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1.
Lactose-limited fermentations of 49 dairy Streptococcus thermophilus strains revealed four distinct fermentation profiles with respect to galactose consumption after lactose depletion. All the strains excreted galactose into the medium during growth on lactose, except for strain IMDOST40, which also displayed extremely high galactokinase (GalK) activity. Among this strain collection eight galactose-positive phenotypes sensu stricto were found and their fermentation characteristics and Leloir enzyme activities were measured. As the gal promoter seems to play an important role in the galactose phenotype, the galR-galK intergenic region was sequenced for all strains yielding eight different nucleotide sequences (NS1 to NS8). The gal promoter played an important role in the Gal-positive phenotype but did not determine it exclusively. Although GalT and GalE activities were detected for all Gal-positive strains, GalK activity could only be detected for two out of eight Gal-positive strains. This finding suggests that the other six S. thermophilus strains metabolize galactose via an alternative route. For each type of fermentation profile obtained, a representative strain was chosen and four complete Leloir gene clusters were sequenced. It turned out that Gal-positive strains contained more amino acid differences within their gal genes than Gal-negative strains. Finally, the biodiversity regarding lactose-galactose utilization among the different S. thermophilus strains used in this study was shown by RAPD-PCR. Five Gal-positive strains that contain nucleotide sequence NS2 in their galR-galK intergenic region were closely related. 相似文献
2.
The galK gene, encoding galactokinase of the Leloir pathway, was insertionally inactivated in Streptococcus mutans UA159. The galK knockout strain displayed only marginal growth on galactose, but growth on glucose or lactose was not affected. In strain UA159, the sugar phosphotransferase system (PTS) for lactose and the PTS for galactose were induced by growth in lactose and galactose, although galactose PTS activity was very low, suggesting that S. mutans does not have a galactose-specific PTS and that the lactose PTS may transport galactose, albeit poorly. To determine if the galactose growth defect of the galK mutant could be overcome by enhancing lactose PTS activity, the gene encoding a putative repressor of the operon for lactose PTS and phospho-β-galactosidase, lacR, was insertionally inactivated. A galK and lacR mutant still could not grow on galactose, although the strain had constitutively elevated lactose PTS activity. The glucose PTS activity of lacR mutants grown in glucose was lower than in the wild-type strain, revealing an influence of LacR or the lactose PTS on the regulation of the glucose PTS. Mutation of the lacA gene of the tagatose pathway caused impaired growth in lactose and galactose, suggesting that galactose can only be efficiently utilized when both the Leloir and tagatose pathways are functional. A mutation of the permease in the multiple sugar metabolism operon did not affect growth on galactose. Thus, the galactose permease of S. mutans is not present in the gal, lac, or msm operons. 相似文献
3.
Galactokinase (GALK), a member the Leloir pathway for normal galactose metabolism, catalyzes the conversion of α-d-galactose to galactose-1-phosphate. For this investigation, we studied the kinetic mechanism and pH profiles of the enzyme from Lactococcus lactis. Our results show that the mechanism for its reaction is sequential in both directions. Mutant proteins D183A and D183N are inactive (<10 000 fold), supporting the role of Asp183 as a catalytic base that deprotonates the C-1 hydroxyl group of galactose. The pH-kcat profile of the forward reaction has a pKa of 6.9 ± 0.2 that likely is due to Asp183. The pH-kcat/KGal profile of the reverse reaction further substantiates this role as it is lacking a key pKa required for a direct proton transfer mechanism. The R36A and R36N mutant proteins show over 100-fold lower activity than that for the wild-type enzyme, thus suggesting that Arg36 lowers the pKa of the C-1 hydroxyl to facilitate deprotonation. 相似文献
4.
Rosa Garcia Sanchez Bärbel Hahn-Hägerdal Marie F Gorwa-Grauslund 《Microbial cell factories》2010,9(1):40
Background
In Saccharomyces cerevisiae galactose is initially metabolized through the Leloir pathway after which glucose 6-phosphate enters glycolysis. Galactose is controlled both by glucose repression and by galactose induction. The gene PGM2 encodes the last enzyme of the Leloir pathway, phosphoglucomutase 2 (Pgm2p), which catalyses the reversible conversion of glucose 1-phosphate to glucose 6-phosphate. Overexpression of PGM2 has previously been shown to enhance aerobic growth of S. cerevisiae in galactose medium. 相似文献5.
Hiroshi Ashihara Atsushi Komamine Masami Shimokoriyama 《Journal of plant research》1974,87(2):121-131
Changes in activities of the glycolytic and pentose phosphate (PP) pathways in glucose catabolism in various parts of the
hypocotyls obtained from 4-day-old etiolatedPhaseolus mungo seedlings were investigated by measuring the inhibition rates of respiration by iodoacetate and malonate, and the release
of14CO2 from [1-14C]- and [6-14C]glucose. The relative activity of the PP pathway in glucose catabolism was higher in the immature part (Part I) and the
aged part (Part V) of the hypocotyls than in the intermediary one (Part III), while the activity of the glycolytic pathway
decreased with aging.
On a fresh weight basis, the enzyme activities of the glycolytic and PP pathways were higher in Part I than in Parts III and
V. On a protein content basis, however, activities of the enzymes of the PP pathway increased with aging and differentiation
of the hypocotyls whereas those of the glycolytic pathway decreased. Levels of nicotinamide adenine nucleotides were found
to be in the following order: Part I>Part III> Part V for NAD++NADH; Part I>Part V>Part III for NADP++NADPH. The stimulative effect of methylene blue on decreasing the C6/C1 ratio was greater in Part III than in Part I, and No effect was observed in Part V.
These data suggest that a decrease in the activity of the glycolytic pathway with aging and differentiation may be due to
the decreasing glycolytic enzyme activities and NAD(H) content. The higher activity of the PP pathway in the immature part
is attributable to larger amounts of NADP(H) and enzymes of the PP pathway. The greater contribution of the PP pathway to
glucose catabolism in the aged part than in the intermediary part seems to results from a more active turnover of NADP and
the relatively higher activity of the enzymes of the PP pathway than those of the glycolytic pathway. 相似文献
6.
Frederik De Bruyn Joeri Beauprez Jo Maertens Wim Soetaert Marjan De Mey 《Applied and environmental microbiology》2013,79(22):7028-7035
The GNB/LNB (galacto-N-biose/lacto-N-biose) pathway plays a crucial role in bifidobacteria during growth on human milk or mucin from epithelial cells. It is thought to be the major route for galactose utilization in Bifidobacterium longum as it is an energy-saving variant of the Leloir pathway. Both pathways are present in B. bifidum, and galactose 1-phosphate (gal1P) is considered to play a key role. Due to its toxic nature, gal1P is further converted into its activated UDP-sugar through the action of poorly characterized uridylyltransferases. In this study, three uridylyltransferases (galT1, galT2, and ugpA) from Bifidobacterium bifidum were cloned in an Escherichia coli mutant and screened for activity on the key intermediate gal1P. GalT1 and GalT2 showed UDP-glucose-hexose-1-phosphate uridylyltransferase activity (EC 2.7.7.12), whereas UgpA showed promiscuous UTP-hexose-1-phosphate uridylyltransferase activity (EC 2.7.7.10). The activity of UgpA toward glucose 1-phosphate was about 33-fold higher than that toward gal1P. GalT1, as part of the bifidobacterial Leloir pathway, was about 357-fold more active than GalT2, the functional analog in the GNB/LNB pathway. These results suggest that GalT1 plays a more significant role than previously thought and predominates when B. bifidum grows on lactose and human milk oligosaccharides. GalT2 activity is required only during growth on substrates with a GNB core such as mucin glycans. 相似文献
7.
Debarati Basu Yan Liang Xiao Liu Klaus Himmeldirk Ahmed Faik Marcia Kieliszewski Michael Held Allan M. Showalter 《The Journal of biological chemistry》2013,288(14):10132-10143
Although plants contain substantial amounts of arabinogalactan proteins (AGPs), the enzymes responsible for AGP glycosylation are largely unknown. Bioinformatics indicated that AGP galactosyltransferases (GALTs) are members of the carbohydrate-active enzyme glycosyltransferase (GT) 31 family (CAZy GT31) involved in N- and O-glycosylation. Six Arabidopsis GT31 members were expressed in Pichia pastoris and tested for enzyme activity. The At4g21060 gene (named AtGALT2) was found to encode activity for adding galactose (Gal) to hydroxyproline (Hyp) in AGP protein backbones. AtGALT2 specifically catalyzed incorporation of [14C]Gal from UDP-[14C]Gal to Hyp of model substrate acceptors having AGP peptide sequences, consisting of non-contiguous Hyp residues, such as (Ala-Hyp) repetitive units exemplified by chemically synthesized (AO)7 and anhydrous hydrogen fluoride-deglycosylated d(AO)51. Microsomal preparations from Pichia cells expressing AtGALT2 incorporated [14C]Gal to (AO)7, and the resulting product co-eluted with (AO)7 by reverse-phase HPLC. Acid hydrolysis of the [14C]Gal-(AO)7 product released 14C-radiolabel as Gal only. Base hydrolysis of the [14C]Gal-(AO)7 product released a 14C-radiolabeled fragment that co-eluted with a Hyp-Gal standard after high performance anion-exchange chromatography fractionation. AtGALT2 is specific for AGPs because substrates lacking AGP peptide sequences did not act as acceptors. Moreover, AtGALT2 uses only UDP-Gal as the substrate donor and requires Mg2+ or Mn2+ for high activity. Additional support that AtGALT2 encodes an AGP GALT was provided by two allelic AtGALT2 knock-out mutants, which demonstrated lower GALT activities and reductions in β-Yariv-precipitated AGPs compared with wild type plants. Confocal microscopic analysis of fluorescently tagged AtGALT2 in tobacco epidermal cells indicated that AtGALT2 is probably localized in the endomembrane system consistent with its function. 相似文献
8.
Gibberellin metabolism in cell-free extracts from spinach leaves in relation to photoperiod 总被引:19,自引:12,他引:7
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Cell-free extracts capable of converting [14C]-labeled gibberellins (GAs) were prepared from spinach (Spinacia oleracea L.) leaves. [14C]-labeled GAs, prepared enzymically from [14C]mevalonic acid, were incubated with these extracts, and products were identified by gas chromatography-mass spectrometry. The following pathway was found to operate in extracts from spinach leaves grown under long day (LD) conditions: GA12 → GA53 → GA44 → GA19 → GA20. The pH optima for the enzymic conversions of [14C]GA53, [14C]GA44 and [14C]GA19 were approximately 7.0, 8.0, and 6.5, respectively. These three enzyme activities required Fe2+, α-ketoglutarate and O2 for activity, and ascorbate stimulated the conversion of [14C]GA53 and [14C]GA19. Extracts from plants given LD or short days (SD) were examined, and enzymic activities were measured as a function of exposure to LD, as well as to darkness following 8 LD. The results indicate that the activities of the enzymes oxidizing GA53 and GA19 are increased in LD and decreased in SD or darkness, but that the enzyme activity oxidizing GA44 remains high irrespective of light or dark treatment. This photoperiodic control of enzyme activity is not due to the presence of an inhibitor in plants grown in SD. These observations offer an explanation for the higher GA20 content of spinach plants in LD than in SD. 相似文献
9.
Metabolic Pathways Leading to Mercury Methylation in Desulfovibrio desulfuricans LS 总被引:2,自引:1,他引:1
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The synthesis of methylmercury by Desulfovibrio desulfuricans LS was investigated on the basis of 14C incorporation from precursors and the measurement of relevant enzyme activities in cell extracts. The previously observed incorporation of C-3 from serine into methylmercury was confirmed by measurement of relatively high activities of serine hydroxymethyltransferase and other enzymes of this pathway. High rates of label incorporation into methylmercury from H14COO- and H14CO3- prompted the assay of enzymes of the acetyl coenzyme A (CoA) synthase pathway. These enzymes were found to be present but at activity levels much lower than those reported for acetogens. Propyl iodide inhibited methylmercury and acetyl-CoA syntheses to similar extents, and methylmercury synthesis was found to compete with acetyl-CoA synthesis for methyl groups. On the basis of these findings, we propose that in methylmercury synthesis by D. desulfuricans LS the methyl group is transferred from CH3-tetrahydrofolate via methylcobalamin. The methyl group may originate from C-3 of serine or from formate via the acetyl-CoA synthase pathway. These pathways are not unique to D. desulfuricans LS, and thus the ability of this bacterium to methylate mercury is most likely associated with the substrate specificity of its enzymes. 相似文献
10.
CO2 production from galactose in galactose-1-phosphate uridyl transferase-deficient Escherichia coli.
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Escherichia coli K-12 deficient in galactose-1-phosphate uridyl transferase is capable of converting significant amounts of d-[1-(14)C]galactose to (14)CO(2), whereas strains deficient in other enzymes of the Leloir pathway cannot do so. 相似文献
11.
12.
Ana R. Neves Wietske A. Pool Ana Solopova Jan Kok Helena Santos Oscar P. Kuipers 《Applied and environmental microbiology》2010,76(21):7048-7060
Accumulation of galactose in dairy products due to partial lactose fermentation by lactic acid bacteria yields poor-quality products and precludes their consumption by individuals suffering from galactosemia. This study aimed at extending our knowledge of galactose metabolism in Lactococcus lactis, with the final goal of tailoring strains for enhanced galactose consumption. We used directed genetically engineered strains to examine galactose utilization in strain NZ9000 via the chromosomal Leloir pathway (gal genes) or the plasmid-encoded tagatose 6-phosphate (Tag6P) pathway (lac genes). Galactokinase (GalK), but not galactose permease (GalP), is essential for growth on galactose. This finding led to the discovery of an alternative route, comprising a galactose phosphotransferase system (PTS) and a phosphatase, for galactose dissimilation in NZ9000. Introduction of the Tag6P pathway in a galPMK mutant restored the ability to metabolize galactose but did not sustain growth on this sugar. The latter strain was used to prove that lacFE, encoding the lactose PTS, is necessary for galactose metabolism, thus implicating this transporter in galactose uptake. Both PTS transporters have a low affinity for galactose, while GalP displays a high affinity for the sugar. Furthermore, the GalP/Leloir route supported the highest galactose consumption rate. To further increase this rate, we overexpressed galPMKT, but this led to a substantial accumulation of α-galactose 1-phosphate and α-glucose 1-phosphate, pointing to a bottleneck at the level of α-phosphoglucomutase. Overexpression of a gene encoding α-phosphoglucomutase alone or in combination with gal genes yielded strains with galactose consumption rates enhanced up to 50% relative to that of NZ9000. Approaches to further improve galactose metabolism are discussed.Lactococcus lactis is a lactic acid bacterium widely used in the dairy industry for the production of fermented milk products. Because of its economic importance, L. lactis has been studied extensively in the last 40 years. A small genome, a large set of genetic tools, a wealth of physiological knowledge, and a relatively simple metabolic potential render L. lactis an attractive model with which to implement metabolic engineering strategies (reviewed in references 21 and 57).In the process of milk fermentation by L. lactis, lactose is taken up and concomitantly phosphorylated at the galactose moiety (C-6) by the lactose-specific phosphoenolpyruvate (PEP)-dependent phosphotransferase system (PTSLac), after which it is hydrolyzed to glucose and galactose 6-phosphate (Gal6P) (64). The glucose moiety enters the glycolytic pathway upon phosphorylation via glucokinase to glucose 6-phosphate (G6P), whereas Gal6P is metabolized to triose phosphates via the d-tagatose 6-phosphate (Tag6P) pathway, encompassing the steps catalyzed by galactose 6-phosphate isomerase (LacAB), Tag6P kinase (LacC), and tagatose 1,6-bisphosphate aldolase (LacD) (Fig. (Fig.1).1). Curiously, during the metabolism of lactose by L. lactis, part of the Gal6P is dephosphorylated and excreted into the growth medium, while the glucose moiety is readily used (2, 7, 51, 56, 60).Open in a separate windowFIG. 1.Schematic overview of the alternative routes for galactose uptake and further catabolism in L. lactis. Galactose can be imported by the non-PTS permease GalP and metabolized via the Leloir pathway (galMKTE) to α-G1P, which is converted to the glycolytic intermediate G6P by α-phosphoglucomutase (pgmH). Alternatively, galactose can be imported by PTSLac (lacFE) and further metabolized to triose phosphates by the Tag6P pathway (lacABCD). Here, we propose a new uptake route consisting of galactose translocation via the galactose PTS, followed by dephosphorylation of the internalized Gal6P to galactose, which is further metabolized via the Leloir pathway (highlighted in the gray box). galP, galactose permease; galM, galactose mutarotase; galK, galactokinase; galT, galactose 1-phosphate uridylyltransferase; galE, UDP-galactose-4-epimerase; pgmH, α-phosphoglucomutase; lacAB, galactose 6-phosphate isomerase; lacC, Tag6P kinase; lacD, tagatose 1,6-bisphosphate aldolase; lacFE, PTSLac; PTSGal, unidentified galactose PTS; Phosphatase; unidentified Gal6P-phosphatase; pgi, phosphoglucose isomerase; pfk, 6-phosphofructo-1-kinase; fba, fructose 1,6-bisphosphate aldolase; tpi, triose phosphate isomerase; α-Gal1P, α-galactose 1-phosphate; α-G1P, α-glucose 1-phosphate; UDP-gal, UDP-galactose; UDP-glc, UDP-glucose; G6P, glucose 6-phosphate; Gal6P, galactose 6-phosphate; Tag6P, tagatose 6-phosphate; TBP, tagatose 1,6-bisphosphate; FBP, fructose 1,6-bisphosphate; DHAP, dihydroxyacetone phosphate; GAP, glyceraldehyde 3-phosphate. The dotted arrow represents the conversions of GAP to pyruvate via the glycolytic pathway. Steps essential to improve galactose consumption are shown in black boxes.As a result of incomplete lactose utilization, some fermented dairy products contain significant residual amounts of galactose. The presence of galactose has been associated with shoddier qualities of the fermented product (6, 27, 43). In particular, galactose is a major contributor to the browning that occurs when dairy products (e.g., yogurt and mozzarella, Swiss, and cheddar cheese) are cooked or heated in the manufacture of pizzas, sauce preparation, or processed cheese. In addition, availability of residual galactose may result in production of CO2 by heterofermentative starters and, consequently, in textural defects such as the development of slits and fractures in cheeses. Therefore, the availability of starter strains with improved galactose utilization capacity is desirable to develop higher-quality dairy products. Additionally, strains with increased galactose metabolism could provide galactose-free foods for individuals and, in particular, children suffering from the rare disease galactosemia (36). To this end, a comprehensive understanding of galactose catabolism is essential.Galactose metabolism in L. lactis was thoroughly studied in the past and has been and still is the subject of some controversy. Indeed, conflicting results regarding the type of PTS involved in galactose uptake have been published. Some authors advocated that galactose is exclusively transported via the plasmid-encoded PTSLac, whereas others proposed transport via a galactose-specific PTS (PTSGal) to the extreme of questioning the contribution of the PTSLac (17, 20, 50, 59). However, a gene encoding PTSGal has never been identified in L. lactis. Independently of the nature of the PTS, it is generally accepted that the resulting Gal6P is metabolized via the Tag6P pathway (lac operon) (Fig. (Fig.1).1). On the other hand, galactose translocated via the highly specific galactose permease (GalP) is metabolized via the Leloir pathway to α-glucose 1-phosphate (α-G1P) through the sequential action of galactose mutarotase (GalM), galactokinase (GalK), and galactose 1-phosphate uridylyltransferase (GalT)/UDP-galactose-4-epimerase (GalE) (gal operon). Entry in glycolysis is preceded by the α-phosphoglucomutase (α-PGM)-catalyzed isomerization of α-G1P to G6P. The use of the Leloir and/or the Tag6P pathway for galactose utilization is currently viewed as being strain dependent (9, 16, 25, 32, 33, 58), but the relative efficacy in the degradation of the sugar has not been established.The ultimate aim of this study was to engineer L. lactis for improved galactose-fermenting capacity as a means to minimize the galactose content in dairy products. To gain insight into galactose catabolism via the Leloir (gal genes) and the Tag6P (lac genes) pathways, a series of L. lactis subsp. cremoris NZ9000 isogenic gal and lac mutants were constructed. Carbon 13 labeling experiments coupled with nuclear magnetic resonance (NMR) spectroscopy were used to investigate galactose metabolism in the gal and lac strains. The data obtained revealed a novel route for galactose dissimilation and provided clues to further enhance galactose utilization. 相似文献
13.
Ana F. Soares Rui A. Carvalho Francisco J. Veiga John G. Jones 《Metabolic engineering》2010,12(6):552-560
Hepatic glycogen is formed by direct and indirect pathways whose activities reflect altered nutrition or disease. Direct/indirect pathway measurements often involve test meals where ~10% of carbohydrate is galactose, but its effects on direct/indirect pathway estimates are unknown. Therefore, direct/indirect pathway contributions in 24-h fasted rats given 2 g/kg 100% glucose (GLU, n=6) or 90% glucose–10% galactose (GLU+GAL, n=6) were measured by [U-13C]glucose dilution and by position-5/position-2 glycogen enrichment (H5/H2) from 2H2O. For GLU+GAL, galactose glycogenesis was independently measured with [1-13C]galactose. Glycogenesis was equivalent in both groups but for GLU+GAL, 23±4% of glycogen was derived from galactose. [U-13C]glucose reported a 30±3% direct pathway contribution to glycogenesis for GLU but only 20±3% for GLU+GAL (p=0.012 vs. GLU). H5/H2 yielded identical direct pathway estimates (32±3% GLU, 29±6% GLU+GAL). Thus, galactose glycogenesis was undetected by H5/H2 while [U-13C]glucose reported a reduced direct/indirect pathway ratio. With [1-13C]galactose also present, correct glycogenic source contributions were obtained. 相似文献
14.
Two galactosyltransferase activities (1 and 2) were measured in the pancreas, liver and gut of the developing rat embryo. 1. N-Acetylglucosamine:Galactosyltransferase. UDP [14C]galactose + N-acetylglucosamine → [14C]galactosyl-β-(1 → 4)-N-acetylglucosamine + UDP. 2. N-Acetylgalactosamine-protein:Galactosyltransferase. UDP [14C]galactose + N-acetylgalactosamine-protein → [14C]galactosyl-β-(1 → 3)-N-acetylgalactosamine-protein + UDP. Galactosyltransferases 1 and 2 increased in the pancreas, about 10- and 40-fold in specific activity, respectively, from 11 to 12 days in utero to birth. During this period the activities of both transferases in the liver were somewhat variable, but showed no definite trend. A drop in the level of galactosyltransferase 1 in the pancreas occurred at birth or shortly thereafter. The “Golgimarker” enzyme for liver, galactosyltransferase 1, may be absent or present at low levels in adult rat pancreas.Zymogen granule membrane preparations apparently are devoid of these galactosyltransferase activities. Bromodeoxyuridine, which inhibits the development of the synthetic capability of the specific exocrine proteins, had essentially no effect on the normal accretion of the galactosyltransferase activities in organ cultures of pancreatic rudiments from 13-day rat embryos. 相似文献
15.
Lactobacillus casei 64H Contains a Phosphoenolpyruvate-Dependent Phosphotransferase System for Uptake of Galactose, as Confirmed by Analysis of ptsH and Different gal Mutants
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Katja Bettenbrock Ulrike Siebers Petra Ehrenreich Carl-Alfred Alpert 《Journal of bacteriology》1999,181(1):225-230
Galactose metabolism in Lactobacillus casei 64H was analyzed by genetic and biochemical methods. Mutants with defects in ptsH, galK, or the tagatose 6-phosphate pathway were isolated either by positive selection using 2-deoxyglucose or 2-deoxygalactose or by an enrichment procedure with streptozotocin. ptsH mutations abolish growth on lactose, cellobiose, N-acetylglucosamine, mannose, fructose, mannitol, glucitol, and ribitol, while growth on galactose continues at a reduced rate. Growth on galactose is also reduced, but not abolished, in galK mutants. A mutation in galK in combination with a mutation in the tagatose 6-phosphate pathway results in sensitivity to galactose and lactose, while a galK mutation in combination with a mutation in ptsH completely abolishes galactose metabolism. Transport assays, in vitro phosphorylation assays, and thin-layer chromatography of intermediates of galactose metabolism also indicate the functioning of a permease/Leloir pathway and a phosphoenolpyruvate-dependent phosphotransferase system (PTS)/tagatose 6-phosphate pathway. The galactose-PTS is induced by growth on either galactose or lactose, but the induction kinetics for the two substrates are different. 相似文献
16.
Presence of Acetyl Coenzyme A (CoA) Carboxylase and Propionyl-CoA Carboxylase in Autotrophic Crenarchaeota and Indication for Operation of a 3-Hydroxypropionate Cycle in Autotrophic Carbon Fixation 总被引:2,自引:1,他引:1
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Castor Menendez Zsuzsa Bauer Harald Huber Nasser Gadon Karl-Otto Stetter Georg Fuchs 《Journal of bacteriology》1999,181(4):1088-1098
The pathway of autotrophic CO2 fixation was studied in the phototrophic bacterium Chloroflexus aurantiacus and in the aerobic thermoacidophilic archaeon Metallosphaera sedula. In both organisms, none of the key enzymes of the reductive pentose phosphate cycle, the reductive citric acid cycle, and the reductive acetyl coenzyme A (acetyl-CoA) pathway were detectable. However, cells contained the biotin-dependent acetyl-CoA carboxylase and propionyl-CoA carboxylase as well as phosphoenolpyruvate carboxylase. The specific enzyme activities of the carboxylases were high enough to explain the autotrophic growth rate via the 3-hydroxypropionate cycle. Extracts catalyzed the CO2-, MgATP-, and NADPH-dependent conversion of acetyl-CoA to 3-hydroxypropionate via malonyl-CoA and the conversion of this intermediate to succinate via propionyl-CoA. The labelled intermediates were detected in vitro with either 14CO2 or [14C]acetyl-CoA as precursor. These reactions are part of the 3-hydroxypropionate cycle, the autotrophic pathway proposed for C. aurantiacus. The investigation was extended to the autotrophic archaea Sulfolobus metallicus and Acidianus infernus, which showed acetyl-CoA and propionyl-CoA carboxylase activities in extracts of autotrophically grown cells. Acetyl-CoA carboxylase activity is unexpected in archaea since they do not contain fatty acids in their membranes. These aerobic archaea, as well as C. aurantiacus, were screened for biotin-containing proteins by the avidin-peroxidase test. They contained large amounts of a small biotin-carrying protein, which is most likely part of the acetyl-CoA and propionyl-CoA carboxylases. Other archaea reported to use one of the other known autotrophic pathways lacked such small biotin-containing proteins. These findings suggest that the aerobic autotrophic archaea M. sedula, S. metallicus, and A. infernus use a yet-to-be-defined 3-hydroxypropionate cycle for their autotrophic growth. Acetyl-CoA carboxylase and propionyl-CoA carboxylase are proposed to be the main CO2 fixation enzymes, and phosphoenolpyruvate carboxylase may have an anaplerotic function. The results also provide further support for the occurrence of the 3-hydroxypropionate cycle in C. aurantiacus. 相似文献
17.
This study compares the activity of galactokinase, galactose-1-phosphate uridyltransferase, and UDPgalactose-4-epimerase, the important enzymes in the pathway of conversion of galactose to glucose in red cells and liver of five inbred strains of mice. In the red cells, galactokinase varied over a four-fold range of activity while the other enzymes varied about two-fold. The activity of each of the enzymes varied independently of the other so that red cells of each strain had a unique pattern for the three enzymes. The red cell activity pattern was not reflected in liver tissue which showed little interstrain variation for each of the three enzymes. The ratio of liver galactokinase to uridyltransferase and epimerase was very similar in all five strains. Oxidation in vivo of 14C galactose to 14CO2 was examined in the two strains of mice with the widest divergence of red cell galactokinase activity and no difference was found in this parameter measuring the physiological disposition of the sugar. The wide variation of the red cell enzyme activity appears to have little metabolic consequence for the animal, the oxidation of the sugar reflecting the relative constancy of the liver enzyme activity. 相似文献
18.
Elicitor-inducible 3-hydroxy-3-methylglutaryl coenzyme a reductase activity is required for sesquiterpene accumulation in tobacco cell suspension cultures 总被引:7,自引:2,他引:5
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Addition of cell wall fragments from Phytophthora species or cellulase from Trichoderma viride, but not pectolyase from Aspergillus japonicus, to tobacco (Nicotiana tabacum) cell suspension cultures induced the accumulation of the extracellular sesquiterpenoid capsidiol. Pulse-labeling experiments with [14C]acetate and [3H]mevalonate suggested that enzymatic steps preceding mevalonate were limiting capsidiol biosynthesis in the pectolyase-treated cell cultures. Treatment of the cell cultures with either Phytophthora cell wall fragments or cellulase induced 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) and sesquiterpene cyclase activities, enzymes of the sesquiterpene biosynthetic pathway, and phenylalanine ammonia lyase activity, an enzyme of the general phenylpropanoid pathway. Pectolyase treatment induced sesquiterpene cyclase and phenylalanine ammonia lyase activities, but not HMGR activity. These results corroborate the importance of inducible HMGR enzyme activity for sesquiterpene accumulation. 相似文献
19.
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Odile Francesca Restaino Ujjwal Bhaskar Priscilla Paul Lingyun Li Mario De Rosa Jonathan S. Dordick Robert J. Linhardt 《Applied microbiology and biotechnology》2013,97(9):3893-3900
A bioengineered heparin, as a replacement for animal-derived heparin, is under development that relies on the fermentative production of heparosan by Escherichia coli K5 and its subsequent chemoenzymatic modification using biosynthetic enzymes. A critical enzyme in this pathway is the mammalian 6-O-sulfotransferase (6-OST-1) which specifically sulfonates the glucosamine residue in a heparin precursor. This mammalian enzyme, previously cloned and expressed in E. coli, is required in kilogram amounts if an industrial process for bioengineered heparin is to be established. In this study, high cell density cultivation techniques were exploited to obtain recombinant 6-OST-1. Physiological studies were performed in shake flasks to establish optimized growth and production conditions. Induction strategies were tested in fed-batch experiments to improve yield and productivity. High cell density cultivation in 7-l culture, together with a coupled inducer strategy using isopropyl β-d-1-thiogalactopyranoside and galactose, afforded 482 mg?l?1 of enzyme with a biomass yield of 16.2 mg?gcdw ?1 and a productivity of 10.5 mg?l?1?h?1. 相似文献