Tony Fawcett

The biochemistry and molecular biology of plant lipid biosynthesis

In the areas of cell biology, biophysics and the biochemistry of signal perception and transduction major advances have come about from an appreciation of the importance of biological membranes. Such membranes not only provide a barrier to the outside of the cell but also define the limits of subcellular organelles contained within them. Subcellular organelles have been fractionated and biochemical compartmentalization of enzyme function deduced by classical work of de Duve and colleagues [16]. Initial electron microscopy observations concentrated on the lipid bilayer description of a biological membrane [18]. Subsequent studies by Singer and Nicholson [114] have led to an appreciation of the importance of the proteins in membranes resulting in the fluid mosaic model. The importance of biological membranes has been additionally highlighted with the development of the chemiosmotic hypothesis of energy generation in biological systems.

Analysis of the structure, substrate specificity, and mechanism of squash glycerol-3-phosphate (1)-acyltransferase.

BACKGROUND Glycerol-3-phosphate (1)-acyltransferase(G3PAT) catalyzes the incorporation of an acyl group from either acyl-acyl carrier proteins (acylACPs) or acyl-CoAs into the sn-1 position of glycerol 3-phosphate to yield 1-acylglycerol-3-phosphate. G3PATs can either be selective, preferentially using the unsaturated fatty acid, oleate (C18:1), as the acyl donor, or nonselective, using either oleate or the saturated fatty acid, palmitate (C16:0), at comparable rates. The differential substrate specificity for saturated versus unsaturated fatty acids seen within this enzyme family has been implicated in the sensitivity of plants to chilling temperatures. RESULTS The three-dimensional structure of recombinant G3PAT from squash chloroplast has been determined to 1.9 A resolution by X-ray crystallography using the technique of multiple isomorphous replacement and provides the first representative structure of an enzyme of this class. CONCLUSIONS The tertiary structure of G3PAT comprises two domains, the larger of which, domain II, features an extensive cleft lined by hydrophobic residues and contains at one end a cluster of positively charged residues flanked by a H(X)(4)D motif, which is conserved amongst many glycerolipid acyltransferases. We predict that these hydrophobic and positively charged residues represent the binding sites for the fatty acyl substrate and the phosphate moiety of the glycerol 3-phosphate, respectively, and that the H(X)(4)D motif is a critical component of the enzymes catalytic machinery.

Fatty Acid and Lipid Biosynthetic Genes Are Expressed at Constant Molar Ratios But Different Absolute Levels during Embryogenesis

In plants, fatty acid and complex lipid synthesis requires the correct spatial and temporal activity of many gene products. Quantitative northern analysis showed that mRNA for the biotin carboxylase subunit of heteromeric acetyl-coenzyme A carboxylase, fatty acid synthase components (3-oxoacyl-acyl carrier protein [ACP] reductase, enoyl-ACP reductase, and acyl-ACP thioesterase), and stearoyl-ACP desaturase accumulate in a coordinate manner duringBrassica napus embryogenesis. The mRNAs were present in a constant molar stoichiometric ratio. Transcript abundance of mRNAs for the catalytic proteins was found to be similar, whereas the number of ACP transcripts was approximately 7-fold higher. The peak of mRNA accumulation of all products was between 20 and 29 d after flowering; by 42 d after flowering, the steady-state levels of all transcripts fell to about 5% of their peak levels, which suggests that the mRNAs have similar stability and kinetics of synthesis. Biotin carboxylase was found to accumulate to a maximum of 59 fmol mg−1 total RNA in embryos, which is in general agreement with the value of 170 fmol mg−1 determined for Arabidopsis siliques (J.S. Ke, T.N. Wen, B.J. Nikolau, E.S. Wurtele [2000] Plant Physiol 122: 1057–1071). Embryos accumulated between 3- and 15-fold more transcripts per unit total RNA than young leaf tissue; the lower quantity of leaf 3-oxoacyl-ACP reductase mRNA was confirmed by reverse transcriptase-polymerase chain reaction. This is in conflict with analysis of B. napus transcripts using an Arabidopsis microarray (T. Girke, J. Todd, S. Ruuska, J. White, C. Benning, J. Ohlrogge [2000] Plant Physiol 124: 1570–1581) where similar leaf to seed levels of fatty acid synthase component mRNAs were reported.

Metabolic control analysis of developing oilseed rape (Brassica napus cv Westar) embryos shows that lipid assembly exerts significant control over oil accumulation.

Metabolic control analysis allows the study of metabolic regulation. We applied both single- and double-manipulation top-down control analysis to examine the control of lipid accumulation in developing oilseed rape (Brassica napus) embryos. The biosynthetic pathway was conceptually divided into two blocks of reactions (fatty acid biosynthesis (Block A), lipid assembly (Block B)) connected by a single system intermediate, the acyl-coenzyme A (acyl-CoA) pool. Single manipulation used exogenous oleate. Triclosan was used to inhibit specifically Block A, whereas diazepam selectively manipulated flux through Block B. Exogenous oleate inhibited the radiolabelling of fatty acids from [1-(14)C]acetate, but stimulated that from [U-14C]glycerol into acyl lipids. The calculation of group flux control coefficients showed that c. 70% of the metabolic control was in the lipid assembly block of reactions. Monte Carlo simulations gave an estimation of the error of the resulting group flux control coefficients as 0.27±0.06 for Block A and 0.73±0.06 for Block B. The two methods of control analysis gave very similar results and showed that Block B reactions were more important under our conditions. This contrasts notably with data from oil palm or olive fruit cultures and is important for efforts to increase oilseed rape lipid yields.

Kinetic mechanism of NADH‐enoyl‐ACP reductase from Brassica napus

Enoyl‐ACP reductase, a component of fatty acid synthase, is a target for anti‐microbial agents and herbicides. Here we demonstrate the kinetic mechanism to be a compulsory‐order ternary complex with NADH binding before the acyl substrate. Matrix‐assisted laser desorption ionisation mass spectrometry analysis of enzymatically and synthesised crotonyl‐ACP substrate showed the former to contain a single acyl group, whereas the latter contained up to four additional crotonylations. The use of authentic crotonyl‐ACP will be important in future kinetic and crystallographic studies.

Functional analyses of differentially expressed isoforms of the Arabidopsis inositol phosphorylceramide synthase.

Sphingolipids are key components of eukaryotic plasma membranes that are involved in many functions, including the formation signal transduction complexes. In addition, these lipid species and their catabolites function as secondary signalling molecules in, amongst other processes, apoptosis. The biosynthetic pathway for the formation of sphingolipid is largely conserved. However, unlike mammalian cells, fungi, protozoa and plants synthesize inositol phosphorylceramide (IPC) as their primary phosphosphingolipid. This key step involves the transfer of the phosphorylinositol group from phosphatidylinositol (PI) to phytoceramide, a process catalysed by IPC synthase in plants and fungi. This enzyme activity is at least partly encoded by the AUR1 gene in the fungi, and recently the distantly related functional orthologue of this gene has been identified in the model plant Arabidopsis. Here we functionally analysed all three predicted Arabidopsis IPC synthases, confirming them as aureobasidin A resistant AUR1p orthologues. Expression profiling revealed that the genes encoding these orthologues are differentially expressed in various tissue types isolated from Arabidopsis.

A homologue of the 65 kDa regulatory subunit of protein phosphatase 2A in early pea (Pisum sativum L.) embryos

A partial cDNA, isolated from an early developing pea (Pisum sativum L.) embryo library, was found to encode a plant homologue of the regulatory subunit (PR65) of protein phosphatase 2A (PP2A). Comparison of the deduced amino acid sequence with a human PR65 sequence showed that the regulatory subunit of PP2A has been highly conserved during evolution. Southern analysis demonstrated that in pea and rape the catalytic and regulatory subunits of PP2A are encoded by multigene families. The levels of the transcripts encoding each subunit are developmentally regulated during pea embryogenesis and expression of the regulatory subunit is not solely restricted to the embryo.

Beta-lactam resistance in Staphylococcus aureus cells that do not require a cell wall for integrity.

ABSTRACT Staphylococcus aureus ATCC 9144 cells with defective cell walls were generated on a medium with elevated osmolality in the presence of sublethal levels of penicillin G. On removal of antibiotic pressure, the cells exhibited stable penicillin and methicillin resistance. The resistance was homogeneous and its acquisition was enhanced following transient cell wall-defective growth. The resistant cells were mecA negative, β-lactamase negative and did not contain any mutations in the coding regions of pbp genes. When penicillin was added back to resistant cells, they continued to grow and produced a diffuse cell wall that was resistant to the action by lysostaphin but was very sensitive to lysis with Triton X-100. These data indicate that the resistant cells are not dependent upon an intact cell wall for osmotic stability and they are able to switch readily to this mode of growth in the presence of penicillin G.

Amino acid sequence analysis of rape seed (Brassica napus) NADH-enoyl ACP reductase

The biosynthesis of fatty acids in plants is catalysed by a type II, dissociable fatty acid synthetase (for review see [ 15]. This enzyme system contains at least seven catalytic domains and a central acyl carrier protein (ACP). The individual catalytic reactions are performed on ACP substrates [6] to which the acyl groups are attached via the 4phosphopantetheine group of the protein. Much is known about the structure of ACP in plants. cDNA [8, 9] and genomic clones have been isolated [3, 5, 7] and the tissue-specific and temporal regulation of the gene has been reported [3]. However, comparatively little is known concerning the structure of catalytic components of the plant fatty acid synthetase complex. Two reductive steps occur in core fatty acid biosynthesis, which are catalysed by enoyl ACP reductase and fl-ketoacyl ACP reductase. Enoyl ACP reductase catalyses the second reductive step in fatty acid biosynthesis and there are two forms of this enzyme, a NADH and a N A D P H enzyme. These are termed type I and type II respectively, and the activities have been separated in Safflower seed [10]. Whilst the type I and type II enzymes are present in seed material, only type I is present in leaf material [ 11 ]. The activity of the type I enoyl ACP reductase has been measured during seed development in oil seed rape. The activity rises prior to major storage lipid synthesis and the shape of the activity profile closely resembles that of lipid deposition [12]. Western blotting has demonstrated that the protein is continually synthesized during lipid deposition, presumably to meet the high demand for fatty acid synthesis in that part of seed development [ 14]. The enzyme is tetrameric and consists of four identical subunits of 33.6 kDa [ 14] . The rape enzyme has two arginine residues which are covalently modified by phenylyglyoxal, following which the enzyme is inactivated. Such inactivation can be protected by pre-incubation with ACP or coenzyme A, indicating that the arginine residues are either at the active site of the enzyme or that substrate binding results in a conformational change which affords protection [2]. This interaction with ACP has been demonstrated further by affinity chromatography of enoyl ACP reductase on an ACP-sepharose column. In this report, we present extensive amino acid sequence data derived from homogeneous NADH-specific enoyl ACP reductase from oil seed rape. Homogeneous 0c-4 NADH-specific enoyl ACP

Fatty acid synthesis in developing leaves of Brassica napus in relation to leaf growth and changes in activity of 3-oxoacyl-ACP reductase

In young expanding leaves of Brassica napus, the demand for fatty acids is met by de novo biosynthesis of fatty acid synthase components, as demonstrated by 3‐oxoacyl‐ACP reductase. Using a novel radio‐chemical assay for 3‐oxoacyl‐ACP reductase and specific antibodies, we have demonstrated a direct relationship between the increase in activity and synthesis of polypeptide. The maximum rate of fatty acid synthesis was between 4 and 7 days post‐emergence, but slowed after this point even though 3‐oxoacyl‐ACP reductase activity was high. Leaf area continued to expand in a linear fashion after reductions in both enzyme activity and the rate of fatty acid synthesis.