Metabolism of 3-deoxy-3-fluoro-D-mannose and 4-deoxy-4-fluoro-D-mannose by Saccharomyces cerevisiae S288C.

Incubation of Saccharomyces cerevisiae S288C with 4-deoxy-4-fluoro-D-[1-14C]-mannose resulted in the formation of three metabolites that were characterized as 4-deoxy-4-fluoro-D-[1-14C]mannose 1,6-bisphosphate, 4-deoxy-4-fluoro-D-[1-14C]-mannose 6-phosphate and GDP-4-deoxy-4-fluoro-D-[1-14C]mannose. In addition, radioactive material was incorporated into a particulate fraction composed primarily of cell-wall polysaccharides. Compared with the 4-fluoro sugar, 3-deoxy-3-fluoro-D-[1-14C]mannose was not transported into yeast cells as well, and its conversion into sugar nucleotide was much less efficient. Metabolites that were isolated after incubation with the 3-fluoro analogue were identified as 3-deoxy-3-fluoro-D-[1-14C]mannose 1,6-bisphosphate, 3-deoxy-3-fluoro-D-[1-14C]mannose 6-phosphate and GDP-3-deoxy-3-fluoro-D-[1-14C]mannose. Little radioactivity was transferred into the cell-wall fraction.     

In vivo metabolism of 3-deoxy-3-fluoro-D-glucose

Recent studies have demonstrated that 3-deoxy-3-fluoro-D-glucose (3-FG) is metabolized to 3-deoxy-3-fluoro-D-sorbitol (3-FS), via aldose reductase, and 3-deoxy-3-fluoro-D-fructose (3-FF), via the sorbitol dehydrogenase reaction with 3-FS, in rat cerebral tissue (Kwee, I. L., Nakada, T., and Card, P. J. (1987) J. Neurochem. 49, 428-433). However, the biochemistry of 3-FG in other mammalian organs has not been investigated making the application of 3-FG as a metabolic tracer uncertain. To address this issue we investigated 3-FG metabolism and distribution in isolated cell lines and in rabbit tissues in vivo with 19F NMR and gas chromatography-mass spectrometry. In general, the production of 3-FS is well correlated with the known distribution of aldose reductase in all the systems studied. Further metabolism of 3-FS to 3-FF was verified to occur in cerebral tissue. Surprisingly, two new fluorinated compounds were found in the liver and kidney cortex. These compounds are identified as 3-deoxy-3-fluoro-D-gluconic acid, which is produced via glucose dehydrogenase activity on 3-FG, and 3-deoxy-3-fluoro-D-gluconate-6-phosphate. Based on enzyme studies, it is argued that the 3-deoxy-3-fluoro-D-gluconate-6-phosphate is derived directly from 3-deoxy-3-fluoro-D-gluconic acid and not as a product of pentose phosphate activity. Direct oxidation and reduction are the major metabolic routes of 3-FG, not metabolism through glycolysis or the pentose phosphate shunt. Thus, 3-FG metabolism coupled with 19F NMR appears to be very useful for monitoring aldose reductase and glucose dehydrogenase activity in vivo.     

Synthesis of 1-deoxy-3-C-methyl-d-fructose and 1-deoxy-3-C-methyl-d-sorbose

Hydroxylation of trans-1,3,4-trideoxy-5,6-O-isopropylidene-3-C-methyl-d-glycero-hex-3-enulose with osmium tetraoxide gave a mixture of 1-deoxy-5,6-O-isopropylidene-3-C-methyl-d-arabino- and -d-xylo-hexulose that was partially resolved by acetonation to give 1-deoxy-2,3:4,5-di-O-isopropylidene-3-C-methyl-β-d-fructopyranose (4), 1-deoxy-3,4:5,6-di-O-isopropylidene-3-C-methyl-keto-d-fructose (5), and 1-deoxy-2,3:4,6-di-O-isopropylidene-3-C-methyl-α-d-sorbofuranose (6). Treatment of a mixture of 4 and 5 with sodium borohydride gave, after column chromatography, 4 and 1-deoxy-3,4:5,6-di-O-isopropylidene-3-C-methyl-d-manno- and -d-gluco-hexitol. Deuterated derivatives corresponding to 4–6 were obtained when isopropylidenation was carried out with acetone-d₆. Deacetonation of 4 and 5 yielded 1-deoxy-3-C-methyl-d-fructose, and 6 similarly afforded 1-deoxy-3-C-methyl-d-sorbose.     

Higher plants express 3-deoxy-D-manno-octulosonate 8-phosphate synthase

Abstract. The enzymatic activity of 3-deoxy- D-manno -octulosonate 8-phosphate (KDOP) synthase was detected in eight diverse plant species, thus providing enzymological data consistent with recent reports of the presence of 3-deoxy- D-manno -octulosonate in plant cell walls. KDOP synthase from spinach was partially purified and characterized. It possessed weak activity as 3-deoxy- D-arabino -heptulosonate 7-phosphate (DAHP) synthase. In the presence of phosphoenolpyruvate, which conferred dramatic thermostability, KDOP synthase had a catalytic temperature optimum of about 53°C. The pH optimum was 6.2, and divalent cations were neither stimulatory nor required for activity. The Km values for arabinose 5-P and phosphoenolpyruvate were 0.27 mol m⁻³ and about 35 mmol m⁻³, respectively. The kinetics of periodate oxidation of KDOP formed by spinach KDOP synthase indicate that the same stereochemical configuration exists as with bacterial KDOP. The possibility that an unregulated species of DAHP synthase found in some bacteria might in fact be a KDOP synthase exhibiting substrate ambiguity of the type seen in higher plants was examined. However, the DAHP synthase isozyme, DS-O, from Acinetobacter calcoaceticus was found to be specific for erythrose 4-P. The KDOP synthase of Acinetobacter calcoaceticus was also found to be specific for arabinose 5-P.     

Preparation of 3-deoxy-3-deuteroestrone and 3-deoxy-4-14C-estrone

3-Deoxy-3-deuteroestrone (1,3,5(10)-estratrien-17-one-3-d) and 3-deoxy-4-¹⁴C-estrone (1 ,3 ,5(10)-estratrien-17-one-¹⁴C) have been prepared. The mechanism of reductive dehalogenation of aryl iodides with lithium aluminum deuteride is discussed. The ¹³C-NMR spectrum of 3-deoxy-3-deuteroestrone is discussed.     

Metabolic pathway of 2-deoxy-2-fluoro-D-glucose studied by F-19 NMR

The behavior of 2-deoxy-2-fluoro-D-glucose (FDG) in mouse has been studied by F-19 NMR method for long period. The F-19 NMR signals of FDG or its metabolites were observed in tissues without serious broadening. FDG was found to be accumulated in organs in the form of FDG or FDG-6-phosphate and 2-deoxy-2-fluoro-D-mannose (FDM) or FDM-6-phosphate, and the latter dominated the former in the heart sampled at 24 hr or later. The fluorine compounds were excreted in urine in both forms. The clearance was rapid from brain, liver, and blood, but was slow from heart.     

Identification of the 22-phosphate esters of ecdysone, 2-deoxyecdysone, 20-hydroxyecdysone and 2-deoxy-20-hydroxyecdysone from newly laid eggs of the desert locust, Schistocerca gregaria.

The four major ecdysteroid (insect moulting hormone) conjugates present in the newly laid eggs of the desert locust, Schistocera gregaria, have been purified by reversed-phase and anion-exchange high-performance liquid chromatography. The steroid moieties were identified as ecdysone, 2-deoxyecdysone, 20-hydroxyecdysone and 2-deoxy-20-hydroxyecdysone. Phosphate analysis of acid-hydrolysed samples showed a steroid:phosphate ratio of approx. 1:1 for all four compounds. The intact conjugates were identified as ecdysone 22-phosphate, 2-deoxyecdysone 22-phosphate, 20-hydroxyecdysone 22-phosphate and 2-deoxy-20-hydroxyecdysone 22-phosphate by fast atom bombardment mass spectrometry and 1H, 13C and 31P n.m.r. The significance of ecdysteroid phosphates as a source of free hormone during embryogenesis is discussed.     

Purification and positional specificity of sn-glycerol-3-phosphate acyltransferase from Escherichia coli membranes.

SN-Glycerol-3-phosphate acyltransferase was solubilized from membranes of Escherichia coli B and K-12 and purified on an affinity column of Sepharose 4B coupled with 6-phosphogluconic acid. Phosphatidylglycerol was required for activation and stabilization of the purified enzyme. The acyl residues were exclusively transferred to the position 1 of sn-glycerol 3-phosphate by the enzyme, regardless of whether the acyl-CoA was saturated or unsaturated.     

L-Aspartate semialdehyde and a 6-deoxy-5-ketohexose 1-phosphate are the precursors to the aromatic amino acids in Methanocaldococcus jannaschii

No orthologs are present in the genomes of the archaea encoding genes for the first two steps in the biosynthesis of the aromatic amino acids leading to 3-dehydroquinate (DHQ). The absence of these genes prompted me to examine the nature of the reactions involved in the archaeal pathway leading to DHQ in Methanocaldococcus jannaschii. Here I report that 6-deoxy-5-ketofructose 1-phosphate and l-aspartate semialdehyde are precursors to DHQ. The sugar, which is derived from glucose 6-P, supplies a "hydroxyacetone" fragment, which, via a transaldolase reaction, undergoes an aldol condensation with the l-aspartate semialdehyde to form 2-amino-3,7-dideoxy-D-threo-hept-6-ulosonic acid. Despite the fact that both hydroxyacetone and hydroxyacetone-P were measured in the cell extracts and confirmed to arise from glucose 6-P, neither compound was found to serve as a precursor to DHQ. This amino sugar then undergoes a NAD dependent oxidative deamination to produce 3,7-dideoxy-d-threo-hept-2,6-diulosonic acid which cyclizes to 3-dehydroquinate. The protein product of the M. jannaschii MJ0400 gene catalyzes the transaldolase reaction and the protein product of the MJ1249 gene catalyzes the oxidative deamination and the cyclization reactions. The DHQ is readily converted into dehydroshikimate and shikimate in M. jannaschii cell extracts, consistent with the remaining steps and genes in the pathway being the same as in the established shikimate pathway.     

Slow-binding inhibition of 2-keto-3-deoxy-6-phosphogluconate (KDPG) aldolase

2-Keto-3-deoxy-6-phosphogluconate (KDPG) aldolase is a key enzyme in the Entner-Doudoroff pathway of bacteria. It catalyzes the reversible production of KDPG from pyruvate and D-glyceraldehyde 3-phosphate through a class I Schiff base mechanism. On the basis of aldolase mechanistic pathway, various pyruvate analogues bearing beta-diketo structures were designed and synthesized as potential inhibitors. Their capacity to inhibit aldolase catalyzed reaction by forming stabilized iminium ion or conjugated enamine were investigated by enzymatic kinetics and UV-vis difference spectroscopy. Depending of the substituent R (methyl or aromatic ring), a competitive or a slow-binding inhibition takes place. These results were examined on the basis of the three-dimensional structure of the enzyme.     

Synthesis and evaluation of non-hydrolyzable d-mannose 6-phosphate surrogates reveal 6-deoxy-6-dicarboxymethyl-d-mannose as a new strong inhibitor of phosphomannose isomerases

Non-hydrolyzable d-mannose 6-phosphate analogues in which the phosphate group was replaced by a phosphonomethyl, a dicarboxymethyl, or a carboxymethyl group were synthesized and kinetically evaluated as substrate analogues acting as potential inhibitors of type I phosphomannose isomerases (PMIs) from Saccharomyces cerevisiae and Escherichia coli. While 6-deoxy-6-phosphonomethyl-d-mannose and 6-deoxy-6-carboxymethyl-d-mannose did not inhibit the enzymes significantly, 6-deoxy-6-dicarboxymethyl-d-mannose appeared as a new strong competitive inhibitor of both S. cerevisiae and E. coli PMIs with K_m/K_i ratios of 28 and 8, respectively. We thus report the first malonate-based inhibitor of an aldose–ketose isomerase to date. Phosphonomethyl mimics of the 1,2-cis-enediolate high-energy intermediate postulated for the isomerization reaction catalyzed by PMIs were also synthesized but behave as poor inhibitors of PMIs. A polarizable molecular mechanics (SIBFA) study was performed on the complexes of d-mannose 6-phosphate and two of its analogues with PMI from Candida albicans, an enzyme involved in yeast infection homologous to S. cerevisiae and E. coli PMIs. It shows that effective binding to the catalytic site occurs with retention of the Zn(II)-bound water molecule. Thus the binding of the hydroxyl group on C1 of the ligand to Zn(II) should be water-mediated. The kinetic study reported here also suggests the dianionic character of the phosphate surrogate as a likely essential parameter for strong binding of the inhibitor to the enzyme active site.     

Simple Procedures for the Preparation of d-Ribose-5-phosphate Ketol-Isomerase,d-Ribulose-5-phosphate 3-Epimerase and d-Sedoheptulose-7-phosphate: d-Glyceraldehyde-3-phosphate Glycolaldehydetransferase

d-Ribose-5-phophate ketol-isomerase (EC 5.3.1,6), d-ribuIose-5-phosphate 3-epimerase (EC 5.1.3.1) and d-sedoheptulose-7-phosphate: d-gIyceraldehyde-3-phosphate glycolaldehyde-transferase (EC 2.2.1,1) have been partially purified. d-Ribose-5-phosphate ketol-isomerase was purified from spinach by column chromatography with DEAE-cellulose and DEAE-Sephadex A-50; d-ribulose-5-phosphate 3-epimerase was purified from baker’s yeast by column chromatography with DEAE-cellulose; and d-sedoheptulose-7-phosphate: d-glyceraldehyde-3-phosphate glycolaldehydetransferase was purified from a Bacillus species No. 102 mutant G3–46–22–6 by column chromatography with DEAE-cellulose. The preparations were used for the determination of the activities of these enzymes in the parent and d-ribose-forming mutants of a Bacillus species.     

Metabolism and actions of 2-deoxy-2-fluoro-D-galactose in vivo

The synthetic D-galactose analog 2-deoxy-2-fluoro-D-galactose (dGalF) offers unique advantages for studies of the D-galactose pathway by non-invasive techniques using 19F-NMR spectroscopy or positron emission from the 18F-labeled compound. The metabolism of 2-deoxy-2-fluoro-D-galactose was studied in rodents using the unlabeled, the 18F-labeled, and the 14C-labeled D-galactose analog. Analyses for the metabolites of 2-deoxy-2-fluoro-D-galactose were performed by HPLC, enzymatic methods, and 19F-NMR spectroscopy in vivo and in vitro. The metabolism of 2-deoxy-2-fluoro-D-galactose was most active in the liver which took up the major part of the administered dose of the 14C-labeled D-galactose analog, but renal excretion was also pronounced. This was confirmed by in vivo scanning of the rat using the 18F-labeled sugar (1.5 microCi/g; 25 nmol/g) and examination by positron-emission tomography and gamma camera. The dose dependence of the levels of the hepatic metabolites of 2-deoxy-2-fluoro-D-galactose was investigated for doses between 25 nmol/g body mass and 1 mumols/g body mass. After 1 h, the major part of the acid-soluble uracil nucleotides consisted of UDP-2-deoxy-2-fluoro-D-hexoses when the dose was at least 0.1 mumols/g. With higher doses, 2-deoxy-2-fluoro-D-galactose 1-phosphate became the predominant initial metabolite. After a dose of 1 mumols/g 2-deoxy-2-fluoro-D-galactose 1-phosphate accumulated rapidly (5.3 +/- 0.4 mumols/g liver after 30 min) followed by the formation of UDP-2-deoxy-2-fluoro-D-galactose and UDP-2-deoxy-2-fluoro-D-glucose (0.7 +/- 0.1 mumols/g and 1.8 +/- 0.1 mumols/g, respectively, after 5 h). The diversion of uridylate, due to the accumulation of UDP-2-deoxy-2-fluoro-D-hexoses, was associated with a rapid depletion of hepatic UTP, UDP-glucose, and UDP-galactose. The UTP content was decreased to 11 +/- 6% of normal within 15 min after administration of 2-deoxy-2-fluoro-D-galactose at a dose of 1 mumols/g. The UTP-depleting action was minimal, however, at a dose of 25 nmols/g or less, indicating that interference in uridylate metabolism would be negligible at the doses required for positron-emission tomography of the liver using the 18F-labeled compound. At higher doses, the UTP deficiency induced by 2-deoxy-2-fluoro-D-galactose could be useful in the chemotherapy of D-galactose-metabolizing tumors such as hepatocellular carcinoma.(ABSTRACT TRUNCATED AT 250 WORDS)     

A practical synthesis of 2-deoxy-2-fluoro-D-arabinofuranose derivatives.

A seven-step synthesis of 1,3-di-O-acetyl-5-O-benzoyl-2-deoxy-2-fluoro-D-arabinofuranose, a versatile intermediate in the synthesis of chemotherapeutically important nucleosides, was achieved from 1,2:5,6-di-O-isopropylidene-3-O-tosyl-alpha-D-allofuranose. The crucial steps were the fluorination by use of potassium fluoride in acetamide and the conversion of 6-O-benzoyl-3-deoxy-3-fluoro-D-glucofuranose into 5-O-benzoyl-2-deoxy-2-fluoro-3-O-formyl-D-arabinofuranose by periodate oxidation. Also described is the synthesis of 1-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)cytosine. This procedure affords good overall yields of products without formation of undesirable, isomeric intermediates and is suitable for large-scale preparations.     

Structural parameters and natural occurrence of 2-deoxy-2,3-didehydro-N-glycoloylneuraminic acid

2-Deoxy-2,3-didehydro-N-glycoloylneuraminic acid has been found to occur in porcine, bovine and equine submandibular glands as well as in the urine of pig, horse and rat. This novel, unsaturated sialic acid was isolated by gel filtration and ion-exchange chromatography. Final purification was achieved by column chromatography or by preparative thin-layer chromatography on cellulose. The structural analysis was performed by combined capillary gas-liquid chromatography/mass spectrometry. The various data were compared with those from synthetic 2-deoxy-2,3-didehydro-N-glycoloylneuraminic acid. Besides of the unsaturated N-glycoloylated sialic acid, also the corresponding N-acetylated derivative was present in the materials analyzed. The inhibitory effect of 2-deoxy-2,3-didehydro-N-glycoloylneuraminic acid on Vibrio cholerae sialidase using N-acetylneuraminyl-(alpha 2----3)-lactose as substrate is slightly higher (50% inhibition at 10 microM) when compared with 2-deoxy-2,3-didehydro-N-acetylneuraminic acid (50% inhibition at 15 microM).     

Study of 1-deoxy-1-(indol-3-yl)-L-sorbose, 1-deoxy-1-(indol-3-yl)-L-tagatose,and their analogs

Alkaline degradation of the ascorbigen 2-C-[(indol-3-yl)methyl]-alpha-L-xylo-hex-3-ulofuranosono-1,4-lactone (1a) led to a mixture of 1-deoxy-1-(indol-3-yl)-L-sorbose (2a) and 1-deoxy-1-(indol-3-yl)-L-tagatose (3a). The mixture of diastereomeric ketoses underwent acetylation and pyranose ring opening under the action of acetic anhydride in pyridine in the presence of 4-dimethylaminopyridine (DMAP) with the formation of a mixture of (E)-2,3,4,5,6-penta-O-acetyl-1-deoxy-1-(indol-3-yl)-L-xylo-hex-1-enitol (4a) and (E)-2,3,4,5,6-penta-O-acetyl-1-deoxy-1-(indol-3-yl)-L-lyxo-hex-1-enitol (5a), which were separated chromatographically. Deacetylation of 4a or 5a afforded cyclised tetrols, tosylation of which in admixture resulted in 1-deoxy-1-(indol-3-yl)-3,5-di-O-tosyl-alpha-L-sorbopyranose (12a) and 1-deoxy-1-(indol-3-yl)-4,5-di-O-tosyl-alpha-L-tagatopyranose (13a). Under alkaline conditions 13a readily formed 2-hydroxy-4-hydroxymethyl-3-(indol-3-yl)cyclopenten-2-one (15a) in 90% yield. Similar transformations were performed for N-methyl- and N-methoxyindole derivatives.     

Methylglyoxal is an intermediate in the biosynthesis of 6-deoxy-5-ketofructose-1-phosphate: a precursor for aromatic amino acid biosynthesis in Methanocaldococcus jannaschii

A biosynthetic pathway is proposed for creating 6-deoxy-5-ketofructose-1-phosphate (DKFP), a precursor sugar for aromatic amino acid biosynthesis in Methanocaldococcus jannaschii. First, two possible routes were investigated to determine if a modified, established biosynthetic pathway could be responsible for generating 6-deoxyhexoses in M. jannaschii. Both the nucleoside diphosphate mannose pathway and a pathway involving nucleoside diphosphate derivatives of fructose-1-P, fructose-2-P, or fructose-1,6-bisP were tested and eliminated. The established pathways did not produce the expected intermediates nor did the anticipated enzymes have the predicted enzymatic activities. Because neither anticipated pathway could produce DKFP, M. jannaschii glucose-6-P metabolism was studied in detail to establish exactly how glucose-6-P is converted into DKFP. This detailed analysis showed that methylglyoxal and a fructose-1-P- or fructose-1,6-bisP-derived dihydroxyacetone-P fragment are key intermediates in DKFP production. Glucose-6-P readily converts to fructose-6-P, which in turn converts to fructose-1,6-bisP. Fructose-6-P and fructose-1,6-bisP convert into glyceraldehyde-3-P (Ga-P-3), which converts into methylglyoxal by a 2,3-elimination of phosphate. The MJ1585-derived enzyme catalyzes the condensation of methylglyoxal with a dihydroxyacetone-P fragment, which is derived from fructose-1-P and/or fructose-1,6-bisP, generating DKFP. The elimination of phosphate from Ga-P-3 proceeds by both enzymatic and chemical routes in cell extracts, producing sufficient concentrations of methylglyoxal to support the reaction. This work is the first report of methylglyoxal functioning in central metabolism.     

Deoxyfluoroketohexoses: 4-deoxy-4-fluoro-D-sorbose and -tagatose and 5-deoxy-5-fluoro-L-sorbose.

4-Deoxy-4-fluoro-alpha-D-sorbose (6) was prepared in crystalline form by the action of potassium hydrogen fluoride on 3,4-anhydro-1,2-O-isopropylidene-beta-D-psicopyranose (3) followed by deacetonation. Under identical conditions, 3,4-anhydro-1,2-O-isopropylidene-beta-D-tagatopyranose (7) underwent epoxide migration to give 4,5-anhydro-1,2-O-isopropylidene-beta-D-fructopyranose (12), which after deacetonation yielded 4-deoxy-4-fluoro-D-tagatose (15) and 5-deoxy-5-fluoro-alpha-L-sorbopyranose (16), the latter as the crystalline, free sugar. The action of glycol-cleavage reagents on the isopropylidene acetals of the deoxyfluoro sugars was consistent with the assigned structures. The structures were established by 13-C n.m.r. studies of the free deoxyfluoro sugars 6 and 16 and of the isopropylidene acetal 13, and by 1-H n.m.r. studies on the acetylated isopropylidene acetals 5 diacetate, 13 diacetate, and 14 diacetate. 5-Deoxy-5-fluoro-L-sorbose (16) was biologically active, producing in mice effects characteristic of deoxyfluorotrioses and of fluoroacetate. 4-Deoxy-4-fluoro-D-tagatose (15) and 4-deoxy-4-fluoro-D-sorbose (6) produced no apparent effects in mice up to a dose of 500mg/kg. The implications of these findings with respect to transport, phosphorylation, and the action of aldolase on ketohexoses are discussed.     

AGPAT6 is a novel microsomal glycerol-3-phosphate acyltransferase

AGPAT6 is a member of the 1-acylglycerol-3-phosphate O-acyltransferase (AGPAT) family that appears to be important in triglyceride biosynthesis in several tissues, but the precise biochemical function of the enzyme is unknown. In the current study, we show that AGPAT6 is a microsomal glycerol-3-phosphate acyltransferase (GPAT). Membranes from HEK293 cells overexpressing human AGPAT6 had higher levels of GPAT activity. Substrate specificity studies suggested that AGPAT6 was active against both saturated and unsaturated long-chain fatty acyl-CoAs. Both glycerol 3-phosphate and fatty acyl-CoA increased the GPAT activity, and the activity was sensitive to N-ethylmaleimide, a sulfhydryl-modifying reagent. Purified AGPAT6 protein possessed GPAT activity but not AGPAT activity. Using [(13)C(7)]oleic acid labeling and mass spectrometry, we found that overexpression of AGPAT6 increased both lysophosphatidic acid and phosphatidic acid levels in cells. In these studies, total triglyceride and phosphatidylcholine levels were not significantly altered, although there were significant changes in the abundance of specific phosphatidylcholine species. Human AGPAT6 is localized to endoplasmic reticulum and is broadly distributed in tissues. Membranes of mammary epithelial cells from Agpat6-deficient mice exhibited markedly reduced GPAT activity compared with membranes from wild-type mice. Reducing AGPAT6 expression in HEK293 cells through small interfering RNA knockdown suggested that AGPAT6 significantly contributed to HEK293 cellular GPAT activity. Our data indicate that AGPAT6 is a microsomal GPAT, and we propose renaming this enzyme GPAT4.     

Intraorganelle localization and substrate specificities of the mitochondrial acyl-CoA: sn-glycerol-3-phosphate O-acyltransferase and acyl-CoA: 1-acyl-sn-glycerol-3-phosphate O-acyltransferase from potato tubers and pea leaves

The mitochondrial sn-glycerol-3-phosphate and 1-acyl-sn-glycerol-3-phosphate O-acyltransferases from potato tubers and pea leaves were investigated with respect to their intraorganelle localization, their positional and substrate specificities, and their fatty acid selectivities. In mitochondria from potato tubers both enzymes were found to be located in the outer membrane. The 1-acyl-sn-glycerol-3-phosphate O-acyltransferase of pea mitochondria showed the same intraorganelle localization whereas the sn-glycerol-3-phosphate O-acyltransferase behaved like a soluble protein of the intermembrane space. The sn-glycerol-3-phosphate O-acyltransferase of both potato and pea mitochondria used sn-glycerol-3-phosphate but not dihydroxyacetone phosphate as acyl acceptor and exclusively catalyzed the formation of 1-acyl-sn-glycerol-3-phosphate which subsequently served as substrate for the second acylation reaction at its C-2 position. Both acyltransferases of potato as well as pea mitochondria showed higher activities with acyl-CoA than with the corresponding acyl-(acyl carrier protein) thioesters. When different acyl-CoA thioesters were offered separately, the sn-glycerol-3-phosphate O-acyltransferase of potato mitochondria displayed no fatty acid specificity whereas the enzyme of pea mitochondria revealed one for saturated acyl groups. On the other hand, the mitochondrial 1-acyl-sn-glycerol-3-phosphate O-acyltransferases from both potato tubers and pea leaves were more active on unsaturated than on saturated acyl-CoA thioesters. Furthermore, these enzymes preferentially used oleoyl- and linoleoyl-CoA when they were offered in a mixture with saturated ones, although the fatty acid selectivity of the pea enzyme was less pronounced than that of the potato enzyme. The sn-glycerol-3-phosphate O-acyltransferase of potato mitochondria displayed a slight preference for saturated acyl groups.