Herbicide sensitivity determinant of wheat plastid acetyl-CoA carboxylase is located in a 400-amino acid fragment of the carboxyltransferase domain

A series of chimeral genes, consisting of the yeast GAL10 promoter, yeast ACC1 leader, wheat acetyl-CoA carboxylase (ACCase; EC 6.4.1.2) cDNA, and yeast ACC1 3'-tail, was used to complement a yeast ACC1 mutation. These genes encode a full-length plastid enzyme, with and without the putative chloroplast transit peptide, as well as five chimeric cytosolic/plastid proteins. Four of the genes, all containing at least half of the wheat cytosolic ACCase coding region at the 5'-end, complement the yeast mutation. Aryloxyphenoxypropionate and cyclohexanedione herbicides, at concentrations below 10 microM, inhibit the growth of haploid yeast strains that express two of the chimeric ACCases. This inhibition resembles the inhibition of wheat plastid ACCase observed in vitro and in vivo. The differential response to herbicides localizes the sensitivity determinant to the third quarter of the multidomain plastid ACCase. Sequence comparisons of different multidomain and multisubunit ACCases suggest that this region includes part of the carboxyltransferase domain, and therefore that the carboxyltransferase activity of ACCase (second half-reaction) is the target of the inhibitors. The highly sensitive yeast gene-replacement strains described here provide a convenient system to study herbicide interaction with the enzyme and a powerful screening system for new inhibitors.     

Single-site mutations in the carboxyltransferase domain of plastid acetyl-CoA carboxylase confer resistance to grass-specific herbicides

Grass weed populations resistant to aryloxyphenoxypropionate (APP) and cyclohexanedione herbicides that inhibit acetyl-CoA carboxylase (ACCase; EC 6.4.1.2) represent a major problem for sustainable agriculture. We investigated the molecular basis of resistance to ACCase-inhibiting herbicides for nine wild oat (Avena sterilis ssp. ludoviciana Durieu) populations from the northern grain-growing region of Australia. Five amino acid substitutions in plastid ACCase were correlated with herbicide resistance: Ile-1,781-Leu, Trp-1,999-Cys, Trp-2,027-Cys, Ile-2,041-Asn, and Asp-2,078-Gly (numbered according to the Alopecurus myosuroides plastid ACCase). An allele-specific PCR test was designed to determine the prevalence of these five mutations in wild oat populations suspected of harboring ACCase-related resistance with the result that, in most but not all cases, plant resistance was correlated with one (and only one) of the five mutations. We then showed, using a yeast gene-replacement system, that these single-site mutations also confer herbicide resistance to wheat plastid ACCase: Ile-1,781-Leu and Asp-2,078-Gly confer resistance to APPs and cyclohexanediones, Trp-2,027-Cys and Ile-2,041-Asn confer resistance to APPs, and Trp-1,999-Cys confers resistance only to fenoxaprop. These mutations are very likely to confer resistance to any grass weed species under selection imposed by the extensive agricultural use of the herbicides.     

Cloning of the yeast FAS3 gene and primary structure of yeast acetyl-CoA carboxylase.

We have isolated and determined the nucleotide sequence of the yeast FAS3 gene, which encodes acetyl-CoA carboxylase (EC 6.4.1.2). The sequence has an open reading frame of 6711 bases coding for a protein of 2237 amino acids with a calculated molecular weight of 250,593. The presence of the unique biotin-binding site, Met-Lys-Met, and the known CNBr peptide and COOH-terminal sequences confirmed the nucleotide-derived amino acid sequence. The yeast, chicken, and rat carboxylases have an overall sequence identity of 34%, suggesting that the eukaryotic carboxylase evolved from a single ancestral gene. The amino acid sequences of yeast fatty acid synthase subunits are least homologous with the animal synthase sequences, whereas carboxylase sequences are highly conserved. The sequences of the ATP, HCO3-, and CoA binding sites of the carboxylases are also well conserved (approximately 50% identical). The sequences surrounding the biotin binding site are poorly conserved, suggesting that this sequence may not be critical as long as the biotin is available for carboxylase reactions. On the basis of this sequence identity, we have defined the putative biotin carboxylase and transcarboxylase domains.     

Insulin stimulates the dephosphorylation and activation of acetyl-CoA carboxylase.

The mechanism underlying the ability of insulin to acutely activate acetyl-CoA carboxylase [acetyl-CoA: carbon-dioxide ligase (ADP-forming), EC 6.4.1.2; AcCoA-Case] has been examined in Fao Reuber hepatoma cells. Insulin promotes the rapid activation of AcCoACase, as measured in cell lysates, and this stimulation persists to the same degree after isolation of AcCoACase by avidin-Sepharose chromatography. The insulin-stimulated enzyme, as compared with control enzyme, exhibits an increase in both citrate-independent and -dependent activity and a decrease in the Ka for citrate. Direct examination of the phosphorylation state of isolated 32P-labeled AcCoACase after insulin exposure reveals a marked decrease in total enzyme phosphorylation coincident with activation. The dephosphorylation due to insulin appears to be restricted to the phosphorylation sites previously shown to regulate AcCoACase activity. All of these effects of insulin are mimicked by a low molecular weight autocrine factor, tentatively identified as an oligosaccharide, present in conditioned medium of hepatoma cells. These data suggest that insulin may activate AcCoACase by inhibiting the activity of protein kinase(s) or stimulating the activity of protein phosphatase(s) that control the phosphorylation state of the enzyme.     

Hotspots and the conservation of evolutionary history

Species diversity is unevenly distributed across the globe, with terrestrial diversity concentrated in a few restricted biodiversity hotspots. These areas are associated with high losses of primary vegetation and increased human population density, resulting in growing numbers of threatened species. We show that conservation of these hotspots is critical because they harbor even greater amounts of evolutionary history than expected by species numbers alone. We used supertrees for carnivores and primates to estimate that nearly 70% of the total amount of evolutionary history represented in these groups is found in 25 biodiversity hotspots.     

Structure and expression of spinach leaf cDNA encoding ribulosebisphosphate carboxylase/oxygenase activase.

Ribulosebisphosphate carboxylase/oxygenase activase is a recently discovered enzyme that catalyzes the activation of ribulose-1,5-bisphosphate carboxylase/oxygenase ["rubisco"; ribulose-bisphosphate carboxylase; 3-phospho-D-glycerate carboxy-lyase (dimerizing), EC 4.1.1.39] in vivo. Clones of rubisco activase cDNA were isolated immunologically from spinach (Spinacea oleracea L.) and Arabidopsis thaliana libraries. Sequence analysis of the spinach and Arabidopsis cDNAs identified consensus nucleotide binding sites, consistent with an ATP requirement for rubisco activase activity. A derived amino acid sequence common to chloroplast transit peptides was also identified. After synthesis of rubisco activase in vitro, the transit peptide was cleaved and the protein was transported into isolated chloroplasts. Analysis of spinach and Arabidopsis nuclear DNA by hybridization indicated a single rubisco activase gene in each species. Leaves of spinach and Arabidopsis wild type contained a single 1.9-kilobase rubisco activase mRNA. In an Arabidopsis mutant lacking rubisco activase protein, mRNA species of 1.7 and 2.1 kilobases were observed under conditions of high-stringency hybridization with a wild-type cDNA probe. This observation indicates that the lesion in the mutant arises from an error in mRNA processing.     

An isoleucine/leucine residue in the carboxyltransferase domain of acetyl-CoA carboxylase is critical for interaction with aryloxyphenoxypropionate and cyclohexanedione inhibitors

cDNA fragments encoding the carboxyltransferase domain of the multidomain plastid acetyl-CoA carboxylase (ACCase) from herbicide-resistant maize and from herbicide-sensitive and herbicide-resistant Lolium rigidum were cloned and sequenced. A Leu residue was found in ACCases from herbicide-resistant plants at a position occupied by Ile in all ACCases from sensitive grasses studied so far. Leu is present at the equivalent position in herbicide-resistant ACCases from other eukaryotes. Chimeric ACCases containing a 1000-aa fragment of two ACCase isozymes found in a herbicide-resistant maize were expressed in a yeast ACC1 null mutant to test herbicide sensitivity of the enzyme in vivo and in vitro. One of the enzymes was resistant/tolerant, and one was sensitive to haloxyfop and sethoxydim, rendering the gene-replacement yeast strains resistant and sensitive to these compounds, respectively. The sensitive enzyme has an Ile residue, and the resistant one has a Leu residue at the putative herbicide-binding site. Additionally, a single Ile to Leu replacement at an equivalent position changes the wheat plastid ACCase from sensitive to resistant. The effect of the opposite substitution, Leu to Ile, makes Toxoplasma gondii apicoplast ACCase resistant to haloxyfop and clodinafop. In this case, inhibition of the carboxyltransferase activity of ACCase (second half-reaction) of a large fragment of the Toxoplasma enzyme expressed in Escherichia coli was tested. The critical amino acid residue is located close to a highly conserved motif of the carboxyltransferase domain, which is probably a part of the enzyme active site, providing the basis for the activity of fop and dim herbicides.     

A Saccharomyces cerevisiae mutant strain defective in acetyl-CoA carboxylase arrests at the G2/M phase of the cell cycle

To elucidate the essential functions of acetyl-CoA carboxylase (ACC1FAS3) in Saccharomyces cerevisiae, a temperature-sensitive mutant (acc1(ts)) was constructed. When the acc1(ts) cells were synchronized in G(1) phase with alpha-factor at the permissive temperature of 24 degrees C and then released from the blockade and incubated at the restrictive temperature of 37 degrees C, 95% of the cell population became arrested at the G(2)M phase of the cell cycle despite the presence of fatty acids (C(14)-C(26)) in the medium. These cells developed large undivided nuclei, and the spindles of the arrested mutant cells were short. Shifting the G(2) arrested cells back to the permissive temperature resulted in a reversal of the cell-cycle arrest, with cells initiating mitosis. However, after 3 h of incubation at 37 degrees C, G(2) arrested mutant cells lost viability and displayed a uniquely altered nuclear envelope. Using [1-(14)C]acetate as a precursor for fatty acids synthesis, we identified the phospholipids and sphingolipids derived from acc1(ts) cells and wild-type cells at 24 degrees C and 37 degrees C, respectively. The levels of inositol-ceramides [IPC, MIPC, and M(IP)(2)C] and very long-chain fatty acids C(24) and C(26) declined sharply in the G(2)M arrested cells because of ACC inactivation. Shifting the acc1(ts) cells to 24 degrees C after 2 h of incubation at 37 degrees C resulted in reactivation of the ACC and elevation of the ceramides and very long-chain fatty acid syntheses with normal cell-cycle progression. In contrast, synthesis of wild-type inositol-ceramides, C(24) and C(26), fatty acids were elevated on incubation at 37 degrees C and declined when the cells shifted to the permissive temperature of 24 degrees C.     

Disorders of pyruvate carboxylase and the pyruvate dehydrogenase complex

Summary The most common defect associated with deficiency of the pyruvate dehydrogenase (PDH) complex occurs in the E₁ component, specifically due to mutations in the X-linked E₁ gene. Clinical sequelae of these mutations, which range from severe neonatal lactic acidosis to carbohydrate-sensitive ataxia, can be different in males and females depending on the nature of the mutation and, in the case of females, on the X-inactivation pattern in different tissues. Males have a high representation of missense mutations among the patient cohort, while females are much more likely to have DNA rearrangements, particularly toward the 3 end of the coding sequence of the gene. Missplicing mutations involving exon 6 deletion have been reported, as has a missense mutation conferring true thiamin-responsiveness of the enzyme and the patient's clinical symptoms.Pyruvate carboxylase deficiency, on the other hand, is a true autosomal recessive disease, though it has high occurrences in particular ethnic groups, especially in Algonkian-speaking Amerindians and in Arabs. In the former group the defect is a simple type in which material cross-reactive to pyruvate carboxylase antibody is present in cultured cells (CRM^+ve). In the latter group, cross-reacting material is rarely present (CRM^–ve). The CRM^+ve patients can survive into teenage years with careful supervision, while the CRM^–ve patients have complications due to hyperammonaemia and dysfunction of the urea cycle and rarely survive beyond 3 months of life.     

Compartmentalization of two forms of acetyl-CoA carboxylase in plants and the origin of their tolerance toward herbicides.

Acetyl-CoA carboxylase (ACCase, EC 6.4.1.2) catalyzes the synthesis of malonyl-CoA, the first intermediate in fatty acid synthesis. We studied the localization of two forms, the prokaryote and the eukaryote forms, of ACCase in pea leaves by comparing the biotin polypeptides of the two ACCases in protein extract from leaves and plastids. We found that the two forms of ACCase were in different cell compartments of pea leaves; the prokaryote form was in the plastids, and the eukaryote form was elsewhere, probably in the cytosol. This result suggested the existence of two sites of malonyl-CoA synthesis. The Gramineae, such as rice and wheat, which lack the accD gene encoding one of the subunits of the prokaryote form of ACCase in their chloroplast genomes, did not have the prokaryote form of the enzyme but had the eukaryote form. The selective grass herbicides of the diphenoxypropionic acid type and the cyclohexanedione type, in vitro, inhibited plastidic ACCase of the eukaryote form from wheat but did not inhibit that of the prokaryote form from pea, suggesting that the origin of the tolerance of intact pea plant toward these herbicides is partly in the insensitivity of the prokaryote form of the enzyme. The origin of the susceptibility of the Gramineae plants toward these herbicides seems to lie in the presence of the herbicide-sensitive eukaryote form and the absence of the insensitive prokaryote form due to the lack of the accD gene in plastid.     

Inhibition of acetyl-CoA carboxylase 2 enhances skeletal muscle fatty acid oxidation and improves whole-body glucose homeostasis in db/db mice

Aims/hypothesis  

Excessive ectopic lipid deposition contributes to impaired insulin action in peripheral tissues and is considered an important link between obesity and type 2 diabetes mellitus. Acetyl-CoA carboxylase 2 (ACC2) is a key regulatory enzyme controlling skeletal muscle mitochondrial fatty acid oxidation; inhibition of ACC2 results in enhanced oxidation of lipids. Several mouse models lacking functional ACC2 have been reported in the literature. However, the phenotypes of the different models are inconclusive with respect to glucose homeostasis and protection from diet-induced obesity.     

Coeliac disease in Middle Eastern countries: a challenge for the evolutionary history of this complex disorder?

About 10,000 years ago domestication and farming of wheat and other cereals developed in the 'Fertile Crescent', an area including modern Turkey, Iraq and Iran. Agriculture then slowly spread from Middle East to Europe. Coeliac disease is the permanent intolerance to dietary gluten, the major protein component of wheat. It has been until relatively recently hypothesised that wheat consumption exerted a negative selective pressure on genes predisposing to coeliac disease, eventually leading to higher coeliac disease frequency in Northeastern Europe because of lack of exposure to cereals. This theory is at variance with recent studies showing that coeliac disease is as common in Middle Eastern countries as in Europe. High prevalence of coeliac disease has been found in Iran, in both the general population and at-risk groups, e.g. patients with irritable bowel syndrome or type 1 diabetes. Clinical manifestations of coeliac disease vary markedly with the age of the patient, the duration and the extent of disease. Clinical studies showed that presentation with non-specific symptoms or no symptoms is as common in the Middle East as in Europe. Wheat represented a major component of the Iranian diet for many centuries and it may be argued that the continuous and high level of exposure to wheat proteins has induced some degree of immune tolerance, leading to milder symptoms that may be misdiagnosed as irritable bowel syndrome or unexplained gastrointestinal disorders. The gluten-free diet represents a real challenge to both patients and clinicians in this area. This is particularly difficult in the absence of any supply for gluten-free diet in Middle Eastern countries.     

Structure of the alpha2epsilon2 Ni-dependent CO dehydrogenase component of the Methanosarcina barkeri acetyl-CoA decarbonylase/synthase complex

Ni-dependent carbon monoxide dehydrogenases (Ni-CODHs) are a diverse family of enzymes that catalyze reversible CO:CO(2) oxidoreductase activity in acetogens, methanogens, and some CO-using bacteria. Crystallography of Ni-CODHs from CO-using bacteria and acetogens has revealed the overall fold of the Ni-CODH core and has suggested structures for the C cluster that mediates CO:CO(2) interconversion. Despite these advances, the mechanism of CO oxidation has remained elusive. Herein, we report the structure of a distinct class of Ni-CODH from methanogenic archaea: the alpha(2)epsilon(2) component from the alpha(8)beta(8)gamma(8)delta(8)epsilon(8) CODH/acetyl-CoA decarbonylase/synthase complex, an enzyme responsible for the majority of biogenic methane production on Earth. The structure of this Ni-CODH component provides support for a hitherto unobserved state in which both CO and H(2)O/OH(-) bind to the Ni and the exogenous FCII iron of the C cluster, respectively, and offers insight into the structures and functional roles of the epsilon-subunit and FeS domain not present in nonmethanogenic Ni-CODHs.