Biochemical characterization of mutant propionyl CoA carboxylases from two minor genetic complementation groups

We have biochemically characterized several parameters of propionyl CoA carboxylase (PCC) activity in fibroblast extracts from PCC-deficient patients belonging to the two minor genetic complementation groups, pcc B and pcc BC. Comparison of PCCs from these groups with those of the two major complementation groups, pcc A and pcc C, has demonstrated that PCCs from both the pcc B and pcc BC groups closely resemble each other as well as PCC from the pcc C group. These results further support the hypothesis that the pcc B and pcc BC lines are interallelic with respect to pcc C and consequently that the structural mutations in the PCCs from these groups involve the same subunit.     

Evaluation of biotin responsiveness in cultured fibroblasts from patients with propionic acidemia: Absence of response by structurally altered carboxylases

We have demonstrated that, although propionyl CoA carboxylase (PCC) activity is deficient in fibroblast extracts from PCC-deficient patients belonging to the two major and two minor genetic complementation groups, the activity of another biotin-dependent carboxylase, -methylcrotonyl CoA carboxylase (MCC), is normal. Moreover, MCC activity is stimulated when the fibroblasts are cultured in high concentrations of biotin, in the same way that it is in normal fibroblasts, whereas the depressed PCC activity remains essentially unchanged. Because these results are parallel with the in vivo failure of high-dose biotin to stimulate PCC activity in peripheral blood leukocytes, we conclude that the biotin responsiveness of PCC in cultured fibroblasts from patients with PCC deficiency may be used to predict or confirm biotin responsiveness in vivo.     

Absence of cross-reacting material in isolated propionyl CoA carboxylase deficiency: nature of residual carboxylating activity.

Fibroblast extracts and fetal liver homogenates from patients with propionic acidemia due to inherited deficiency of propionyl CoA carboxylase (PCC) were analyzed for the presence of immunologically cross-reactive PCC protein. Using several rabbit antisera raised against homogeneous human liver PCC, homogeneous pig heart PCC, or the individual non-identical subunits of the human liver enzyme, we found no detectable cross-reacting material by direct or competitive immunotitration in several cell lines from patients in either major complementation group (pcc A; pcc C) with isolated PCC deficiency. In contrast, cells of a patient from the bio complementation group contained normal amounts of immunoreactive PCC. Further analysis of the pcc A and pcc C mutants revealed that their residual propionyl CoA carboxylating activity varied greatly depending on the concentration of extract or homogenate protein used in the PCC assay. When propionyl CoA carboxylation was assayed at high protein concentration in a fetal liver homogenate from a pcc C patient, the apparent PCC activity was comparable to that found in normal human fetal liver. Significantly, the specific activity in the mutant, but not in the control, extract declined steeply as protein concentration was lowered, and this loss could not be prevented by adding PCC substrates, bovine serum albumin, glycerol, or 2-mercaptoethanol. Moreover, detailed analyses of immunotitration curves of control fibroblasts extracts showed that fresh extracts contained an amount of nonimmunotitratable carboxylating activity corresponding to the residual activity present in fresh extracts of mutant cell lines. We conclude that the residual propionyl CoA carboxylating activity found in isolated PCC deficiency represents another carboxylase that can utilize propionyl CoA as a substrate rather than a mutant form of PCC with markedly different immunochemical and physicochemical properties.     

Kinetic analysis of genetic complementation in heterokaryons of propionyl CoA carboxylase-deficient human fibroblasts.

We studied genetic complementation of propionyl CoA carboxylase (PCC) deficiency in cultures of polyethylene glycol (PEG)-induced heterokaryons, using mutant fibroblast lines assigned to five mutant classes, designated bio, pcc A, pcc B, pcc C, and pcc BC. By measuring PCC activity directly in extracts of fused cells or indirectly in intact cells by [1-14C]propionate utilization, we confirmed the nonlinear nature of the PCC deficiency complementation map described by Gravel et al. [1]. When we studied the kinetics of complementation, we detected three distinct patterns using the [1-14C]propionate utilization assay. When either pcc A or pcc C lines were fused to bio cells, 14C-fixation increased to half of the maximally restored values within 4 hrs. In pcc A x pcc C crosses or in pcc A x pcc B crosses, however, complementation was much slower. In fusions between pcc B and pcc C cells, a third pattern was elicited; complementation was incomplete, maximum restoration of PCC activity begin less than 20% of that observed in other complementing crosses. From these data and previous biochemical evidence, we suggest (1) that the bio and pcc mutations affect different genes; (2) that complementation between pcc A and either pcc B, pcc C, or pcc BC lines is intergenic and involves subunit exchange and synthesis of new PCC molecules; and (3) that complementation between pcc B and pcc C mutants is interallelic.     

Biochemical evidence for diverse etiologies in biotin-responsive multiple carboxylase deficiency

Biotin-responsive multiple carboxylase deficiency can be categorized by clinical criteria into a neonatal-onset disorder and a distinct syndrome of infantile onset. Pedigrees in each instance are consistent with autosomal recessive inheritance. For a neonatal-onset proband, the sensitivity to relative biotin deprivation and the rapid clinical response to biotin supplementation are reflected by in vitro studies. Specific activities of biotin-dependent pyruvate carboxylase, propionyl CoA carboxylase, and 3-methylcrotonyl CoA carboxylase are 0.8 to 16% of mean control values after growth of fibroblasts in intermediate and very low biotin concentrations. Following relative biotin depletion, pyruvate carboxylase activity returns to normal after only 14 hr of growth in biotin-supplemented medium. In contrast, carboxylase activities in fibroblasts of an infantile-onset proband remain normal at very low biotin concentrations, even when avidin is added to the growth medium. The clinical heterogeneity, taken together with the distinct responses of cultured skin fibroblasts to biotin deprivation in vitro, probably reflect fundamentally different etiologies for the two categories of biotin-responsive multiple carboxylase deficiency.This work was supported by USPHS Grants GM28838 and AM25884.     

Investigating the role of native propionyl‐CoA and methylmalonyl‐CoA metabolism on heterologous polyketide production in Escherichia coli

6‐Deoxyerythronolide B (6dEB) is the macrocyclic aglycone precursor of the antibiotic natural product erythromycin. Heterologous production of 6dEB in Escherichia coli was accomplished, in part, by designed over‐expression of a native prpE gene (encoding a propionyl‐CoA synthetase) and heterologous pcc genes (encoding a propionyl‐CoA carboxylase) to supply the needed propionyl‐CoA and (2S)‐methylmalonyl‐CoA biosynthetic substrates. Separate E. coli metabolism includes three enzymes, Sbm (a methylmalonyl‐CoA mutase), YgfG (a methylmalonyl‐CoA decarboxylase), and YgfH (a propionyl‐CoA:succinate CoA transferase), also involved in propionyl‐CoA and methylmalonyl‐CoA metabolism. In this study, the sbm, ygfG, and ygfH genes were individually deleted and over‐expressed to investigate their effect on heterologous 6dEB production. Our results indicate that the deletion and over‐expression of sbm did not influence 6dEB production; ygfG over‐expression reduced 6dEB production by fourfold while ygfH deletion increased 6dEB titers from 65 to 129 mg/L in shake flask experiments. It was also found that native E. coli metabolism could support 6dEB biosynthesis in the absence of exogenous propionate and the substrate provision pcc genes. Lastly, the effect of the ygfH deletion was tested in batch bioreactor cultures in which 6dEB titers improved from 206 to 527 mg/L. Biotechnol. Bioeng. 2010; 105: 567–573. © 2009 Wiley Periodicals, Inc.     

Essential arginine residues in the active sites of propionyl CoA carboxylase and beta-methylcrotonyl CoA carboxylase

At least one arginine residue is essential for substrate binding in or near the active sites of propionyl CoA carboxylase (PCC) and beta-methylcrotonyl CoA carboxylase (beta MCC) in cultured human fibroblasts. This conclusion is based on studies of enzyme inhibition by phenylglyoxal, a reagent which specifically modifies arginine residues. Human fibroblast PCC both in extracts and in a 20-fold purified preparation was nearly completely protected from phenylglyoxal inhibition following incubation with propionyl CoA or ATP. It appears that a phosphate group from either ATP or the CoA moiety of propionyl CoA reacts with the essential arginine residue(s). beta MCC which was similarly inhibited by phenylglyoxal was protected by beta-methylcrotonyl CoA and ATP. Thus phenylglyoxal may be used to label specific arginine residues within the active sites of previously sequenced carboxylases.     

Molecular basis for genetic complementation in propionyl CoA carboxylase deficiency

The extent of genetic complementation in polyethylene glycol-induced heterokaryons of propionyl CoA carboxylase deficient fibroblast lines was determined by comparing enzyme activity changes over time in pairwise fusions of the three major complementation groups, bio, pcc A and pcc C, with the activity changes in similarly mixed but unfused cultures. Maximum complementation between bio and pcc. A or pcc C lines was attained within 24 h after fusion and was not inhibited by cycloheximide. In contrast, the complementation between pcc A and pcc C lines only attained 50% of the maximum restored carboxylase activity by 24–36 h and the increase was 93% inhibited by cycloheximide. Maximum restoration of activity was not achieved until 72–96 h after fusion. Removal of cycloheximide at 24 h permitted complementation to take place. Our studies suggest that intergenic complementation between the bio and pcc A or pcc C lines is due to the contribution within the heterokaryon of normal enzymes from each of the respective lines, resulting in almost immediate, protein synthesis-independent, partial restoration of carboxylase activity. Complementation between pcc A and pcc C lines also appears to be intergenic but probably results from the de novo synthesis of normal subunits and protomers which assemble into normal or stabilized propionyl CoA carboxylase molecules with restored enzyme activity.     

Purification and characterization of a <Emphasis Type="Italic">Galactomyces reessii</Emphasis> hydratase that converts 3-methylcrotonic acid to 3-hydroxy-3-methylbutyric acid

Cell free extracts of Galactomyces reessii contain a hydratase as the key enzyme for the transformation of 3-methylcrotonic acid to 3-hydroxy-3-methylbutyric acid. Highest levels of hydratase activity were obtained during growth on isovaleric acid. The enzyme, an enoyl CoA hydratase, was purified 147-fold by precipitation with ammonium sulphate and successive chromatography over columns of DE-52, Blue Sepharose CL-6B and Sephacryl S-200. During purification, hydratase activity was measured spectrophotometrically (OD change at 263 nm) for 3-methylcrotonyl CoA and crotonyl CoA as substrates. The enzyme displayed highest activity with crotonyl CoA with a K _cat of 1,050,000 min⁻¹. The ratio of crotonyl CoA to 3-methylcrotonyl CoA activities was constant (20:1) during all steps of purification. The K _cat for crotonyl CoA was also about 20 times greater than the K _cat for 3-methylcrotonyl CoA (51,700 min⁻¹). The enzyme had pH and temperature optima at 7.0 and 35°C, a native M _r of 260±4.5 kDa and a subunit M _r of 65 kDa, suggesting that the enzyme was a homotetramer. The pI of the purified hydratase was 5.5, and the N-terminal amino acid sequence was VPEGYAEDLLKGKMMRFFDS. Hydratase activity for 3-methylcrotonyl CoA was competitively inhibited by acetyl CoA, propionyl CoA and acetoacetyl CoA. Journal of Industrial Microbiology & Biotechnology (2002) 28, 81–87 DOI: 10.1038/sj/jim/7000215 Received 27 June 2001/ Accepted in revised form 17 September 2001     

Biochemical characterization of biotin-responsive multiple carboxylase deficiency: heterogeneity within the bio genetic complementation group

Three biotin-dependent enzymes, pyruvate carboxylase (PC), propionyl CoA carboxylase (PCC), and beta-methylcrotonyl CoA carboxylase (beta MCC), were biochemically characterized in fibroblasts from two patients with neonatal multiple carboxylase deficiency. Genetic complementation analyses indicated that both cell lines, designated lines 1 and 2, were deficient in the various carboxylase activities and belonged to the bio complementation group. The activities of the three carboxylases became normal when line 2 cells were incubated in medium supplemented with biotin (1 mg/l) for 24 hrs, whereas 4-6 days were required to achieve maximum activities of PC, PCC, and beta MCC (57%, 46%, and 29% of mean normal enzyme activity, respectively) in line 1 cells incubated in medium containing up to 10 mg/1 biotin. Furthermore, PC activity in line 2 continued to increase under apparent gluconeogenic conditions in culture, but not in line 1. Thermostability studies suggested that biotin stabilizes PC and beta MCC in both cell lines. PC in line 1 cells incubated with or without biotin was less stable than that in normal or line 2 cells, and the less than normal increase of enzyme activities in line 1, especially that of PC, may represent incomplete biotination. These results indicate that there is biochemical heterogeneity within the bio complementation group. Immunotitration with antibodies prepared against purified pig heart PCC demonstrated normal quantities of cross-reacting material in both lines and no differences in the amount of this material after incubation with supplemental biotin, despite the seven- to 20-fold increase in PCC activity. Thus, the increase in carboxylase activity in both bio lines appears to represent activation of rpe-existing apocarboxylase rather than de novo enzyme synthesis. The primary defect in this form of multiple carboxylase deficiency may be in a common holocarboxylase synthetase or in biotin transport. If the defect is in the synthetase, the differences noted between the two bio lines could be explained by a difference in the enzyme's Km for biotin.     

Evidence for two genetic complementation groups in pyruvate carboxylase-deficient human fibroblast cell lines

We have examined genetic complementation in pyruvate carboxylase deficiency by comparing the enzyme activity in polyethylene glycol-induced heterokaryons with that in unfused mixtures of fibroblasts from three affected children. Complementation, manifested as a three- to sevenfold increase in pyruvate carboxylase activity, was observed in fusions between a biotin-responsive multiple carboxylase (pyruvate carboxylase, propionyl CoA carboxylase, and -methylcrotonyl CoA carboxylase) deficient fibroblast line and two other lines deficient only in pyruvate carboxylase activity. Kinetic analysis of complementing pyruvate carboxylase deficient lines, measured by the rate of restoration of enzyme activity as a function of time, revealed that maximum restoration was achieved within 10–24 hr after fusion. This profile is similar to those observed for fusions between the multiple carboxylase deficient line and two lines deficient in propionyl CoA carboxylase activity that are known to represent different gene mutations. Although the patients with pyruvate carboxylase deficiency had similar clinical findings, our studies indicate that pyruvate carboxylase deficiency is genetically heterogeneous, with at least two distinct, probably intergenic, complementation groups.This work was supported by an NIH research grant (AM 25675) and an A. D. Williams research grant (6-48360). B. Wolf is the recipient of an NIH Research Career Development Award (AM 00677) and is aided by a Basil O'Connor Starter Research Grant from The National Foundation-March of Dimes (5-263). G. Feldman is the recipient of an NIH predoctoral training grant (GM 07492). This article is No. 100 from the Department of Human Genetics at the Medical College of Virginia.     

LC-MS/MS quantification of short-chain acyl-CoA's in Escherichia coli demonstrates versatile propionyl-CoA synthetase substrate specificity

Aims: This paper utilized quantitative LC‐MS/MS to profile the short‐chain acyl‐CoA levels of several strains of Escherichia coli engineered for heterologous polyketide production. To further compare and potentially expand the levels of available acyl‐CoA molecules, a propionyl‐CoA synthetase gene from Ralstonia solanacearum (prpE‐RS) was synthesized and expressed in the engineered strain BAP1. Methods and Results: Upon feeding propionate, the engineered E. coli strains had increased the levels of both propionyl‐ and methylmalonyl‐CoA of 6‐ to 30‐fold and 3·7‐ to 6·8‐fold, respectively. Expression of prpE‐RS resulted in no significant increases in acetyl‐, butyryl‐ and propionyl‐CoA when fed the corresponding substrates (sodium acetate, butyrate or propionate). More interesting, however, were the results from strain BAP1 engineered for native prpE overexpression, which indicated increases in the same range of acyl‐CoA formation. Conclusions: The increased acyl‐CoA levels across the strains profiled in this study reflect the genetic modifications implemented for improved polyketide production and also indicate flexibility of the native PrpE. Significance and Impact of the Study: The results provide direct evidence of enhanced acyl‐CoA levels correlating to those strains engineered for polyketide biosynthesis. This information and the inherent flexibility of the native PrpE enzyme support future efforts to characterize, engineer and extend acyl‐CoA precursor supply for additional heterologous biosynthetic attempts.     

Genetics and ontogeny of butyryl CoA dehydrogenase in the mouse and linkage of Bcd-1 with Dao-1

A zymogram method has been developed for fatty acyl CoA dehydrogenase and used to examine the electrophoretic properties of butyryl CoA dehydrogenase (BCD) from mouse tissues. A single form of BCD is present in extracts of liver, kidney, heart, and intestine. Ontogenetic, tissue distribution, and subcellular fractionation results are consistent with the mitochondrial origin previously reported for this enzyme. A genetic variant for BCD-1 was used to provide evidence for a locus determining the electrophoretic properties of this enzyme (designated Bcd-1), which is linked to Dao-1 (encoding d-amino acid oxidase).This research was funded in part by the Australian Research Grants Committee.     

Regulation of Acetyl CoA Carboxylase and Carnitine Palmitoyl Transferase‐1 in Rat Adipocytes

Objective: Acetyl CoA carboxylase (ACC) is a key enzyme in energy balance. It controls the synthesis of malonyl‐CoA, an allosteric inhibitor of carnitine palmitoyltransferase‐1 (CPT‐I). CPT‐I is the gatekeeper of free fatty acid (FFA) oxidation. To test the hypothesis that both enzymes play critical roles in regulation of FFA partitioning in adipocytes, we compared enzyme mRNA expression and specific activity from fed, fasted, and diabetic rats. Research Methods and Procedures: Direct effects of nutritional state, insulin, and FFAs on CPT‐I and ACC mRNA expression were assessed in adipocytes, liver, and cultured adipose tissue explants. We also determined FFA partitioning in adipocytes from donors exposed to different nutritional conditions. Results: CPT‐I mRNA and activity decreased in adipocytes but increased in liver in response to fasting. ACC mRNA and activity decreased in both adipocytes and liver during fasting. These changes were not caused directly by fasting‐associated changes in plasma insulin and FFA concentrations because insulin suppressed CPT‐I mRNA and did not affect ACC mRNA in vitro, whereas exogenous oleate had no effect on either. Despite the decrease in adipocyte CPT‐I mRNA and specific activity, CO₂ production from endogenous FFAs increased, suggesting increased FFA transport through CPT‐I for β‐oxidation. Discussion: Stimulation of FFA transport through CPT‐I occurs in both tissues, but CPT‐I mRNA and specific activity correlate with FFA transport in liver and not in adipocytes. We conclude that the mechanism responsible for increasing FFA oxidation in adipose tissue during fasting involves mainly allosteric regulation, whereas altered gene expression may play a central role in the liver.     

Hydrogen-stimulated CO2 fixation and coordinate induction of hydrogenase and ribulosebiphosphate carboxylase in a H2-uptake positive strain of Rhizobium japonicum

H₂-uptake positive strains (122 DES and SR) and H₂-uptake negative strains SR2 and SR3 of Rhizobium japonicum were examined for ribulosebisphosphate (RuBP) carboxylase and H₂-uptake activities during growth conditions which induced formation of the hydrogenase system. The rate of ¹⁴CO₂ uptake by hydrogenase-derepressed cells was about 6-times greater in the presence than in the absence of H₂. RuBP carboxylase activity was observed in free-living R. japonicum strains 122 DES or SR only when the cells were derepressed for their hydrogenase system. Hydrogenase and RuBP carboxylase activities were coordinately induced by H₂ and both were repressed by added succinate. Hydrogenase-negative mutant strains SR2 and SR3 derived from R. japonicum SR showed no detecyable RuBP carboxylase activities under hydrogenase derepression conditions. No detectable RuBP carboxylase was observed in bacteroids formed by H₂-uptake positive strains R. japonicum 122 DES or SR. Propionyl CoA carboxylase activity was consistently observed in extracts of cells from free-living cultures of R. japonicum but activity was not appreciably influenced by the addition of H₂. Neither phosphoenolpyruvate carboxylase nor phosphoenolpyruvate carboxykinase activity was detected in extracts of R. japonicum.Abbreviations RuBP Ribulose 1,5-bisphosphate - (Na₂EDTA) (Ethylenedinitrilo)-tetraacetic acid, disodium salt - (propionyl CoA) Propionyl coenzyme A - (PEP) Phosphoenolpyruvate - (GSH) Reduced glutathione - (Tricine) N-tris(hydroxymethyl)-methylglycine     

A spectrophotometric assay for measuring acetyl–coenzyme A carboxylase

Acetyl–coenzyme A (CoA) carboxylase catalyzes the first step in the biosynthesis of fatty acids in bacteria and eukaryota. This enzyme is the target of drug design for treatment of human metabolic diseases and of herbicides acting specifically on the eukaryotic form of the enzyme in grasses. Acetyl–CoA carboxylase activity screening in drug and herbicide design depends mostly on a time-consuming enzyme assay that is based on the incorporation of radiolabeled bicarbonate into the product malonyl–CoA. Here we describe a new simple, continuous, and quick photometric assay avoiding radioactive substrate. It couples the carboxylation of acetyl–CoA to the nicotinamide adenine dinucleotide phosphate (NADPH)-dependent reduction of malonyl–CoA, which is catalyzed by recombinant malonyl–CoA reductase of Chloroflexus aurantiacus. This assay can be adapted for high-throughput screening.     

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.     

ErbB-2 Induces Bilateral Adrenal Pheochromocytoma Formation in Mice

Pheochromocytoma (PCC) is a rare catecholamine-producing tumor that arises from the adrenal medulla and is often familial. The genetic basis for familial PCC involves mutations of RET, VHL, SHDx or NF-1 in more than 20% of cases. Additional genes may be important in pathogenesis of both familial and sporadic PCC. ErbB-2/Her2/Neu is a growth factor receptor tyrosine kinase that is frequently overexpressed in tumors and there is clinical evidence suggesting that enhanced ErbB-2 growth factor receptor signaling may play a role in PCC. In the present study, ectopic expression of an activated ErbB-2 transgene resulted in bilateral adrenal PCC. Analyses of tumor samples and normal adrenal tissue revealed that levels of the Pten tumor suppressor protein were greatly reduced in PCCs, while levels of the cell cycle regulatory protein cyclin D1 were usually increased. In addition, levels of phospo-AKT were increased in PCCs versus normal adrenal tissue. Biochemical analyses established that PCC’s were functionally active, producing abundant levels of the catecholamines, epinephrine and norepinephrine. These data establish that increased ErbB-2 growth factor receptor signaling in the adrenal medulla can lead to PCC through combined influences on Pten, AKT and cyclin     

Acetyl coenzyme A carboxylase in species of Triticum of different ploidy

The cellular amounts and cellular activities of acetyl CoA carboxylase (ACC; EC 6.4.1.2.) were determined in the first leaves of diploid, tetraploid and hexaploid species of Triticum (wheat). Per leaf the ACC activities were very similar in T. monococcum (2 ), T. dicoccum (4 ) and T. aestivum (6 ). The ACC activity per chloroplast also showed little variation between species of different ploidy but since chloroplast number increases with ploidy, the ACC activities and ACC amounts per cell also increased with ploidy. These cellular increases in ACC amounts associated with increases in gene dosage were highly co-ordinated in the diploids T. monococcum and T. tauschii and their respective autotetraploids so the specific activity of ACC was highly conserved in these plants. The relevance of these findings to attempts to genetically manipulate lipid biosynthesis in chloroplasts is discussed.Abbreviation ACC acetyl CoA carboxylase We are very grateful to Dr. Kevin Pyke and Miss Jo Marrison for many helpful discussions and to Dr. Collin Law for the generous gift of seeds.