Regulation of Plant Acetyl-CoA Carboxylase by Adenylate Nucleotides

The assay of acetyl-CoA carboxylase (EC 6.4.1.2) does not follow ideal zero-order kinetics when assayed in a crude extract from wheat (Triticum aestivum L.) germ. Our results show that the lack of ideality is the consequence of contamination by ATPase and adenylate kinase. These enzyme activities generate significant amounts of ADP and AMP in the assay mixture, thus limiting the availability of ATP for the carboxylase reaction. Moreover, ADP and AMP are competitive inhibitors, with respect to ATP, of acetyl-CoA carboxylase. Similar relationships between adenylate nucleotides and acetyl-CoA carboxylase are found in isolated chloroplasts. There is no evidence that acetyl-CoA carboxylase activity in the extracts of the plant systems examined is altered by covalent modification, such as a phosphorylation-dephosphorylation cycle. A scheme is presented that illustrates the dependency of acetyl-CoA carboxylase and fatty acid synthesis on the energy demands of the chloroplasts in vivo.     

Phosphorylation of pea chloroplast acetyl-CoA carboxylase

We have examined whether chloroplast acetyl-CoA carboxylase is a phosphoprotein. Pea ( Pisum sativum ) chloroplasts were incubated in the presence of [γ- ³³ P]-ATP and radiolabeled proteins were examined after immunoprecipitation with antibodies against all four known subunits of heteromeric chloroplast acetyl-CoA carboxylase. The β-subunit of the carboxyltransferase was found to be labeled by ³³ P. Phosphoamino acid analysis of the immunoprecipitated β-subunit of the carboxyltransferase indicates that it is phosphorylated on serine residues. Incorporation of ³³ P into carboxyltransferase β-subunit decreased in chloroplasts transferred to dark conditions after labeling in the light. Dephosphorylation of pea chloroplast extracts by an alkaline phosphatase-agarose conjugate reduced in vitro acetyl-CoA carboxylase activity by 67%. Furthermore, while acetyl-CoA carboxylase activity and its carboxyltransferase half-reaction were reduced in dephosphorylated extracts, the biotin carboxylase half-reaction was not inhibited. The evidence presented here points to the carboxyltransferase β-subunit of chloroplast acetyl-CoA carboxylase as a candidate for regulation by protein phosphorylation/dephosphorylation.     

Acetyl-coenzyme A carboxylase in maize leaves

Purified chloroplasts from mesophyll and bundle sheath cells of maize leaves have been shown to be the location of acetyl-CoA carboxylase. In disrupted chloroplasts the enzyme was recovered in the stromal fraction, along with protein-bound biotin; acetyl-CoA carboxylase activity did not require a membrane component. Mg²⁺ and ATP are required for activity and sulfhydryl protecting agents enhance stability of the enzyme. Acetyl-CoA carboxylase activity was independent of leaf development in cell-free extracts of maize. Comparison of acetyl-CoA carboxylase activity with [¹⁴C]acetate incorporation into lipids, in isolated chloroplasts from developing leaves of maize, indicate that acetyl-CoA carboxylase is not limiting fatty acid synthesis.     

Studies on wheat acetyl CoA carboxylase and the cloning of a partial cDNA

Wheat germ acetyl CoA carboxylase (ACCase) was purified by liquid chromatography and electroelution. During purification bovine serum albumin (BSA) was used to coat Amicon membranes used to concentrate partially pure ACCase. Despite further SDS-PAGE/electroelution and microbore HPLC steps BSA remained associated. This presented serious protein sequencing artefacts which may reflect the affinity of BSA for fatty acids bound to ACCase. To avoid these artefacts the enzyme was digested in gel with Endoproteinase LysC protease without the presence of BSA, and the resulting peptides blotted and sequenced.A partial cDNA (1.85 kb) encoding ACCase from a wheat embryo library was cloned, which hybridised to a 7.5 kb RNA species on northern blot of wheat leaf poly(A)⁺ RNA. The partial cDNA therefore represents about 0.25 of the full-length cDNA. The clone was authenticated by ACCase peptide sequencing and immuno cross-reactivity of the overexpressed clone. The derived amino acid sequence showed homology with both rat and yeast ACCase sequences (62%).Antibodies raised against wheat acetyl CoA carboxylase were specific for a 220 kDa protein from both wheat embryo and leaf. In addition, by using a novel quick assay for ACCase that utilised ¹²⁵I-streptavidin, we showed the major biotin containing protein to be 220 kDa in both leaf and germ. This is in marked contrast to the previously published molecular mass of 75 kDa allocated to wheat leaf ACCase.     

Inhibition of Acetyl-CoA Carboxylase Activity by Haloxyfop and Tralkoxydim

Acetyl-coenzyme A (CoA) carboxylase from maize (Zea mays L.) is inhibited by nanomolar concentrations of both haloxyfop, an aryloxyphenoxypropionate, and tralkoxydim, a cyclohexanedione herbicide. These results suggest that acetyl-CoA carboxylase, which catalyzes the first committed step in fatty acid biosynthesis, may be the target of these herbicides, contrary to an earlier report suggesting that aryloxyphenoxypropionate herbicides do not inhibit acetyl-CoA carboxylase.     

Comparison of acetyl-CoA carboxylases from parsley cell cultures and wheat germ

The activity of acetyl-CoA carboxylase of suspension-cultured cells of parsley (Petroselinum hortense Hoffm.) is greatly stimulated by light soon after transferring cells to new culture medium. Parsley acetyl-CoA carboxylase has been purified from frozen cells by treatment of the crude protein extract with Dowex 1 × 2 and polyethyleneimine, precipitation with (NH₄)₂SO₄, chromatography on DEAE-cellulose and blue Sepharose CL-6B, and gel filtration on Sepharose 6B. A recovery of about 8% has been achieved with a 300-fold increase in specific activity. Wheat germ acetyl-CoA carboxylase has been purified 2180-fold by a similar procedure. The two carboxylases have the following characteristics: Molecular weights of 840,000 for the parsley carboxylase and 700,000 for the wheat germ carboxylase have been estimated from the elution volumes of a calibrated Sepharose 6B column. Analysis by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate showed that the carboxylases from parsley and wheat each are composed of one large subunit (M_r = 210,000 and 240,000, respectively) and possibly one smaller polypeptide component (M_r = 105,000 and 98,000, respectively). Avidin-binding experiments demonstrated that the 240,000 — M_r component of wheat germ carboxylase is the biotin-containing subunit of this enzyme. No isoenzymes of the parsley carboxylase could be demonstrated.     

Propionyl-CoA carboxylase of Myxococcus xanthus: catalytic properties and function in developing cells

An acyl-coenzyme A carboxylase that carboxylates acetyl-CoA, butyryl-CoA, propionyl-CoA, and succinyl-CoA was purified from Myxococcus xanthus. Since the enzyme showed maximal rates of carboxylation with propionyl-CoA, the enzyme is thought to be propionyl-CoA carboxylase. The apparent K _m values for acetyl-CoA, butyryl-CoA, propionyl-CoA, and succinyl-CoA were found to be 0.2, 0.2, 0.03, and 1.0 mM, respectively. The native enzyme has a molecular mass of 605–615 kDa and is composed of nonidentical subunits (α and β) with molecular masses of 53 and 56 kDa, respectively. The enzyme showed maximal activity at pH 7.0–7.5 and at 25–30°C, and was affected by variation in concentrations of ATP and Mg²⁺. During development of M. xanthus, the propionyl-CoA carboxylase activity increased gradually, with maximum activity observed during the sporulation stage. Previous work has shown that a propionyl-CoA-carboxylase-deficient mutant of M. xanthus reduces levels of long-chain fatty acids. These results suggest that the propionyl-CoA carboxylase is also responsible for the carboxylation of acetyl-CoA to malonyl-CoA used for the synthesis of long-chain fatty acids during development. Received: 24 February 1998 / Accepted: 25 May 1998     

Acetyl coenzyme a carboxylase activity in developing seedlings and chloroplasts of barley and its virescens mutant

Acetyl coenzyme A (CoA) carboxylase activity of whole tissue homogenates and chloroplast preparations was analyzed as the acetyl-CoA-dependent incorporation of [¹⁴C]bicarbonate into an acid-stable product. The absolute requirement for ATP and MgCl₂, the complete inhibition with avidin, and end-product analysis were consistent with the presence of acetyl-CoA carboxylase activity. Little difference was found between the mutant and normal tissue homogenates from the 1- to 3-day growth stages, during which period both showed a 3-fold increase. However, by 4 days, the activity of the mutant exceeded that of the normal. Fractionation studies showed that the enzyme was a soluble protein present in the stromal fraction of chloroplasts. The biotin content was also highest in the stroma, although it was found in the lamellar fraction as well. For both the mutant and the normal, the highest acetyl-CoA carboxylase activities were obtained in the stromal preparations from 4-day seedlings (54 and 31 nmoles per milligram protein per minute for the mutant and the normal, respectively) with a progressive decline by 6 and 8 days. The difference between the mutant and the normal was not due to the accumulation of an inhibitor in the normal.     

Purification and Characterization of Acetyl-CoA Carboxylase from the Diatom Cyclotella cryptica

Acetyl-CoA carboxylase from the diatom Cyclotella cryptica has been purified to near homogeneity by the use of ammonium sulfate fractionation, gel filtration chromatography, and affinity chromatography with monomeric avidin-agarose. The specific activity of the final preparation was as high as 14.6 micromoles malonyl-CoA formed per milligram protein per minute, indicating a 600-fold purification. Native acetyl-CoA carboxylase has a molecular weight of approximately 740 kilodaltons and appears to be composed of four identical biotin-containing subunits. The enzyme has maximal activity at pH 8.2, but enzyme stability is greater at pH 6.5. K_m values for MgATP, acetyl-CoA, and HCO₃- were determined to be 65, 233, and 750 micromolar, respectively. The purified enzyme is strongly inhibited by palmitoyl-CoA, and is inhibited to a lesser extent by malonyl-CoA, ADP, and phosphate. Pyruvate stimulates enzymatic activity to a slight extent. Acetyl-CoA carboxylase from Cyclotella cryptica is not inhibited by cyclohexanedione or aryloxyphenoxypropionic acid herbicides as strongly as monocot acetyl-CoA carboxylases; 50% and 0% inhibition was observed in the presence of 23 micromolar clethodim and 100 micromolar haloxyfop, respectively.     

Synthesis of proteins by chloroplasts from iron-deficient Euglena gracilis

Chloroplasts isolated from Euglena gracilis made iron deficient by growth on 0.5 μm iron show distinct qualitative and quantitative changes in their polypeptide composition in comparison with iron-sufficient (40 μm) chloroplasts. These changes were noted in the stromal, thylakoid, and envelope subfractions. Iron-deficient chloroplasts have a sedimentation behavior similar to that of iron-sufficient chloroplasts and also contain substantial amounts of ribulose-1,5-bisphosphate carboxylase. In addition, iron-deficient chloroplasts incorporate [³H]leucine into polypeptides at rates about one-third of those from control chloroplasts (40 μm Fe) on a per-microgram-chlorophyll basis. Incorporation of [³H]leucine into specific polypeptides, resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, shows relatively normal synthesis of the large subunit of ribulose-1,5-bisphosphate carboxylase and two of the three major chloroplast-derived polypeptides of the thylakoids. No incorporation was detected, however, into a polypeptide of ca. 33 kd which is synthesized by normal plastids. Iron-deficient chloroplasts also synthesize a stromal polypeptide of ca. 85 kd not seen in chloroplasts from normal cells. This evidence is consistent with a direct or indirect role for iron in the regulation of synthesis of specific proteins in the chloroplast.     

Queuine in plants and plant tRNAs: Differences between embryonic tissue and mature leaves

In eubacterial and eukaryotic tRNAs specific for Asn, Asp, His and Tyr the modified deazaguanosinederivative queuosine occurs in position 34, the first position of the anticodon. Analysis of unfractionated tRNAs from wheat and from tobacco leaves shows that these tRNAs contain high amounts of guanosine (G) in place of queuosine (Q). This was measured by the exchange of G34 for [³H]guanine catalysed by the specific tRNA guanine transglycosylase from E. coli. Upon gel electrophoretic separation of the labeled tRNAs, seven Q-deficient tRNA species including isoacceptors are detectable. Two are identified as cytoplasmic tRNAs^Tyr and tRNA^Asp and two represent chloroplast tRNA^Tyr isoacceptors. In contrast to leaf cytoplasm and chloroplasts, wheat germ has low amounts of tRNAs with G34 in place of Q.A new enzymatic assay is described for quantitation of free queuine in cells and tissues. Analysis of queuine in plant tissues shows that wheat germ contains about 200 ng queuine per g wet weight. In wheat and tobacco leaves queuine is present, if at all, in amounts lower than 10 ng/g wet weight. The absence of Q in tRNAs from plant leaves is therefore caused by a deficiency of queuine. Tobacco cells cultivated in a synthetic medium without added queuine do not contain Q in tRNA, indicating that these rapidly growing cells do not synthesize queuine de novo.     

Fat metabolism in higher plants. Further characterization of wheat germ acetyl coenzyme A carboxylase.

Wheat germ acetyl CoA carboxylase was purified 600-fold over the crude homogenate. The purified enzyme gave rise to complex electrophoretic patterns in dissociating gels. As isolated, the activity of wheat germ acetyl CoA carboxylase exhibited profound dependence on the composition of the reaction mixture. In addition to the substrates MgATP, HCO₃, and acetyl CoA, the enzyme required both free Mg²⁺ and K⁺ for optimal activity. The effects of the two ions were additive. At pH 8.5, Mg²⁺ activated the carboxylase by adding to the enzyme prior to the other reactants in an equilibrium ordered reaction mechanism.     

Temperature-sensitivity of D1 protein metabolism in isolated Zea mays chloroplasts

The effects of low temperature on the synthesis and stability of the 32 kDa D1 protein of photosystem II were investigated in chloroplasts isolated from maize (Zea mays cv. LG11) leaves. The synthesis of D1 by intact chloroplasts in vitro was strongly dependent on temperature; the Q₁₀ for the initial rate of incorporation of [³⁵S]-methionine into D1 was ca. 2.6 over the range 13–25°C. The synthesis of other thylakoid polypeptides exhibited a similar temperature dependence, whilst synthesis of stromal proteins was considerably less temperature-dependent, with the exception of two polypeptides of ca. 56 and 59.5 kDa. The stability of newly-synthesized D1 in the thylakoid membranes was dependent both on the temperature at which the plants were grown and on the temperature during the pulse-labelling period when the protein was synthesized. In chloroplasts isolated from maize leaves grown at 25°C, D1 that was synthesized and assembled at 25 °C in vitro was rapidly degraded during the chase period. At lower chase temperatures the protein was more stable. When chloroplasts from 25°C-grown leaves were pulse-labelled at 13°C, the stability of D1 was markedly enhanced at all temperatures during the chase period. This effect was even more pronounced in chloroplasts isolated from plants grown at 14°C. The implications of these results are discussed with regard to the ability of maize to recover from photoinhibitory damage at low temperatures.     

Purification and immunochemical characterization of ascitic fluid glycoproteins containing certain tumor-associated and blood group antigen markers

Ascitic fluids from patients with various types of cancer were screened for the CA 19-9 and CA 125 tumor-associated antigenic activities. Two fluids exhibiting the highest activities were tested for their binding to various lectin-Sepharose columns resulting in both being bound best to wheat germ agglutinin (WGA) Sepharose. The WGA column eluate of one fluid was further chromatographed by HPLC and three peaks were obtained with approximate molecular weights of 3.65 MDa, 664 kDa and 330 kDa, of which only the largest fraction contained the CA 19-9 activity. The fluids were also fractionated on a Sephacryl S-400 column with most of the activity being present in or near the void volume.Monoclonal antibodies were used to demonstrate that the purified glycoproteins also contained the blood group A determinant, the four Lewis determinants Le^a, Le^b, Le^x and Le^y, and the sialylated-Le^x determinant, while other antibody analyses failed to detect other blood group and/or carbohydrate sequence determinants. Some of the blood group expressions could be separated from the CA 19-9 and CA 125 active glycoproteins by adsorption with various lectins other than the WGA.Abbreviations used NeuAc N-acetyl-D-neuraminic acid - Gal galactose,D-galactopyranose - Fuc fucose,L-fucopyranose - GlcNAc N-acetyl-D-glucosamine - GalNAc N-acetyl-D-galactosamine - WGA wheat germ agglutinin - PBS phosphate buffered saline     

The purification and properties of a protein kinase and the partial purification of a phosphoprotein phosphatase that inactivate and activate acetyl-CoA carboxylase

the occurrence of a soluble fraction from rat liver that inactivates acetyl-CoA carboxylase was previously reported by this laboratory (1). The purification of this fraction is now reported, and we show that it behaves as a cAMP-independent kinase that inactivates acetyl-CoA carboxylase by phosphorylation. The kinase has a molecular weight of 160,000 and it requires ATP and Mg²⁺ for activity. A partial purification from rat liver cytosol of a Mg²⁺-requiring phosphoprotein phosphatase of high molecular weight (greater than 200,000) which dephosphorylates phosphorylated acetyl-CoA carboxylase with the regeneration of enzyme activity is also reported. The kinase, phosphatase, and acetyl-CoA carboxylase are separable from each other by a combination of ammonium sulfate precipitation, DEAE-cellulose chromatography, and gel filtration.     

Control of acetyl-CoA carboxylase by covalent modification

Summary In this review, various experiments which establish the occurrence of covalent modification mechanisms, both in vivo and in vitro, in the control of acetyl-CoA carboxylase have been presented. It is interesting to note that phosphorylation of the carboxylase results in disaggregation of the active species. These studies indicate that aggregation and disaggregation of the enzyme are involved in the control of carboxylase activity. Our covalent modification mechanism and the allosteric control mechanism share a common ground in that both mechanisms affect the equilibrium between protomers and polymers of the enzyme. However, it is clear that the allosteric control mechanism cannot functon alone under normal physiological conditions. Covalent modification of the carboxylase is prerequiste for efficient functioning of the allosteric mechanism.There are many aspects of the regulation of acetyl-CoA carboxylase which require further clarification. However, it is now established that short-term control of acetyl-CoA carboxylase involves the covalent modification mechanism.This research was supported by a grant from National Institutes of Health (AM 12865).This is Journal Paper No. 7701 from Purdue Agriculture Experiment Station.     

Regulation of plant Fatty Acid biosynthesis : analysis of acyl-coenzyme a and acyl-acyl carrier protein substrate pools in spinach and pea chloroplasts

In previous work (D. Post-Beittenmiller, J.G. Jaworski, J.B. Ohlrogge [1991] J Biol Chem 266: 1858-1865), the in vivo acyl-acyl carrier protein (ACP) pools were measured in spinach (Spinacia oleracea) leaves and changes in their levels were compared to changes in the rates of fatty acid biosynthesis. To further examine the pools of substrates and cofactors for fatty acid biosynthesis and to evaluate metabolic regulation of this pathway, we have now examined the coenzyme A (CoA) and short chain acyl-CoA pools, including acetyl- and malonyl-CoA, in isolated spinach and pea (Pisum sativum) chloroplasts. In addition, the relationships of the acetyl- and malonyl-CoA pools to the acetyl- and malonyl-ACP pools have been evaluated. These studies have led to the following conclusions: (a) Essentially all of the CoA (31-54 μm) in chloroplasts freshly isolated from light-grown spinach leaves or pea seedling was in the form of acetyl-CoA. (b) Chloroplasts contain at least 77% of the total leaf acetyl-CoA, based on comparison of acetyl-CoA levels in chloroplasts and total leaf. (c) CoA-SH was not detected either in freshly isolated chloroplasts or in incubated chloroplasts and is, therefore, less than 2 μm in the stroma. (d) The malonyl-CoA:ACP transacylase reaction is near equilibrium in both light- and dark-incubated chloroplasts, whereas the acetyl-CoA:ACP transacylase reaction is far from equilibrium in light-incubated chloroplasts. However, the acetyl-CoA:ACP transacylase reaction comes nearer to equilibrium when chloroplasts are incubated in the dark. (e) Malonyl-CoA and -ACP could be detected in isolated chloroplasts only during light incubations, and increased with increased rates of fatty acid biosynthesis. In contrast, both acetyl-CoA and acetyl-ACP were detectable in the absence of fatty acid biosynthesis, and acetyl-ACP decreased with increased rates of fatty acid biosynthesis. Together these data have provided direct in situ evidence that acetyl-CoA carboxylase plays a regulatory role in chloroplast fatty acid biosynthesis.     

Molecular cloning of a chloroplastic proteinassociated with both the envelope and thylakoid membranes

Chloroplasts consist of six morphologically distinct compartments. Each compartment has a specific set of polypeptides that perform distinct biochemical functions. We report here the identification of a membrane-associated protein with a novel localization. This protein was synthesized as a 37 kDa precursor and was processed to a mature protein of 30 kDa after being imported into isolated pea chloroplasts. Fractionation of chloroplasts showed that the 30 kDa mature protein was associated with both of the envelope membranes as well as with thylakoid membranes. Immunocyto-chemical localization of the 30 kDa protein revealed that the protein occurred in clusters in the vicinity of both the envelope and the thylakoid. Possible functions of this 30 kDa protein, inferred from its novel localization pattern, are discussed.Abbreviations CAB light-harvesting chlorophyll a/b-binding protein of photosystem II - prCAB precursor protein to CAB - SS small subunit of ribulose-1,5-bisphosphate carboxylase - prSS precursor protein to SS - RCF relative centrifugation force     

Occurrence,biochemistry and possible biotechnological application of the 3-hydroxypropionate cycle

The 3-hydroxypropionate cycle, a pathway for autotrophic carbon dioxide fixation, is reviewed with special emphasis on the biochemistry of CO₂ fixing enzymes in Acidianus brierleyi, a thermophilic and acidophilic archeon. In the 3-hydroxypropionate cycle, two enzymes, acetyl-CoA carboxylase and propionyl-CoA carboxylase, catalyze CO₂ fixation. It has been shown in A. brierleyi, and subsequently in Metallosphaera sedula, that acetyl-CoA carboxylase is promiscuous, acting equally well on acetyl-CoA and propionyl-CoA. The subunit structure of the acyl-CoA carboxylase was shown to be ₄₄₄. Gene cloning revealed that the genes encoding the three subunits are adjacent to each other. accC encodes the -subunit (59 kDa subunit, biotin carboxylase subunit), accB encodes the -subunit (20 kDa subunit, biotin carboxyl carrier protein), and pccB encodes the -subunit (62 kDa subunit, carboxyltransferase subunit). Sequence analyses showed that accC and accB are co-transcribed and that pccB is transcribed separately. Potential biotechnological applications for the 3-hydroxypropionate cycle are also presented.     

A Corynebacterium glutamicum gene encoding a two-domain protein similar to biotin carboxylases and biotin-carboxyl-carrier proteins

Following the analysis of transposon Tn5432-induced mutants of Corynebacterium glutamicum ATCC 13032, a gene encoding a protein with a biotin-binding motif was cloned. The DNA sequence of this gene revealed an open reading frame encoding 591 amino acids with a calculated mol. mass of 63.4 kDa. The protein is composed of two domains, an N-terminal biotin carboxylase and a C-terminal biotin-carboxyl-carrier protein, that are highly similar to corresponding subunits from prokaryotic and eukaryotic biotin enzymes. Over 70% identity was found to a protein from Mycobacterium leprae proposed to be part of an acyl-CoA carboxylase. Since it was not possible to inactivate the C. glutamicum gene, the gene most likely encodes a subunit of the essential acetyl-CoA carboxylase, which catalyzes the committed step in fatty acid synthesis. Received: 2 December 1995 / Accepted: 20 May 1996