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1.
Jean-Luc Décout Marie-Christine Maurel 《Origins of life and evolution of the biosphere》1993,23(5-6):299-306
In our search for primitive RNA catalysts, we noticed that N6-ribosyl-adenine, a compound easily synthesized under presumed prebiotic conditions, has a free imidazole group. We showed that it is, as a catalyst, a potential analogue of histidine. Furthermore, among the chemical groups involved in protein catalysis, the imidazole ring of histidine has no equivalent in the RNA world. We have synthesized aliphatic amino groups containing polymers with adenine rings linked to macromolecules by their 6-amino group. These polymers exhibit pronounced catalytic activities in the hydrolysis of p-nitrophenylacetate. We discuss here the fact that in primitive catalysis the imidazole group could have been replaced by N6-substituted adenine derivatives. 相似文献
2.
The pentose phosphate cycle is considered as a major source of NADPH and pentose needed for nucleic acid biosynthesis. 6-Phosphogluconate dehydrogenase (6PGD), an enzyme participating in this cycle, catalyzes the oxidative decarboxylation of 6PGD to ribulose 5-phosphate with the subsequent release of CO2 and the reduction of NADP. We have determined the amino acid sequence of 6PGD of Bactrocera oleae and constructed a three-dimensional model based on the homologous known sheep structure. In a comparative study of 6PGD sequences from numerous species, all the conserved and variable regions of the enzyme were analyzed and the regions of functional importance were localized, in an attempt promoted also by the direct involvement of the enzyme in various human diseases. Thus, analysis of amino acid variability of 37 6PGD sequences revealed that all regions important for the catalytic activity, such as those forming the substrate and coenzyme binding sites, are highly conserved in all species examined. Moreover, several amino acid residues responsible for substrate and coenzyme specificity were also found to be identical in all species examined. The higher percentage of protein divergence is observed at two regions that accumulate mutations, located at the distant parts of the two domains of the enzyme with respect to their interface. These peripheral regions of nonfunctional importance are highly variable and are predicted as antigenic, thus reflecting possible regions for antibody recognition. Furthermore, locating the differences between diptera 6PGD sequences on the three-dimensional model suggests probable positions of different amino acid residues appearing at B. oleae fast, intermediate, and slow allozymic variants.
Abbreviations used:
6Pgd, 6-Phosphogluconate dehydrogenase gene; 6PGD, 6-Phosphogluconate dehydrogenase enzyme; NADPH, nicotinamide adenine dinucleotide phosphate; ADP, adenine dinucleotide phosphate; TNBS, 2,4,6 trinitrobenzensulfonic acid. 相似文献
3.
Bassam Al Balaa Kristof Brijs Kurt Gebruers Jean Vandenhaute Johan Wouters Isabelle Housen 《Bioresource technology》2009,100(24):6465-6471
The role of residues Asp60, Tyr35 and Glu141 in the pH-dependent activity of xylanase XYL1p from Scytalidium acidophilum was investigated by site-directed mutagenesis. These amino acids are highly conserved among the acidophilic family 11 xylanases and located near the catalytic site. XYL1p and its single mutants D60N, Y35W and E141A and three combined mutants DN/YW, DN/EA and YW/EA were over-expressed in Pichia pastoris and purified. Xylanase activities at different pH’s and temperatures were determined. All mutations increased the pH optimum by 0.5–1.5 pH units. All mutants have lower specific activities except the E141A mutant that exhibited a 50% increase in specific activity at pH 4.0 and had an overall catalytic efficiency higher than the wild-type enzyme. Thermal unfolding experiments show that both the wild-type and E141A mutant proteins have a Tm maximum at pH 3.5, the E141A mutant being slightly less stable than the wild-type enzyme. These mutations confirm the importance of these amino acids in the pH adaptation. Mutant E141A with its enhanced specific activity at pH 4.0 and improved overall catalytic efficiency is of possible interest for biotechnological applications. 相似文献
4.
Hydrogen-exchange studies of I · C and G · C double helices were carried out to test the generality of conclusions reached previously in studies of adenine-containing polymers (preceding paper). The cytosine amino group shows hydrogen-exchange behavior similar to the analogous group in adenine; a pH-independent pathway and a parallel general catalysis pathway require prior separation of the base-pair and pre-equilibrium protonation at the ring N. The cytosine amino group does, however, display greater sensitivity to specific and to general catalysis than found for adenine. In the G · C helix, the ring NH proton of guanine exchanges at the opening-limited rate, as does the analogous proton in A · U and A · T pairs, while the guanine amino protons exchange without a prior opening of structure. From the observed exchange rates and the known chemistry for the pH-independent reaction, one can calculate equilibrium opening constants of 4 × 10−3 for poly(rI) · poly(rC) and perhaps one tenth of that for poly(rG) · poly(rC). Also the opening rate constant for the G · C helix is 0.01 s−1.These results, when applied to published exchange curves for DNA, indicate an equilibrium opening constant of 0.005, an opening rate constant of 0.04 s−1, and a closing rate constant of 10 s−1. (All values refer to studies at 0 °C.) These values point to the same kind of traveling-loop model for base-pair opening discussed previously for the opening reactions in adenine-containing double helices. 相似文献
5.
Ravindra Tewari 《Journal of biomolecular structure & dynamics》2013,31(3):675-686
Abstract Conformational preferences of the hypermodified nucleic acid bases N6-methyl-N6-(N- threonylcarbonyl) Adenine, m6 tc6 Ade, and 2-methylthio-N6-(N-threonylcarbonyl) Adenine, mS2 tc6 Ade, have been studied theoretically using the quantum chemical PCILO (Perturbative Configuration Interaction using Localized Orbitals) method. The multidimensional conformational space has been searched using selected grid points formed by combining the various torsion angles which take the favoured values obtained from energy variation with respect to each torsion angle individually. In m6 tc6 Ade and mS2 tc6 Ade alike the threonylcar- bonyl substituent preferably orients away (distal) from the imidazole moiety of the adenine ring. And as in the simpler N6-(N-threonylcarbonyl) Adenine, tc6 Ade, the atoms in the ureido group as well as the amino acid carbon atoms C(12) and C(13) remain coplanar with the purine base. As in tc6 Ade, this conformation is stabilized by the intramolecular hydrogen bond between N(11)H of the amino acid and N(l) of the adenine base. The N6-methyl protons, in m6 tc6 Ade, take trans-staggered orientation with respect to the C(6)-N(6) bond. The preferred orientation of the 2-methylthio group is cis to the C(2)-N(3) bond in mS2 tc6 Ade. This is in marked contrast to the modified nucleic acid base 2-methylthio-N6-(Δ2-isopentenyl) Adenine, mS2 i6 Ade, where the 2-methylthio group orients trans to the C(2)- N(3) bond, causing a change in the preferred orientation of the isopentenyl component on methylthiolation. The present results thus indicate that unlike in the isopentenyl adenine the role of further chemical substitutions in threonylcarbonyl adenine may be indirect and less pronounced. 相似文献
6.
拟南芥谷胱甘肽S-转移酶Zeta类(AtGSTZ)是一种与细胞代谢和环境净化密切相关的多功能酶.应用易错PCR和多轮DNA洗牌技术构建了AtGSTZ随机突变文库;再利用pH指示剂颜色改变法对突变文库进行筛选,获得了9个二氯乙酸脱氯活性提高的突变子.其中,NN23含25个氨基酸突变,比活力提高120%,NN20含24个氨基酸突变,比活力提高102%,EC1含2个氨基酸突变,比活力提高47%,其他6个为单点突变,比活力分别提高9%~60%.酶学分析显示,所有进化酶对底物二氯乙酸的催化效率和对谷胱甘肽的亲和力以及个别进化酶的复性能力都得到不同程度的提高,但热稳定性均没有明显改善.同时,对一系列与AtGSTZ空间折叠及催化活性相关位点进行了讨论. 相似文献
7.
Modified purines are found in all organisms in the tRNA, rRNA, and even DNA, raising the possibility of an early role for
these compounds in the evolution of life. These include N
6-methyladenine, 1-methyladenine, N
6,N
6-dimethyladenine, 1-methylhypoxanthine, 1-methylguanine, and N
2-methylguanine. We find that these bases as well as a number of nonbiological modified purines can be synthesized from adenine
and guanine by the simple reaction of an amine or an amino group with adenine and guanine under the concentrated conditions
of the drying-lagoon or drying-beach model of prebiotic synthesis with yields as high as 50%. These compounds are therefore
as prebiotic as adenine and guanine and could have played an important role in the RNA world by providing additional functional
groups in ribozymes, especially for the construction of hydrophobic binding pockets.
Received: 7 August 1998 / Accepted: 31 December 1998 相似文献
8.
Mannose-6-phosphate isomerase catalyzes the interconversion of mannose-6-phosphate and fructose-6-phosphate. The gene encoding a putative mannose-6-phosphate isomerase from Thermus thermophilus was cloned and expressed in Escherichia coli. The native enzyme was a 29 kDa monomer with activity maxima for mannose 6-phosphate at pH 7.0 and 80 °C in the presence of 0.5 mM Zn2+ that was present at one molecule per monomer. The half-lives of the enzyme at 65, 70, 75, 80, and 85 °C were 13, 6.5, 3.7, 1.8, and 0.2 h, respectively. The 15 putative active-site residues within 4.5 Å of the substrate mannose 6-phosphate in the homology model were individually replaced with other amino acids. The sequence alignments, activities, and kinetic analyses of the wild-type and mutant enzymes with amino acid changes at His50, Glu67, His122, and Glu132 as well as homology modeling suggested that these four residues are metal-binding residues and may be indirectly involved in catalysis. In the model, Arg11, Lys37, Gln48, Lys65 and Arg142 were located within 3 Å of the bound mannose 6-phosphate. Alanine substitutions of Gln48 as well as Arg142 resulted in increase of Km and dramatic decrease of kcat, and alanine substitutions of Arg11, Lys37, and Lys65 affected enzyme activity. These results suggest that these 5 residues are substrate-binding residues. Although Trp13 was located more than 3 Å from the substrate and may not interact directly with substrate or metal, the ring of Trp13 was essential for enzyme activity. 相似文献
9.
James C. Lacey Jr. Dail W. Mullins Jr. Charles L. Watkins 《Journal of biomolecular structure & dynamics》2013,31(4):783-793
Abstract We have synthesized the free amino acid adenylate anydrides of phenylalanine, leucine, isoleucine and valine. These activated compounds are very labile at high pH, but at low pH they become more stable. Proton NMR spectra of these adenylates show that, in every case, the hydrophobic side chains, even in these small molecules at low pH and low concentration, are associated with the “face” of the adenine ring. Although aromatic rings are known to associate with adenine in this fashion, to our knowledge this is the first report of an intercalative-type interaction of aliphatic side chains with nucleic acid bases. Since adenine is the most hydrophobic base, these interactions are of a hydrophobic character, and occur in spite of the fact that the adenine ring is protonated. These results may have implications regarding recognition processes in DNA-protein and RNA-protein interactions. 相似文献
10.
The kinetic and molecular properties of cyanobacterial glucose-6-phosphate dehydrogenase, partly purified from Anabaena sp. ATCC 27893, show that it undergoes relatively slow, reversible transitions between different aggregation states which differ in catalytic activity. Sucrose gradient centrifugation and polyacrylamide gel electrophoresis reveal three principal forms, with approximate molecular weights of 120 000 (M
1), 240 000 (M
2) and 345 000 (M
3). The relative catalytic activities are: M
1M
2<M
3. In concentrated solutions of the enzyme, the equilibrium favors the more active, oligomeric forms. Dilution in the absence of effectors shifts the equilibrium in favor of the M
1 form, with a marked diminution of catalytic activity. This transition is prevented by a substrate, glucose-6-phosphate, and also by glutamine. The other substrate, nicotinamide adenine dinucleotide phosphate (NADP+), and (in crude cell-free extracts) ribulose-1,5-diphosphate are negative effectors, which tend to maintain the enzyme in the M
1 form. The equilibrium state between different forms of the enzyme is also strongly dependent on hydrogen ion concentration. Although the optimal pH for catalytic activity is 7.4, dissociation to the hypoactive M
1 form is favored at pH values above 7; a pH of 6.5 is optimal for maintenace of the enzyme in the active state. Reduced nicotamide adenine dinucleotide phosphate (NADPH) and adenosine 5-triphosphate (ATP), inhibit catalytic activity, but do not significantly affect the equilibrium state. The relevance of these findings to the regulation of enzyme activity in vivo is discussed.Abbreviations G6PD
glucose-6-phosphate dehydrogenase
- 6PGD
6-phosphogluconate dehydrogenase
- RUDP
ribulose-1,5-diphosphate
- G6P
glucose-6-phosphate
- 6PG
6-phosphogluconate 相似文献
11.
Leucine dehydrogenase was inhibited by p-chioromercuribenzoate and HgCl2, but not by 5,5′-dithiobis(2-nitrobenzoic acid), 4,4′-dithiopyridine and N-ethylmaleimide. Modification of sulfhydryl groups of the enzyme with p-chloromercuribenzoate and HgCl2 was accompanied with a loss of the enzyme activity. The 6 reactive sulfhydryl groups per enzyme molecule play an essential role for catalysis. Approximately 12 sulfhydryl groups were titrated per molecule in the presence of 8 m urea: the enzyme contains 2 sulfhydryl groups per subunit, and one of them participates in the catalytic action. Fluorometric and gel filtration studies on binding of NADH to the enzyme revealed that the enzyme contains 6 coenzyme binding sites per molecule.These results are compatible with the hexameric structure of leucine dehydrogenase composed of identical subunits, showing that each subunit has one catalytic site and one indispensable sulfhydryl group. 相似文献
12.
Two molecular orbitals: MO7 (29a) and MO13 (23a) have been identified using dual space analysis (DSA) as the signatures of adenine non-planarity (C1 point group symmetry). The non-planarity of adenine has been demonstrated to be from the attachment of the amino group (NH2) to purine rings as well as the non-rigid deformability of the purine ring of adenine. Orbital 29a (3a″ in the planar case), a π-like orbital, is the direct result of the attachment of the amino group to the purine ring. Orbital 23a (23a′ in the planar case) is the result of the deformability of the purine ring in non-planar adenine (NP) and will be experimentally challenging to resolve. 相似文献
13.
Fukuda T Kato-Murai M Kadonosono T Sahara H Hata Y Suye S Ueda M 《Applied microbiology and biotechnology》2007,76(5):1027-1033
Recently, in family 3 β-glucosidase (BGL), the catalytically important Asp nucleophile has been identified in the SDW segment
of the SDWG sequence by site-directed mutagenesis. However, the details about the roles of each amino acid residue of the
SDWG sequence have not been investigated. W293 of the SDW segment, which is the residue next to the nucleophile (D292) in
family 3 BGL, is very important for hydrolytic reaction as a binder to a substrate. G294 of the SDWG sequence might play an
important role in catalysis. In this study, to obtain a functional BGL1 mutant by the substitution of G294 using a genetic
engineering method, the library of mutant BGL1 from Aspergillus oryzae was rapidly constructed by yeast cell surface engineering, and the hydrolytic activities of mutants were comprehensively
detected. Consequently, G294F, G294W, and G294Y, in which G was substituted with aromatic amino acids, showed higher activities
for substrate recognition than the parent strain (1.5-, 1.5-, and 1.6-fold, respectively). These results suggest the presence
of some interaction between the sugar rings and aromatic ring of W293 at the entrance of the catalytic pocket, which enhances
the substrate recognition of β-glucosidase. 相似文献
14.
Juan A. Castelán-Vega Alicia Jiménez-Alberto Rosa M. Ribas-Aparicio 《Journal of molecular modeling》2010,16(7):1213-1222
M.HgiDII is a methyltransferase (MTase) from Herpetosiphon giganteus that recognizes the sequence GTCGAC. This enzyme belongs to a group of MTases that share a high degree of amino acid similarity,
albeit none of them has been thoroughly characterized. To study the catalytic mechanism of M.HgiDII and its interactions with
DNA, we performed molecular dynamics simulations with a homology model of M.HgiDII complexed with DNA and S-adenosyl-methionine.
Our results indicate that M.HgiDII may not rely only on Glu119 to activate the cytosine ring, which is an early step in the
catalysis of cytosine methylation; apparently, Arg160 and Arg162 may also participate in the activation by interacting with
cytosine O2. Another residue from the catalytic site, Val118, also played a relevant role in the catalysis of M.HgiDII. Val118
interacted with the target cytosine and kept water molecules from accessing the region of the catalytic pocket where Cys79
interacts with cytosine, thus preventing water-mediated disruption of interactions in the catalytic site. Specific recognition
of DNA was mediated mainly by amino acids of the target recognition domain, although some amino acids (loop 80–88) of the
catalytic domain may also contribute to DNA recognition. These interactions involved direct contacts between M.HgiDII and
DNA, as well as indirect contacts through water bridges. Additionally, analysis of sequence alignments with closely related
MTases helped us to identify a motif in the TRD of M.HgiDII that may be relevant to specific DNA recognition. 相似文献
15.
Expanding the scope of stereoselectivity is of current interest in enzyme catalysis. In this study, using error-prone polymerase
chain reaction (PCR), a thermostable adenosine diphosphate (ADP)-glucose pyrophosphorylase (AGPase) from Thermus caldophilus GK-24 has been altered to improve its catalytic activity toward enatiomeric substrates including [glucose-1-phosphate (G-1-P)
+ uridine triphosphate (UTP)] and [N-acetylglucosamine-1-phosphate (GlcNAc) + UTP] to produce uridine diphosphate (UDP)-glucose and UDP-N-acetylglucosamine, respectively. To elucidate the amino acids responsible for catalytic activity, screening for UDP-glucose
pyrophosphorylase (UGPase) and UDP-N-acetylglucosamine pyrophosphorylase (UNGPase) activities was carried out. Among 656 colonies, two colonies showed UGPase
activities and three colonies for UNGPase activities. DNA sequence analyses and enzyme assays showed that two mutant clones
(H145G) specifically have an UGPase activity, indicating that the changed glycine residue from histidine has the base specificity
for UTP. Also, three double mutants (H145G/A325V) showed a UNGPase, and A325 was associated with sugar binding, conferring
the specificity for the sugar substrates and V325 of the mutant appears to be indirectly involved in the binding of the N-acetylamine group of N-acetylglucosmine-1-phosphate.
The authors Hosung Sohn and Yong-Sam Kim equally contributed to the study. 相似文献
16.
A cel44C-man26A gene of endophytic Paenibacillus
polymyxa GS01 has multi-glycosyl hydrolases in two catalytic domains 总被引:2,自引:0,他引:2
Cho KM Hong SY Lee SM Kim YH Kahng GG Kim H Yun HD 《Applied microbiology and biotechnology》2006,73(3):618-630
A bacterial strain Paenibacillus polymyxa GS01 was isolated from the interior of the roots of Korean cultivars of ginseng (Panax ginseng C. A. Meyer). The cel44C-man26A gene was cloned from this endophytic strain. This 4,056-bp gene encodes for a 1,352-aa protein which, based on BLAST search homologies, contains a glycosyl hydrolase family 44 (GH44) catalytic domain, a fibronectin domain type 3, a glycosyl hydrolase family 26 (GH26) catalytic domain, and a cellulose-binding module type 3. The multifunctional enzyme domain GH44 possesses cellulase, xylanase, and lichenase activities, while the enzyme domain GH26 possesses mannanase activity. The Cel44C enzyme expressed in and purified from Escherichia coli has an optimum pH of 7.0 for cellulase and lichenase activities, but is at an optimum pH of 5.0 for xylanase and mannanase activities. The optimum temperature for enzymatic activity was 50°C for all substrates. No detectable enzymatic activity was detected for the Cel44C-Man26A mutants E91A and E222A. These results suggest that the amino acid residues Glu91 and Glu222 may play an important role in the glycosyl hydrolases activity of Cel44C-Man26A. 相似文献
17.
Our understanding of how life emerged on Earth has much to do with speculations about the ways in which prebiotic catalysts could have been formed. Since enzymes, the contemporary biological catalysts, are polymers of amino acids, we looked at the possible activity of free amino acids as catalysts. In this study it is shown experimentally that mixtures of free amino acids exert catalytic activities of -galactosidase, carbonic anhydrase, and catalase. We also observed different levels of catalytic activty of individual amino acids: some were more efficient than others. Apparently, assemblies of amino acids which were formed around substrate molecules through weak interactions, could, in principle, catalyze many prebiotic reactions. This might have been one step in the emergence of biological enzymes. 相似文献
18.
In this work, a fundamental regulatory role of formate on thuringiensin production by resting cell of Bacillus thuringiensis YBT-032 was investigated. Nicotinamide adenine dinucleotide (NADH) production and formate dehydrogenase activity increased
with formate addition from 0.5 to 2.0 g/L, respectively. However, with the formate addition of 1.5 g/L, the activities of
pyruvate kinase and glucose 6-phosphate dehydrogenase reached a peak and increased by 316 and 150% relative to those of the
control, respectively. In addition, intracellular production of pyruvate, aspartate, citrate and adenine were significantly
enhanced by 75, 66, 32 and 78% as well. An improvement (90%) of thuringiensin production was also successfully obtained. Interestingly
to point out, thuringiensin yield was closely correlative with adenine production, and the linear relationship was also observed.
The results suggest that appropriate formate addition did act as a modulator and facilitate carbon flux in glycolysis and
pentose phosphate pathway to synthesize adenine and thuringiensin via intracellular NADH availability. 相似文献
19.
Two different types of polymers containing nucleic acid bases in the backbone or in the pendant groups were prepared. The polymers of the first type were polyureas obtained by the polyaddition reaction of uracil and adenine with hexamethylene diisocyanate (HMD1). The second type that is cationic polyurethanes containing nucleic acid bases in pendant groups, were obtained by the Menschutkin reaction of halogenated derivatives of uracil and adenine with a linear polyurethane containing tertiary nitrogen atoms which was based on HMD1 and N-methyldiethanolamine. Base base interactions were studied for the polymers by ultraviolet (u.r.), optical rotary dispersion (o.r.d.), and nuclear magnetic resonance (n.m.r.) spectra. A relatively high value of hypochromicity, ≈17% was observed for the mixture of the ionic polyurethane with uracil pendant and herring-sperm DNA. Complementary hydrogen bonding interactions were detected for the mixture of the ionic polyurethane with adenine pendant and with uracil pendant. The non-thrombogenic character of the polymers was examined according to the modified Lee White method. The ionic polyurethanes with adenine and uracil pendant exhibited a fairly good anti-clotting properly. 相似文献
20.
RNA molecules that are assembled from the four standard nucleotides contain a limited number of chemical functional groups, a characteristic that is generally thought to restrict the potential for catalysis by ribozymes. Although polypeptides carry a wider range of functional groups, many contemporary protein-based enzymes employ coenzymes to augment their capabilities. The coenzymes possess additional chemical moieties that can participate directly in catalysis and thereby enhance catalytic function. In this work, we demonstrate a mechanism by which ribozymes can supplement their limited repertoire of functional groups through RNA-catalyzed incorporation of various coenzymes and coenzyme analogues. The group I ribozyme of Tetrahymena thermophila normally mediates a phosphoester transfer reaction that results in the covalent attachment of guanosine to the ribozyme. Here, a shortened version of the ribozyme is shown to catalyze the self-incorporation of coenzymes and coenzyme analogues, such as NAD+ and dephosphorylated CoA-SH. Similar ribozyme activities may have played an important role in the RNA world, when RNA enzymes are thought to have maintained a complex metabolism in the absence of proteins and would have benefited from the inclusion of additional functional groups.Correspondence to: G.F. Joyce 相似文献