Alun D. McCarthy

Fatty acid synthase- an example of protein evolution by gene fusion

Abstract Although the mechanism of fatty acid synthesis is essentially the same in all organisms, the fatty acid synthase complex exists in a remarkable variety of structural forms in different organisms. This demonstrates how new proteins can arise during evolution by fusion of genes coding for smaller, existing proteins.

Effect of squalestatin 1 on the biosynthesis of the mevalonate pathway lipids

The effects of squalestatin 1 on rat brain and liver homogenates and on Chinese hamster ovary tissue culture cells have been investigated. This compound effectively inhibits squalene biosynthesis in a highly selective manner. Cytoplasmic farnesyl pyrophosphate and geranylgeranyl pyrophosphate synthases are not affected, which is also the case for microsomal cis-prenyltransferase. In tissue culture cells, squalestatin 1 inhibits cholesterol biosynthesis completely, but does not alter dolichol synthesis or protein isoprenylation to a great extent. Incorporation of [3H]mevalonate into ubiquinone-9 and -10 increases 3-4-fold, probably as a result of increased synthesis of this lipid. Squalestatin 1 appears not only to be an effective inhibitor of cholesterol biosynthesis, but also to be more specific than other inhibitors used earlier in various in vitro and in vivo systems.

Esterification and absorption of cholesterol: in vitro and in vivo observations in the rat

Activity of the enzyme acyl-CoA:cholesterol acyltransferase (ACAT) in isolated rat enterocytes was reduced by approx. 75% following a single oral dose of Sandoz compound 58-035 (30 mg.kg-1). Despite this, the formation of [14C]cholesteryl esters from [1-14C]oleic acid remained unaffected in ACAT-inhibited cell preparations. The increase in serum cholesterol concentrations observed after overnight cholesterol/cholic acid (1%/0.5%) feeding to rats was abolished by pre-treatment with Sandoz compound 58-035 (30 mg.kg-1). These results can be reconciled with a previously proposed model for the transmembrane movement of cholesterol which implicates ACAT-independent esterification and hydrolysis as a transport mechanism for the movement of cholesterol across the enterocyte apical membrane.

Reaction of chloroacetyl-CoA with rabbit fatty acid synthase: A new method to label specifically and quantify pantetheine prosthetic groups

The substrate analogue chloroacetyl‐CoA inhibits fatty acid synthase by reacting with the ‘central’ or pantetheine thiol and not the ‘peripheral’ or β‐ketoacylsynthase thiol as previously reported. This was demonstrated by the isolation of [14C]carboxymethylcysteamine after acid hydrolysis of enzyme labelled with chloro[14C]acetyl‐CoA, and by the demonstration that more than one of the partial reactions is inhibited. This reagent now represents a simple and convenient tool both for quantification of the pantetheine thiol and for labelling this site for peptide mapping and isolation.

Amino acid sequence around the active serine in the acyl transferase domain of rabbit mammary fatty acid synthase

Rabbit mammary fatty acid synthase was labelled in the acyl transferase domain(s) by the formation of the O‐ester intermediates after incubation with [14C]acetyl‐ or malonyl‐CoA. Elastase peptides containing the labelled acyl groups were isolated using high performance liquid chromatography and sequenced by fast atom bombardment mass spectrometry. An identical peptide (acyl‐Ser‐Leu‐Gly‐Glu‐Val‐Ala) was obtained after labelling with acetyl‐ or malonyl‐CoA. This confirms the hypothesis that, unlike Escherichia coli or yeast, a single transferase catalyses the transfer of both acetyl‐ and malonyl‐groups in the mammalian complex. The sequence at this site is compared with that around the active serine in other acyl transferases and hydrolases.

Determination of lipid hydroperoxides in native low-density lipoprotein by a chemiluminescent flow-injection assay

Lipid hydroperoxides have been implicated in the pathogenesis of atherosclerosis. This work was therefore set up to obtain a fast and specific chemiluminescent assay for measuring hydroperoxides in native low-density lipoprotein (LDL). The apparatus was a complete HPLC system including two pumps, an autosampler, a computer and a chemiluminescent detector with a T-mixing coil in the place of the column. Samples were injected from the autosampler and mixed with luminescent reagent (3 microM luminol and 1 microM microperoxidase in 0.1 M carbonate buffer (pH 10)) in the T-piece. To generate a calibration curve, linoleic acid hydroperoxide was obtained by incubating soybean lipoxygenase with linoleic acid. The calculated conjugated diene concentration was in good agreement with the nominal linoleic acid hydroperoxide concentration. The chemiluminescence was linear with the amount of linoleic acid hydroperoxide injected and the detection limit was about 3 pmol linoleic acid hydroperoxide. The chemiluminescence induced by copper-oxidized LDL was linear with concentration; the detection limit, when compared with linoleic acid hydroperoxide, was similar. The reproducibility of the linoleic acid hydroperoxide and of oxidized LDL hydroperoxide was examined in single pools. The coefficient of variation on the triplicates of each pool was about 3%. The titre of the linoleic acid hydroperoxide and oxidized LDL peroxides was quite stable for at least 10 days when stored under argon at 4 degrees C in the presence of EDTA. The mean value of the LDL hydroperoxides in 16 control subjects was 145.20 +/- 98.81 pmol/mg LDL protein. In conclusion, the microperoxidase-luminol-dependent chemiluminescence flow-injection assay is a rapid, sensitive and selective method for measuring lipid hydroperoxides in native LDL.

Amino acid sequence around the reactive serine residue of the thioesterase domain of rabbit fatty acid synthase

Two thermolytic peptides containing the reactive serine residue of the thioesterase domain of rabbit fatty acid synthase have been isolated and sequenched by Edman degradation and fast atom bombardment mass spectrometry. The sequence (V-A-G-Y-S-Y-G) contains the motif G-X-S-X-G found around the reactive serine residue of all known serine proteinases and esterases.

Evidence that the multifunctional polypeptides of vertebrate and fungal fatty acid synthases have arisen by independent gene fusion events

The enoyl reductase (NADPH binding site) of rabbit mammary fatty acid synthase has been radioactively labelled using pyridoxal phosphate and sodium [3H]borohydride. Using this method we have been able to add this site to the four sites whose location has already been mapped within the multifunctional polypeptide chain of the protein. The results show that the enoyl reductase lies between the 3‐oxoacylsynthase and the acyl carrier. This confirms that the active sites occur in a different order on the single multifunctional polypeptide of vertebrate fatty acid synthase and the two multifunctional polypeptides of fungal fatty acid synthase, and suggests that these two systems have arisen by independent gene fusion events.

Evidence that the acyl-O-esters are intermediates in the catalysis. The mechanism of rabbit mammary fatty acid synthase.

The sequence acetyl‐CoA → acetyl‐O‐enzyme → acetyl‐S‐acyl carrier protein has for the first time been demonstrated directly with a multifunctional (mammalian) fatty acid synthase. This was achieved by blocking of the active‐site thiols of rabbit mammary fatty acid synthase with iodoacetamide. The modified enzyme was incubated with [14C]acetyl‐CoA to form acetyl‐O‐enzyme, and acetyl‐CoA was removed rapidly by centrifuge desalting. We were then able to demonstrate transfer of the acetyl group from [14C]acetyl‐O‐enzyme to the pantetheine thiol in a fragment of rabbit mammary fatty acid synthase containing the phosphopantetheine group, and to E. coli acyl carrier protein.