevisiae/genética , Talaromyces/enzimologíaRESUMEN
Control of the expression levels of multiple enzymes in transgenic yeasts is essential for the effective production of complex molecules through fermentation. Here, we propose a tunable strategy for the control of expression levels based on the design of terminator regions and other gene-expression control elements in Saccharomyces cerevisiae. Our genome-integrated system, which is capable of producing high expression levels over a wide dynamic range, will broadly enable metabolic engineering and synthetic biology. We demonstrated that the activities of multiple cellulases and the production of ethanol were doubled in a transgenic yeast constructed with our system compared with those achieved with a standard expression system.
Asunto(s)
Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/genética , Celulasas/genética , Celulasas/metabolismo , Celulosa/metabolismo , Etanol/metabolismo , Fermentación , Expresión Génica , Genes Sintéticos , Ingeniería de Proteínas , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Biología Sintética , Transactivadores/genética , Transactivadores/metabolismo , Transformación GenéticaRESUMEN
Strong terminator regions could be used to improve metabolically engineered yeasts by increasing the target enzyme protein yields above those achieved with traditional terminator regions. We recently identified five strong terminator regions (RPL41Bt, RPL15At, DIT1t, RPL3t, and IDP1t) in a comprehensive analysis of Saccharomyces cerevisiae. The effect of the terminator regions was analyzed by measuring the protein production of a linked transgene, and was shown to be twice that of a traditional terminator region (PGK1t). Here, we investigated whether the activity of the terminator regions is affected by exchange of a strong promoter or reporter in the linked transgene, carbon source for cell growth, stress factors, host yeast strain, or stage of the growth phase. Our results indicate that the activities of all five terminator regions were twice that of PGK1t in all conditions tested. In addition, we demonstrated that the strong activity of these terminator regions could be used to improve secretory production of endoglucanase II derived from Tricoderma ressei, and that the DIT1t strain was the best of the five strains for this purpose. We therefore propose that DIT1t, and the four other terminator regions, could be applied to the development of improved metabolically engineered yeasts.
Asunto(s)
Celulasa/química , Ingeniería Metabólica , Biosíntesis de Proteínas , Regiones Terminadoras Genéticas , Regiones no Traducidas 3'/genética , Reactores Biológicos , Carbono/química , Carbono/metabolismo , Celulasa/biosíntesis , Celulasa/genética , Celulasa/metabolismo , Genoma Fúngico , Regiones Promotoras Genéticas , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crecimiento & desarrollo , Saccharomyces cerevisiae/metabolismo , TransgenesRESUMEN
The terminator regions of eukaryotes encode functional elements in the 3' untranslated region (3'-UTR) that influence the 3'-end processing of mRNA, mRNA stability, and translational efficiency, which can modulate protein production. However, the contribution of these terminator regions to gene expression remains unclear, and therefore their utilization in metabolic engineering or synthetic genetic circuits has been limited. Here, we comprehensively evaluated the activity of 5302 terminator regions from a total of 5880 genes in the budding yeast Saccharomyces cerevisiae by inserting each terminator region downstream of the P TDH3 - green fluorescent protein (GFP) reporter gene and measuring the fluorescent intensity of GFP. Terminator region activities relative to that of the PGK1 standard terminator ranged from 0.036 to 2.52, with a mean of 0.87. We thus could isolate the most and least active terminator regions. The activities of the terminator regions showed a positive correlation with mRNA abundance, indicating that the terminator region is a determinant of mRNA abundance. The least active terminator regions tended to encode longer 3'-UTRs, suggesting the existence of active degradation mechanisms for those mRNAs. The terminator regions of ribosomal protein genes tended to be the most active, suggesting the existence of a common regulator of those genes. The â³terminatomeâ³ (the genome-wide set of terminator regions) thus not only provides valuable information to understand the modulatory roles of terminator regions on gene expression but also serves as a useful toolbox for the development of metabolically and genetically engineered yeast.
Asunto(s)
Genoma Fúngico , Saccharomyces cerevisiae/genética , Regiones Terminadoras Genéticas/genética , Regiones no Traducidas 3' , Gliceraldehído-3-Fosfato Deshidrogenasa (Fosforilante)/genética , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Ingeniería Metabólica , Fosfoglicerato Quinasa/genética , Fosfoglicerato Quinasa/metabolismo , Regiones Promotoras Genéticas , Estabilidad del ARN , ARN Mensajero/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
Coenzyme B(12) dependent diol dehydratase undergoes mechanism-based inactivation by glycerol, accompanying the irreversible cleavage of the coenzyme Co-C bond. Bachovchin et al. [Biochemistry16, 1082-1092 (1977)] reported that glycerol bound in the G(S) conformation, in which the pro-S-CH(2) OH group is oriented to the hydrogen-abstracting site, primarily contributes to the inactivation reaction. To understand the mechanism of inactivation by glycerol, we analyzed the X-ray structure of diol dehydratase complexed with cyanocobalamin and glycerol. Glycerol is bound to the active site preferentially in the same conformation as that of (S)-1,2-propanediol, i.e. in the G(S) conformation, with its 3-OH group hydrogen bonded to Serα301, but not to nearby Glnα336. k(inact) of the Sα301A, Qα336A and Sα301A/Qα336A mutants with glycerol was much smaller than that of the wild-type enzyme. k(cat) /k(inact) showed that the Sα301A and Qα336A mutants are substantially more resistant to glycerol inactivation than the wild-type enzyme, suggesting that Serα301 and Glnα336 are directly or indirectly involved in the inactivation. The degree of preference for (S)-1,2-propanediol decreased on these mutations. The substrate activities towards longer chain 1,2-diols significantly increased on the Sα301A/Qα336A double mutation, probably because these amino acid substitutions yield more space for accommodating a longer alkyl group on C3 of 1,2-diols. Database Structural data are available in the Protein Data Bank under the accession number 3AUJ. Structured digital abstract ⢠Diol dehydrase gamma subunit, Diol dehydrase beta subunit and Diol dehydrase alpha subunit physically interact by X-ray crystallography (View interaction).
Asunto(s)
Cobamidas/metabolismo , Resistencia a Medicamentos , Glicerol/metabolismo , Klebsiella oxytoca/enzimología , Propanodiol Deshidratasa/química , Propanodiol Deshidratasa/metabolismo , Propilenglicol/metabolismo , Catálisis , Dominio Catalítico , Cristalización , Cristalografía por Rayos X , Cinética , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Mutación/genética , Propanodiol Deshidratasa/genética , Propilenglicol/química , Conformación Proteica , Estereoisomerismo , Vitamina B 12/metabolismoRESUMEN
The control of metabolic flow is a prerequisite for efficient chemical production in transgenic microorganisms. Exogenous genes required for the biosynthesis of target chemicals are expressed under strong promoters, while the endogenous genes of the original metabolic pathway are repressed by disruption or mutation. These genetic manipulations occasionally cause harmful effects to the host. In the lactate-producing yeast Saccharomyces cerevisiae, where endogenous pyruvate decarboxylase (PDC) is disrupted and exogenous lactate dehydrogenase (LDH) is introduced, PDC deletion is extremely detrimental to cell growth but is required for efficient production of lactate. A suitable means to dynamically control the metabolic flow from ethanol fermentation during the growth phase to lactate fermentation during the production phase is needed. Here, we demonstrated that this flow can be controlled by the exclusive expression of PDC and LDH with a Cre-lox genetic switch. This switch was evaluated with a gene cassette that encoded two different fluorescence proteins and enabled changes in genotype and phenotype within 2 and 10 h, respectively. Transgenic yeast harboring this switch and the PDC-LDH cassette showed a specific growth rate (0.45 h (-1)) that was almost the same as that of wild-type (0.47 h (-1)). Upon induction of the genetic switch, the transgenic yeast produced lactate from up to 85.4% of the glucose substrate, while 91.7% of glucose went to ethanol before induction. We thus propose a "metabolic shift" concept that can serve as an alternative means to obtain gene products that are currently difficult to obtain by using conventional methodologies.
Asunto(s)
Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Etanol/metabolismo , Fermentación , Genes Fúngicos , Glucosa/metabolismo , L-Lactato Deshidrogenasa/genética , L-Lactato Deshidrogenasa/metabolismo , Ácido Láctico/biosíntesis , Ingeniería Metabólica , Redes y Vías Metabólicas/genética , Piruvato Descarboxilasa/genética , Piruvato Descarboxilasa/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Biología SintéticaRESUMEN
We report that several tryptophan-rich peptides exhibit an affinity for a hydrophobic ionic liquid (IL) (1-ethyl-3-methylimidazolium bis-trifluoromethanesulfonyl imide), and that green fluorescent protein (GFP) fused to a peptides, "SSSWWSWWWW" (SW1) or "SWWWWSWWWW" (SW2), containing serine (S) and tryptophan (W) at the C terminus localized at the IL/water interface. While GFPs without W-rich peptide distributed only in water phase, SW1- and SW2-GFPs were accumulated at the interface. The localization of SW1-GFP showed biphasic behavior, and most distinctive localization was observed at 7.1 µM. The localization of SW2-GFP presumably occurred at largely lower concentration (≤0.5 µM) than that of SW1-GFP, which difference was due to the higher hydrophobicity of SW2 peptide.
Asunto(s)
Proteínas Fluorescentes Verdes/análisis , Imidazoles/química , Líquidos Iónicos/química , Péptidos/química , Sulfonamidas/química , Triptófano/química , Proteínas Fluorescentes Verdes/genética , Péptidos/análisis , Péptidos/genética , Proteínas Recombinantes de Fusión/análisis , Proteínas Recombinantes de Fusión/química , Agua/químicaRESUMEN
Deoxyuridine triphosphatase (dUTPase) is a ubiquitous enzyme that has been widely studied owing to its function and evolutionary significance. The gene coding for the dUTPase from the Chlorella alga was codon-optimized and synthesized. The synthetic gene was expressed in Escherichia coli and recombinant core Chlorella dUTPase (chdUTPase) was purified. Crystallization of chdUTPase was performed by the repetitive hanging-drop vapor-diffusion method at 298 K with ammonium sulfate as the precipitant. In the presence of 2'-deoxyuridine-5'-[(α,ß)-imido]triphosphate and magnesium, the enzyme produced die-shaped hexagonal R3 crystals with unit-cell parameters a = b = 66.9, c = 93.6 Å, γ = 120°. X-ray diffraction data for chdUTPase were collected to 1.6 Å resolution. The crystallization of chdUTPase with manganese resulted in very fragile clusters of needles.
Asunto(s)
Chlorella/enzimología , Pirofosfatasas/química , Cristalización , Difracción de Rayos XRESUMEN
Both terminators and promoters regulate gene expression. In Saccharomyces cerevisiae, the TPS1 terminator (TPS1t), coupled to a gene encoding a fluorescent protein, produced more transgenic mRNA and protein than did similar constructs containing other terminators, such as CYC1t, TDH3t, and PGK1t. This suggests that TPS1t can be used as a general terminator in the development of metabolically engineered yeast in high-yield systems.
Asunto(s)
Regulación Fúngica de la Expresión Génica , Glucosiltransferasas/metabolismo , Biosíntesis de Proteínas/genética , ARN Mensajero/biosíntesis , Proteínas de Saccharomyces cerevisiae/biosíntesis , Saccharomyces cerevisiae/genética , Regiones Terminadoras Genéticas , Citocromos c/genética , Citocromos c/metabolismo , Glucosiltransferasas/genética , Gliceraldehído-3-Fosfato Deshidrogenasa (Fosforilante)/genética , Gliceraldehído-3-Fosfato Deshidrogenasa (Fosforilante)/metabolismo , Regiones Promotoras Genéticas , ARN Mensajero/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , TransgenesRESUMEN
Here we report a significant enhancement of galactose response without altering the characteristics of glucose repression. To improve the galactose response, we fabricated transgenic yeasts harboring HIS3pro-GAL1, HIS3pro-GAL2 and GAL10pro-GAL4, and evaluated the synergistic effects of these three genes by immunoblot and flow cytometry analyses.
Asunto(s)
Galactosa/metabolismo , Regulación Fúngica de la Expresión Génica , Saccharomyces cerevisiae/genética , Medios de Cultivo , Galactosa/genética , Genes Fúngicos , Glucosa/metabolismo , Regiones Promotoras Genéticas , Transcripción GenéticaRESUMEN
The methods of homologous high-level expression and simple large-scale purification for coenzyme B(12)-dependent ethanolamine ammonia-lyase of Escherichia coli were developed. The eutB and eutC genes in the eut operon encoded the large and small subunits of the enzyme, respectively. The enzyme existed as the heterododecamer alpha(6)beta(6). Upon active-site titration with adeninylpentylcobalamin, a strong competitive inhibitor for coenzyme B(12), the binding of 1 mol of the inhibitor per mol of the alphabeta unit caused complete inhibition of enzyme, in consistent with its subunit structure. EPR spectra indicated the formation of substrate-derived radicals during catalysis and the binding of cobalamin in the base-on mode, i.e. with 5,6-dimethylbenzimidazole coordinating to the cobalt atom. The purified wild-type enzyme underwent aggregation and inactivation at high concentrations. Limited proteolysis with trypsin indicated that the N-terminal region is not essential for catalysis. His-tagged truncated enzymes were similar to the wild-type enzyme in catalytic properties, but more resistant to p-chloromercuribenzoate than the wild-type enzyme. A truncated enzyme was highly soluble even in the absence of detergent and resistant to aggregation and oxidative inactivation at high concentrations, indicating that a short N-terminal sequence is sufficient to change the solubility and stability of the enzyme.
Asunto(s)
Escherichia coli/enzimología , Etanolamina Amoníaco-Liasa/aislamiento & purificación , Etanolamina Amoníaco-Liasa/metabolismo , Catálisis , Estabilidad de Enzimas , Etanolamina Amoníaco-Liasa/química , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , SolubilidadRESUMEN
The reactions of diol dehydratase with 3-unsaturated 1,2-diols and thioglycerol were investigated. Holodiol dehydratase underwent rapid and irreversible inactivation by either 3-butene-1,2-diol, 3-butyne-1,2-diol or thioglycerol without catalytic turnovers. In the inactivation, the Co-C bond of adenosylcobalamin underwent irreversible cleavage forming unidentified radicals and cob(II)alamin that resisted oxidation even in the presence of oxygen. Two moles of 5'-deoxyadenosine per mol of enzyme was formed as an inactivation product from the coenzyme adenosyl group. Inactivated holoenzymes underwent reactivation by diol dehydratase-reactivating factor in the presence of ATP, Mg(2+) and adenosylcobalamin. It was thus concluded that these substrate analogues served as mechanism-based inactivators or pseudosubstrates, and that the coenzyme was damaged in the inactivation, whereas apoenzyme was not damaged. In the inactivation by 3-unsaturated 1,2-diols, product radicals stabilized by neighbouring unsaturated bonds might be unable to back-abstract the hydrogen atom from 5'-deoxyadenosine and then converted to unidentified products. In the inactivation by thioglycerol, a product radical may be lost by the elimination of sulphydryl group producing acrolein and unidentified sulphur compound(s). H(2)S or sulphide ion was not formed. The loss or stabilization of product radicals would result in the inactivation of holoenzyme, because the regeneration of the coenzyme becomes impossible.
Asunto(s)
Butileno Glicoles/farmacología , Cobamidas/metabolismo , Glicerol/análogos & derivados , Propanodiol Deshidratasa/antagonistas & inhibidores , Cobamidas/química , Espectroscopía de Resonancia por Spin del Electrón , Inhibidores Enzimáticos/farmacología , Escherichia coli/enzimología , Glicerol/farmacología , Glicoles/farmacología , Cinética , Modelos Moleculares , Propanodiol Deshidratasa/química , Propanodiol Deshidratasa/metabolismoRESUMEN
Diol dehydratase of Klebsiella oxytoca contains an essential histidine residue. Its X-ray structure revealed that the migrating hydroxyl group on C2 of substrate is hydrogen-bonded to Hisalpha143. Mutant enzymes in which Hisalpha143 was mutated to another amino acid residue were expressed in Escherichia coli, purified, and examined for enzymatic activity. The Halpha143Q mutant was 34% as active as the wild-type enzyme. Halpha143A and Halpha143L showed only a trace of activity. Kinetic analyses indicated that the hydrogen bonding interaction between the hydroxyl group on C2 of substrate and the side chain of residue alpha143 is important not only for catalysis but also for protecting radical intermediates. Halpha143E and Halpha143K that did not exist as (alphabetagamma) 2 complexes were inactive. The deuterium kinetic isotope effect on the overall reaction suggested that a hydrogen abstraction step is fully rate-determining for the wild type and Halpha143Q and partially rate-determining for Halpha143A. The preference for substrate enantiomers was reversed by the Halpha143Q mutation in both substrate binding and catalysis. Upon the inactivation of the Halpha143A holoenzyme by 1,2-propanediol, cob(II)alamin without an organic radical coupling partner accumulated, 5'-deoxyadenosine was quantitatively formed from the coenzyme adenosyl group, and the apoenzyme itself was not damaged. This inactivation was thus concluded to be a mechanism-based inactivation. The holoenzyme of Halpha143Q underwent irreversible inactivation by O 2 in the absence of substrate at a much lower rate than the wild type.
Asunto(s)
Cobamidas/metabolismo , Histidina/metabolismo , Propanodiol Deshidratasa/metabolismo , Cristalografía por Rayos X , Espectroscopía de Resonancia por Spin del Electrón , Histidina/química , Histidina/genética , Enlace de Hidrógeno , Concentración de Iones de Hidrógeno , Cinética , Klebsiella oxytoca/enzimología , Klebsiella oxytoca/genética , Klebsiella oxytoca/metabolismo , Modelos Biológicos , Estructura Molecular , Mutagénesis Sitio-Dirigida , Mutación , Propanodiol Deshidratasa/química , Propanodiol Deshidratasa/genética , Relación Estructura-Actividad , Especificidad por SustratoRESUMEN
A protein crystal lattice consists of surface contact regions, where the interactions of specific groups play a key role in stabilizing the regular arrangement of the protein molecules. In an attempt to control protein incorporation in a crystal lattice, a leucine zipper-like hydrophobic interface (comprising four leucine residues) was introduced into a helical region (helix 2) of the human pancreatic ribonuclease 1 (RNase 1) that was predicted to form a suitable crystallization interface. Although crystallization of wild-type RNase 1 has not yet been reported, the RNase 1 mutant having four leucines (4L-RNase 1) was successfully crystallized under several different conditions. The crystal structures were subsequently determined by X-ray crystallography by molecular replacement using the structure of bovine RNase A. The overall structure of 4L-RNase 1 is quite similar to that of the bovine RNase A, and the introduced leucine residues formed the designed crystal interface. To characterize the role of the introduced leucine residues in crystallization of RNase 1 further, the number of leucines was reduced to three or two (3L- and 2L-RNase 1, respectively). Both mutants crystallized and a similar hydrophobic interface as in 4L-RNase 1 was observed. A related approach to engineer crystal contacts at helix 3 of RNase 1 (N4L-RNase 1) was also evaluated. N4L-RNase 1 also successfully crystallized and formed the expected hydrophobic packing interface. These results suggest that appropriate introduction of a leucine zipper-like hydrophobic interface can promote intermolecular symmetry for more efficient protein crystallization in crystal lattice engineering efforts.
Asunto(s)
Cristalografía por Rayos X/métodos , Leucina Zippers , Ribonucleasa Pancreática/química , Secuencia de Aminoácidos , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Modelos Moleculares , Datos de Secuencia Molecular , Mutación , Ingeniería de Proteínas , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Ribonucleasa Pancreática/genética , Ribonucleasa Pancreática/metabolismo , Homología de Secuencia de AminoácidoRESUMEN
Human cystathionine beta-synthase plays a key role in maintaining low intracellular levels of homocysteine and is unique in being a pyridoxal phosphate-dependent enzyme that is a hemeprotein. It catalyzes the beta-replacement of serine and homocysteine to generate the condensation product, cystathionine. While the structure of a truncated catalytic core of the protein has been determined by crystallography, a model for the full-length enzyme has been developed guided by hydrogen-deuterium exchange mass spectrometric and docking studies. In this review, we have utilized the available structural models for human cystathionine beta-synthase to conduct a structure-function analysis of a select group of pathogenic mutations described in patients with hereditary hyperhomocysteinemia.
Asunto(s)
Cistationina betasintasa/química , Hemo/metabolismo , Mutación , Catálisis , Cistationina betasintasa/genética , Cistationina betasintasa/metabolismo , Humanos , Modelos Moleculares , Conformación ProteicaRESUMEN
The importance of each active-site residue in adenosylcobalamin-dependent diol dehydratase of Klebsiella oxytoca was estimated using mutant enzymes in which one of the residues interacting with substrate and/or K(+) was mutated to Ala or another amino acid residue. The Ealpha170A and Dalpha335A mutants were totally inactive, and the Halpha143A mutant showed only a trace of activity, indicating that Glu-alpha170, Asp-alpha335, and His-alpha143 are catalytic residues. The Qalpha141A, Qalpha296A, and Salpha362A mutants showed partial activity. It was suggested from kinetic parameters that Gln-alpha296 is important for substrate binding and Gln-alpha296 and Gln-alpha141 for preventing the enzyme from mechanism-based inactivation. The Ealpha221A, Ealpha170H, and Dalpha335A did not form the (alphabetagamma)(2) complex, suggesting that these mutations indirectly disrupt subunit contacts. Among other Glu-alpha170 and Asp-alpha335 mutants, Ealpha170D and Ealpha170Q were 2.2 +/- 0.3% and 0.02% as active as the wild-type enzyme, respectively, whereas Dalpha335N was totally inactive. Kinetic analysis indicated that the presence and the position of a carboxyl group in the residue alpha170 are essential for catalysis as well as for the continuous progress of catalytic cycles. It was suggested that the roles of Glu-alpha170 and Asp-alpha335 are to participate in the binding of substrate and intermediates and keep them appropriately oriented and to function as a base in the dehydration of the 1,1-diol intermediate. In addition, Glu-alpha170 seems to stabilize the transition state for the hydroxyl group migration from C2 to C1 by accepting the proton of the spectator hydroxyl group on C1.
Asunto(s)
Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Klebsiella oxytoca/enzimología , Propanodiol Deshidratasa/química , Propanodiol Deshidratasa/metabolismo , Ácido Aspártico , Proteínas Bacterianas/genética , Dominio Catalítico/genética , Cobamidas/metabolismo , Ácido Glutámico , Cinética , Mutagénesis Sitio-Dirigida , Propanodiol Deshidratasa/genética , Relación Estructura-Actividad , Especificidad por SustratoRESUMEN
The crystal structures of ADP bound and nucleotide-free forms of molecular chaperone-like diol dehydratase-reactivating factor (DDR) were determined at 2.0 and 3.0 A, respectively. DDR exists as a dimer of heterodimer (alphabeta)2. The alpha subunit has four domains: ATPase domain, swiveling domain, linker domain, and insert domain. The beta subunit, composed of a single domain, has a similar fold to the beta subunit of diol dehydratase (DD). The binding of an ADP molecule to the nucleotide binding site of DDR causes a marked conformational change of the ATPase domain of the alpha subunit, which would weaken the interactions between the DDR alpha and beta subunits and make the displacement of the DDR beta subunit by DD through the beta subunit possible. The binding of the DD beta subunit to the DDR alpha subunit induces steric repulsion between the DDR alpha and DD alpha subunits that would lead to the release of a damaged cofactor from inactivated holoDD.