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Microbial 3-hydroxypropionic acid (3-HP) production can potentially replace petroleum-based production methods for acrylic acid. Here, we constructed a yeast strain that expressed enzymes related to 3-HP biosynthesis within the mitochondria. This approach aimed to enhance the 3-HP production by utilizing the mitochondrial acetyl-CoA, an important intermediate for synthesizing 3-HP. The strain that expressed 3-HP-producing enzymes in the mitochondria (YPH-mtA3HP) showed improved production of 3-HP compared to that shown by the strain expressing 3-HP-producing enzymes in the cytosol (YPH-cyA3HP). Additionally, cMCR was overexpressed, which regulates a rate-limiting reaction in synthesizing 3-HP. In this study, we aimed to further enhance 3-HP production by expressing multiple copies of cMCR in the mitochondria using the δ-integration strategy to optimize the expression level of cMCR (YPH-mtA3HPx*). The results of flask-scale cultivation showed that 3-HP production by cMCR δ-integration was significantly higher, exhibiting a yield of 160 mg/L in YPH-mtA3HP6* strain and 257 mg/L in YPH-mtA3HP22* strain. Notably, YPH-mtA3HP22*, exhibited the highest 3-HP titer, which was 3.2-fold higher than that of YPH-cyA3HP. Our results demonstrated the potential of utilizing the mitochondrial compartment within S. cerevisiae for enhancing 3-HP production. • Enzyme localization within mitochondria enhanced 3-HP production. • The overexpression of cMCR within mitochondria enhanced 3-HP production. • YPH-mtA3HP22* exhibited 3.2-fold higher 3-HP titer than that of YPH-cyA3HP. [ABSTRACT FROM AUTHOR]

Lignocelluloytic enzymes are industrially applied as biocatalysts for the deconstruction of recalcitrant plant biomass. To study their biocatalytic and physiological function, the assessment of their binding behavior and spatial distribution on lignocellulosic material is a crucial prerequisite. In this study, selected hydrolases and oxidoreductases from the white rot fungus Phanerochaete chrysosporium were localized on model substrates as well as poplar wood by confocal laser scanning microscopy. Two different detection approaches were investigated: direct tagging of the enzymes and tagging specific antibodies generated against the enzymes. Site-directed mutagenesis was employed to introduce a single surface-exposed cysteine residue for the maleimide site-specific conjugation. Specific polyclonal antibodies were produced against the enzymes and were labeled using N-hydroxysuccinimide (NHS) ester as a cross-linker. Both methods allowed the visualization of cell wall-bound enzymes but showed slightly different fluorescent yields. Using native poplar thin sections, we identified the innermost secondary cell wall layer as the preferential attack point for cellulose-degrading enzymes. Alkali pretreatment resulted in a partial delignification and promoted substrate accessibility and enzyme binding. The methods presented in this study are suitable for the visualization of enzymes during catalytic biomass degradation and can be further exploited for interaction studies of lignocellulolytic enzymes in biorefineries. [ABSTRACT FROM AUTHOR]

Acetyl-CoA is a key metabolic intermediate with an important role in transcriptional regulation. The nuclear-cytosolic acetyl-CoA synthetase 2 (ACSS2) was found to sustain the growth of hypoxic tumor cells. It generates acetyl-CoA from acetate, but exactly which pathways it supports is not fully understood. Here, quantitative analysis of acetate metabolism reveals that ACSS2 fulfills distinct functions depending on its cellular location. Exogenous acetate uptake is controlled by expression of both ACSS2 and the mitochondrial ACSS1, and ACSS2 supports lipogenesis. The mitochondrial and lipogenic demand for two-carbon acetyl units considerably exceeds the uptake of exogenous acetate, leaving it to only sparingly contribute to histone acetylation. Surprisingly, oxygen and serum limitation increase nuclear localization of ACSS2. We find that nuclear ACSS2 recaptures acetate released from histone deacetylation for recycling by histone acetyltransferases. Our work provides evidence for limited equilibration between nuclear and cytosolic acetyl-CoA and demonstrates that ACSS2 retains acetate to maintain histone acetylation.

The objective of this study was to elucidate the roles of sugar in the formation of root systems. Several parts of theseminal root were investigated to determine their sucrose, glucose and fructose contents, and the activity and the in situ localization of the activities of two kinds of metabolic enzymes, invertase and sucrose synthase, whichhydrolyze sucrose. The sucrose, glucose and fructose concentrations in the 0-1 cm section from the root apex were three to five times those in the other sections. The invertase and sucrose synthase activities were also higher in the apical section. The in situ localization of invertase activity was detected in the cell elongation zone of the seminal root using histochemical method. The sucrose synthase activity was detected in the cell elongation zone of the seminal root and the root apices of lateral roots. These results suggested that sucrose is transported to the root elongation zone and the surrounding tissue of the lateral root primordia, and is cleaved into glucose, fructose, and UDP-glucose by invertase or sucrose synthase. This suggested that sucrose contributes to root formation by serving as the energy source, the carbon source for cell wall synthesis, and as a compatible solute for cell elongation.

Summary Acetyl-CoA is a key metabolic intermediate with an important role in transcriptional regulation. The nuclear-cytosolic acetyl-CoA synthetase 2 (ACSS2) was found to sustain the growth of hypoxic tumor cells. It generates acetyl-CoA from acetate, but exactly which pathways it supports is not fully understood. Here, quantitative analysis of acetate metabolism reveals that ACSS2 fulfills distinct functions depending on its cellular location. Exogenous acetate uptake is controlled by expression of both ACSS2 and the mitochondrial ACSS1, and ACSS2 supports lipogenesis. The mitochondrial and lipogenic demand for two-carbon acetyl units considerably exceeds the uptake of exogenous acetate, leaving it to only sparingly contribute to histone acetylation. Surprisingly, oxygen and serum limitation increase nuclear localization of ACSS2. We find that nuclear ACSS2 recaptures acetate released from histone deacetylation for recycling by histone acetyltransferases. Our work provides evidence for limited equilibration between nuclear and cytosolic acetyl-CoA and demonstrates that ACSS2 retains acetate to maintain histone acetylation. [ABSTRACT FROM AUTHOR]

Rice endosperm enzyme activity affects the qualities of cooked rice. In this study, we aimed to investigate the differential localization of various enzymes in the endosperm of milled rice. Using hydrolytic enzyme analysis, we confirmed the activities of amylase, protease, and lipase. We milled brown rice to yields of 30 - 90%, at intervals of 10%, and then extracted enzymes from the milled rice. Total protein content was reduced by about 43% with 60% removal of the outer layer, resulting from the decrease in milling yield from 90 - 30%. However, the watersoluble protein content was only approximately 0.3%) in both the inner and outer layers. Moreover, the activities of esterase (C4), acidic and alkaline phosphatase, leucine allyl amidase, naphthol-AS-BI-phosphodiester hydrolase, α-and β-galactosidases, N-acetyl-β-glucosaminidase, debranching enzyme, β-amylase, and a-glucosidasc were similar between the inner and outer layers. In contrast, the activities of esterase lipase (C8), valine allyl amidase, cystine allyl amidase, trypsin, β-glucosidase, α-mannosidase, α-amylase, and polygalacturonase were greater in the outer layer. Thus, these results showed that measurement of rice endosperm enzyme activity could be used to assess enzyme localization. [ABSTRACT FROM AUTHOR]

To better understand how enzyme localization affects enzyme activity we studied the cellular localization of the glycosyltransferase MurG, an enzyme necessary for cell wall synthesis at the spore during sporulation in the bacterium Bacillus subtilis . During sporulation MurG was gradually enriched to the membrane at the forespore and point mutations in a MurG helical domain disrupting its localization to the membrane caused severe sporulation defects, but did not affect localization nor caused detectable defects during exponential growth. We found that this localization is dependent on the phospholipid cardiolipin, as in strains where the cardiolipin-synthesizing genes were deleted, MurG levels were diminished at the forespore. Furthermore, in this cardiolipin-less strain, MurG localization during sporulation was rescued by external addition of purified cardiolipin. These results support localization as a critical factor in the regulation of proper enzyme function and catalysis. [ABSTRACT FROM AUTHOR]

This article reports a comparison of the ease of disruption of gram-positive (Lactobacillus acidophilus) and gram-negative (Pectobacterium carotovorum) bacteria using high-pressure homogenization (HPH). The location factor for both enzymes calculated from enzyme release kinetics together with localization studies identified them as cytoplasmic enzymes. The results showed that release of β-galactosidase by HPH ofL. acidophiluswas more difficult than the release ofL-asparaginase fromP. carotovorum. It took nine passes at 55.14 MPa for maximum release of β-galactosidase (0.949 IU/mL) as compared to six passes at 41.35 MPa forL-asparaginase (4.653 IU/mL); 1.7 IU of β-galactosidase was released as against 11.5 IU ofL-asparaginase per MJ of energy during high-pressure homogenization. [ABSTRACT FROM AUTHOR]

The effect of phosphate starvation on growth and acid phosphatases (APases) localization and activity in oat tissues was investigated. Oat cultivars ( Avena sativa L.-Arab, Polar, Szakal) were grown for 1-3 weeks in complete nutrient medium (+P) and without phosphate (−P). Pi concentration in plant tissues decreased strongly after culturing on −P medium. Pi deficit reduced shoot growth, stimulated root elongation and increased ratio of root/shoot in all oat cultivars. Pi deficit had a greater impact on growth of oat cv. Polar than other varieties. A decrease in the internal Pi status led to an increase of acid phosphatase activities in extracts from shoots and roots, and in root exudates. The highest activity of secreted APases was observed for oat cv. Arab, during the third week of growth under Pi-deficient conditions. The activity of extracellular APase was high in young, growing zones of roots of −P plants. Histochemical visualization indicated high activity of APases in the epidermis and vascular tissues of −P plants. Pi deficiency increased intracellular APase activity in shoot mainly in oat cv. Polar, whereas APase activity in roots was the highest in oat cv. Szakal. Protein extracts from roots and shoots were run on native discontinuous PAGE to determine which isoform(s) may be affected by Pi deficiency. Three major APase isoforms were detected in all oat plants; one was strongly induced by Pi deficit. The studied oat cultivars differed in terms of acclimation to deficiency of phosphate-used various pools of APases to acquire Pi from external or internal sources. [ABSTRACT FROM AUTHOR]

Hydrolytic enzymes involved in rotifer digestive processes were investigated directly at the sites of enzyme action using the ELF (Enzyme Labelled Fluorescence) technique. After enzymatic hydrolysis of an artificial ELF substrate, the fluorescent product ELF alcohol (ELFA) marked the sites of enzyme action. The time development of ELFA labelling was studied at different incubation times. Phosphatases, β- N-acetylhexosaminidases and lipases were examined in Brachionus angularis, B. calyciflorus, Keratella cochlearis and Lecane closterocerca from fed-batch cultures. We detected activities of all studied enzymes mostly in the stomach and intestine of rotifers. L. closterocerca was the only species showing enzyme activity at the mastax. Lipase activity was observed in the stomach and intestine of all species and in the mastax of L. closterocerca. Phosphatases were frequently located at the corona of B. calyciflorus. In other cases, both phosphatases and β- N-acetylhexosaminidases were rarely detected at the corona, and on the lorica and epidermis of some species. [ABSTRACT FROM AUTHOR]

Abstract: A novel human dual-specific protein phosphatase (DSP), designated DUSP27, is here described. The DUSP27 gene contains three exons, rather than the predicted 4–14 exons, and encodes a 220 amino acid protein. DUSP27 is structurally similar to other small DSPs, like VHR and DUSP13. The location of DUSP27 on chromosome 10q22, 50kb upstream of DUSP13, suggests that these two genes arose by gene duplication. DUSP27 is an active enzyme, and its kinetic parameters and were determined. DUSP27 is a cytosolic enzyme, expressed in skeletal muscle, liver and adipose tissue, suggesting its possible role in energy metabolism. [Copyright &y& Elsevier]

Sulfurospirillum multivorans is a dehalorespiring organism, which is able to utilize tetrachloroethene as terminal electron acceptor in an anaerobic respiratory chain. The localization of the tetrachloroethene reductive dehalogenase in dependence on different growth substrates was studied using the freeze-fracture replica immunogold labeling technique. When the cells were grown with pyruvate plus fumarate, a major part of the enzyme was either localized in the cytoplasm or membrane associated facing the cytoplasm. In cells grown on pyruvate or formate as electron donors and tetrachloroethene as electron acceptor, most of the enzyme was detected at the periplasmic side of the cytoplasmic membrane. These results were confirmed by immunoblots of the enzyme with and without the twin arginine leader peptide. Trichloroethene exhibited the same effect on the enzyme localization as tetrachloroethene. The data indicated that the localization of the enzyme was dependent on the electron acceptor utilized. [ABSTRACT FROM AUTHOR]

A proteomic approach was used to identify the digestive enzymes secreted by exocytosis and by microapocrine vesicles and enzyme midgut compartmentalization in Spodoptera frugiperda larvae. For this, proteomic analyses were performed in isolated midgut enterocyte microvillar membrane, in a fraction enriched in microapocrine vesicles (separated in soluble and membrane fractions), in the washings of the peritrophic membrane to isolate its loosely- and tightly-bound proteins, and in the peritrophic membrane contents. PM washings correspond to proteins extracted from the mucus layer surrounding PM. Serine endopeptidases (trypsins, chymotrypsins and serine endopeptidase homologs that have substitutions in the catalytic residues) and lipases are mainly secreted by exocytosis. Aminopeptidases are mainly microvillar enzymes and some are secreted membrane-bound to microapocrine vesicles, whereas carboxypeptidase isoforms follow different secretory routes. The results also showed that most polymer hydrolases (such as amylase and endopeptidases) are not retained in the ectoperitrophic fluid (found in PM washings but absent from PM contents). On the contrary, most enzymes involved in intermediate digestion (exemplified by carboxypeptidase and aminopeptidase) do not pass through the peritrophic membrane. Finally, the data revealed that the protein composition of PM includes peritrophins classified as peritrophic membrane proteins, PMP, and chitin deacetylase. [Display omitted] • Proteomic allowed identification of secretion routes and midgut location of proteins. • Enzymes attacking polymers are mainly secreted by exocytosis and pass through PM • Glycoside hydrolases are mainly secreted in microapocrine vesicles. • Intermediate and final digestion enzymes are retained in the ectoperitrophic fluid. • PM proteins are mainly peritrophins with 3 or more chitin binding domains. [ABSTRACT FROM AUTHOR]

Prevotella bryantii B14 is a strictly anaerobic, Gram-negative, polysaccharides-degrading ruminal bacterium. EDTA/sphaeroplasting and osmotic shock procedure were used in present work to localise endoxylanolytic, b -xylosidase and a -L-arabinofuranosidase activities of P. bryantii B14. Late exponential phase cells released most of the endoxylanolytic and CMC-ase activity by osmotic shock, showing the periplasmic location of both enzymatic activities, while b -xylosidase and a -L-arabinofuranosidase activities were recovered largely in membrane cell fraction. About 23 % of the total culture endoxylanase activity and about 30 % of the CMC-ase activity were found to be extracellular. No b -xylosidase and a -L-arabinofuranosidase activities were detected in culture supernatant of P. bryantii B14.

Mesophyll protoplasts isolated from peeled oat (Avena sativa L. cv Victory) leaves with 1% (w/v) Cellulysin in 20 mM KPO4, pH 5.5 and 0.6 M sorbitol retain about 6% of the polyamine oxidase (PAO, EC 1.4.3.4) activity of the whole peeled leaf. However, more than 99% of the oat leaf PAO activity is apoplastic and can be extracted by vacuum infiltration with 200 mM NaCl and this procedure extracts no activity for the cytoplasmic marker enzyme glucose-6-phosphate dehydrogenase (G6PD, EC 1.1.1.49). By these criteria we consider PAO in oat leaves to be totally apoplastic and PAO found in the isolated protoplast to be contamination. The degree of protoplast contamination by PAO depends on the pH and ionic strength of the isolating and washing medium. It can be eliminated by washing protoplasts in 0.6 M sorbitol with 100 mM KPO4, pH 6.5. Pellets of lysed protoplasts incubated with dialyzed apoplastic enzymes in 5 mM KPO4, pH 5.5 adsorb about 87% of the added PAO activity but only about 25% of the added peroxidase (EC 1.11.1.7) activity. The adsorbed activity can be solubilized from the pellet by extraction with 1 M NaCl. The results demonstrate that weakly ionically bound cell wall enzymes may contaminate protoplasts isolated and purified by conventional techniques. [ABSTRACT FROM AUTHOR]

Cellular localization of cytosolic glutamine synthetase (GS1; EC 6.3.1.2) in vascular bundles of leaf blades of rice ( Oryza sativa L.), at the stage at which leaf blades 6 (the lowest position) to 10 were fully expanded, was investigated immunocytologically with an affinity-purified anti-GS1 immunoglobulin G. Strong signals for GS1 protein were detected in companion cells of large vascular bundles when blades 6-8 were tested. Signals for GS1 were also observed in vascular-parenchyma cells of both large and small vascular bundles. The results further support our hypothesis that GS1 is important for the export of leaf nitrogen from senescing leaves. The signals in companion cells were less striking in the younger green leaves and were hardly detected in the non-green portion of the 11th blade. In the non-green blades, strong signals for GS1 protein were detected in sclerenchyma and xylemparenchyma cells. When total GS extracts prepared from the 6th,10th, and the non-green 11th blades were subjected to anion-exchange chromatography, the activity of GS1 was clearly separated from that of chloroplastic GS, indicating that GS1 proteins detected in the vascular tissues were able to synthesize glutamine. The function of GS1 detected in the developing leaves is discussed. [ABSTRACT FROM AUTHOR]

The cellular distribution of the 36000-M polypeptide of NADPH-protochlorophyllide oxidoreductase has been determined in ultrathin sections of barley leaves by the method of immunogold labelling. In leaves of etiolated seedlings a large portion of the immunoreactive protein was localized within the prolamellar body. However, approximately one third of the total immunoreactive protein was present outside the plastid in the area of the plasmalemma. During illumination of etiolated seedlings the two polypeptide populations were differentially affected by light. While the concentration of the plastid-localized immunoreactive protein rapidly decreased and was hardly detectable after 16 h of continuous white-light treatment, the concentration of the extraplastidic polypeptide did not decline significantly during this illumination period. A similar distribution pattern of the immunoreactive polypeptide was also found in maize and rye. The chlorophyll-deficient barley mutant xantha-l contained the immunoreactive 36000-M polypeptide, even though the prolamellar body was not detectable in etioplasts of this mutant. All of the immunoreactive polypeptide was localized outside the plastid in the area of the plasmalemma. Despite the apparent absence of the enzyme protein from the plastid, dark-grown mutant plants contained the same relative concentration of mRNA activity for the NADPH-protochlorophyllide oxidoreductase, which declined rapidly during illumination, as in wild-type plants. The antigenic properties and the apparent molecular weight of the plastid-localized NADPH-protochlorophyllide oxidoreductase and the 36000-M immunoreactive polypeptide outside the plastid were so similar as to indicate that the two proteins may be of common origin. [ABSTRACT FROM AUTHOR]

Glycolytic enzyme activities and enzyme activities of the oxidative pentose-phosphate pathway were measured in intact chloroplasts from Chlamydomonas reinhardii. By comparison with the total enzyme activities of intact protoplasts of the alga the data were used to locate these enzymes quantitatively in the algal chloroplast. It was found that the glycolytic chain in C. reinhardii is roughly split into a plastidic and an extraplastidic part. More than 90% of the first part of glycolysis (from fructose-6-phosphate to triose-phosphate is located in the plastid while more than 95% of the second part (from glycerate-3-phosphate to pyruvate) is outside. Around 70% of glucose-6-phosphate dehydrogenase and gluconate-6-phosphate dehydrogenase, two key enzymes of the oxidative pentose phosphate pathway, are located in the plastid. It is concluded that in C. reinhardii the major part of hexose breakdown to triose-phosphate occurs in the chloroplast and that a tight cooperation between the plastid and the cytoplasm is required for appreciable sugar breakdown to occur in the algal cell. [ABSTRACT FROM AUTHOR]

The localization of APS reductase and bisulfite reductase in Desulfovibrio gigas, D. vulgaris Hildenborough and D. thermophilus was studied by immunoelectron microscopy. Polyclonal antibodies were raised against the purified enzymes from each strain. Cells fixed with formaldehyde/glutaraldehyde were embedded and ultrathin sections were incubated with antibodies and subsequently labeled with protein A-gold. The bisulfite reductase in all three strains and APS reductase in d. gigas and D. vulgaris were found in the cytoplasm. The labeling of d. thermophilus with APS reductase antibodies resulted in a distribution of gold particles over the cytoplasmic membrane region. The localization of the two enzymes is discussed with respect to the mechanism and energetics of dissimilatory sulfate reduction. [ABSTRACT FROM AUTHOR]

A metal-salt precipitation method with p-nitrophenyl phosphate as substrate has been used to localize in the electron microscope acid phosphatase activity in isolated aleurone layers of barley ( Hordeum vulgare L.), treated for 16 h in the presence or absence of gibberellic acid (GA). The paper confirms results obtained earlier with an azo-dye precipitation method of enzyme localization. In addition the results show for the first time that in GA-treated tissue enzyme activity is associated with the endoplasmic reticulum (ER), there being reaction product deposited in the ER cisternae. It is suggested that this activity represents new enzyme synthesized on ER in response to GA and probably destined for secretion. [ABSTRACT FROM AUTHOR]

During growth of the primary leaves of Avena sativa L., the distribution of extractable L-phenylalanine ammonia-lyase (PAL, EC 4.3.1.5) and chalcone-flavanone isomerase (CFI, EC 5.5.1.6) activities in distinct leaf sections (top section, medium section and meristematic basal section) and in the epidermal and mesophyll tissues were investigated in relation to C-glycosylflavone accumulation. Characteristic changes have been observed in the levels of PAL and CFI activities within the three leaf sections, depending upon their stage of development. An increase in both enzyme activities accompanies a strong flavone accumulation in the section of the leaf that derives from the basal meristem. Highest specific PAL activity is localized in the meristem itself, which is poor in both flavones and CFI activity. Total flavone accumulation was found to be nearly the same in all three leaf tissues, lower and upper epidermis and mesophyll. Similarly, PAL activity is distributed about equally in these tissues in young leaves; in older ones, activity is relatively higher in the lower leaf epidermis. In contrast, CFI is found to be localized almost entirely in the mesophyll and not in the epiderms. Therefore the question arises whether CFI is involved at all in flavone metabolism and whether it may represent, as a marker enzyme, the localization of other specific C-enzymes of the flavonoid biosynthetic pathway in oat primary leaves. [ABSTRACT FROM AUTHOR]

Subpopulations of primary sensory neurons in mammalian dorsal root ganglion (DRG) exhibit carbonic anhydrase (CA) activity. To identify these subpopulations in DRG cells of mouse and chicken, the reliability of the cytochemical localization of the enzyme requires that several conditions be fulfilled: (1) Preservation of the enzyme activity in glutaraldehyde-containing fixative; (2) accessibility of the cytoenzymatic reaction throughout 20-μm thick Vibratome sections; (3) retention of the reaction product in situ during OsO post-fixation; (4) specificity of the cytoenzymatic reaction for CA activity as corroborated by the immunocytochemical detection with antibodies anti-CA II in mouse DRG; (5) strict correlation between the CA activity and the cytological characteristics in a given subclass of neurons. On the basis of these criteria, it is concluded that the CA activity may be used as a cell marker to identify cytologically defined neuronal subpopulations and their axons in mouse DRG. In chicken DRG, CA activity is not consistently expressed in a given subclass of ganglion cells and their axons. Hence, it is assumed that the expression of CA activity by DRG cells in chicken is modulated by functional or environmental conditions. [ABSTRACT FROM AUTHOR]

Isolated nuclei of Physarum contain endogenous RNA polymerase activity. We provide evidence for four different states of RNA polymerase B: 1. free enzyme (85%); 2. weakly bound enzyme (10%) and 3. tightly bound enzyme (0-4%), which can be solubilized from isolated nuclei with 0.5 M and 1.5 M NaCl respectively; 4. 'initiated' enzyme. The latter fraction (1-5% of the total RNA polymerase B) is not soluble in salt extractions, does not accept external templates, shows high salt optimum for transcription (0.4 M NaCl) and produces by elongation RNA molecules of mainly 10 S. Treatment of isolated nuclei from differentiating cultures with Triton X-100 increases the proportion of the 'initiated' enzyme at the expense of the tightly bound enzyme fraction. This indicates a potential transcription control mechanism which operates at the chromatin level and results in variable proportions of silent and transcribing RNA polymerase B molecules during differentiation of Physarum. [ABSTRACT FROM AUTHOR]