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1.
Yoshida S Inui M Yukawa H Kanao T Tomizawa K Atomi H Imanaka T 《Journal of biotechnology》2006,124(3):532-544
The hyperthermophilic archaeon, Thermococcus kodakaraensis KOD1 harbors a structurally novel, Type III Rubisco (Rbc(Tk)). In terms of protein engineering of Rubiscos, the enzyme may provide an alternative target to the conventional Type I and Type II enzymes. With a future aim to improve the catalytic properties of Rbc(Tk), here we examined whether or not the enzyme could support growth of a mesophilic organism dependent on CO2 fixation. Via double-crossover homologous recombination, we first deleted three Rubisco genes present on the chromosome of the photosynthetic mesophile Rhodopseudomonas palustris No. 7. The mutant strain (delta3) could neither grow under photoautotrophic nor photoheterotrophic conditions. We introduced the rbc(Tk) gene into strain delta3 either on a plasmid, or by integrating the gene onto the chromosome. The two transformant strains harboring rbc(Tk) displayed growth under photoautotrophic and photoheterotrophic conditions, both dependent on CO2 fixation. Specific growth rates and Rubisco activity levels were compared under photoheterotrophic conditions among the two transformants and the wild-type strain. We observed that the levels of Rubisco activity in the respective cell-free extracts correlated well with the specific growth rates. Immunoprecipitation experiments revealed that Rubisco activity detected in the transformants was derived solely from Rbc(Tk). These results demonstrated that the Type III Rbc(Tk) from a hyperthermophile could support CO2 fixation in a mesophilic organism, and that the specific growth rate of the transformant can be used as a convenient parameter for selection of engineered proteins with improved Rubisco activity. 相似文献
2.
Engineering of a Type III Rubisco from a Hyperthermophilic Archaeon in Order To Enhance Catalytic Performance in Mesophilic Host Cells
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The hyperthermophilic archaeon Thermococcus kodakaraensis harbors a type III ribulose 1,5-bisphosphate carboxylase/oxygenase (RbcTk). It has previously been shown that RbcTk is capable of supporting photoautotrophic and photoheterotrophic growth in a mesophilic host cell, Rhodopseudomonas palustris Δ3, whose three native Rubisco genes had been disrupted. Here, we have examined the enzymatic properties of RbcTk at 25°C and have constructed mutant proteins in order to enhance its performance in mesophilic host cells. Initial sites for mutagenesis were selected by focusing on sequence differences in the loop 6 and α-helix 6 regions among RbcTk and the enzymes from spinach (mutant proteins SP1 to SP7), Galdieria partita (GP1 and GP2), and Rhodospirillum rubrum (RR1). Loop 6 of RbcTk is one residue longer than those found in the spinach and G. partita enzymes, and replacing RbcTk loop 6 with these regions led to dramatic decreases in activity. Six mutant enzymes retaining significant levels of Rubisco activity were selected, and their genes were introduced into R. palustris Δ3. Cells harboring mutant protein SP6 displayed a 31% increase in the specific growth rate under photoheterotrophic conditions compared to cells harboring wild-type RbcTk. SP6 corresponds to a complete substitution of the original α-helix 6 of RbcTk with that of the spinach enzyme. Compared to wild-type RbcTk, the purified SP6 mutant protein exhibited a 30% increase in turnover number (kcat) of the carboxylase activity and a 17% increase in the kcat/Km value. Based on these results, seven further mutant proteins were designed and examined. The results confirmed the importance of the length of loop 6 in RbcTk and also led to the identification of specific residue changes that resulted in an increase in the turnover number of RbcTk at ambient temperatures. 相似文献
3.
Nishitani Y Yoshida S Fujihashi M Kitagawa K Doi T Atomi H Imanaka T Miki K 《The Journal of biological chemistry》2010,285(50):39339-39347
The Calvin-Benson-Bassham cycle is responsible for carbon dioxide fixation in all plants, algae, and cyanobacteria. The enzyme that catalyzes the carbon dioxide-fixing reaction is ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco). Rubisco from a hyperthermophilic archaeon Thermococcus kodakarensis (Tk-Rubisco) belongs to the type III group, and shows high activity at high temperatures. We have previously found that replacement of the entire α-helix 6 of Tk-Rubisco with the corresponding region of the spinach enzyme (SP6 mutant) results in an improvement of catalytic performance at mesophilic temperatures, both in vivo and in vitro, whereas the former and latter half-replacements of the α-helix 6 (SP4 and SP5 mutants) do not yield such improvement. We report here the crystal structures of the wild-type Tk-Rubisco and the mutants SP4 and SP6, and discuss the relationships between their structures and enzymatic activities. A comparison among these structures shows the movement and the increase of temperature factors of α-helix 6 induced by four essential factors. We thus supposed that an increase in the flexibility of the α-helix 6 and loop 6 regions was important to increase the catalytic activity of Tk-Rubisco at ambient temperatures. Based on this structural information, we constructed a new mutant, SP5-V330T, which was designed to have significantly greater flexibility in the above region, and it proved to exhibit the highest activity among all mutants examined to date. The thermostability of the SP5-V330T mutant was lower than that of wild-type Tk-Rubisco, providing further support on the relationship between flexibility and activity at ambient temperatures. 相似文献
4.
Mutation design of a thermophilic Rubisco based on three‐dimensional structure enhances its activity at ambient temperature
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Riku Aono Takaaki Sato Tomoyuki Takai Kenta Tagashira Wakao Fukuda Haruyuki Atomi Tadayuki Imanaka Kunio Miki 《Proteins》2016,84(10):1339-1346
Ribulose‐1,5‐bisphosphate carboxylase/oxygenase (Rubisco) plays a central role in carbon dioxide fixation on our planet. Rubisco from a hyperthermophilic archaeon Thermococcus kodakarensis (Tk‐Rubisco) shows approximately twenty times the activity of spinach Rubisco at high temperature, but only one‐eighth the activity at ambient temperature. We have tried to improve the activity of Tk‐Rubisco at ambient temperature, and have successfully constructed several mutants which showed higher activities than the wild‐type enzyme both in vitro and in vivo. Here, we designed new Tk‐Rubisco mutants based on its three‐dimensional structure and a sequence comparison of thermophilic and mesophilic plant Rubiscos. Four mutations were introduced to generate new mutants based on this strategy, and one of the four mutants, T289D, showed significantly improved activity compared to that of the wild‐type enzyme. The crystal structure of the Tk‐Rubisco T289D mutant suggested that the increase in activity was due to mechanisms distinct from those involved in the improvement in activity of Tk‐Rubisco SP8, a mutant protein previously reported to show the highest activity at ambient temperature. Combining the mutations of T289D and SP8 successfully generated a mutant protein (SP8‐T289D) with the highest activity to date both in vitro and in vivo. The improvement was particularly pronounced for the in vivo activity of SP8‐T289D when introduced into the mesophilic, photosynthetic bacterium Rhodopseudomonas palustris, which resulted in a strain with nearly two‐fold higher specific growth rates compared to that of a strain harboring the wild‐type enzyme at ambient temperature. Proteins 2016; 84:1339–1346. © 2016 Wiley Periodicals, Inc. 相似文献
5.
H Sugawara H Yamamoto N Shibata T Inoue S Okada C Miyake A Yokota Y Kai 《The Journal of biological chemistry》1999,274(22):15655-15661
Ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco, EC 4.1.1. 39) obtained from a thermophilic red alga Galdieria partita has the highest specificity factor of 238 among the Rubiscos hitherto reported. Crystal structure of activated Rubisco from G. partita complexed with the reaction intermediate analogue, 2-carboxyarabinitol 1,5-bisphosphate (2-CABP) has been determined at 2.4-A resolution. Compared with other Rubiscos, different amino residues bring the structural differences in active site, which are marked around the binding sites of P-2 phosphate of 2-CABP. Especially, side chains of His-327 and Arg-295 show the significant differences from those of spinach Rubisco. Moreover, the side chains of Asn-123 and His-294 which are reported to bind the substrate, ribulose 1,5-bisphosphate, form hydrogen bonds characteristic of Galdieria Rubisco. Small subunits of Galdieria Rubisco have more than 30 extra amino acid residues on the C terminus, which make up a hairpin-loop structure to form many interactions with the neighboring small subunits. When the structures of Galdieria and spinach Rubiscos are superimposed, the hairpin region of the neighboring small subunit in Galdieria enzyme and apical portion of insertion residues 52-63 characteristic of small subunits in higher plant enzymes are almost overlapped to each other. 相似文献
6.
The loop between alpha-helix 6 and beta-strand 6 in the alpha/beta-barrel active site of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco, EC 4.1.1.39) plays a key role in discriminating between gaseous substrates CO(2) and O(2). Based on numerous x-ray crystal structures, loop 6 is either closed or open depending on the presence or absence, respectively, of substrate ligands. The carboxyl terminus folds over loop 6 in the closed conformation, prompting speculation that it may trigger or latch loop 6 closure. Because an x-ray crystal structure of tobacco Rubisco revealed that phosphate is located at a site in the open form that is occupied by the carboxyl group of Asp-473 in the closed form, it was proposed that Asp-473 may serve as the latch that holds the carboxyl terminus over loop 6. To assess the essentiality of Asp-473 in catalysis, we used directed mutagenesis and chloroplast transformation of the green alga Chlamydomonas reinhardtii to create D473A and D473E mutant enzymes. The D473A and D473E mutant strains can grow photoautotrophically, indicating that Asp-473 is not essential for catalysis. However, both substitutions caused 87% decreases in carboxylation catalytic efficiency (V(max)/K(m)) and approximately 16% decreases in CO(2)/O(2) specificity. If the carboxyl terminus is required for stabilizing loop 6 in the closed conformation, there must be additional residues at the carboxyl terminus/loop 6 interface that contribute to this mechanism. Considering that substitutions at residue 473 can influence CO(2)/O(2) specificity, further study of interactions between loop 6 and the carboxyl terminus may provide clues for engineering an improved Rubisco. 相似文献
7.
Okano Y Mizohata E Xie Y Matsumura H Sugawara H Inoue T Yokota A Kai Y 《FEBS letters》2002,527(1-3):33-36
Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) catalyzes the reactions of carboxylation and oxygenation of ribulose-1,5-bisphosphate. These reactions require that the active site should be closed by a flexible loop (loop 6) of the large subunit. Rubisco from a red alga, Galdieria partita, has the highest specificity for carboxylation reaction among the Rubiscos hitherto reported. The crystal structure of unactivated Galdieria Rubisco has been determined at 2.6 A resolution. The electron density map reveals that a sulfate binds only to the P1 anion-binding site of the active site and the loop 6 is closed. Galdieria Rubisco has a unique hydrogen bond between the main chain oxygen of Val332 on the loop 6 and the epsilon-amino group of Gln386 of the same large subunit. This interaction is likely to be crucial to understanding for stabilizing the loop 6 in the closed state and to making a higher affinity for anionic ligands. 相似文献
8.
Weimin Ma Lanzhen Wei Zongjuan Long Liping Chen Quanxi Wang 《Acta Physiologiae Plantarum》2008,30(6):897-904
The goal of this study was to investigate the contribution of increased activity of individual non-regulated enzymes in the
Calvin cycle to improve photosynthetic yield. Two non-regulated enzymes, rice fructose-1,6-bisphosphate aldolase (FBA) and
spinach triosephosphate isomerase (TPI), were individually cloned and overexpressed in the cyanobacterium Anabaena sp. strain PCC 7120 cells. The enzyme activity and the photosynthetic yield, as reflected by the cell growth rate, photosynthetic
oxygen evolution and dry cellular weight, were measured and compared between the wild-type and transgenic cells harboring
either FBA or TPI. Though the activity of these two individual non-regulated enzymes was similarly increased in the corresponding
transgenic cells, the contributions of each enzyme on the amount of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco),
reflected by the levels of Rubisco large subunit, and the photosynthetic yield were different. Transgenic cells, carrying
FBA, showed an evident increase in Rubisco amount and photosynthetic yield, while there was no increase in cells harboring
TPI. This indicates that the contributions of non-regulated enzymes in the Calvin cycle on photosynthetic yield differed and
firstly reveals that increased activity of only a single non-regulated enzyme in transgenic cells markedly improves the photosynthetic
yield via stimulating the amount of Rubisco and consequently accelerating the ribulose-1,5-bisphosphate (RuBP) regeneration
rate. 相似文献
9.
Hepatic storage of oligosaccharides and glycolipids in a cat affected with GM1 gangliosidosis.
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1. Glycopeptides and glycolipids were isolated from normal cat liver and liver from a cat affected with GM1 gangliosidosis. 2. Bio-Gel P-6 chromatography of the crude glycopeptide fractions demonstrated three major peaks of hexose-containing compounds that were greatly increased in the mutant liver sample; these peaks contained oligosaccharides that comprised over 2% of the liver wet weight. 3. Two of the major pathological oligosaccharides, GP5 and GP6, were purified by chromatography on charcoal/Celite and Sephadex G-25. Oligosaccharides GP5 and GP6 had apparent mol.wts. of 1800 +/- 200 and 1350+/-200 respectively, and contained galactose, mannose and N-acetylglucosamine in molar proportions of 2.0:3.1:4.1 (GP5) and 1.0:2.2:2.7 (GP6). Periodate oxidation studies demonstrated the presence of galactose in a non-reducing terminal position. 4. The neutral glycolipid fraction from the mutant cat liver has a 1.3-fold increase in hexose content accompanied by an increased concentration of asialo-(ganglioside GM1). 5. There was a 2-fold increase of gangliosides in the mutant cat liver compared with normal cats. Ganglioside GM1 and a compound tentatively identified as N-glycolloyl-(ganglioside GM1) were the major glycolipids accumulated. 相似文献
10.
Karkehabadi S Peddi SR Anwaruzzaman M Taylor TC Cederlund A Genkov T Andersson I Spreitzer RJ 《Biochemistry》2005,44(29):9851-9861
Comparison of subunit sequences and X-ray crystal structures of ribulose-1,5-bisphosphate carboxylase/oxygenase indicates that the loop between beta-strands A and B of the small subunit is one of the most variable regions of the holoenzyme. In prokaryotes and nongreen algae, the loop contains 10 residues. In land plants and green algae, the loop is comprised of approximately 22 and 28 residues, respectively. Previous studies indicated that the longer betaA-betaB loop was required for the assembly of cyanobacterial small subunits with plant large subunits in isolated chloroplasts. In the present study, chimeric small subunits were constructed by replacing the loop of the green alga Chlamydomonas reinhardtii with the sequences of Synechococcus or spinach. When these engineered genes were transformed into a Chlamydomonas mutant that lacks small-subunit genes, photosynthesis-competent colonies were recovered, indicating that loop size is not essential for holoenzyme assembly. Whereas the Synechococcus loop causes decreases in carboxylation V(max), K(m)(O(2)), and CO(2)/O(2) specificity, the spinach loop causes complementary decreases in carboxylation V(max), K(m)(O(2)), and K(m)(CO(2)) without a change in specificity. X-ray crystal structures of the engineered proteins reveal remarkable similarity between the introduced betaA-betaB loops and the respective loops in the Synechococcus and spinach enzymes. The side chains of several large-subunit residues are altered in regions previously shown by directed mutagenesis to influence CO(2)/O(2) specificity. Differences in the catalytic properties of divergent Rubisco enzymes may arise from differences in the small-subunit betaA-betaB loop. This loop may be a worthwhile target for genetic engineering aimed at improving photosynthetic CO(2) fixation. 相似文献
11.
Rubisco activase is an AAA(+) protein, a superfamily with members that use a "Sensor 2" domain for substrate recognition. To determine whether the analogous domain of activase is involved in recognition of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco, EC 4.1.1.39), two chimeric activases were constructed, interchanging a Sensor 2-containing region between activases from spinach and tobacco. Spinach chimeric activase was a poor activator of both spinach and tobacco Rubisco. In contrast, tobacco chimeric activase activated spinach Rubisco far better than tobacco Rubisco, similar to spinach activase. A point mutation, K311D, in the Sensor 2 domain of the tobacco chimeric activase abolished its ability to better activate spinach Rubisco. The opposite mutation, D311K, in wild type tobacco activase produced an enzyme that activated both spinach and tobacco Rubisco, whereas a second mutation, D311K/L314V, shifted the activation preference toward spinach Rubisco. The involvement of these two residues in substrate selectivity was confirmed by introducing the analogous single and double mutations in cotton activase. The ability of the two tobacco activase mutants to activate wild type and mutant Chlamydomonas Rubiscos was also examined. Tobacco D311K activase readily activated wild type and P89R but not D94K Rubisco, whereas the tobacco L314V activase only activated D94K Rubisco. The tobacco activase double mutant D311K/L314V activated wild type Chlamydomonas Rubisco better than either the P89R or D94K Rubisco mutants, mimicking activation by spinach activase. The results identified a substrate recognition region in activase in which two residues may directly interact with two residues in Rubisco. 相似文献
12.
Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco)is methylated at the alpha-amino group of the N-terminal methionine of the processed form of the small subunit (SS), and at the epsilon-amino group of lysine-14 of the large subunit (LS) in some species. The Rubisco LS methyltransferase (LSMT) gene has been cloned and expressed from pea and specifically methylates lysine-14 of the LS of Rubisco. We determine here that both pea and tobacco Rubisco LSMT also exhibit (alpha)N-methyltransferase activity toward the SS of Rubisco, suggesting that a single gene product can produce a bifunctional protein methyltransferase capable of catalyzing both (alpha)N-methylation of the SS and (epsilon)N-methylation of the LS. A homologue of the Rubisco LSMT gene (rbcMT-S) has also been identified in spinach that is closely related to Rubisco LSMT sequences from pea and tobacco. Two mRNAs are produced from rbcMT-S, and both long and short forms of the spinach cDNAs were expressed in Escherichia coli cells and shown to catalyze methylation of the alpha-amino group of the N-terminal methionine of the SS of Rubisco. Thus, the absence of lysine-14 methylation in species like spinach is apparently a consequence of a monofunctional protein methyltransferase incapable of methylating Lys-14, with activity limited to methylation of the SS. 相似文献
13.
Orthophosphate (Pi) stimulates the activation of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) while paradoxically inhibiting its catalysis. Of three Pi-binding sites, the roles of the 5P- and latch sites have been documented, whereas that of the 1P-site remained unclear. Conserved residues at the 1P-site of Rubisco from the cyanobacterium Synechocystis PCC6803 were substituted and the kinetic properties of the enzyme derivatives and effects on cell photosynthesis and growth were examined. While Pi-stimulated Rubisco activation diminished for enzyme mutants T65A/S and G404A, inhibition of catalysis by Pi remained unchanged. Together with previous studies, the results suggest that all three Pi-binding sites are involved in stimulation of Rubisco activation, whereas only the 5P-site is involved in inhibition of catalysis. While all the mutations reduced the catalytic turnover of Rubisco (K(cat)) between 6- and 20-fold, the photosynthesis and growth rates under saturating irradiance and inorganic carbon (Ci) concentrations were only reduced 40-50% (in the T65A/S mutants) or not at all (G404A mutant). Analysis of the mutant cells revealed a 3-fold increase in Rubisco content that partially compensated for the reduced K(cat) so that the carboxylation rate per chlorophyll was one-third of that in the wild type. Correlation between the kinetic properties of Rubisco and the photosynthetic rate (P(max)) under saturating irradiance and Ci concentrations indicate that a >60% reduction in K(cat) can be tolerated before P(max) in Synechocystsis PCC6803 is affected. These results indicate that the limitation of Rubisco activity on the rate of photosynthesis in Synechocystis is low. Determination of Calvin cycle metabolites revealed that unlike in higher plants, cyanobacterial photosynthesis is constrained by phosphoglycerate reduction probably due to limitation of ATP or NADPH. 相似文献
14.
Changes in Activities of Enzymes of Carbon Metabolism in Leaves during Exposure of Plants to Low Temperature 总被引:30,自引:12,他引:18
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The aim of this study was to determine the response of photosynthetic carbon metabolism in spinach and bean to low temperature. (a) Exposure of warm-grown spinach and bean plants to 10°C for 10 days resulted in increases in the total activities of a number of enzymes, including ribulose 1,5-bisphosphate carboxylase (Rubisco), stromal fructose 1,6 bisphosphatase (Fru 1,6-P2ase), sedoheptulose 1,7-bisphosphatase (Sed 1,7-P2ase), and the cytosolic Fru 1,6-P2ase. In spinach, but not bean, there was an increase in the total activity of sucrose-phosphate synthase. (b) The CO2-saturated rates of photosynthesis for the cold-acclimated spinach plants were 68% greater at 10°C than those for warm-acclimated plants, whereas in bean, rates of photosynthesis at 10°C were very low after exposure to low temperature. (c) When spinach leaf discs were transferred from 27 to 10°C, the stromal Fru 1,6-P2ase and NADP-malate dehydrogenase were almost fully activated within 8 minutes, and Rubisco reached 90% of full activation within 15 minutes of transfer. An initial restriction of Calvin cycle fluxes was evident as an increase in the amounts of ribulose 1,5-bisphosphate, glycerate-3-phosphate, Fru 1,6-P2, and Sed 1,7-P2. In bean, activation of stromal Fru 1,6-P2ase was weak, whereas the activation state of Rubisco decreased during the first few minutes after transfer to low temperature. However, NADP-malate dehydrogenase became almost fully activated, showing that no loss of the capacity for reductive activation occurred. (d) Temperature compensation in spinach evidently involves increases in the capacities of a range of enzymes, achieved in the short term by an increase in activation state, whereas long-term acclimation is achieved by an increase in the maximum activities of enzymes. The inability of bean to activate fully certain Calvin cycle enzymes and sucrose-phosphate synthase, or to increase nonphotochemical quenching of chlorophyll fluorescence at 10°C, may be factors contributing to its poor performance at low temperature. 相似文献
15.
Olufsen M Smalås AO Moe E Brandsdal BO 《The Journal of biological chemistry》2005,280(18):18042-18048
Uracil DNA glycosylase (UDG) is a DNA repair enzyme in the base excision repair pathway and removes uracil from the DNA strand. Atlantic cod UDG (cUDG), which is a cold-adapted enzyme, has been found to be up to 10 times more catalytically active in the temperature range 15-37 degrees C as compared with the warm-active human counterpart. The increased catalytic activity of cold-adapted enzymes as compared with their mesophilic homologues are partly believed to be caused by an increase in the structural flexibility. However, no direct experimental evidence supports the proposal of increased flexibility of cold-adapted enzymes. We have used molecular dynamics simulations to gain insight into the structural flexibility of UDG. The results from these simulations show that an important loop involved in DNA recognition (the Leu(272) loop) is the most flexible part of the cUDG structure and that the human counterpart has much lower flexibility in the Leu(272) loop. The flexibility in this loop correlates well with the experimental k(cat)/K(m) values. Thus, the data presented here add strong support to the idea that flexibility plays a central role in adaptation to cold environments. 相似文献
16.
A temperature-conditional, photosynthesis-deficient mutant of the green alga Chlamydomonas reinhardtii, previously recovered by genetic screening, results from a leucine 290 to phenylalanine (L290F) substitution in the chloroplast-encoded large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco, EC ). Rubisco purified from mutant cells grown at 25 degrees C has a reduction in CO(2)/O(2) specificity and is inactivated at lower temperatures than those that inactivate the wild-type enzyme. Second-site alanine 222 to threonine (A222T) or valine 262 to leucine (V262L) substitutions were previously isolated via genetic selection for photosynthetic ability at the 35 degrees C restrictive temperature. These intragenic suppressors improve the CO(2)/O(2) specificity and thermal stability of L290F Rubisco in vivo and in vitro. In the present study, directed mutagenesis and chloroplast transformation were used to create the A222T and V262L substitutions in an otherwise wild-type enzyme. Although neither substitution improves the CO(2)/O(2) specificity above the wild-type value, both improve the thermal stability of wild-type Rubisco in vitro. Based on the x-ray crystal structure of spinach Rubisco, large subunit residues 222, 262, and 290 are far from the active site. They surround a loop of residues in the nuclear-encoded small subunit. Interactions at this subunit interface may substantially contribute to the thermal stability of the Rubisco holoenzyme. 相似文献
17.
Effect of Thiosulfate on the Photosynthetic Growth of Rhodopseudomonas palustris 总被引:4,自引:3,他引:1
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Cell yields of Rhodopseudomonas palustris grown photoheterotrophically in pyruvate-mineral salts medium were increased by the photooxidation of added thiosulfate. However, thiosulfate had no effect on cell yields of cultures grown aerobically in darkness, although thiosulfate was also oxidized. The presence of thiosulfate increased photosynthetic cell yields on a variety of other organic substrates. Growth of cells in thiosulfate-containing medium, or the addition of thiosulfate to cells grown in thiosulfate-free medium, induced the formation of a thiosulfate-oxidizing system which quantitatively photooxidized thiosulfate to sulfate. R. palustris grew photoautotrophically with thiosulfate as an oxidizable substrate. Large amounts of supplemental bicarbonate carbon were incorporated when cells were grown photosynthetically in pyruvate-thiosulfate medium. Cells harvested after photoautotrophic or photoheterotrophic growth in fumarate-thiosulfate medium fixed (14)CO(2) at an 8- to 10-fold greater rate when provided with thiosulfate. The evolution of (14)CO(2) from pyruvate-1-(14)C during photoassimilation by R. palustris was greatly suppressed by the presence of thiosulfate. The increase in photoheterotrophic cell yields of R. palustris caused by the oxidation of thiosulfate may result from assimilation of substrate carbon which is normally evolved as carbon dioxide. 相似文献
18.
Miyazaki K Wintrode PL Grayling RA Rubingh DN Arnold FH 《Journal of molecular biology》2000,297(4):1015-1026
We have used laboratory evolution methods to enhance the thermostability and activity of the psychrophilic protease subtilisin S41, with the goal of investigating the mechanisms by which this enzyme can adapt to different selection pressures. A combined strategy of random mutagenesis, saturation mutagenesis and in vitro recombination (DNA shuffling) was used to generate mutant libraries, which were screened to identify enzymes that acquired greater thermostability without sacrificing low-temperature activity. The half-life of seven-amino acid substitution variant 3-2G7 at 60 degrees C is approximately 500 times that of wild-type and far surpasses those of homologous mesophilic subtilisins. The dependence of half-life on calcium concentration indicates that enhanced calcium binding is largely responsible for the increased stability. The temperature optimum of the activity of 3-2G7 is shifted upward by approximately 10 degrees C. Unlike natural thermophilic enzymes, however, the activity of 3-2G7 at low temperatures was not compromised. The catalytic efficiency, k(cat)/K(M), was enhanced approximately threefold over a wide temperature range (10 to 60 degrees C). The activation energy for catalysis, determined by the temperature dependence of k(cat)/K(M) in the range 15 to 35 degrees C, is nearly identical to wild-type and close to half that of its highly similar mesophilic homolog, subtilisin SSII, indicating that the evolved S41 enzyme retained its psychrophilic character in spite of its dramatically increased thermostability. These results demonstrate that it is possible to increase activity at low temperatures and stability at high temperatures simultaneously. The fact that enzymes displaying both properties are not found in nature most likely reflects the effects of evolution, rather than any intrinsic physical-chemical limitations on proteins. 相似文献
19.
Cold-active enzymes have received little research attention although they are very useful in industries. Since the structure bases of cold adaptation of enzymes are still unclear, it is also very difficult to obtain cold-adapted enzymes for industrial applications using routine protein engineering methods. In this work, we employed directed evolution method to randomly mutate a mesophilic cellulase, endoglucanase Ⅲ (EG Ⅲ) from Trichoderma reesei, and obtained a cold-adapted mutant, designated as w-3. DNA sequence analysis indicates that w-3 is a truncated form of native EG Ⅲ with a deletion of 25 consecutive amino acids at C-terminus. Further examination of enzymatic kinetics and thermal stability shows that mutant w-3 has a higher Kcat value and becomes more thermolabile than its parent. In addition, activation energies of w-3 and wild type EG Ⅲ calculated from Arrhenius equation are 13.3 kJ· mol-1 and 26.2 kJ ·mol-1, respectively. Therefore, the increased specific activity of w-3 at lower tempera 相似文献
20.
Probing the role of a mobile loop in substrate binding and enzyme activity of human salivary amylase
Mammalian amylases harbor a flexible, glycine-rich loop 304GHGAGGA(310), which becomes ordered upon oligosaccharide binding and moves in toward the substrate. In order to probe the role of this loop in catalysis, a deletion mutant lacking residues 306-310 (Delta306) was generated. Kinetic studies showed that Delta306 exhibited: (1) a reduction (>200-fold) in the specific activity using starch as a substrate; (2) a reduction in k(cat) for maltopentaose and maltoheptaose as substrates; and (3) a twofold increase in K(m) (maltopentaose as substrate) compared to the wild-type (rHSAmy). More cleavage sites were observed for the mutant than for rHSAmy, suggesting that the mutant exhibits additional productive binding modes. Further insight into its role is obtained from the crystal structures of the two enzymes soaked with acarbose, a transition-state analog. Both enzymes modify acarbose upon binding through hydrolysis, condensation or transglycosylation reactions. Electron density corresponding to six and seven fully occupied subsites in the active site of rHSAmy and Delta306, respectively, were observed. Comparison of the crystal structures showed that: (1) the hydrophobic cover provided by the mobile loop for the subsites at the reducing end of the rHSAmy complex is notably absent in the mutant; (2) minimal changes in the protein-ligand interactions around subsites S1 and S1', where the cleavage would occur; (3) a well-positioned water molecule in the mutant provides a hydrogen bond interaction similar to that provided by the His305 in rHSAmy complex; (4) the active site-bound oligosaccharides exhibit minimal conformational differences between the two enzymes. Collectively, while the kinetic data suggest that the mobile loop may be involved in assisting the catalysis during the transition state, crystallographic data suggest that the loop may play a role in the release of the product(s) from the active site. 相似文献