Purification and characterizations of beta-Ketoacyl-[acyl-carrier-protein] reductase, beta-hydroxyacyl-[acyl-carrier-protein] dehydrase, and enoyl-[acyl-carrier-protein] reductase from Spinacia oleracea leaves

β-Ketoacyl-[acyl-carrier-protein] (ACP) reductase, β-hydroxyacyl-ACP dehydrase, and enoyl-ACP reductase have been purified to homogeneity from extracts of spinach leaves. Based on sodium dodecyl sulfate-polyacrylamide gel eletrophoresis studies, the monomeric molecular weights of the β-ketoacyl-ACP reductase, β-hydroxyacyl-ACP dehydrase, and enoyl-ACP reductase were 24,200, 19,000, and 32,500, respectively, and by gel filtration, their molecular weights were 97,000, 85,000, and 115,000, respectively, suggesting that these three enzymes exist as tetramers. The β-ketoacyl-ACP reductase, the β-hydroxyacyl-ACP dehydrase, and the enoyl-ACP reductase contained two, one, and two cystein residues per monomer. β-Ketoacyl-ACP reductase preferably utilized NADPH as the reductant, whereas enoyl-ACP reductase was absolutely specific to NADH. β-Ketoacyl-ACP reductase reversibly catalyzed the reduction of acetoacetyl-ACP to d-β-hydroxybutyryl-ACP and β-hydroxyacyl-ACP dehydrase catalyzed the dehydration of d-β-hydroxyacyl-ACP to 2-enoyl-ACP. Both β-hydroxyacyl-ACP dehydrase and enoyl-ACP reductase were active with 2-enoyl-ACPs having chain lengths from C₄ to C₁₆, with 2-hexenoyl-ACP and 2-octenoyl-ACP being the most effective substrate. CoA esters served as substrates with the β-ketoacyl-ACP reductase and the enoyl-ACP reductase but were inert with β-hydroxyacyl-ACP dehydrase. These enzymes were inhibited by p-chloromercuribenzoate but not by N-ethylmaleimide.     

Fatty Acid Synthetase of Spinacia oleracea Leaves

The molecular organization of fatty acid synthetase system in spinach (Spinacia oleracea L. var. Viroflay) leaves was examined by a procedure similar to that employed for the safflower system (Carthamus tinctorius var. UC-1). The crude extract contained all the component activities (acetyl-CoA:ACP transacylase, malonyl-CoA:ACP transacylase, β-ketoacyl-ACP synthetase, β-ketoacyl-ACP reductase, β-hydroxyacyl-ACP dehydrase, and enoyl-ACP reductase [I]) involved in the synthesis of fatty acids, but enoyl-ACP reductase (II) present in safflower seeds extract could not be detected spectrophotometrically. By polyethylene glycol fractionation followed by several chromatographic procedures, i.e. Sephadex G-200, hydroxyapatite, and blue-agarose, the component enzymes were clearly separated from one another. Properties of β-ketoacyl-ACP reductase, β-hydroxyacyl-ACP dehydrase, and enoyl-ACP reductase (I) from spinach were compared with the same enzymes in safflower seeds and Escherichia coli.     

Cloning and sequence analysis of putative type II fatty acid synthase genes from <Emphasis Type="Italic">Arachis hypogaea</Emphasis> L.

The cultivated peanut is a valuable source of dietary oil and ranks fifth among the world oil crops. Plant fatty acid biosynthesis is catalysed by type II fatty acid synthase (FAS) in plastids and mitochondria. By constructing a full-length cDNA library derived from immature peanut seeds and homology-based cloning, candidate genes of acyl carrier protein (ACP), malonyl-CoA:ACP transacylase, β-ketoacyl-ACP synthase (I, II, III), β-ketoacyl-ACP reductase, β-hydroxyacyl-ACP dehydrase and enoyl-ACP reductase were isolated. Sequence alignments revealed that primary structures of type II FAS enzymes were highly conserved in higher plants and the catalytic residues were strictly conserved in Escherichia coli and higher plants. Homologue numbers of each type II FAS gene expressing in developing peanut seeds varied from 1 in KASII, KASIII and HD to 5 in ENR. The number of single-nucleotide polymorphisms (SNPs) was quite different in each gene. Peanut type II FAS genes were predicted to target plastids except ACP2 and ACP3. The results suggested that peanut may contain two type II FAS systems in plastids and mitochondria. The type II FAS enzymes in higher plants may have similar functions as those in E. coli.     

Photocontrol of gibberellin metabolism in situ in maize

Two forms of spinach acyl carrier protein (ACP-I and ACP-II) have recently been characterized and found to be expressed in a tissue-specific manner (JB Ohlrogge, TM Kuo, 1985 J Biol Chem 260: 8032). To examine possible different functions for these ACP isoforms, we have tested purified preparations of spinach leaf ACP-I and ACP-II and Escherichia coli ACP in several in vitro reactions of fatty acid metabolism. Total de novo fatty acid synthesis and malonyl-CoA:ACP transacylase do not appear to discriminate between acyl carrier protein isoforms. In contrast, the K_m of oleoyl-ACP thioesterase for oleoyl-ACP-II is 10-fold higher than for oleoyl-ACP-I, whereas the K_m of acyl-ACP glycerol-3-phosphate acyl transferase is 5-fold higher for oleoyl-ACP-I than for oleoyl-ACP-II. A characterization of these reactions and a possible role for ACP isoforms in regulation of fatty acid metabolism in plants are described.     

Comparative studies of sheep lung and liver nitrofurantoin reductase

1. Nitrofurantoin reductase which catalyzes the bioactivation of nitrofurantoin was purified to electrophoretic homogenity from sheep liver and lung microsomes, with a yield of 15% and 35%, respectively. The specific activity of both reductases was found to be similar (140 nmol/min/mg protein).2. The effects of nitrofurantoin and NADPH concentrations, pH, ionic strength, amount of enzyme and reaction period, on the enzyme activity were studied and the optimum conditions for maximum activity of purified liver and lung nitrofurantoin reductases were determined.3. The enzyme concentration was found proportional with the square root of the rate of nitrofurantoin reduction up to approximately 15 μg protein/ml and 25 μg protein/ml incubation mixture for liver and lung nitrofurantoin reductases, respectively.4. The plots of inverse of the nitrofurantoin concentration against the inverse of the square root of the velocity for the reduction of nitrofurantoin by liver and lung enzymes gave K_m values as 27.78 μM and 32.25 μM, respectively.5. The purified liver and lung enzymes were also saturated by NADPH at similar concentrations and the K_m values were calculated as 29.4 μM and 35.5 μM, respectively.6. The effects of magnesium, nickel, cadmium and copper ions on the nitrofurantoin reductase activity were examined. Magnesium ion was found to have almost no effect, whereas the other ions inhibited the activity of both liver and lung reductases.     

Combined effect of epigallocatechin gallate and triclosan on enoyl-ACP reductase of Mycobacterium tuberculosis

Among the various inhibitors known for enoyl-acyl carrier protein (ACP) reductases, triclosan and green tea catechins are two promising candidates. In the present study, we show, for the first time that epigallocatechin gallate (EGCG), a major component of green tea catechins, inhibits InhA, the enoyl-ACP reductase of Mycobacterium tuberculosis with an IC50 of 17.4 μM. EGCG interferes with the binding of NADH to InhA. We also demonstrate that EGCG increased the inhibitory activity of triclosan towards InhA and vice versa. Direct binding assay using [³H]EGCG and fluorescence titration assay support the spectrophotometric/kinetic inhibition data. The biochemical data has been explained by docking simulation studies.     

Stereospecificity of hydrogen transfer by pyridine nucleotide-dependent enoyl reductases in fatty acid synthesis: Studies with enzymes obtained from developing castor bean seeds and Chlorella vulgaris

The mechanism of hydrogen incorporation into fatty acids wasinvestigated with fatty acid synthetase systems from developingcastor bean seeds and Chlorella vulgaris. Fatty acids synthesizedin the presence of D₂O or stereospecifically deuterium-labeledNADPH or NADH were isolated and analyzed by mass spectrometryto examine the localization of deuterium atoms in the molecule.The stereospecificities of ß-ketoacyl-acyl carrierprotein (ACP) reductase and enoyl-ACP reductase for reducedpyridine nucleotide were determined with acetoacetyl-ACP andcrotonyl-ACP as substrates. The products were also analyzedby gas chromatography-mass spectrometry. The following resultswere obtained:

1. ß-Ketoacyl-ACP reductases from both castor bean seedsand C. vulgaris used the B-side hydrogen of NADPH.
2. Enoyl-ACPreductase from C. vulgaris required NADH for the activity.
3. Enoyl-ACPreductase from castor bean seeds used the A-side hydrogenofNADPH, whereas that from C. vulgaris used the B-side hydrogenof NADH.
4. When stearate was synthesized with the crude fattyacid synthetasefraction from castor bean seeds, hydrogen atomsfrom water werefound on the even-numbered methylene carbonatoms (two hydrogenatoms per carbon atom) and some were foundon the odd-numberedmethylene carbon atoms. Hydrogen atoms fromthe B-side of NADPHwere found on the odd-numbered methylenecarbon atoms (one hydrogenatom per carbon atom). Hydrogen atomsfrom the A-side of NADPHwere also found on the odd-numberedmethylene carbon atoms,but the number of incorporated hydrogenatoms was less thanexpected.

(Received October 17, 1979; )     

流产布氏杆菌烯脂酰ACP还原酶的鉴定

烯脂酰ACP还原酶是细菌脂肪酸合成的关键酶之一．流产布氏杆菌基因组有2个注释为烯脂酰ACP还原酶基因fabI的同源基因：fabI1和fabI2．由这2个fabI同源基因编码的蛋白质分别与大肠杆菌FabI有50%和51%的同源性,且都拥有与大肠杆菌FabI一样的催化中心Tyr-(Xaa)₆-Lys序列．分别用携带这2个同源基因的质粒载体转化大肠杆菌fabI温度敏感突变菌株JP1111．转化子能在42℃生长,表明这2个基因均能遗传互补大肠杆菌fabI突变,并使此菌株恢复脂肪酸的合成．另外,体外酶学分析显示,由这2个同源基因编码的蛋白质都拥有烯脂酰ACP还原酶活性,均能参与细菌脂肪酸合成．上述结果证实,流产布氏杆菌同时拥有2个同种类型的烯脂酰ACP还原酶,是一种新的烯脂酰ACP多样性的表现．     

Nature of the Fatty Acid Synthetase Systems in Parenchymal and Epidermal Cells of Allium porrum L. Leaves

Fatty acid synthesis was compared in cell-free extracts of epidermis and parenchyma of Allium porrum L. leaves. Parenchyma extracts had the major fatty acid synthetase (FAS) activity (70-90%) of the whole leaf; palmitic acid was also the major fatty acid synthesized when acetyl-coenzyme A (CoA) was the primer, but when acetyl-acyl carrier protein (ACP) was employed, C_18:0 and C_16:0 were synthesized in equal proportion. With the epidermal FAS system when either acetyl-CoA or acetyl-ACP was tested in the presence of labeled malonyl-CoA, palmitic acid was the only product synthesized. Specific activities of the FAS enzyme activities were determined in both tissue extracts.

The properties of malonyl-CoA:ACP transacylase were examined from the two different tissues. The molecular weights estimated by Sephadex G-200 chromatography were 38,000 for the epidermal enzyme and 45,000 for parenchymal enzyme. The optimal pH was for both enzymes 7.8 to 8.0 and the maximal velocity 0.4 to 0.5 micromoles per milligram protein per minute. These enzymes had different affinities for malonyl-CoA and ACP. For the malonyl-CoA:ACP transacylase of epidermis, the K_m values were 5.6 and 13.7 micromolar for malonyl-CoA and ACP, respectively, and 4.2 and 21.7 micromolar for the parenchymal enzyme. These results suggest that the FAS system in both tissues are nonassociated, that the malonyl-CoA:ACP transacylases are isozymes, and that both in epidermis and in parenchyma tissue two independent FAS system occur. Evidence would suggest that β-ketoacyl-ACP synthase II is present in the parenchymal cells but missing in the epidermal cell.

     

Comparison of the properties of detergent-solubilized NADPH-cytochrome P-450 reductases from pig liver and kidney immunochemical,kinetic, and reconstitutive properties

NADPH-cytochrome P-450 reductases from pig liver and kidney and rabbit liver microsomes were purified to a specific activity of 50–62 μmol cytochrome c reduced/min/mg. All reductase preparations were separated into one major and one minor fraction on Sephadex G-200 columns. The molecular weights of the major fractions of the reductases were estimated to be 74,000, 75,000, and 75,500 for rabbit liver, pig kidney, and liver reductases, respectively, whereas the molecular weight of the minor fractions of these reductases, 67,000, was the same as that of the steapsin-solubilized pig liver reductase on SDS-polyacrylamide gel electrophoresis. K_m values for NADPH and cytochrome c were: 20 and 29 μm or 14 and 28 μm for the pig kidney or liver reductase, respectively. Immunochemical studies, including Ouchterlony double diffusion experiments and inhibition of benzphetamine N-demethylation activity in microsomes by antibody against pig liver NADPH-cytochrome P-450 reductase, indicated the similarity of the purified liver and kidney reductases. There were no differences in the ability to reconstitute NADPH-mediated benzphetamine N-demethylation and laurate hydroxylation in reconstituted systems between the pig liver and kidney reductases, indicating that the reductase did not determine substrate specificity in these systems.     

Increased acylation of lysophosphatidylcholine in microsomes of trembler mouse peripheral nerves

We report here the presence of a Stearoyl-CoA: [1-palmitoyl]-glycerophosphorylcholine (LPC) transacylase activity in the microsomal fraction of normal and Trembler mouse sciatic nerves. Under the experimental conditions studied as a function of incubation time, protein concentration, acyl-CoA and LPC concentrations, the transacylase specific activity was 2–3 times higher in the microsomes of the mutant's nerves than in those of the control. The addition of 5 mM ATP-Mg to the incubation medium, in the absence of bovine serum albumin, leads to a 90% decrease of the stearoyl-CoA thioesterase activity, but increases the transacylation by only 10–20%. This may be due to the low value (10 μM) of the apparent K_m for C₁₈-CoA observed for the mutant's transacylase. In microsomes from control nerves, transacylation requires exogenous LPC, whereas in Trembler mouse sciatic nerve microsomes, the transacylase can use endogenous LPC.     

Purification and properties of two 2,5-diketo-d-gluconate reductases from a mutant strain derived from Corynebacterium sp.

Two 2,5-diketo-d-gluconate reductases, I and II, were purified respectively 918-fold and 28-fold from a mutant strain derived from Corynebacterium sp. SHS 0007. The enzymes appeared to be homogeneous on polyacrylamide gel electrophoresis. Both reductases converted 2,5-diketo-d-gluconate to 2-keto-l-gulonate in the presence of NADPH and seemed to be active only for reduction. The molecular weights of reductases I and II were estimated to be 29,000 and 34,000, respectively; and both were monomeric. Their isoelectric points were respectively pH 4.3 and pH 4.1. The optimum pH was 6.0 to 7.0 for reductase I, and 6.0 to 7.5 for reductase II. The K_m values (pH 7.0, 30°C) of reductase I for 2,5-diketo-d-gluconate and for NADPH were 1.8 mM and 12 μM, respectively; and the corresponding values of reductase II were 13.5 mM and 13 μM. Both reductases converted 5-keto-d-fructose to l-sorbose in the presence of NADPH.     

Purification and Characterization of NAD(P)H:Nitrate Reductase and NADH:Nitrate Reductase from Corn Roots

The nitrate reductase activity of 5-day-old whole corn roots was isolated using phosphate buffer. The relatively stable nitrate reductase extract can be separated into three fractions using affinity chromatography on blue-Sepharose. The first fraction, eluted with NADPH, reduces nearly equal amounts of nitrate with either NADPH or NADH. A subsequent elution with NADH yields a nitrate reductase which is more active with NADH as electron donor. Further elution with salt gives a nitrate reductase fraction which is active with both NADH and NADPH, but is more active with NADH. All three nitrate reductase fractions have pH optima of 7.5 and Stokes radii of about 6.0 nanometers. The NADPH-eluted enzyme has a nitrate K_m of 0.3 millimolar in the presence of NADPH, whereas the NADH-eluted enzyme has a nitrate K_m of 0.07 millimolar in the presence of NADH. The NADPH-eluted fraction appears to be similar to the NAD(P)H:nitrate reductase isolated from corn scutellum and the NADH-eluted fraction is similar to the NADH:nitrate reductases isolated from corn leaf and scutellum. The salt-eluted fraction appears to be a mixture of NAD(P)H: and NADH:nitrate reductases.     

Subunits of fatty acid synthetase complexes. Comparative study of enzyme activities and properties of the half-molecular weight nonidentical subunits of fatty acid synthetase complexes obtained from rat, human, and chicken liver and yeast.

Rat, human, and chicken liver and yeast fatty acid synthetase complexes were dissociated into half-molecular weight nonidentical subunits of molecular weight 225,000–250,000 under the same conditions as used previously for the pigeon liver fatty acid synthetase complex [Lornitzo, F. A., Qureshi, A. A., and Porter, J. W. (1975) J. Biol. Chem.250, 4520–4529]. The separation of the half-molecular weight nonidentical subunits I and II of each fatty acid synthetase was then achieved by affinity chromatography on Sepharose ?-aminocaproyl pantetheine. The separations required, as with the pigeon liver fatty acid synthetase, a careful control of temperature, ionic strength, pH, and column flow rate for success, along with the freezing of the enzyme at ?20 °C prior to the dissociation of the complex and the loading of the subunits onto the column. The separated subunit I (reductase) from each fatty acid synthetase contained β-ketoacyl and crotonyl thioester reductases. Subunit II (transacylase) contained acetyl- and malonyl-coenzyme A: pantetheine transacylases. Each subunit of each complex also contained activities for the partial reactions, β-hydroxyacyl thioester dehydrase (crotonase), and palmitoyl-CoA deacylase. The specific activities of a given partial reaction did not vary in most cases more than twofold from one fatty acid synthetase species to another. The rat and human liver fatty acid synthetases required a much higher ionic strength for stability of their complexes and for the reconstitution of their overall synthetase activity from subunits I and II than did the pigeon liver enzyme. On reconstitution by dialysis in high ionic strength potassium phosphate buffer of subunits I and II of each complex, 65–85% of the control fatty acid synthetase activity was recovered. The rat and human liver fatty acid synthetases cross-reacted on immunoprecipitation with antisera. Similarly, chicken and pigeon liver fatty acid synthetases crossreacted with their antisera. There was, however, no cross-reaction between the mammalian and avian liver fatty acid synthetases and the yeast fatty acid synthetase did not cross-react with any of the liver fatty acid synthetase antisera.     

Functional analysis of an interspecies chimera of acyl carrier proteins indicates a specialized domain for protein recognition

The nodulation protein NodF of Rhizobium shows 25% identity to acyl carrier protein (ACP) from Escherichia coli (encoded by the gene acpP). However, NodF cannot be functionally replaced by AcpP. We have investigated whether NodF is a substrate for various E. coli enzymes which are involved in the synthesis of fatty acids. NodF is a substrate for the addition of the 4′-phosphopantetheine prosthetic group by holo-ACP synthase. The K_m value for NodF is 61?μM, as compared to 2?μM for AcpP. The resulting holo-NodF serves as a substrate for coupling of malonate by malonyl-CoA:ACP transacylase (MCAT) and for coupling of palmitic acid by acyl-ACP synthetase. NodF is not a substrate for β-keto-acyl ACP synthase III (KASIII), which catalyses the initial condensation reaction in fatty acid biosynthesis. A chimeric gene was constructed comprising part of the E.coliacpP gene and part of the nodF gene. Circular dichroism studies of the chimeric AcpP-NodF (residues 1–33 of AcpP fused to amino acids 43–93 of NodF) protein encoded by this gene indicate a similar folding pattern to that of the parental proteins. Enzymatic analysis shows that AcpP-NodF is a substrate for the enzymes holo-ACP synthase, MCAT and acyl-ACP synthetase. Biological complementation studies show that the chimeric AcpP-NodF gene is able functionally to replace NodF in the root nodulation process in Vicia sativa. We therefore conclude that NodF is a specialized acyl carrier protein whose specific features are encoded in the C-terminal region of the protein. The ability to exchange domains between such distantly related proteins without affecting conformation opens exciting possibilities for further mapping of the functional domains of acyl carrier proteins (i. e., their recognition sites for many enzymes).     

The KC Channel in the cbb3-Type Respiratory Oxygen Reductase from Rhodobacter capsulatus Is Required for Both Chemical and Pumped Protons

The heme-copper superfamily of proton-pumping respiratory oxygen reductases are classified into three families (A, B, and C families) based on structural and phylogenetic analyses. Most studies have focused on the A family, which includes the eukaryotic mitochondrial cytochrome c oxidase as well as many bacterial homologues. Members of the C family, also called the cbb₃-type oxygen reductases, are found only in prokaryotes and are of particular interest because of their presence in a number of human pathogens. All of the heme-copper oxygen reductases require proton-conducting channels to convey chemical protons to the active site for water formation and to convey pumped protons across the membrane. Previous work indicated that there is only one proton-conducting input channel (the K^C channel) present in the cbb₃-type oxygen reductases, which, if correct, must be utilized by both chemical protons and pumped protons. In this work, the effects of mutations in the K^C channel of the cbb₃-type oxygen reductase from Rhodobacter capsulatus were investigated by expressing the mutants in a strain lacking other respiratory oxygen reductases. Proton pumping was evaluated by using intact cells, and catalytic oxygen reductase activity was measured in isolated membranes. Two mutations, N346M and Y374F, severely reduced catalytic activity, presumably by blocking the chemical protons required at the active site. One mutation, T272A, resulted in a substantially lower proton-pumping stoichiometry but did not inhibit oxygen reductase activity. These are the first experimental data in support of the postulate that pumped protons are taken up from the bacterial cytoplasm through the K^C channel.     

The use of a hybrid genetic system to study the functional relationship between prokaryotic and plant multi-enzyme fatty acid synthetase complexes

Fatty acid synthesis in bacteria and plants is catalysed by a multi-enzyme fatty acid synthetase complex (FAS II) which consists of separate monofunctional polypeptides. Here we present a comparative molecular genetic and biochemical study of the enoyl-ACP reductase FAS components of plant and bacterial origin. The putative bacterial enoyl-ACP reductase gene (envM) was identified on the basis of amino acid sequence similarities with the recently cloned plant enoyl-ACP reductase. Subsequently, it was unambiguously demonstrated by overexpression studies that theenvM gene encodes the bacterial enoyl-ACP reductase. An anti-bacterial agent called diazaborine was shown to be a specific inhibitor of the bacterial enoyl-ACP reductase, whereas the plant enzyme was insensitive to this synthetic antibiotic. The close functional relationship between the plant and bacterial enoyl-ACP reductases was inferred from genetic complementation of anenvM mutant ofEscherichia coli. Ultimately,envM gene-replacement studies, facilitated by the use of diazaborine, demonstrated for the first time that a single component of the plant FAS system can functionally replace its counterpart within the bacterial multienzyme complex. Finally, lipid analysis of recombinantE. coli strains with the hybrid FAS system unexpectedly revealed that enoyl-ACP reductase catalyses a rate-limiting step in the elongation of unsaturated fatty acids.     

Conversion of a NADPH-dependent aldehyde reducing enzyme into aldose reductase

• 1.1. Aldose reductase, aldehyde reductase and high-K_m, aldose reductase were purified from the inner medulla of dog kidney.
• 2.2. Compared with aldose reductase, high-K_m aldose reductase had a lower isoelectric point, a lower activity for aldo-sugars and a lower sensitivity for aldose reductase inhibitors, and it was not activated by sulfate ions. Both reductases had the same molecular weight (38,500) and immunochemical properties.
• 3.3. High-K_m aldose reductase was easily converted into an aldose reductase-like enzyme, namely a generated reductase upon incubation in neutral buffer solution.
• 4.4. The generated reductase was identical with aldose reductase with respect to the isoelectric point, substrate specificity, activation by sulfate ions and IC₅₀ values for aldose reductase inhibitors. The generated reductase revealed immunochemical identity with aldose reductase as well as high-K_m aldose reductase.