RESUMEN
Affinity chromatography utilizing specific interactions between therapeutic proteins and bead-immobilized capturing agents is a standard method for protein purification, but its scalability is limited by long purification times, activity loss by the capturing molecules and/or purified protein, and high costs. Here, we report a platform for purifying therapeutic antibodies via affinity precipitation using the endogenous calcium ion-binding protein, calsequestrin (CSQ), which undergoes a calcium ion-dependent phase transition. In this method, ZZ-CSQ fusion proteins with CSQ and an affinity protein (Z domain of protein A) capture antibodies and undergo multimerization and subsequent aggregation in response to calcium ions, enabling the antibody to be collected by affinity precipitation. After robustly validating and optimizing the performance of the platform, the ZZ-CSQ platform can rapidly purify therapeutic antibodies from industrial harvest feedstock with high purity (>97%) and recovery yield (95% ± 3%). In addition, the ZZ-CSQ platform outperforms protein A-based affinity chromatography (PAC) in removing impurities, yielding â¼20-fold less DNA and â¼4.8-fold less host cell protein (HCP) contamination. Taken together, this platform is rapid, recyclable, scalable, and cost-effective, and it shows antibody-purification performance superior or comparable to that of the standard affinity chromatography method.
Asunto(s)
Calcio , Calsecuestrina , Anticuerpos/metabolismo , Calcio/metabolismo , Proteínas de Unión al Calcio , Calsecuestrina/química , Calsecuestrina/genética , Calsecuestrina/metabolismo , Cromatografía de Afinidad/métodos , Proteína Estafilocócica A/metabolismoRESUMEN
ß-Lactam antibiotics that inhibit penicillin-binding proteins (PBPs) have been widely used in the treatment of bacterial infections. However, the molecular basis underlying the different inhibitory potencies of ß-lactams against specific PBPs is not fully understood. Here, we present the crystal structures of penicillin-binding protein D2 (PBPD2) from Listeria monocytogenes, a Gram-positive foodborne bacterial pathogen that causes listeriosis in humans. The acylated structures in complex with four antibiotics (penicillin G, ampicillin, cefotaxime, and cefuroxime) revealed that the ß-lactam core structures were recognized by a common set of residues; however, the R1 side chains of each antibiotic participate in different interactions with PBPD2. In addition, the structural complementarities between the side chains of ß-lactams and the enzyme were found to be highly correlated with the relative reactivities of penam or cephem antibiotics against PBPD2. Our study provides the structural basis for the inhibition of PBPD2 by clinically important ß-lactam antibiotics that are commonly used in listeriosis treatment. Our findings imply that the modification of ß-lactam side chains based on structural complementarity could be useful for the development of potent inhibitors against ß-lactam-resistant PBPs.
Asunto(s)
Antibacterianos/metabolismo , Proteínas Bacterianas/metabolismo , Listeria monocytogenes/metabolismo , Proteínas de Unión a las Penicilinas/metabolismo , beta-Lactamas/metabolismo , Ampicilina/metabolismo , Cefotaxima/metabolismo , Cefuroxima/metabolismo , Cristalografía por Rayos X , Humanos , Listeria monocytogenes/efectos de los fármacos , Penicilina G/metabolismoRESUMEN
Ketosteroid isomerase (3-oxosteroid Δ(5)-Δ(4)-isomerase, KSI) from Pseudomonas putida catalyzes allylic rearrangement of the 5,6-double bond of Δ(5)-3-ketosteroid to 4,5-position by stereospecific intramolecular transfer of a proton. The active site of KSI is formed by several hydrophobic residues and three catalytic residues (Tyr14, Asp38, and Asp99). In this study, we investigated the role of a hydrophobic Met112 residue near the active site in the catalysis, steroid binding, and stability of KSI. Replacing Met112 with alanine (yields M112A) or leucine (M112L) decreased the kcat by 20- and 4-fold, respectively. Compared with the wild type (WT), M112A and M112L KSIs showed increased KD values for equilenin, an intermediate analogue; these changes suggest that loss of packing at position 112 might lead to unfavorable steroid binding, thereby resulting in decreased catalytic activity. Furthermore, M112A and M112L mutations reduced melting temperature (Tm) by 6.4°C and 2.5°C, respectively. These changes suggest that favorable packing in the core is important for the maintenance of stability in KSI. The M112K mutation decreased kcat by 2000-fold, compared with the WT. In M112K KSI structure, a new salt bridge was formed between Asp38 and Lys112. This bridge could change the electrostatic potential of Asp38, and thereby contribute to the decreased catalytic activity. The M112K mutation also decreased the stability by reducing Tm by 4.1°C. Our data suggest that the Met112 residue may contribute to the catalytic activity and stability of KSI by providing favorable hydrophobic environments and compact packing in the catalytic core.
Asunto(s)
Cetosteroides/metabolismo , Metionina/genética , Esteroide Isomerasas/genética , Secuencia de Aminoácidos , Sustitución de Aminoácidos/genética , Catálisis , Dominio Catalítico/genética , Enlace de Hidrógeno , Interacciones Hidrofóbicas e Hidrofílicas , Mutación/genética , Pseudomonas putida/genética , Pseudomonas putida/metabolismo , Alineación de Secuencia , Temperatura de TransiciónRESUMEN
We analyzed the structure of horseradish peroxidase (HRP) under denaturing conditions of 9 M urea or 6 M guanidine hydrochloride (GdnHCl). Far-UV circular dichroism (CD) spectra indicated the existence of native-like secondary structure of holo-HRP in 9 M urea. In addition, slight changes in near-UV and Soret region CD spectra of holo-HRP in 9 M urea suggest that the tertiary structure of holo-HRP and the binding of heme remain partially intact in this condition. A transition in the thermal unfolding transition curve of holo-HRP in 9 M urea indicated the existence of a considerable amount of secondary structure. However, no secondary structure, tertiary structure, or interaction between heme and HRP were observed in holo-HRP in 6 M GdnHCl. Small-angle X-ray scattering indicated that although distal and proximal domains of holo-HRP in 9 M urea might be partially unfolded, the central region that contains the heme might maintain its tertiary structure. Our results suggest that retention of the heme is essential for maintenance of the structure of HRP under highly denaturing conditions.
Asunto(s)
Hemo/química , Peroxidasa de Rábano Silvestre/química , Dicroismo Circular , Guanidina/química , Modelos Moleculares , Desnaturalización Proteica , Estructura Terciaria de Proteína , Desplegamiento Proteico , Temperatura , Urea/química , Difracción de Rayos XRESUMEN
The crystal structure of Ton1535, a hypothetical protein from Thermococcus onnurineus NA1, was determined at 2.3 Å resolution. With two antiparallel α-helices in a helix-turn-helix motif as a repeating unit, Ton1535 consists of right-handed coiled N- and C-terminal regions that are stacked together using helix bundles containing a left-handed helical turn. One left-handed helical turn in the right-handed coiled structure produces two unique structural properties. One is the presence of separated concave grooves rather than one continuous concave groove, and the other is the contribution of α-helices on the convex surfaces of the N-terminal region to the extended surface of the concave groove of the C-terminal region and vice versa.
Asunto(s)
Proteínas Arqueales/química , Thermococcus , Secuencia de Aminoácidos , Cristalografía por Rayos X , Interacciones Hidrofóbicas e Hidrofílicas , Modelos Moleculares , Datos de Secuencia Molecular , Dominios y Motivos de Interacción de Proteínas , Estructura Secundaria de ProteínaRESUMEN
EgtD is an S-adenosyl-l-methionine (SAM)-dependent histidine N,N,N-methyltransferase that catalyzes the formation of hercynine from histidine in the ergothioneine biosynthetic process of Mycobacterium smegmatis. Ergothioneine is a secreted antioxidant that protects mycobacterium from oxidative stress. Here, we present three crystal structures of EgtD in the apo form, the histidine-bound form, and the S-adenosyl-l-homocysteine (SAH)/histidine-bound form. The study revealed that EgtD consists of two distinct domains: a typical methyltransferase domain and a unique substrate binding domain. The histidine binding pocket of the substrate binding domain primarily recognizes the imidazole ring and carboxylate group of histidine rather than the amino group, explaining the high selectivity for histidine and/or (mono-, di-) methylated histidine as substrates. In addition, SAM binding to the MTase domain induced a conformational change in EgtD to facilitate the methyl transfer reaction. The structural analysis provides insights into the putative catalytic mechanism of EgtD in a processive trimethylation reaction.
Asunto(s)
Betaína/análogos & derivados , Histidina/análogos & derivados , Histidina/química , Modelos Químicos , Modelos Moleculares , Mycobacterium smegmatis/enzimología , Proteína Metiltransferasas/química , Proteína Metiltransferasas/ultraestructura , Betaína/química , Sitios de Unión , Metilación , Unión Proteica , Conformación Proteica , Estructura Terciaria de ProteínaRESUMEN
The bromodomain and extraterminal domain (BET) family proteins recognize acetyl-lysine (Kac) at the histone tail through two tandem bromodomains, i.e., BD1 and BD2, to regulate gene expression. BET proteins are attractive therapeutic targets in cancer due to their involvement in oncogenic transcriptional activation, and bromodomains have defined Kac-binding pockets. Here, we present DW-71177, a potent BET inhibitor that selectively interacts with BD1 and exhibits strong antileukemic activity. X-ray crystallography, isothermal titration calorimetry, and molecular dynamic studies have revealed the robust and specific binding of DW-71177 to the Kac-binding pocket of BD1. DW-71177 effectively inhibits oncogenes comparable to the pan-BET inhibitor OTX-015, but with a milder impact on housekeeping genes. It efficiently blocks cancer-associated transcriptional changes by targeting genes that are highly enriched with BRD4 and histone acetylation marks, suggesting that BD1-selective targeting could be an effective and safe therapeutic strategy against leukemia.
Asunto(s)
Leucemia Mieloide Aguda , Factores de Transcripción , Humanos , Factores de Transcripción/metabolismo , Histonas , Proteínas Nucleares , Quinoxalinas/farmacología , Leucemia Mieloide Aguda/tratamiento farmacológico , Proteínas de Ciclo Celular/metabolismo , Proteínas que Contienen BromodominioRESUMEN
Connexin family proteins assemble into hexameric hemichannels in the cell membrane. The hemichannels dock together between two adjacent membranes to form gap junction intercellular channels (GJIChs). We report the cryo-electron microscopy structures of Cx43 GJICh, revealing the dynamic equilibrium state of various channel conformations in detergents and lipid nanodiscs. We identify three different N-terminal helix conformations of Cx43-gate-covering (GCN), pore-lining (PLN), and flexible intermediate (FIN)-that are randomly distributed in purified GJICh particles. The conformational equilibrium shifts to GCN by cholesteryl hemisuccinates and to PLN by C-terminal truncations and at varying pH. While GJIChs that mainly comprise GCN protomers are occluded by lipids, those containing conformationally heterogeneous protomers show markedly different pore sizes. We observe an α-to-π-helix transition in the first transmembrane helix, which creates a side opening to the membrane in the FIN and PLN conformations. This study provides basic structural information to understand the mechanisms of action and regulation of Cx43 GJICh.
Asunto(s)
Conexina 43 , Canales Iónicos , Humanos , Microscopía por Crioelectrón , Subunidades de Proteína , Uniones ComunicantesRESUMEN
AKT1 signaling pathway is important for the regulation of protein synthesis and cell survival with implications in carcinogenesis. In this study, we explored the prognostic significance of AKT1 pathway in intrahepatic cholangiocarcinomas. We investigated the status of phosphatase and tensin homolog deleted on chromosome 10 (PTEN), phosphorylated (p) AKT1 (p-AKT1), p-mammalian target of rapamycin (p-MTOR), p-p70 ribosomal protein S6 kinase (p-RPS6KB2) and p-eukaryotic initiation factor 4E-binding protein-1 (p-EIF4EBP1) in 101 intrahepatic cholangiocarcinomas by immunohistochemistry. Western blot analysis was performed to verify the expression levels of p-AKT1 and p-MTOR. The relationship of protein expression with clinicopathological data and the correlations of protein expression levels were explored. The overexpression of p-AKT1, p-MTOR, and PTEN was associated with a better survival in patients with intrahepatic cholangiocarcinoma (P=0.0137, 0.0194, and 0.0337, respectively). In a multivariate analysis, PTEN was an independent prognostic factor, and p-AKT1 showed tendency (P=0.032 and 0.051, respectively). The overexpression of p-MTOR was correlated with well-to-moderately differentiated tumors (P<0.001) and tumors without metastasis (P=0.046). Expression levels of the AKT1 signaling pathway proteins in this study showed positive correlations with each other, except for PTEN. Aberrant expressions of p-AKT1 and p-MTOR in intrahepatic cholangiocarcinoma were associated with a favorable prognosis, possibly in a PTEN-independent manner. Our results indicate that dysregulation of the AKT1 pathway may have an important role in the development of intrahepatic cholangiocarcinoma, but not necessarily in the progression of the disease.
Asunto(s)
Neoplasias de los Conductos Biliares/enzimología , Conductos Biliares Intrahepáticos/enzimología , Biomarcadores de Tumor/análisis , Colangiocarcinoma/enzimología , Fosfohidrolasa PTEN/análisis , Proteínas Proto-Oncogénicas c-akt/análisis , Serina-Treonina Quinasas TOR/análisis , Proteínas Adaptadoras Transductoras de Señales/análisis , Adolescente , Adulto , Anciano , Neoplasias de los Conductos Biliares/mortalidad , Neoplasias de los Conductos Biliares/patología , Conductos Biliares Intrahepáticos/patología , Western Blotting , Proteínas de Ciclo Celular , Distribución de Chi-Cuadrado , Colangiocarcinoma/mortalidad , Colangiocarcinoma/patología , Femenino , Humanos , Inmunohistoquímica , Estimación de Kaplan-Meier , Masculino , Persona de Mediana Edad , Fosfoproteínas/análisis , Fosforilación , Pronóstico , Modelos de Riesgos Proporcionales , República de Corea , Proteínas Quinasas S6 Ribosómicas 70-kDa/análisis , Regulación hacia Arriba , Adulto JovenRESUMEN
PURPOSE: The aim of this study was to investigate the roles of renal tumor antigen (RAGE) in the progression and clinical outcome of hepatocellular carcinoma (HCC). METHODS: RAGE mRNA levels in 350 cases of HCC were investigated by quantitative real-time reverse transcription polymerase chain reaction. We analyzed the relationship of RAGE mRNA level with clinicopathologic parameters and clinical outcome. To identify the possible role of RAGE on cellular invasion, we performed in vitro analyses using small interfering RNAs (siRNAs). RESULTS: RAGE mRNA level was significantly higher in HCC than in noncancerous hepatic tissues (P < 0.001). Overexpression of RAGE was significantly correlated with the presence of multiple tumors (P = 0.021), high alfa-fetoprotein level (P = 0.042), and advanced tumor stage (P = 0.016). Higher levels of RAGE expression were associated with significantly shorter overall survival time (P = 0.029). Knockdown of RAGE expression by siRNAs suppressed the invasive ability of HCC cells and the expression and secretion of matrix metalloproteinase-9 (MMP-9). We found that RAGE and MMP-9 expressions were correlated in HCCs, and furthermore, the combination of RAGE and MMP-9 expression was associated with the survival of patients (P = 0.0066). CONCLUSIONS: Our results suggest that RAGE may be important in tumor invasion and could be a potential predictor for the prognosis of HCC patients.
Asunto(s)
Antígenos de Neoplasias/metabolismo , Carcinoma Hepatocelular/enzimología , Carcinoma Hepatocelular/patología , Neoplasias Hepáticas/enzimología , Neoplasias Hepáticas/patología , Metaloproteinasa 9 de la Matriz/metabolismo , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Recurrencia Local de Neoplasia/enzimología , Adulto , Anciano , Antígenos de Neoplasias/genética , Supervivencia sin Enfermedad , Femenino , Técnicas de Silenciamiento del Gen , Células Hep G2 , Humanos , Estimación de Kaplan-Meier , Hígado/metabolismo , Masculino , Persona de Mediana Edad , Proteínas Quinasas Activadas por Mitógenos/genética , Invasividad Neoplásica , Estadificación de Neoplasias , Modelos de Riesgos Proporcionales , ARN Mensajero/metabolismo , ARN Interferente Pequeño , Carga Tumoral , Adulto Joven , alfa-Fetoproteínas/metabolismoRESUMEN
Coproduction of multiple proteins at high levels in a single human cell line would be extremely useful for basic research and medical applications. Here, a novel strategy for the stable expression of multiple proteins by integrating the genes into defined transcriptional hotspots in the human genome is presented. As a proof-of-concept, it is shown that EYFP is expressed at similar levels from hotspots and that the EYFP expression increases proportionally with the copy number. It is confirmed that three different fluorescent proteins, encoded by genes integrated at different loci, can be coexpressed at high levels. Further, a stable cell line is generated, producing antigens from different human coronaviruses: MERS-CoV and HCoV-OC43. Antibodies raised against these antigens, which contain human N-glycosylation, show neutralizing activities against both viruses, suggesting that the coexpression system provides a quick and predictable way to produce multiple coronavirus antigens, such as the recent 2019 novel human coronavirus.
Asunto(s)
Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , Antígenos Virales , Coronavirus Humano OC43 , Expresión Génica , Coronavirus del Síndrome Respiratorio de Oriente Medio , Animales , Antígenos Virales/genética , Antígenos Virales/inmunología , Chlorocebus aethiops , Coronavirus Humano OC43/genética , Coronavirus Humano OC43/inmunología , Femenino , Células HEK293 , Humanos , Ratones , Coronavirus del Síndrome Respiratorio de Oriente Medio/genética , Coronavirus del Síndrome Respiratorio de Oriente Medio/inmunología , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Células VeroRESUMEN
RNA demethylase has recently been known to be associated with cancer development but its selective inhibitors as anti-cancer agents have rarely been investigated to date. Herein, we have developed a fluorescent nanobiosensor which enables efficient quantitative analysis of RNA demethylase ALKBH5 activity and shows a high potential for robust inhibitor screening.
Asunto(s)
Desmetilasa de ARN, Homólogo 5 de AlkB/química , Técnicas Biosensibles , Proteínas de Unión al ADN/química , Endorribonucleasas/química , Proteínas de Escherichia coli/química , Grafito/química , Nanopartículas/química , Desmetilación , Espectrometría de FluorescenciaRESUMEN
Direct-acting agents against viral components are considered as the most promising candidates for the successful antiviral therapeutics. To date, no direct-acting drugs exist for the treatment against dengue virus (DV) infection, which can develop into life-threatening diseases. RNA-dependent RNA polymerase (RdRp), an RNA virus-specific enzyme highly conserved among various viral families, has been known as the broad-range antiviral drug target. Here, we developed an RNA-based graphene biosensor system [RNA nano-graphene oxide system (RANGO)] to enable the fluorescence-based quantitative analysis of the RdRp enzyme activity. We used the RANGO system to a high-throughput chemical screening to identify novel direct-acting antiviral drug candidates targeting DV RdRp from the FDA-approved small-molecule library. RANGO accelerated the massive selection of drug candidates. We found that one of the selected hit compounds, montelukast, showed antiviral activity in vitro and in vivo by directly inhibiting replication of DV and thus relieved related symptoms.
RESUMEN
The backbone dynamics of Y14F mutant of Delta(5)-3-ketosteroid isomerase (KSI) from Comamonas testosteroni has been studied in free enzyme and its complex with a steroid analogue, 19-nortestosterone hemisuccinate (19-NTHS), by 15N NMR relaxation measurements. Model-free analysis of the relaxation data showed that the single-point mutation induced a substantial decrease in the order parameters (S2) in free Y14F KSI, indicating that the backbone structures of Y14F KSI became significantly mobile by mutation, while the chemical shift analysis indicated that the structural perturbations of Y14F KSI were more profound than those of wild-type (WT) KSI upon 19-NTHS binding. In the 19-NTHS complexed Y14F KSI, however, the key active site residues including Tyr14, Asp38 and Asp99 or the regions around them remained flexible with significantly reduced S2 values, whereas the S2 values for many of the residues in Y14F KSI became even greater than those of WT KSI upon 19-NTHS binding. The results thus suggest that the hydrogen bond network in the active site might be disrupted by the Y14F mutation, resulting in a loss of the direct interactions between the catalytic residues and 19-NTHS.
Asunto(s)
Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Esteroide Isomerasas/química , Esteroide Isomerasas/genética , Sustitución de Aminoácidos , Sitios de Unión , Comamonas testosteroni/enzimología , Enlace de Hidrógeno , Modelos Moleculares , Mutación , Nandrolona/análogos & derivados , Nandrolona/química , Isótopos de Nitrógeno , Resonancia Magnética Nuclear Biomolecular , Tirosina/química , Tirosina/genéticaRESUMEN
Multidimensional NMR was employed to investigate the structural changes in the urea-induced equilibrium unfolding of the dimeric ketosteroid isomerase (KSI) from Pseudomonas putida biotype B. Sequence specific backbone assignments for the native KSI and the protein with 3.5 M urea were carried out using various 3D NMR experiments. Hydrogen exchange measurements indicated that the secondary structures of KSI were not affected significantly by urea up to 3.5 M. However, the chemical shift analysis of 1H-(15)N HSQC spectra at various urea concentrations revealed that the residues in the dimeric interface region, particularly around the beta5-strand, were significantly perturbed by urea at low concentrations, while the line-width analysis indicated the possibility of conformational exchange at the interface region around the beta6-strand. The results thus suggest that the interface region primarily around the beta5- and beta6-strands could play an important role as the starting positions in the unfolding process of KSI.
Asunto(s)
Esteroide Isomerasas/química , Urea/química , Dimerización , Modelos Moleculares , Resonancia Magnética Nuclear Biomolecular , Desnaturalización Proteica , Pliegue de ProteínaRESUMEN
Failure to detect the intermediate in spite of its existence often leads to the conclusion that two-state transition in the unfolding process of the protein can be justified. In contrast to the previous equilibrium unfolding experiment fitted to a two-state model by circular dichroism and fluorescence spectroscopies, an equilibrium unfolding intermediate of a dimeric ketosteroid isomerase (KSI) could be detected by small angle X-ray scattering (SAXS) and analytical ultracentrifugation. The sizes of KSI were determined to be 18.7A in 0M urea, 17.3A in 5.2M urea, and 25.1A in 7M urea by SAXS. The size of KSI in 5.2M urea was significantly decreased compared with those in 0M and 7M urea, suggesting the existence of a compact intermediate. Sedimentation velocity as obtained by ultracentrifugation confirmed that KSI in 5.2M urea is distinctly different from native and fully-unfolded forms. The sizes measured by pulse field gradient nuclear magnetic resonance (NMR) spectroscopy were consistent with those obtained by SAXS. Discrepancy of equilibrium unfolding studies between size measurement methods and optical spectroscopies might be due to the failure in detecting the intermediate by optical spectroscopic methods. Further characterization of the intermediate using (1)H NMR spectroscopy and Kratky plot supported the existence of a partially-folded form of KSI which is distinct from those of native and fully-unfolded KSIs. Taken together, our results suggest that the formation of a compact intermediate should precede the association of monomers prior to the dimerization process during the folding of KSI.
Asunto(s)
Pliegue de Proteína , Pseudomonas putida/enzimología , Esteroide Isomerasas/química , Dicroismo Circular , Dimerización , Desnaturalización Proteica , Dispersión de Radiación , Urea/químicaRESUMEN
KSI (ketosteroid isomerase) catalyses an allylic isomerization reaction at a diffusion-controlled rate. A hydrogen bond network, Asp(99).Water(504).Tyr(14).Tyr(55).Tyr(30), connects two critical catalytic residues, Tyr(14) and Asp(99), with Tyr(30), Tyr(55) and a water molecule in the highly apolar active site of the Pseudomonas putida KSI. In order to characterize the interactions among these amino acids in the hydrogen bond network of KSI, double-mutant cycle analysis was performed, and the crystal structure of each mutant protein within the cycle was determined respectively to interpret the coupling energy. The DeltaDeltaG(o) values of Y14F/D99L (Tyr(14)-->Phe/Asp(99)-->Leu) KSI, 25.5 kJ/mol for catalysis and 28.9 kJ/mol for stability, were smaller than the sums (i.e. 29.7 kJ/mol for catalysis and 34.3 kJ/mol for stability) for single mutant KSIs respectively, indicating that the effect of the Y14F/D99L mutation was partially additive for both catalysis and stability. The partially additive effect of the Y14F/D99L mutation suggests that Tyr(14) and Asp(99) should interact positively for the stabilization of the transition state during the catalysis. The crystal structure of Y14F/D99L KSI indicated that the Y14F/D99L mutation increased the hydrophobic interaction while disrupting the hydrogen bond network. The DeltaDeltaG(o) values of both Y30F/D99L and Y55F/D99L KSIs for the catalysis and stability were larger than the sum of single mutants, suggesting that either Tyr(30) and Asp(99) or Tyr(55) and Asp(99) should interact negatively for the catalysis and stability. These synergistic effects of both Y30F/D99L and Y55F/D99L mutations resulted from the disruption of the hydrogen bond network. The synergistic effect of the Y55F/D99L mutation was larger than that of the Y30F/D99L mutation, since the former mutation impaired the proper positioning of a critical catalytic residue, Tyr(14), involved in the catalysis of KSI. The present study can provide insight into interpreting the coupling energy measured by double-mutant cycle analysis based on the crystal structures of the wild-type and mutant proteins.
Asunto(s)
Pseudomonas putida/enzimología , Esteroide Isomerasas/química , Sitios de Unión , Catálisis , Cristalización , Enlace de Hidrógeno , Cinética , Modelos Moleculares , Mutación , Desnaturalización Proteica , Proteínas Recombinantes/química , Esteroide Isomerasas/metabolismo , TermodinámicaRESUMEN
Low-barrier hydrogen bonds (LBHBs) have been proposed to have important influences on the enormous reaction rate increases achieved by many enzymes. Δ(5)-3-ketosteroid isomerase (KSI) catalyzes the allylic isomerization of Δ(5)-3-ketosteroid to its conjugated Δ(4)-isomers at a rate that approaches the diffusion limit. Tyr14, a catalytic residue of KSI, has been hypothesized to form an LBHB with the oxyanion of a dienolate steroid intermediate generated during the catalysis. The unusual chemical shift of a proton at 16.8 ppm in the nuclear magnetic resonance spectrum has been attributed to an LBHB between Tyr14 Oη and C3-O of equilenin, an intermediate analogue, in the active site of D38N KSI. This shift in the spectrum was not observed in Y30F/Y55F/D38N and Y30F/Y55F/Y115F/D38N mutant KSIs when each mutant was complexed with equilenin, suggesting that Tyr14 could not form LBHB with the intermediate analogue in these mutant KSIs. The crystal structure of Y30F/Y55F/Y115F/D38N-equilenin complex revealed that the distance between Tyr14 Oη and C3-O of the bound steroid was within a direct hydrogen bond. The conversion of LBHB to an ordinary hydrogen bond in the mutant KSI reduced the binding affinity for the steroid inhibitors by a factor of 8.1-11. In addition, the absence of LBHB reduced the catalytic activity by only a factor of 1.7-2. These results suggest that the amount of stabilization energy of the reaction intermediate provided by LBHB is small compared with that provided by an ordinary hydrogen bond in KSI.
Asunto(s)
Equilenina/metabolismo , Pseudomonas putida/enzimología , Esteroide Isomerasas/química , Esteroide Isomerasas/genética , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Biocatálisis , Dominio Catalítico , Cristalografía por Rayos X , Equilenina/química , Enlace de Hidrógeno , Modelos Moleculares , Mutación , Unión Proteica , Espectroscopía de Protones por Resonancia Magnética , Pseudomonas putida/genética , Esteroide Isomerasas/metabolismo , Especificidad por SustratoRESUMEN
YgjG is a putrescine aminotransferase enzyme that transfers amino groups from compounds with terminal primary amines to compounds with an aldehyde group using pyridoxal-5'-phosphate (PLP) as a cofactor. Previous biochemical data show that the enzyme prefers primary diamines, such as putrescine, over ornithine as a substrate. To better understand the enzyme's substrate specificity, crystal structures of YgjG from Escherichia coli were determined at 2.3 and 2.1 Å resolutions for the free and putrescine-bound enzymes, respectively. Sequence and structural analyses revealed that YgjG forms a dimer that adopts a class III PLP-dependent aminotransferase fold. A structural comparison between YgjG and other class III aminotransferases revealed that their structures are similar. However, YgjG has an additional N-terminal helical structure that partially contributes to a dimeric interaction with the other subunit via a helix-helix interaction. Interestingly, the YgjG substrate-binding site entrance size and charge distribution are smaller and more hydrophobic than other class III aminotransferases, which suggest that YgjG has a unique substrate binding site that could accommodate primary aliphatic diamine substrates, including putrescine. The YgjG crystal structures provide structural clues to putrescine aminotransferase substrate specificity and binding.
Asunto(s)
Escherichia coli/enzimología , Putrescina/metabolismo , Transaminasas/química , Secuencia de Aminoácidos , Cristalografía por Rayos X , Dimerización , Datos de Secuencia Molecular , Conformación Proteica , Desnaturalización Proteica , Fosfato de Piridoxal/metabolismo , Homología de Secuencia de Aminoácido , Especificidad por Sustrato , Transaminasas/metabolismoRESUMEN
Penicillin-binding proteins (PBPs), which mediate the peptidoglycan biosynthetic pathway in the bacterial cell wall, have been intensively investigated as a target for the design of antibiotics. In this study, PBPD2, a low-molecular-weight PBP encoded by lmo2812 from Listeria monocytogenes, was overexpressed in Escherichia coli, purified and crystallized at 295â K using the sitting-drop vapour-diffusion method. The crystal belonged to the primitive orthorhombic space group P212121, with unit-cell parameters a = 37.7, b = 74.7, c = 75.1â Å, and diffracted to 1.55â Å resolution. There was one molecule in the asymmetric unit. The preliminary structure was determined by the molecular-replacement method.