RESUMEN
Hyperthermophilic ('superheat-loving') archaea found in high-temperature environments such as Pyrobaculum aerophilum contain multicopper oxidases (MCOs) with remarkable efficiency for oxidizing cuprous and ferrous ions. In this work, directed evolution was used to expand the substrate specificity of P. aerophilum McoP for organic substrates. Six rounds of error-prone PCR and DNA shuffling followed by high-throughput screening lead to the identification of a hit variant with a 220-fold increased efficiency (kcat/Km) than the wild-type for 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) without compromising its intrinsic activity for metal ions. The analysis of the X-ray crystal structure reveals four proximal mutations close to the T1Cu active site. One of these mutations is within the 23-residues loop that occludes this site, a distinctive feature of prokaryotic MCOs. The increased flexibility of this loop results in an enlarged tunnel and one additional pocket that facilitates bulky substrate-enzyme interactions. These findings underscore the synergy between mutations that modulate the dynamics of the active-site loop enabling enhanced catalytic function. This study highlights the potential of targeting loops close to the T1Cu for engineering improvements suitable for biotechnological applications.
Asunto(s)
Dominio Catalítico , Oxidorreductasas , Especificidad por Sustrato , Oxidorreductasas/metabolismo , Oxidorreductasas/química , Oxidorreductasas/genética , Pyrobaculum/enzimología , Pyrobaculum/genética , Modelos Moleculares , Cristalografía por Rayos XRESUMEN
The gene encoding a repressor open reading frame sugar kinase (ROK) family protein from hyperthermophilic crenarchaeon Pyrobaculum calidifontis, Pcal-HK, was cloned and expressed in Escherichia coli. The recombinant protein was produced in soluble and highly active form. Purified Pcal-HK was highly thermostable and existed in a monomeric form in solution. The enzyme was specific to ATP as phosphoryl donor but showed broad specificity to phosphoryl acceptors. It catalyzed the phosphorylation of a number of hexoses, including glucose, glucosamine, N-acetyl glucosamine, fructose and mannose, at nearly the same rate and similar affinity. The enzyme was metal ion dependent exhibiting highest activity at 90-95 °C and pH 8.5. Mg2+ was most effective metal ion, which could be partially replaced by Mn2+, Ni2+ or Zn2+. Kinetic parameters were determined at 90 °C and the enzyme showed almost similar catalytic efficiency (kcat/Km) towards the above mentioned hexoses. To the best of our knowledge, Pcal-HK is the most active thermostable ROK family hexokinase characterized to date which catalyzes the phosphorylation of various hexoses with nearly similar affinity.
Asunto(s)
Hexoquinasa , Pyrobaculum , Estabilidad de Enzimas , Hexoquinasa/genética , Hexoquinasa/metabolismo , Cinética , Fosforilación , Pyrobaculum/enzimología , Pyrobaculum/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidad por Sustrato , Escherichia coli/genéticaRESUMEN
Biofuel cells generate electric energy using an enzyme as a catalyst for an electrode but their stability and low battery output pose problems for practical use. To solve these problems, this study aimed to build a long-lasting and high-output biocathode as a catalyst using a highly stable hyperthermophilic archaeal enzyme, multi-copper oxidase, from Pyrobaculum aerophilum (McoP). To increase output, McoP was oriented and immobilized on single-walled carbon nanotubes (SWCNT) with a high specific surface area, and the electrode interface was designed to achieve highly efficient electron transfer between the enzyme and electrode. Type 1 copper (T1Cu), an electron-accepting site in the McoP molecule, is located near the C-terminus. Therefore, McoP was prepared by genetically engineering a CNT-binding peptide with the sequence LLADTTHHRPWT, at the C-terminus of McoP (McoP-CBP). We then constructed an electrode using a complex in which McoP-CBP was aligned and immobilized on SWCNT, and then clarified the effect of CBP. The amounts of immobilized enzymes on McoP-SWCNT and (McoP-CBP)-SWCNT complexes were almost equal. CV measurement of the electrode modified with both complexes showed 5.4 times greater current density in the catalytic reaction of the (McoP-CBP)-SWCNT/GC electrode than in the McoP-SWCNT/GC electrode. This is probably because CBP fusion immobilize the enzyme on SWCNTs in an orientational manner, and T1Cu, the oxidation-reduction site in McoP, is close to the electrode, which improves electron transfer efficiency.
Asunto(s)
Fuentes de Energía Bioeléctrica , Enzimas Inmovilizadas/metabolismo , Nanotubos de Carbono/química , Oxidorreductasas/metabolismo , Péptidos/metabolismo , Pyrobaculum/enzimología , Catálisis , Electrodos , Enzimas Inmovilizadas/química , Oxidorreductasas/química , Péptidos/químicaRESUMEN
Enzymes from hyperthermophilic archaea are potential candidates for industrial use because of their superior pH, thermal, and long-term stability, and are expected to improve the long-term stability of biofuel cells (BFCs). However, the reported multicopper oxidase (MCO) from hyperthermophilic archaea has lower redox potential than MCOs from other organisms, which leads to a decrease in the cell voltage of BFCs. In this study, we attempted to positively shift the redox potential of the MCO from hyperthermophilic archaeon Pyrobaculum aerophilum (McoP). Mutations (M470L and M470F) were introduced into the axial ligand of the T1 copper atom of McoP, and the enzymatic chemistry and redox potentials were compared with that of the parent (M470). The redox potentials of M470L and M470F shifted positively by about 0.07 V compared with that of M470. In addition, the catalytic activity of the mutants towards 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) increased 1.2-1.3-fold. The thermal stability of the mutants and the electrocatalytic performance for O2 reduction of M470F was slightly reduced compared with that of M470. This research provides useful enzymes for application as biocathode catalysts for high-voltage BFCs.
Asunto(s)
Proteínas Arqueales , Fuentes de Energía Bioeléctrica , Mutagénesis Sitio-Dirigida , Mutación Missense , Oxidorreductasas , Pyrobaculum , Sustitución de Aminoácidos , Proteínas Arqueales/química , Proteínas Arqueales/genética , Oxidorreductasas/química , Oxidorreductasas/genética , Pyrobaculum/enzimología , Pyrobaculum/genéticaRESUMEN
The gene encoding Pcal_0842, annotated as a cellulase (accession no. ABO08268), was cloned and expressed in Escherichia coli. The gene product was produced in insoluble form in E. coli in high amounts even without addition of the inducer isopropyl ß-D-1-thiogalactopyranoside. The recombinant protein was solubilized in 8 M urea and refolded by gradual removal of urea. The refolded protein exhibited both α-1,4- and ß-1,4-glycosidic cleavage activities. The enzyme activity increased with the increase in temperature till 120 °C. Apart from very high optimal temperature, recombinant Pcal_0842 was extremely thermostable. There was no significant loss in activity even after heating for 100 h at 100 °C. The half-lives of Pcal_0842 were 6 and 2.5 h at 110 and 120 °C, respectively. To the best of our knowledge, Pcal_0842 is the most thermostable glycosidase characterized to date and this is the first report on cloning and characterization of an enzyme from archaea that displays both α-1,4- and ß-1,4-glycosidic cleavage activities.
Asunto(s)
Proteínas Arqueales/metabolismo , Glicósido Hidrolasas/metabolismo , Glicósidos/metabolismo , Pyrobaculum/enzimología , Temperatura , Secuencia de Aminoácidos , Dicroismo Circular , Estabilidad de Enzimas , Expresión Génica , Glicósido Hidrolasas/química , Glicósido Hidrolasas/genética , Concentración de Iones de Hidrógeno , Hidrólisis , Cinética , Modelos Moleculares , Pyrobaculum/genética , Proteínas Recombinantes/aislamiento & purificación , Solubilidad , Especificidad por SustratoRESUMEN
Pcal_0768 gene encoding an amylomaltase, a 4-α-glucanatransferase belonging to family 77 of glycosyl hydrolases, from Pyrobaculum calidifontis was cloned and expressed in Escherichia coli. The recombinant protein was produced in E. coli in soluble and active form. However, the expression level was not very high. Analysis of the mRNA of initial seven codons at the 5'-end of the gene revealed the presence of a hair pin like secondary structure. This secondary structure was removed by site directed mutagenesis, without altering the amino acids, which resulted in enhanced expression of the cloned gene. Recombinant Pcal_0768 exhibited optimal amylomaltase activity at 80 °C and pH 6.9. Under these conditions, the specific activity was 690 U/ mg. Recombinant Pcal_0768 was highly thermostable with a half-life of 6 h at 100 °C. It exhibited the highest kcat value among the characterized glucanotransferases. No metal ions were required for activity or stability of the enzyme. Recombinant Pcal_0768 was successfully employed in the synthesis of modified starch for producing thermoreversible gel. To the best of our knowledge, till now this is the most thermostable enzyme among the characterized amylomaltases. High thermostability and starch modification potential make it a novel and distinct amylomaltase.
Asunto(s)
Proteínas Arqueales , Clonación Molecular , Expresión Génica , Sistema de la Enzima Desramificadora del Glucógeno , Calor , Pyrobaculum , Proteínas Arqueales/biosíntesis , Proteínas Arqueales/química , Proteínas Arqueales/genética , Proteínas Arqueales/aislamiento & purificación , Estabilidad de Enzimas , Escherichia coli/enzimología , Escherichia coli/genética , Sistema de la Enzima Desramificadora del Glucógeno/biosíntesis , Sistema de la Enzima Desramificadora del Glucógeno/química , Sistema de la Enzima Desramificadora del Glucógeno/genética , Sistema de la Enzima Desramificadora del Glucógeno/aislamiento & purificación , Pyrobaculum/enzimología , Pyrobaculum/genética , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificaciónRESUMEN
Protein glycosylation constitutes a critical post-translational modification that supports a vast number of biological functions in living organisms across all domains of life. A seemingly boundless number of enzymes, glycosyltransferases, are involved in the biosynthesis of these protein-linked glycans. Few glycan-biosynthetic glycosyltransferases have been characterized in vitro, mainly due to the majority being integral membrane proteins and the paucity of relevant acceptor substrates. The crenarchaeote Pyrobaculum calidifontis belongs to the TACK superphylum of archaea (Thaumarchaeota, Aigarchaeota, Crenarchaeota, Korarchaeota) that has been proposed as an eukaryotic ancestor. In archaea, N-glycans are mainly found on cell envelope surface-layer proteins, archaeal flagellins and pili. Archaeal N-glycans are distinct from those of eukaryotes, but one noteworthy exception is the high-mannose N-glycan produced by P. calidifontis, which is similar in sugar composition to the eukaryotic counterpart. Here, we present the characterization and crystal structure of the first member of a crenarchaeal membrane glycosyltransferase, PcManGT. We show that the enzyme is a GDP-, dolichylphosphate-, and manganese-dependent mannosyltransferase. The membrane domain of PcManGT includes three transmembrane helices that topologically coincide with "half" of the six-transmembrane helix cellulose-binding tunnel in Rhodobacter spheroides cellulose synthase BcsA. Conceivably, this "half tunnel" would be suitable for binding the dolichylphosphate-linked acceptor substrate. The PcManGT gene (Pcal_0472) is located in a large gene cluster comprising 14 genes of which 6 genes code for glycosyltransferases, and we hypothesize that this cluster may constitute a crenarchaeal N-glycosylation (PNG) gene cluster.
Asunto(s)
Manosiltransferasas/química , Manosiltransferasas/metabolismo , Polisacáridos/metabolismo , Pyrobaculum/enzimología , Proteínas Arqueales/química , Proteínas Arqueales/metabolismo , Cristalografía por Rayos X , Glicosilación , Modelos Moleculares , Conformación Proteica , Procesamiento Proteico-Postraduccional , Pyrobaculum/químicaRESUMEN
NDP-forming type acyl-CoA synthetase superfamily proteins are known to have six essential subdomains (1, 2, 3, a, b, c) of which partition and order are varied, suggesting yet-to-be-defined subdomain rearrangement happened in its evolution. Comparison in physicochemical and biochemical characteristics between the recombinant proteins which we made from fragmented subdomains and wild-type protein, acetate-CoA ligase in a hyperthermophilic archaeon, consisting of two distinct subunits (α1-2-3 and ßa-b-c) provided a clue to the mystery of its molecular evolutionary passage. Although solubility and thermostability of each fragmented subdomain turned out to be lower than that of wild-type, mixture of the three synthetic subunits of α1-2, α3, and ßa-b-c had quaternary structure, thermostability, and enzymatic activity comparable to those of the wild-type. This suggests that substantial independence and mobility of subdomain 3 have enabled rearrangement of the subdomains; and thermostability of the subdomains has constrained the composition of the subunits.
Asunto(s)
Acetato CoA Ligasa/química , Acetato CoA Ligasa/metabolismo , Estabilidad de Enzimas , Dominios Proteicos , Pyrobaculum/enzimología , TemperaturaRESUMEN
Micro total analysis system (µTAS) is expected to be applied in various fields. In particular, since electrochemical measurement is inexpensive and easy, electrochemical measurement can be integrated with a microchannel. However, electrochemical detection sensitivity in a microchannel is lowered because the diffusion of the detection target is limited. In an ordinary electrochemical detection system, using a stirrer in a beaker can overcome limited diffusion. We previously proposed a new detection system that combines a microliquid solution agitation technology using surface acoustic waves (SAWs) with the µTAS. The SAWs function as microstirrers, thus making electrochemical detection possible by overcoming limited diffusion of the sample. However, when the solution is stirred by the SAWs, the temperature of the solution increases to 70°C due to vibrational energy. This leads to enzyme inactivation and impaired electrochemical response. Therefore, in this study, we used a hyperthermophile-derived enzyme. Temperature and electrochemical characteristics of the detection system using SAWs and a multi-copper oxidase (MCO) derived from the hyperthermophilic archaea Pyrobaculum aerophilum were studied. Laccase, which is an MCO derived from the thermophilic fungus Trametes versicolor, was also studied. We also characterized the enzyme-electrochemical reaction using SAWs by comparing the magnitude of the reduction current obtained using the two enzymes with different heat resistances. We observed an increase in the electrochemical response with the SAWs, without impaired enzyme activity. Thus, the use of a thermostable enzyme is suitable for the development of a biosensor that uses SAWs for agitation.
Asunto(s)
Técnicas Electroquímicas , Lacasa/metabolismo , Oxidorreductasas/metabolismo , Sonido , Temperatura , Tamaño de la Partícula , Polyporaceae/enzimología , Pyrobaculum/enzimología , Propiedades de SuperficieRESUMEN
A gene encoding galactose 1-phosphate uridylyltransferase (GalT) was identified in the hyperthermophilic archaeon Pyrobaculum aerophilum. The gene was overexpressed in Escherichia coli, after which its product was purified and characterized. The expressed enzyme was highly thermostable and retained about 90% of its activity after incubation for 10 minutes at temperatures up to 90°C. Two different crystal structures of P. aerophilum GalT were determined: the substrate-free enzyme at 2.33 Å and the UDP-bound H140F mutant enzyme at 1.78 Å. The main-chain coordinates of the P. aerophilum GalT monomer were similar to those in the structures of the E. coli and human GalTs, as was the dimeric arrangement. However, there was a striking topological difference between P. aerophilum GalT and the other two enzymes. In the E. coli and human enzymes, the N-terminal chain extends from one subunit into the other and forms part of the substrate-binding pocket in the neighboring subunit. By contrast, the N-terminal chain in P. aerophilum GalT extends to the substrate-binding site in the same subunit. Amino acid sequence alignment showed that a shorter surface loop in the N-terminal region contributes to the unique topology of P. aerophilum GalT. Structural comparison of the substrate-free enzyme with UDP-bound H140F suggests that binding of the glucose moiety of the substrate, but not the UDP moiety, gives rise to a large structural change around the active site. This may in turn provide an appropriate environment for the enzyme reaction.
Asunto(s)
Proteínas Arqueales/química , Galactosafosfatos/química , Subunidades de Proteína/química , Pyrobaculum/química , UTP-Hexosa-1-Fosfato Uridililtransferasa/química , Secuencia de Aminoácidos , Proteínas Arqueales/genética , Proteínas Arqueales/metabolismo , Sitios de Unión , Clonación Molecular , Cristalografía por Rayos X , Estabilidad de Enzimas , Escherichia coli/genética , Escherichia coli/metabolismo , Galactosafosfatos/metabolismo , Expresión Génica , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Calor , Humanos , Cinética , Modelos Moleculares , Mutación , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Multimerización de Proteína , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Pyrobaculum/enzimología , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Especificidad por Sustrato , UTP-Hexosa-1-Fosfato Uridililtransferasa/genética , UTP-Hexosa-1-Fosfato Uridililtransferasa/metabolismoRESUMEN
Porphobilinogen synthase (PBG synthase) gene from Pyrobaculum calidifontis was cloned and expressed in E. coli. The recombinant enzyme was purified as an octamer and was found by mass spectrometry to have a subunit Mr of 37676.59 (calculated, 37676.3). The enzyme showed high thermal stability and retained almost all of its activity after incubation at 70⯰C for 16â¯h in the presence of ß-mercaptoethanol (ß-ME) and zinc chloride. However, in the absence of the latter the enzyme was inactivated after 16â¯h although it regained full activity in the presence of ß-ME and zinc chloride. The protein contained 4â¯mol of tightly bound zinc per octamer. Further, 4â¯mol of low affinity zinc could be incorporated following incubation with exogenous zinc salts. The enzyme was inactivated by incubation with levulinic acid followed by treatment with sodium borohydride. Tryptic digest of the modified enzyme and mass spectrometric analysis showed that Lys257 was the site of modification, which has previously been shown to be the site for the binding of 5-aminolevulinic acid giving rise to the propionate-half of porphobilinogen. P. calidifontis PBG synthase was inactivated by 5-chlorolevulinic acid and the residue modified was shown to be the central cysteine (Cys127) of the zinc-binding cysteine-triad, comprising Cys125, 127, 135. The present results in conjunction with earlier findings on zinc containing PBG synthases, are discussed which advocate that the catalytic role of zinc in the activation of the 5-aminolevulinic acid molecule forming the acetate-half of PBG is possible.
Asunto(s)
Porfobilinógeno Sintasa/metabolismo , Pyrobaculum/enzimología , Relación Dosis-Respuesta a Droga , Ácidos Levulínicos/farmacología , Estructura Molecular , Porfobilinógeno Sintasa/antagonistas & inhibidores , Porfobilinógeno Sintasa/química , Relación Estructura-ActividadRESUMEN
LeLoir glycosyltransferases are important biocatalysts for the production of glycosidic bonds in natural products, chiral building blocks, and pharmaceuticals. Trehalose transferase (TreT) is of particular interest since it catalyzes the stereo- and enantioselective α,α-(1â1) coupling of a nucleotide sugar donor and monosaccharide acceptor for the synthesis of disaccharide derivatives. Heterologously expressed thermophilic trehalose transferases were found to be intrinsically aggregation prone and are mainly expressed as catalytically active inclusion bodies in Escherichia coli To disfavor protein aggregation, the thermostable protein mCherry was explored as a fluorescent protein tag. The fusion of mCherry to trehalose transferase from Pyrobaculum yellowstonensis (PyTreT) demonstrated increased protein solubility. Chaotropic agents like guanidine or the divalent cations Mn(II), Ca(II), and Mg(II) enhanced the enzyme activity of the fusion protein. The thermodynamic equilibrium constant, Keq, for the reversible synthesis of trehalose from glucose and a nucleotide sugar was determined in both the synthesis and hydrolysis directions utilizing UDP-glucose and ADP-glucose, respectively. UDP-glucose was shown to achieve higher conversions than ADP-glucose, highlighting the importance of the choice of nucleotide sugars for LeLoir glycosyltransferases under thermodynamic control.IMPORTANCE The heterologous expression of proteins in Escherichia coli is of great relevance for their functional and structural characterization and applications. However, the formation of insoluble inclusion bodies is observed in approximately 70% of all cases, and the subsequent effects can range from reduced soluble protein yields to a complete failure of the expression system. Here, we present an efficient methodology for the production and analysis of a thermostable, aggregation-prone trehalose transferase (TreT) from Pyrobaculum yellowstonensis via its fusion with mCherry as a thermostable fluorescent protein tag. This fusion strategy allowed for increased enzyme stability and solubility and could be applied to other (thermostable) proteins, allowing rapid visualization and quantification of the mCherry-fused protein of interest. Finally, we have demonstrated that the enzymatic synthesis of trehalose from glucose and a nucleotide sugar is reversible by approaching the thermodynamic equilibrium in both the synthesis and hydrolysis directions. Our results show that uridine establishes an equilibrium constant which is more in favor of the product trehalose than when adenosine is employed as the nucleotide under identical conditions. The influence of different nucleotides on the reaction can be generalized for all LeLoir glycosyltransferases under thermodynamic control as the position of the equilibrium depends solely on the reaction conditions and is not affected by the nature of the catalyst.
Asunto(s)
Estabilidad de Enzimas , Proteínas Recombinantes de Fusión/metabolismo , Transferasas/metabolismo , Trehalosa/metabolismo , Adenosina Difosfato Glucosa , Aniones , Catálisis , Cationes , Activación Enzimática , Estabilidad de Enzimas/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Vectores Genéticos , Glucosa/metabolismo , Glicosiltransferasas/metabolismo , Cinética , Agregado de Proteínas , Pyrobaculum/enzimología , Pyrobaculum/genética , Proteínas Recombinantes de Fusión/genética , Solubilidad , Transferasas/genética , Uridina Difosfato GlucosaRESUMEN
The genome sequence of Pyrobaculum calidifontis contains two open reading frames, Pcal_0144 and Pcal_0970, exhibiting homology with L-asparaginases. In search of a thermostable L-asparaginase with no glutaminase activity, we have cloned and expressed the gene encoding Pcal_0970 in Escherichia coli. Recombinant Pcal_0970 was produced in insoluble and inactive form which was solubilized and refolded into enzymatically active form. The refolded Pcal_0970 showed the highest activity at or above 100 °C. Optimum pH for the enzyme activity was 6.5. Addition of divalent metal cations or EDTA had no significant effect on the activity. The enzyme was capable of hydrolyzing D-asparagine with a 20% activity as compared to 100% with L-asparagine. Pcal_0970 did not show any detectable activity when L-glutamine or D-glutamine was used as substrate. Pcal_0970 exhibited a Km value of 4.5 ± 0.4 mmol/L and Vmax of 355 ± 13 µmol min-1 mg-1 towards L-asparagine. The activation energy, from the linear Arrhenius plot, was determined as 39.9 ± 0.6 kJ mol-1. To the best of our knowledge, Pcal_0970 is the most thermostable L-asparaginase with a half-life of more than 150 min at 100 °C and this is the first report on characterization of an L-asparaginase from phylum Crenarchaeota.
Asunto(s)
Asparaginasa/metabolismo , Glutaminasa/metabolismo , Pyrobaculum/enzimología , Asparaginasa/aislamiento & purificación , Clonación Molecular , Estabilidad de Enzimas , Glutamina/metabolismo , Semivida , Concentración de Iones de Hidrógeno , Cinética , Pyrobaculum/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidad por Sustrato , TemperaturaRESUMEN
The genome of the hyperthermophilic archaeon Pyrobaculum calidifontis contains an open reading frame, Pcal_0041, annotated as encoding a PfkB family ribokinase, consisting of phosphofructokinase and pyrimidine kinase domains. Among the biochemically characterized enzymes, the Pcal_0041 protein was 37% identical to the phosphofructokinase (Ape_0012) from Aeropyrum pernix, which displayed kinase activity toward a broad spectrum of substrates, including sugars, sugar phosphates, and nucleosides, and 36% identical to a phosphofructokinase from Desulfurococcus amylolyticus To examine the biochemical function of the Pcal_0041 protein, we cloned and expressed the gene and purified the recombinant protein. Although the Pcal_0041 protein contained a putative phosphofructokinase domain, it exhibited only low levels of phosphofructokinase activity. The recombinant enzyme catalyzed the phosphorylation of nucleosides and, to a lower extent, sugars and sugar phosphates. Surprisingly, among the substrates tested, the highest activity was detected with ribose 1-phosphate (R1P), followed by cytidine and uridine. The catalytic efficiency (kcat/Km ) toward R1P was 11.5 mM-1 · s-1 ATP was the most preferred phosphate donor, followed by GTP. Activity measurements with cell extracts of P. calidifontis indicated the presence of nucleoside phosphorylase activity, which would provide the means to generate R1P from nucleosides. The study suggests that, in addition to the recently identified ADP-dependent ribose 1-phosphate kinase (R1P kinase) in Thermococcus kodakarensis that functions in the pentose bisphosphate pathway, R1P kinase is also present in members of the Crenarchaeota.IMPORTANCE The discovery of the pentose bisphosphate pathway in Thermococcus kodakarensis has clarified how this archaeon can degrade nucleosides. Homologs of the enzymes of this pathway are present in many members of the Thermococcales, suggesting that this metabolism occurs in these organisms. However, this is not the case in other archaea, and degradation mechanisms for nucleosides or ribose 1-phosphate are still unknown. This study reveals an important first step in understanding nucleoside metabolism in Crenarchaeota and identifies an ATP-dependent ribose 1-phosphate kinase in Pyrobaculum calidifontis The enzyme is structurally distinct from previously characterized archaeal members of the ribokinase family and represents a group of proteins found in many crenarchaea.
Asunto(s)
Fosfofructoquinasas/genética , Nucleósidos de Pirimidina/metabolismo , Pyrobaculum/enzimología , Pyrobaculum/genética , Ribosamonofosfatos/metabolismo , Estabilidad de Enzimas , Fosfofructoquinasas/metabolismo , Fosforilación , Proteínas Recombinantes , Especificidad por SustratoRESUMEN
A gene encoding L-serine dehydrogenase (L-SerDH) that exhibits extremely low sequence identity to the Agrobacterium tumefaciens L-SerDH was identified in the hyperthermophilic archaeon Pyrobaculum calidifontis. The predicted amino acid sequence showed 36% identity with that of Pseudomonas aeruginosa L-SerDH, suggesting that P. calidifontis L-SerDH is a novel type of L-SerDH, like Ps. aeruginosa L-SerDH. The overexpressed enzyme appears to be the most thermostable L-SerDH described to date, and no loss of activity was observed by incubation for 30 min at temperatures up to 100 °C. The enzyme showed substantial reactivity towards D-serine, in addition to L-serine. Two different crystal structures of P. calidifontis L-SerDH were determined using the Se-MAD and MR method: the structure in complex with NADP+/sulfate ion at 1.18 Å and the structure in complex with NADP+/L-tartrate (substrate analog) at 1.57 Å. The fold of the catalytic domain showed similarity with that of Ps. aeruginosa L-SerDH. However, the active site structure significantly differed between the two enzymes. Based on the structure of the tartrate, L- and D-serine and 3-hydroxypropionate molecules were modeled into the active site and the substrate binding modes were estimated. A structural comparison suggests that the wide cavity at the substrate binding site is likely responsible for the high reactivity of the enzyme toward both L- and D-serine enantiomers. This is the first description of the structure of the novel type of L-SerDH with bound NADP+ and substrate analog, and it provides new insight into the substrate binding mechanism of L-SerDH. The results obtained here may be very informative for the creation of L- or D-serine-specific SerDH by protein engineering.
Asunto(s)
Oxidorreductasas de Alcohol/química , Proteínas Arqueales/química , Simulación del Acoplamiento Molecular , Pyrobaculum/enzimología , Oxidorreductasas de Alcohol/metabolismo , Proteínas Arqueales/metabolismo , Dominio Catalítico , Cristalografía por Rayos X , Estabilidad de Enzimas , Calor , NADP/química , NADP/metabolismo , Unión Proteica , Serina/química , Serina/metabolismo , Especificidad por Sustrato , Tartratos/química , Tartratos/metabolismoRESUMEN
tRNAHis guanylyltransferase (Thg1) has unique reverse (3'-5') polymerase activity occurring in all three domains of life. Most eukaryotic Thg1 homologs are essential genes involved in tRNAHis maturation. These enzymes normally catalyze a single 5' guanylation of tRNAHis lacking the essential G-1 identity element required for aminoacylation. Recent studies suggest that archaeal type Thg1, which includes most archaeal and bacterial Thg1 enzymes is phylogenetically distant from eukaryotic Thg1. Thg1 is evolutionarily related to canonical 5'-3' forward polymerases but catalyzes reverse 3'-5'polymerization. Similar to its forward polymerase counterparts, Thg1 encodes the conserved catalytic palm domain and fingers domain. Here we investigate the minimal requirements for reverse polymerization. We show that the naturally occurring minimal Thg1 enzyme from Ignicoccus hospitalis (IhThg1), which lacks parts of the conserved fingers domain, is catalytically active. And adds all four natural nucleotides to RNA substrates, we further show that the entire fingers domain of Methanosarcina acetivorans Thg1 and Pyrobaculum aerophilum Thg1 (PaThg1) is dispensable for enzymatic activity. In addition, we identified residues in yeast Thg1 that play a part in preventing extended polymerization. Mutation of these residues with alanine resulted in extended reverse polymerization. PaThg1 was found to catalyze extended, template dependent tRNA repair, adding up to 13 nucleotides to a truncated tRNAHis substrate. Sequencing results suggest that PaThg1 fully restored the near correct sequence of the D- and acceptor stem, but also produced incompletely and incorrectly repaired tRNA products. This research forms the basis for future engineering efforts towards a high fidelity, template dependent reverse polymerase.
Asunto(s)
Desulfurococcaceae/enzimología , Methanosarcina/enzimología , Nucleotidiltransferasas/metabolismo , Pyrobaculum/enzimología , ARN de Transferencia de Histidina/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimología , Secuencia de Aminoácidos , Sitios de Unión , Dominio Catalítico , Secuencia Conservada , Desulfurococcaceae/genética , Expresión Génica , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Cinética , Methanosarcina/genética , Modelos Moleculares , Mutación , Nucleotidiltransferasas/química , Nucleotidiltransferasas/genética , Polimerizacion , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Ingeniería de Proteínas/métodos , Pyrobaculum/genética , ARN de Transferencia de Histidina/química , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Alineación de Secuencia , Homología de Secuencia de AminoácidoRESUMEN
Genome sequence of the hyperthermophilic archaeon Pyrobaculum calidifontis contains an open reading frame, Pcal_0632, annotated as glyceraldehyde-3-phosphate dehydrogenase, which is partially overlapped with phosphoglycerate kinase. In the phylogenetic tree, Pcal_0632 clustered with phosphorylating glyceraldehyde-3-phosphate dehydrogenases characterized from hyperthermophilic archaea and exhibited highest identity of 54% with glyceraldehyde-3-phosphate dehydrogenase from Sulfolobus tokodaii. To examine biochemical function of the protein, Pcal_0632 gene was expressed in Escherichia coli and the gene product was purified. The recombinant enzyme catalyzed the conversion of glyceraldehyde 3-phosphate and inorganic phosphate into 1,3-bisphosphoglycerate utilizing both NAD and NADP as cofactor with a marked preference for NADP. The enzyme was highly stable against temperature and denaturants. Half-life of the enzyme was 60 min at 100 °C. It retained more than 60% of its activity even after an incubation of 72 h at room temperature in the presence of 6 M urea. High thermostability and resistance against denaturants make Pcal_0632 a novel glyceraldehyde-3-phosphate dehydrogenase.
Asunto(s)
Proteínas Arqueales/metabolismo , Gliceraldehído-3-Fosfato Deshidrogenasa (Fosforilante)/metabolismo , Pyrobaculum/enzimología , Termotolerancia , Proteínas Arqueales/química , Estabilidad de Enzimas , Gliceraldehído-3-Fosfato Deshidrogenasa (Fosforilante)/química , Desnaturalización Proteica , Especificidad por SustratoRESUMEN
Pyrobaculum islandicum is a hyperthermophilic archaeon that grows optimally at 95-100 °C. In the previous study, we extensively purified a serine racemase from this organism and cloned the gene for overexpression in Escherichia coli (Ohnishi et al. 2008). This enzyme also exhibits highly thermostable L-serine/L-threonine dehydratase activity. In the present study, we aimed to elucidate the molecular mechanisms underlying the high thermostability of this enzyme. A recombinant variant of this enzyme, PiSRvt, constructed by truncating the C-terminal 72 amino acids, was compared with the native enzyme, PiSR. The dehydratase activity of PiSR and PiSRvt was found to owe to a homotrimer and a monomer, respectively, that demonstrated high and moderate thermostability, respectively. These observations reveal that the C-terminal region contributes to monomer trimerization that provides the extreme thermostability.
Asunto(s)
Proteínas Arqueales/química , Racemasas y Epimerasas/química , Termotolerancia , Proteínas Arqueales/genética , Proteínas Arqueales/metabolismo , Estabilidad de Enzimas , Desnaturalización Proteica , Dominios Proteicos , Pyrobaculum/enzimología , Racemasas y Epimerasas/genética , Racemasas y Epimerasas/metabolismoRESUMEN
The genome of the hyperthermophilic archaeon Pyrobaculum calidifontis contains an open reading frame, Pcal_1032, annotated as glucokinase. Amino acid sequence analysis showed that Pcal_1032 belonged to ROK (repressor, open reading frame, and kinase) family of sugar kinases. To examine the properties of Pcal_1032, the coding gene was cloned and expressed in Escherichia coli. However, expression of the gene was low resulting in a poor yield of the recombinant protein. A single site directed mutation in Pcal_1032 gene, without altering the amino acid sequence, resulted in approximately tenfold higher expression. Purified recombinant Pcal_1032 efficiently phosphorylated various hexoses with a marked preference for glucose. ATP was the most preferred phosphoryl group donor. Optimum temperature and pH for the glucokinase activity of Pcal_1032 were 95 °C and 8.5, respectively. Catalytic efficiency (k cat/K m) towards glucose was 437 mM-1 s-1. The recombinant enzyme was highly stable against temperature with a half-life of 25 min at 100 °C. In addition, Pcal_1032 was highly stable in the presence of denaturants. There was no significant change in the CD spectra and enzyme activity of Pcal_1032 even after overnight incubation in the presence of 8 M urea. To the best of our knowledge, Pcal_1032 is the most active and highly stable glucokinase characterized to date from archaea, and this is the first description of the characterization of a glucokinase from genus Pyrobaculum.
Asunto(s)
Proteínas Arqueales/genética , Glucoquinasa/genética , Pyrobaculum/enzimología , Proteínas Arqueales/metabolismo , Estabilidad de Enzimas , Escherichia coli/genética , Glucoquinasa/metabolismo , Pyrobaculum/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidad por SustratoRESUMEN
Genome sequence of Pyrobaculum calidifontis, a hyperthermophilic archaeon, harbors three open-reading frames annotated as alcohol dehydrogenases. One of them, Pcal_1311, does not display a significantly high homology with any of the characterized alcohol dehydrogenases. Highest homology of 38% was found with the characterized counterpart from Geobacillus stearothermophilus. To examine the biochemical properties of Pcal_1311, we have cloned and functionally expressed the gene in Escherichia coli. Purified recombinant Pcal_1311 catalyzed the NAD(H)-dependent oxidation of various alcohols and reduction of aldehydes, with a marked preference for substrates with functional group at the terminal carbon. Highest activity for the oxidation reaction (3 µmol min-1 mg-1) was found with 1,4-butanediol and for the reduction reaction (150 µmol min-1 mg-1) with glutaraldehyde. Both the oxidation and reduction activities increased with the increase in temperature up to 80 °C. Recombinant Pcal_1311 was highly stable and retained more than 90% activity even after incubation of 180 min at 90 °C. In addition to the thermostabilty, Pcal_1311 was highly stable in the presence of known denaturants including urea and guanidine hydrochloride. The high stability, particularly thermostability, and the NADH-dependent aldehyde reduction activity make Pcal_1311 a unique member in the alcohol dehydrogenase family.