scholarly journals 083 Is HMGR the Key Regulator of α-Farnesene Biosynthesis of Apple?

HortScience ◽  
2000 ◽  
Vol 35 (3) ◽  
pp. 403A-403
Author(s):  
H.P.V. Rupasinghe ◽  
K.C. Almquist ◽  
G. Paliyath ◽  
D.P. Murr
Keyword(s):  
Mevalonate Pathway ◽  
Higher Plants ◽  
Competitive Inhibitor ◽  
The Novel ◽  
Rate Limiting Step ◽  
Ethylene Action ◽  
Hmgr Activity ◽  
Coa Reductase ◽  
Rate Limiting

We tested the hypothesis that conversion of 3-hydroxy-3-methylglutaryl co-enzyme A (HMG CoA) to mevalonate (MVA) catalyzed by HMG CoA reductase (HMGR) is the rate limiting step for α-farnesene biosynthesis of apples. In higher plants, isopentenyl pyrophosphate (IPP) is derived via two pathways: 1) the classical mevalonate pathway, and 2) the novel glyceraldehyde-3-phosphate (GAP)/pyruvate pathway independent of HMGR action. When apple skin discs were incubated with MVA, or GAP and pyruvate, MVA increased α-farnesene levels in the skin but not GAP and pyruvate. Treating apple fruits with Lovastatin (1000 ppm), a competitive inhibitor of HMGR, inhibited α-farnesene accumulation in the skin by 20% to 50% during storage. Content of α-farnesene in the skin increased during the first 2 to 4 months in storage, and then decreased. In contrast, HMGR activity, as determined by the conversion of [4-3H]HMG CoA to MVA in the total membrane and soluble fraction, was the highest at the time of harvest and gradually decreased during 5 months of storage in air at 0 °C. The potent ethylene action inhibitor 1-MCP inhibited ethylene production and α-farnesene evolution by 99% and 97%, respectively. The effect of 1-MCP on in vitro activity of HMGR was marginal (≈30% inhibition). 1-MCP inhibited respiratory CO2 evolution by 50%, which suggests also that inhibition by 1-MCP of α-farnesene synthesis in apple could be regulated by the acetyl CoA pool. In plants, HMGR is encoded by a small gene family and differentially expressed. As the first step of studying the molecular mechanism of HMGR regulation, we have isolated a 444-bp fragment of apple hmgr gene using apple skin mRNA and degenerate oligonucleotides designed against conserved regions of plant hmgr genes.

scholarly journals Sterol-induced dislocation of 3-hydroxy-3-methylglutaryl coenzyme A reductase from membranes of permeabilized cells

2013 ◽  
Vol 24 (21) ◽  
pp. 3300-3308 ◽  
Author(s):  
Rania Elsabrouty ◽  
Youngah Jo ◽  
Tammy T. Dinh ◽  
Russell A. DeBose-Boyd
Keyword(s):  
Deubiquitinating Enzymes ◽  
Intact Cells ◽  
Permeabilized Cells ◽  
Rate Limiting Step ◽  
Viable System ◽  
Coa Reductase ◽  
Rate Limiting ◽  
Induced Dislocation ◽  
Pharmacologic Inhibition

The polytopic endoplasmic reticulum (ER)–localized enzyme 3-hydroxy-3-methylglutaryl CoA reductase catalyzes a rate-limiting step in the synthesis of cholesterol and nonsterol isoprenoids. Excess sterols cause the reductase to bind to ER membrane proteins called Insig-1 and Insig-2, which are carriers for the ubiquitin ligases gp78 and Trc8. The resulting gp78/Trc8-mediated ubiquitination of reductase marks it for recognition by VCP/p97, an ATPase that mediates subsequent dislocation of reductase from ER membranes into the cytosol for proteasomal degradation. Here we report that in vitro additions of the oxysterol 25-hydroxycholesterol (25-HC), exogenous cytosol, and ATP trigger dislocation of ubiquitinated and full-length forms of reductase from membranes of permeabilized cells. In addition, the sterol-regulated reaction requires the action of Insigs, is stimulated by reagents that replace 25-HC in accelerating reductase degradation in intact cells, and is augmented by the nonsterol isoprenoid geranylgeraniol. Finally, pharmacologic inhibition of deubiquitinating enzymes markedly enhances sterol-dependent ubiquitination of reductase in membranes of permeabilized cells, leading to enhanced dislocation of the enzyme. Considered together, these results establish permeabilized cells as a viable system in which to elucidate mechanisms for postubiquitination steps in sterol-accelerated degradation of reductase.

scholarly journals Determining the Rate-Limiting Step for Light-Responsive Redox Regulation in Chloroplasts

Antioxidants ◽  
2018 ◽  
Vol 7 (11) ◽  
pp. 153 ◽  
Author(s):  
Keisuke Yoshida ◽  
Toru Hisabori
Keyword(s):  
Redox Regulation ◽  
Reducing Power ◽  
Rubisco Activase ◽  
Power Transfer ◽  
Target Proteins ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
Rate Limiting

Thiol-based redox regulation ensures light-responsive control of chloroplast functions. Light-derived signal is transferred in the form of reducing power from the photosynthetic electron transport chain to several redox-sensitive target proteins. Two types of protein, ferredoxin-thioredoxin reductase (FTR) and thioredoxin (Trx), are well recognized as the mediators of reducing power. However, it remains unclear which step in a series of redox-relay reactions is the critical bottleneck for determining the rate of target protein reduction. To address this, the redox behaviors of FTR, Trx, and target proteins were extensively characterized in vitro and in vivo. The FTR/Trx redox cascade was reconstituted in vitro using recombinant proteins from Arabidopsis. On the basis of this assay, we found that the FTR catalytic subunit and f-type Trx are rapidly reduced after the drive of reducing power transfer, irrespective of the presence or absence of their downstream target proteins. By contrast, three target proteins, fructose 1,6-bisphosphatase (FBPase), sedoheptulose 1,7-bisphosphatase (SBPase), and Rubisco activase (RCA) showed different reduction patterns; in particular, SBPase was reduced at a low rate. The in vivo study using Arabidopsis plants showed that the Trx family is commonly and rapidly reduced upon high light irradiation, whereas FBPase, SBPase, and RCA are differentially and slowly reduced. Both of these biochemical and physiological findings suggest that reducing power transfer from Trx to its target proteins is a rate-limiting step for chloroplast redox regulation, conferring distinct light-responsive redox behaviors on each of the targets.

scholarly journals The rate-limiting step of protein synthesis in vivo and in vitro and the distribution of growing peptides between the puromycinlabile and puromycin-non-labile sites on polyribosomes

1971 ◽  
Vol 122 (3) ◽  
pp. 267-276 ◽  
Author(s):  
D. C. N. Earl ◽  
Susan T. Hindley
Keyword(s):  
Amino Acids ◽  
Protein Synthesis ◽  
Donor Site ◽  
Peptide Bond ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
Donor And Acceptor ◽  
Rate Limiting

1. At 3 min after an intravenous injection of radioactive amino acids into the rat, the bulk of radioactivity associated with liver polyribosomes can be interpreted as growing peptides. 2. In an attempt to identify the rate-limiting step of protein synthesis in vivo and in vitro, use was made of the action of puromycin at 0°C, in releasing growing peptides only from the donor site, to study the distribution of growing peptides between the donor and acceptor sites. 3. Evidence is presented that all growing peptides in a population of liver polyribosomes labelled in vivo are similarly distributed between the donor and acceptor sites, and that the proportion released by puromycin is not an artifact of methodology. 4. The proportion released by puromycin is about 50% for both liver and muscle polyribosomes labelled in vivo, suggesting that neither the availability nor binding of aminoacyl-tRNA nor peptide bond synthesis nor translocation can limit the rate of protein synthesis in vivo. Attempts to alter this by starvation, hypophysectomy, growth hormone, alloxan, insulin and partial hepatectomy were unsuccessful. 5. Growing peptides on liver polyribosomes labelled in a cell-free system in vitro or by incubating hemidiaphragms in vitro were largely in the donor site, suggesting that either the availability or binding of aminoacyl-tRNA, or peptide bond synthesis, must be rate limiting in vitro and that the rate-limiting step differs from that in vivo. 6. Neither in vivo nor in the hemidiaphragm system in vitro was a correlation found between the proportion of growing peptides in the donor site and changes in the rate of incorporation of radioactivity into protein. This could indicate that the intracellular concentration of amino acids or aminoacyl-tRNA limits the rate of protein synthesis and that the increased incorporation results from a rise to a higher but still suboptimum concentration.

scholarly journals Mechanism of d-fructose isomerization by Arthrobacterd-xylose isomerase

1992 ◽  
Vol 283 (1) ◽  
pp. 223-233 ◽  
Author(s):  
M Rangarajan ◽  
B S Hartley
Keyword(s):  
Ring Opening ◽  
Ph Dependence ◽  
Xylose Isomerase ◽  
Competitive Inhibitor ◽  
Apparent Dissociation Constant ◽  
Ph Values ◽  
Rate Limiting Step ◽  
Competitive Inhibitors ◽  
Rate Limiting ◽  
Very High

The mechanism of D-fructose isomerization by Arthrobacter D-xylose isomerase suggested from X-ray-crystallographic studies was tested by detailed kinetic analysis of the enzyme with various metal ions at different pH values and temperatures. At D-fructose concentrations used in commercial processes Mg2+ is the best activator with an apparent dissociation constant of 63 microM; Co2+ and Mn2+ bind more strongly (apparent Kd 20 microM and 10 microM respectively) but give less activity (45% and 8% respectively). Ca2+ is a strict competitive inhibitor versus Mg2+ (Ki 3 microM) or Co2+ (Ki 105 microM). The kinetics show a compulsory order of binding; Co2+ binds first to Site 2 and then to Site 1; then D-fructose binds at Site 1. At normal concentrations Mg2+ binds at Site 1, then D-fructose and then Mg2+ at Site 2. At very high Mg2+ concentrations (greater than 10 mM) the order is Mg2+ at Site 1, Mg2+ at Site 2, then D-fructose. The turnover rate (kcat.) is controlled by ionization of a residue with apparent pKa at 30 degrees C of 6.0 +/- 0.07 (Mg2+) or 5.3 +/- 0.08 (Co2+) and delta H = 23.5 kJ/mol. This appears to be His-219, which is co-ordinated to M[2]; protonation destroys isomerization by displacing M[2]; Co2+ binds more strongly at Site 2 than Mg2+, so competes more strongly against H+. The inhibition constant (Ki) for the two competitive inhibitors 5-thio-alpha-D-glucopyranose and D-sorbitol is invariant with pH, but Km(app.) in the Mg[1]-enzyme is controlled by ionization of a group with pKa 6.8 +/- 0.07 and delta H = 27 kJ/mol, which appears to be His-53. This shows that Km(app.) is a complex constant that includes the rate of the ring-opening step catalysed by His-53, which explains the pH-dependence. In the Mg[1]Mg[2]-enzyme or Co[1]Co[2]-enzyme, the pKa is lower (6.2 +/- 0.1 or 5.6 +/- 0.08) because of the extra adjacent cation. Hence the results fit the previously proposed pathway, but show that the mechanisms differ for Mg2+ and Co2+ and that the rate-limiting step is isomerization and not ring-opening as previously postulated.

scholarly journals Escherichia coli Peptidase A, B, or N Can Process Translation Inhibitor Microcin C

2008 ◽  
Vol 190 (7) ◽  
pp. 2607-2610 ◽  
Author(s):  
Teymur Kazakov ◽  
Gaston H. Vondenhoff ◽  
Kirill A. Datsenko ◽  
Maria Novikova ◽  
Anastasia Metlitskaya ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Cell Growth ◽  
Trna Synthetase ◽  
Methionine Residue ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
Translation Inhibitor ◽  
Rate Limiting ◽  
Broad Specificity

ABSTRACT The heptapeptide-nucleotide microcin C (McC) targets aspartyl-tRNA synthetase. Upon its entry into a susceptible cell, McC is processed to release a nonhydrolyzable aspartyl-adenylate that inhibits aspartyl-tRNA synthetase, leading to the cessation of translation and cell growth. Here, we surveyed Escherichia coli cells with singly, doubly, and triply disrupted broad-specificity peptidase genes to show that any of three nonspecific oligopeptidases (PepA, PepB, or PepN) can effectively process McC. We also show that the rate-limiting step of McC processing in vitro is deformylation of the first methionine residue of McC.

scholarly journals Coassembly of SecYEG and SecA Fully Restores the Properties of the Native Translocon

2018 ◽  
Vol 201 (1) ◽  
Author(s):  
Priya Bariya ◽  
Linda L. Randall
Keyword(s):  
Lipid Bilayers ◽  
Rate Constant ◽  
Apparent Rate Constant ◽  
Membrane Vesicles ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
Secretory System ◽  
Rate Limiting

ABSTRACTIn all cells, a highly conserved channel transports proteins across membranes. InEscherichia coli, that channel is SecYEG. Many investigations of this protein complex have used purified SecYEG reconstituted into proteoliposomes. How faithfully do activities of reconstituted systems reflect the properties of SecYEG in the native membrane environment? We investigated by comparing threein vitrosystems: the native membrane environment of inner membrane vesicles and two methods of reconstitution. One method was the widely used reconstitution of SecYEG alone into lipid bilayers. The other was our method of coassembly of SecYEG with SecA, the ATPase of the translocase. For nine different precursor species we assessed parameters that characterize translocation: maximal amplitude of competent precursor translocated, coupling of energy to transfer, and apparent rate constant. In addition, we investigated translocation in the presence and absence of chaperone SecB. For all nine precursors, SecYEG coassembled with SecA was as active as SecYEG in native membrane for each of the parameters studied. Effects of SecB on transport of precursors faithfully mimicked observations madein vivo. From investigation of the nine different precursors, we conclude that the apparent rate constant, which reflects the step that limits the rate of translocation, is dependent on interactions with the translocon of portions of the precursors other than the leader. In addition, in some cases the rate-limiting step is altered by the presence of SecB. Candidates for the rate-limiting step that are consistent with our data are discussed.IMPORTANCEThis work presents a comprehensive quantification of the parameters of transport by the Sec general secretory system in the threein vitrosystems. The standard reconstitution used by most investigators can be enhanced to yield six times as many active translocons simply by adding SecA to SecYEG during reconstitution. This robust system faithfully reflects the properties of translocation in native membrane vesicles. We have expanded the number of precursors studied to nine. This has allowed us to conclude that the rate constant for translocation varies with precursor species.

Regulation of the purine salvage pathway in rat liver

1992 ◽  
Vol 262 (3) ◽  
pp. E344-E352 ◽  
Author(s):  
Y. A. Kim ◽  
M. T. King ◽  
W. E. Teague ◽  
G. A. Rufo ◽  
R. L. Veech ◽  
...  
Keyword(s):  
Rat Liver ◽  
De Novo ◽  
Equilibrium Constants ◽  
Purine Metabolism ◽  
Purine Salvage ◽  
Rate Limiting Step ◽  
Delta G ◽  
Salvage Pathways ◽  
Rate Limiting

The regulation of purine metabolism in rat liver has been examined under conditions that alter the flux through the pathway. Rats were given intraperitoneal injections of ethanol, sodium acetate, or sodium phosphate to attain body water concentrations of approximately 70, 20, and 10 mM, respectively. The livers were freeze-clamped after 30 min, and extracts were made for the analysis of metabolites, cofactors, purine bases, and nucleosides; homogenates were made for the measurement of the activities and kinetic parameters of seven enzymes that participate in purine salvage. The values of the equilibrium constants of nine reactions were determined in vitro and compared with the ratios of the reactants measured in liver. The changes in phosphoribosylpyrophosphate (PRPP), a key intermediate in both the de novo and salvage pathways of purine metabolism, were directly correlated with the changes in ribose 5-phosphate (ribose-5-P); ([PRPP] = 1.7[ribose-5-P] - 7.4 mumol/kg). Ribose-5-P concentrations in turn could be predicted from the liver content of fructose 6-phosphate and glyceraldehyde 3-phosphate by calculation from the known equilibria. The maximum velocities in the tissue of the seven enzymes measured were calculated from the measured substrate values in the liver and with consideration of other effectors of enzyme activity. PRPP synthetase was the least active of the enzymes measured, indicating a possible rate-limiting step. The delta G of the enzyme steps differed from equilibrium values by factors ranging from 4 (nucleoside phosphorylase) to 10(5) (PRPP synthetase and purine transferase reactions). The regulation of purine salvage appeared to depend on the levels of PRPP and ribose-5-P.

Altering potato isoprenoid metabolism increases biomass and induces early flowering

2020 ◽  
Vol 71 (14) ◽  
pp. 4109-4124
Author(s):  
Moehninsi ◽  
Iris Lange ◽  
B Markus Lange ◽  
Duroy A Navarre
Keyword(s):  
Metabolic Regulation ◽  
Transcriptional Control ◽  
Mevalonate Pathway ◽  
Early Flowering ◽  
Bioenergy Crops ◽  
Isoprenoid Metabolism ◽  
Transgenic Lines ◽  
Coa Reductase ◽  
Rate Limiting

Abstract Isoprenoids constitute the largest class of plant natural products and have diverse biological functions including in plant growth and development. In potato (Solanum tuberosum), the regulatory mechanism underlying the biosynthesis of isoprenoids through the mevalonate pathway is unclear. We assessed the role of 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) homologs in potato development and in the metabolic regulation of isoprenoid biosynthesis by generating transgenic lines with down-regulated expression (RNAi-hmgr) or overexpression (OE) of one (StHMGR1 or StHMGR3) or two genes, HMGR and farnesyl diphosphate synthase (FPS; StHMGR1/StFPS1 or StHMGR3/StFPS1). Levels of sterols, steroidal glycoalkaloids (SGAs), and plastidial isoprenoids were elevated in the OE-HMGR1, OE-HMGR1/FPS1, and OE-HMGR3/FPS1 lines, and these plants exhibited early flowering, increased stem height, increased biomass, and increased total tuber weight. However, OE-HMGR3 lines showed dwarfism and had the highest sterol amounts, but without an increase in SGA levels, supporting a rate-limiting role for HMGR3 in the accumulation of sterols. Potato RNAi-hmgr lines showed inhibited growth and reduced cytosolic isoprenoid levels. We also determined the relative importance of transcriptional control at regulatory points of isoprenoid precursor biosynthesis by assessing gene–metabolite correlations. These findings provide novel insights into specific end-products of the sterol pathway and could be important for crop yield and bioenergy crops.

Enhancement of hepatic glycogen by gluconeogenic precursors: substrate flux or metabolic control?

1990 ◽  
Vol 258 (6) ◽  
pp. E899-E906 ◽  
Author(s):  
J. H. Youn ◽  
R. N. Bergman
Keyword(s):  
Glycogen Metabolism ◽  
Hepatic Glycogen ◽  
Glycogen Accumulation ◽  
Major Mechanism ◽  
Rate Limiting Step ◽  
Key Enzyme ◽  
Glucose Phosphorylation ◽  
Rate Limiting ◽  
Gluconeogenic Substrates

After a meal or glucose load, most carbons of hepatic glycogen are derived from gluconeogenesis. In vitro, hepatic glycogen accumulation is sluggish with glucose alone but markedly enhanced in the presence of gluconeogenic substrates. These findings conflict with the classical view that glucose is the major precursor of hepatic glycogen and have been termed the "glucose paradox." In this review, we attempt to elucidate the central mechanism underlying the glucose paradox by critically examining the in vitro data of hepatic glycogen accumulation. Our analysis is inconsistent with the current hypothesis that glucose phosphorylation is rate limiting for hepatic glycogen accumulation from glucose and that gluconeogenesis enhances glycogen accumulation primarily by increasing substrate flux to the hepatic glucose 6-phosphate pool. Instead, our analysis leads us to the conclusion that the rate-limiting step is the net incorporation of glucose 6-phosphate into glycogen, which is synergistically facilitated with glucose and gluconeogenic substrates. Thus gluconeogenic substrates are involved in the regulation of key enzyme(s) of glycogen metabolism. In addition, in the livers from fasted rats there is substantial cycling through glycogen, and that suppression of glycogen degradation may be a major mechanism in the enhancement of glycogen accumulation by gluconeogenic substrates. Thus we propose a specific hypothesis of the role of gluconeogenic substrates in glycogen metabolism (i.e., inhibition of phosphorylase), which can be tested by future studies.

scholarly journals The Heme Oxygenase(s)-Phytochrome System ofPseudomonas aeruginosa

2004 ◽  
Vol 279 (44) ◽  
pp. 45791-45802 ◽  
Author(s):  
Rosalina Wegele ◽  
Ronja Tasler ◽  
Yuhong Zeng ◽  
Mario Rivera ◽  
Nicole Frankenberg-Dinkel
Keyword(s):  
Heme Oxygenase ◽  
Pathogenic Bacteria ◽  
Nmr Studies ◽  
Biliverdin Reductase ◽  
Heme Oxygenases ◽  
Rate Limiting Step ◽  
Phytochrome System ◽  
Rate Limiting ◽  
C Nmr

For many pathogenic bacteria likePseudomonas aeruginosaheme is an essential source of iron. After uptake, the heme molecule is degraded by heme oxygenases to yield iron, carbon monoxide, and biliverdin. The heme oxygenase PigA is only induced under iron-limiting conditions and produces the unusual biliverdin isomers IXβ and IXδ. The gene for a second putative heme oxygenase inP. aeruginosa,bphO, occurs in an operon with the genebphPencoding a bacterial phytochrome. Here we provide biochemical evidence thatbphOencodes for a second heme oxygenase inP. aeruginosa. HPLC,1H, and13C NMR studies indicate that BphO is a “classic” heme oxygenase in that it produces biliverdin IXα. The data also suggest that the overall fold of BphO is likely to be the same as that reported for other α-hydroxylating heme oxygenases. Recombinant BphO was shown to prefer ferredoxins or ascorbate as a source of reducing equivalentsin vitroand the rate-limiting step for the oxidation of heme to biliverdin is the release of product. In eukaryotes, the release of biliverdin is driven by biliverdin reductase, the subsequent enzyme in heme catabolism. BecauseP. aeruginosalacks a biliverdin reductase homologue, data are presented indicating an involvement of the bacterial phytochrome BphP in biliverdin release from BphO and possibly from PigA.

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