Malonyl coenzyme A synthetase. Purification and properties

Malonyl coenzyme A synthetase (EC 6.2.1.14) was induced in Pseudomonas fluorescens grown on malonate as a sole carbon source. This enzyme was purified, for the first time, over 30-fold by the combination of ammonium sulfate precipitation, Sephadex G-150 gel filtration, DEAE-Sephacel ion exchange chromatography, and hydroxylapatite chromatography. The purified enzyme, which had a specific activity of about 0.512 mumol/min/mg, appeared to be electrophoretically homogeneous. The molecular size of the enzyme was determined to be 98,000 Da which is composed of two 49,000-Da subunits. The optimum pH for the enzyme was 7.5. Malonyl coenzyme A synthetase requires ATP, CoA, and Mg2+ for the full enzyme activity. With succinate or acetate, the synthetic rate of CoA derivative was 40% of that observed with malonate. The malonyl coenzyme A synthetase showed typical Michaelis-Menten kinetics for the substrate, malonate, ATP, and coenzyme A, from which the Km values were calculated to be 3.8 X 10(-4) M, 2 X 10(-3) M, and 10(-4) M and Vmax values to be 0.117 mumol/min/mg, 0.111 mumol/min/mg, and 0.142 mumol/min/mg, respectively. The purified malonyl coenzyme A synthetase was immunogenic in the rabbit and Ouchterlony double diffusion analysis revealed a single precipitant line with the enzyme. The antiserum inhibited the enzyme activity and the extent of inhibition was dependent on the amount of the serum added.     

Malonate, Malonyl-Coenzyme A, and Acetyl-Coenzyme A in Developing Rat Brain

Abstract: Free malonate, malonyl-coenzyme A (malonyl-CoA), and acetyl-CoA were assayed in rat brain at developmental ages from the 20th day of gestation to 60 days of postnatal life. The determination of malonate was based on its conversion to malonyl-CoA and decarboxylation to acetyl-CoA by enzyme extracts from Pseudo-monas fluorescens. The resulting acetyl-CoA reacted with [4-¹⁴C]oxaloacetate to form [5-¹⁴C]citrate, which was isolated by TLC. Malonyl-CoA in perchloric acid extracts from brain was converted to acetyl-CoA by rat liver mitochondrial malonyl-CoA decarboxylase (EC 4.1.1.9). Acetyl-CoA derived from this step was assayed by a modified CoA-cycling procedure. Brain acetyl-CoA was also assayed by CoA cycling. Prenatal brain contained no free malonate but malonyl-CoA was present. The acetyl-CoA level was relatively high just prior to birth and declined slightly with growth. Malonate concentrations after birth rose rapidly to reach 192 nmol/g wet weight at 60 days. Adult levels for malonyl-CoA and acetyl-CoA were 1.83 and 1.90 nmol/g wet weight, respectively. The origin and natural role of free malonate in brain are not known but deacylation of malonyl-CoA by reversal of the malonyl-CoA synthetase reaction is postulated. Rat liver and kidney also contain substantial concentrations of free malonate.     

New experiments of biotin enzymes.

The objects of structural studies on biotin-enzymes were acetyl CoA-carboxylase and pyruvate carboxylase of Saccharomyces cerevisiae and beta-methylcrotonyl CoA-carboxylase and acetyl CoA-carboxylase of Achromobacter IV S. It was found that these enzymes can be arranged in three groups. In the first group, as represented by acetyl CoA-carboxylase of Achromobacter, the active enzyme could be resolved in three types of functional components: (1) the biotin-carboxyl carrier protein, (2) the biotin carboxylase, and (3) the carboxyl transferase. In the second group, as represented by beta-methylcrotonyl CoA-carboxylase from Achromobacter only two types of polypeptides are present. The one carries the biotin carboxylase activity together with the biotin-carboxyl-carrier protein, the other one carries the carboxyl transferase activity. In this third group, as represented by the two enzymes of yeast, all three catalytic functions are incorporated in one multifunctional polypeptide chain. The evolution of the different enzymes is discussed. The animal tissues acetyl CoA-carboxylase is under metabolic control, as known from previous studies. It thus has to be expected that the levels of malonyl CoA in livers of rats in all states of depressed fatty acid synthesis are much lower than under normal conditions because the carboxylation of acetyl CoA is strongly reduced and cannot keep pace with the consumption of malonyl CoA by fatty acid synthetase. A new highly sensitive assay method for malonyl CoA was developed which uses tritiated NADPH and measures the incorporation of radioactivity into the fatty acids formed from malonyl CoA in the presence of purified fatty acid synthetase. The application of this method to liver extracts showed that the level of malonyl CoA which amounts to about 7 nmoles per gram of wet liver drops to less than 10% within a starvation period of 24 hr and even further if the starvation period is extended to 48 hr. A low malonyl CoA concentration is also found in the alloxan diabetic animals and in animals being fed a fatty diet after starvation. On the other hand, feeding a carbohydrate rich diet leads to malonyl CoA levels surpassing the levels found after feeding a balanced diet. These observations reconfirm the concept that fatty acid synthesis is principally regulated by the carboxylation of acetyl CoA.     

Substrate-dependent dissociation of malate thiokinase.

Malate thiokinase has been purified to apparent homogeneity by employing conventional purification techniques along with affinity chromatography. The enzyme is composed of two nonidentical subunits (alpha subunit Mr=34,000, beta subunit Mr=42,500) to yield an alpha 4 beta 4 structure for the native enzyme. Phosphorylation of the enzyme by ATP occurs exclusively on the alpha subunit. The phosphorylated enzyme is acid labile and base stable consistent with phosphorylation of a histidine residue. Dephosphorylation of the enzyme is promoted by ADP, succinate, malate, and coenzyme A plus inorganic phosphate. Phosphorylation of the enzyme leads to a reversible change in the sedimentation properties of the enzyme; the native enzyme exhibits an S20,w of approximately 10, whereas the phosphoenzyme exhibits an S20,w of approximately 7. Formation of the 7 S form of the enzyme is also observed when coenzyme A and succinyl-CoA interact with the enzyme. The ratio of alpha to beta subunits in both the 10 S and 7 S forms of the enzyme is approximately 1.0, suggesting that the 7 S form of the enzyme has an alpha 2 beta 2 structure.     

Crystal structure of two quaternary complexes of dethiobiotin synthetase, enzyme-MgADP-AlF3-diaminopelargonic acid and enzyme-MgADP-dethiobiotin-phosphate; implications for catalysis.

The crystal structures of two complexes of dethiobiotin synthetase, enzyme-diaminopelargonic acid-MgADP-AlF3 and enzyme-dethiobiotin-MgADP-Pi, respectively, have been determined to 1.8 A resolution. In dethiobiotin synthetase, AlF3 together with carbamylated diaminopelargonic acid mimics the phosphorylated reaction intermediate rather than the transition state complex for phosphoryl transfer. Observed differences in the binding of substrate, diaminopelargonic acid, and the product, dethiobiotin, suggest considerable displacements of substrate atoms during the ring closure step of the catalytic reaction. In both complexes, two metal ions are observed at the active site, providing evidence for a two-metal mechanism for this enzyme.     

Phosphorylated and dephosphorylated structures of pig heart, GTP-specific succinyl-CoA synthetase

Succinyl-CoA synthetase (SCS) catalyzes the reversible phosphorylation/dephosphorylation reaction:???rm succinyl ?hbox ?-?CoA+NDP+P_i?leftrightarrow succinate+CoA+NTP??where N denotes adenosine or guanosine. In the course of the reaction, an essential histidine residue is transiently phosphorylated. We have crystallized and solved the structure of the GTP-specific isoform of SCS from pig heart (EC 6.2.1.4) in both the dephosphorylated and phosphorylated forms. The structures were refined to 2.1 A resolution. In the dephosphorylated structure, the enzyme is stabilized via coordination of a phosphate ion by the active-site histidine residue and the two "power" helices, one contributed by each subunit of the alphabeta-dimer. Small changes in the conformations of residues at the amino terminus of the power helix contributed by the alpha-subunit allow the enzyme to accommodate either the covalently bound phosphoryl group or the free phosphate ion. Structural comparisons are made between the active sites in these two forms of the enzyme, both of which can occur along the catalytic path. Comparisons are also made with the structure of Escherichia coli SCS. The domain that has been shown to bind ADP in E. coli SCS is more open in the pig heart, GTP-specific SCS structure.     

Purification and physicochemical properties of fatty acid synthetase and acetyl-CoA carboxylase from lactating rabbit mammary gland.

Fatty acid synthetase was purified 13-fold from lactating rabbit mammary glands by a procedure which involved chromatography on DEAE-cellulose, ammonium sulphate precipitation and gel filtration on Sepharose 4B. The preparation was completed within two days and over 100 mg of enzyme was isolated from 100--150 g of mammary tissue, which represented a yield of over 40%. The preparation was homogeneous by the criteria of polyacrylamide gel electrophoresis and ultracentrifugal analysis. The sedimentation constant, S20,w was 13.3 S, the absorption coefficient, A280nm1%, measured refractometrically was 10.0 +/- 0.1, and the amino acid composition was determined. The subunit molecular weight determined by gel electrophoresis in the presence of sodium dodecyl sulphate was 252,000 +/- 6,000, and the molecular weight of the native enzyme measured by sedimentation equilibrium was 515,000. These experiments indicate that at the concentrations which exist in mammary tissue (2--4 mg/ml) fatty acid synthetase is a dimer. The purified enzyme did however show a tendency to dissociate to a monomeric 9-9S species on storage for several days or following exposure to a low ionic strength buffer at pH 8.3. There was only a small quantity of alkali labile phosphate (0.2 molecules per subunit) bound covalently to the purified enzyme. Acetyl-CoA carboxylase was purified 300-fold in a 50% yield within 24 h by ammonium sulphate and polyethylene glycol precipitations [Hardie, D.G. and Cohen, P. (1978) FEBS Lett. 91, 1--7]. The preparation was in a state approaching homogeneity as judged by polyacrylamide gel electrophoresis, gel filtration on Sepharose 4B and ultracentrifugal analysis. The sedimentation constant, S20,w, was 50.5 S, the absorption index, A280nm1%, was 14.5 +/- 0.7, and the amino acid composition was determined. The subunit molecular weight of acetyl-CoA carboxylase determined by gel electrophoresis in the presence of sodium dodecyl sulphate was identical to that of fatty acid synthetase (252,000) as shown by electrophoresis of a mixture of the two proteins. The preparations also contained two minor components of molecular weight 235,000 and 225,000, which appear to be derived from the major species of mol. wt 252,000. A large emount of phosphate (3.2 molecules per subunit) was found to be bound covalently to the purified enzyme. The properties of fatty acid synthetase and acetyl-CoA carboxylase are compared to those obtained by other workers.     

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.     

Roles of active site residues in Pseudomonas aeruginosa phosphomannomutase/phosphoglucomutase

In Pseudomonas aeruginosa, the dual-specificity enzyme phosphomannomutase/phosphoglucomutase catalyzes the transfer of a phosphoryl group from serine 108 to the hydroxyl group at the 1-position of the substrate, either mannose 6-P or glucose 6-P. The enzyme must then catalyze transfer of the phosphoryl group on the 6-position of the substrate back to the enzyme. Each phosphoryl transfer is expected to require general acid-base catalysis, provided by amino acid residues at the enzyme active site. An extensive survey of the active site residues by site-directed mutagenesis failed to identify a single key residue that mediates the proton transfers. Mutagenesis of active site residues Arg20, Lys118, Arg247, His308, and His329 to residues that do not contain ionizable groups produced proteins for which V(max) was reduced to 4-12% of that of the wild type. The fact that no single residue decreased catalytic activity more significantly, and that several residues had similar effects on V(max), suggested that the ensemble of active site amino acids act by creating positive electrostatic potential, which serves to depress the pK of the substrate hydroxyl group so that it binds in ionized form at the active site. In this way, the necessity of positioning the reactive hydroxyl group near a specific amino acid residue is avoided, which may explain how the enzyme is able to promote catalysis of both phosphoryl transfers, even though the 1- and 6-positions do not occupy precisely the same position when the substrate binds in the two different orientations in the active site. When Ser108 is mutated, the enzyme retains a surprising amount of activity, which has led to the suggestion that an alternative residue becomes phosphorylated in the absence of Ser108. (31)P NMR spectra of the S108A protein confirm that it is phosphorylated. Although the S108A/H329N protein had no detectable catalytic activity, the (31)P NMR spectra were not consistent with a phosphohistidine residue.     

Identification of Ser424 as the protein kinase A phosphorylation site in CTP synthetase from Saccharomyces cerevisiae.

The URA7-encoded CTP synthetase [EC 6.3.4.2, UTP:ammonia ligase (ADP-forming)] in the yeast Saccharomyces cerevisiae is phosphorylated on a serine residue and stimulated by cAMP-dependent protein kinase (protein kinase A) in vitro. In vivo, the phosphorylation of CTP synthetase is mediated by the RAS/cAMP pathway. In this work, we examined the hypothesis that amino acid residue Ser424 contained in a protein kinase A sequence motif in the URA7-encoded CTP synthetase is the target site for protein kinase A. A CTP synthetase synthetic peptide (SLGRKDSHSA) containing the protein kinase A motif was a substrate (Km = 30 microM) for protein kinase A. This peptide also inhibited (IC50 = 45 microM) the phosphorylation of purified wild-type CTP synthetase by protein kinase A. CTP synthetase with a Ser424 --> Ala (S424A) mutation was constructed by site-directed mutagenesis. The mutated enzyme was not phosphorylated in response to the activation of protein kinase A activity in vivo. Purified S424A mutant CTP synthetase was not phosphorylated and stimulated by protein kinase A. The S424A mutant CTP synthetase had reduced Vmax and elevated Km values for ATP and UTP when compared with the protein kinase A-phosphorylated wild-type enzyme. The specificity constants for ATP and UTP for the S424A mutant CTP synthetase were 4.2- and 2.9-fold lower, respectively, when compared with that of the phosphorylated enzyme. In addition, the S424A mutant enzyme was 2.7-fold more sensitive to CTP product inhibition when compared with the phosphorylated wild-type enzyme. These data indicated that the protein kinase A target site in CTP synthetase was Ser424 and that the phosphorylation of this site played a role in the regulation of CTP synthetase activity.     

Multiple phosphorylation of acetyl-CoA carboxylase in chick liver cells. A cyclic AMP-independent process.

When chick liver cells in monolayer culture were incubated with 32Pi in the presence of insulin, acetyl-CoA carboxylase became extensively labeled with 32Pi reaching a stoichiometry of 9 to 10 mol of phosphoryl group per mol of 240,000-dalton enzyme subunit. The covalently bound phosphate was found to be metabolically labile, turning over with a t1/2 of approximately 2 h (enzyme t1/2 approximately equal to 24 h). Addition of Bt2cAMP altered neither the rate nor extent of phosphorylation. Contrary to other reports, the fully phosphorylated acetyl-CoA carboxylase appears to be catalytically active.     

Active site organization of bacterial type I fatty acid synthetase

Four kinds of active sites of bacterial fatty acid synthetase were mapped on distinct regions within a subunit. Active sites were specifically labeled with radioactive substrates and active-site-directed inhibitors. Labeled enzymes were cleaved with proteases, and the fragments thus produced were identified with respect to specific labels by SDS-polyacrylamide gel electrophoresis and a fluorographic technique. The linear alignment of such fragments in the original subunit was established and when the results were combined with those of our previous work, five active sites were located in three regions as follows. Starting from the N-terminal of the subunit, we located acetyl, malonyl and palmitoyl transferases in the first region, the acyl carrier site in the second region (Morishima & Ikai (1985) Biochim. Biophys. Acta 832, 297-307), and beta-ketoacyl synthetase in the third region. The observed order of active sites of bacterial fatty acid synthetase can be correlated with that of the yeast enzyme, which has two kinds of subunits.     

The isolation of the two subunits of yeast fatty acid synthetase.

The two subunits that comprise the yeast fatty acid synthetase (designated α and β) have been isolated. The separation was performed using DEAE Biogel A chromatography after first treating yeast fatty acid synthetase with 3,4,5,6 tetrahydrophthalic anhydride. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of the fractions eluted from the ion exchange column indicated that the separation of the subunits was essentially complete. It was possible to remove the 3,4,5,6 tetrahydrophthalate derivative from the subunits and regenerate certain of the partial activities. The α subunit was found to have the β-keto reductase activity as well as the acyl carrier protein component associated with it. The β subunit had the acetyl and malonyl transacylases and the palmitoyl transferase activity associated with it. The different extent to which the malonyl and acetyl transacylase activities were regained indicated that these two catalytic sites have separate domains in the β subunit.     

The architecture of the animal fatty acid synthetase complex. IV. Mapping of active centers and model for the mechanism of action

The fatty acid synthetase of animal tissue consists of two subunits, each containing seven catalytic centers and an acyl carrier site. Proteolytic cleavage patterns indicate that the subunit is arranged into three major domains, I, II, and III. Domain I contains the NH2-terminal end of the polypeptide and the catalytic sites of beta-ketoacyl synthetase (condensing enzyme) and the acetyl-and malonyl-transacylases. This domain, therefore, functions as a site for acetyl and malonyl substrate entry into the process of fatty acid synthesis and acts in part as the site of carbon-carbon condensation, resulting in chain elongation. Domain II is the medial domain and contains the beta-ketoacyl and enoyl reductases, probably the dehydratase, and the 4'-phosphopantetheine prosthetic group of the acyl carrier protein site. Domain II, therefore, is designated as the reduction domain where the keto carbon is reduced to methylene carbon by sequential processes of reduction, dehydration, and reduction again. Throughout these processes, the acyl group is attached to the pantetheine-SH of the acyl carrier protein. The latter site is distal to the cysteine-SH of the beta-ketoacyl synthetase, constitutes the 15000-dalton polypeptide at the COOH-terminal end of Domain II, and connects to Domain III. When the growing chain reaches C16 carbon length, the fatty acyl group is released by the thioesterase activity, which is contained in Domain III. A functional model is proposed based on the aforementioned results and the recent evidence that the synthetase subunits are arranged in a head-to-tail fashion, such that the pantetheine-SH of the acyl carrier protein of one subunit and the cysteine-SH of the beta-ketoacyl synthetase of the second subunit are juxtaposed. In this model, a palmitate synthesizing site contains Domain I of one subunit and Domains II and III of the second subunit. Therefore, even though each subunit contains all of the partial activities of the reaction sequence, the actual palmitate synthesizing unit consists of one-half of a subunit interacting with the complementary half of the other subunit.     

Thiophosphorylation as a probe for subunit interactions in Escherichia coli succinyl coenzyme A synthetase. Further evidence for catalytic cooperativity and substrate synergism

Succinyl-CoA synthetase has an (alpha beta)2 subunit structure and shows half-of-the-sites reactivity with respect to the formation of the phosphohistidyl residues that acts as a catalytic intermediate. Adenosine 5'-O-(3-thio)triphosphate has been found to be a substrate, but the overall maximum velocity is 3 orders of magnitude lower than that seen with ATP. Moreover, steps of the reaction involving thiophosphoryl transfer are much slower than the corresponding phosphoryl transfers. These properties of adenosine 5'-O-(3-thio)triphosphate as a substrate have been exploited to test the concept of alternating sites catalytic cooperativity proposed earlier as a rationale for the subunit structure of succinyl-CoA synthetase. As predicted by this model for catalysis, the rate of discharge of thiophosphate from the enzyme in the presence of succinate and CoA is stimulated by ATP. Neither of two nonhydrolyzable analogs of ATP has an equivalent effect. The results indicate that the transfer of the thiophosphoryl group from the enzyme to succinate at one active site is not favored until the neighboring active site is phosphorylated by ATP, with accompanying reciprocal changes in the conformations of the two halves of the enzyme molecule.     

Hybridization studies of chicken liver fatty acid synthetase. Evidence for the participation in palmitate synthesis of cysteine and phosphopantetheine sulfhydryl groups on adjacent subunits

Enzymatically inactive variants of chicken liver fatty acid synthetase have been prepared by specific chemical modification of the active cysteine SH group with iodoacetamide, and the phosphopantetheine SH group with chloroacetyl-CoA. Hybridization of each of these variants with the unmodified enzyme yielded (modified)-(unmodified) hybrid dimers which possessed 50% synthetase activity. A 50% active (iodoacetamide-modified)-(chloroacetyl-CoA-modified) hybrid dimer was also demonstrated by recombination of these variants with each other. These results indicate that the two functional sites on the synthetase are independently active, and that each is comprised of a cysteine SH group from one subunit and a complementary phosphopantetheine SH group from the other subunit as depicted by the head-to-tail arrangement proposed by Wakil and co-workers (Wakil, S. J., Stoops, J. K., and Joshi, V.C.     

羧酸盐对鸭肝脂肪酸合成酶的抑制作用

本文研究了一系列羧酸盐对鸭肝脂肪酸合成酶的抑制规律,抑制动力学以及对几个含羧基的巯基试剂修饰该酶的保护动力学,并计算了相应的在酶上结合的平衡常数。提出羧酸盐结合于该酶缩合中心区域一个可结合羧基负离子的部位,该部位也是丙二酰基的结合部位,它可使携带了丙二酰基的活臂向缩合中心定向运动,并为缩合活性所必需。     

On the role of cyclic AMP-independent protein kinases in the modification of yeast ribosomal proteins in vivo

Two proteins of yeast 40S ribosome subunit and four proteins of the 60S ribosome subunit were labelled in vivo with [32P]orthophosphate. Five of these proteins were phosphorylated by protein kinase 3, an enzyme which is cyclic AMP-independent and uses ATP and GTP as phosphoryl donors. Two proteins, belonging to the 60S ribosome subunit were phosphorylated by another, highly specific, cyclic AMP-independent protein kinase 1 B. Both in vivo and in vitro the most extensively phosphorylated protein species were acidic proteins, L44, L45 (according to the nomenclature of Kruiswijk & Planta, Molec. Biol. Rep., 1, 409-415, 1974) possibly corresponding to bacterial L7 and L12 proteins. The 40S ribosomal protein, S9, analogous to mammalian S6 protein, was phosphorylated in vivo but was not phosphorylated in vitro by either of the cyclic AMP-independent protein kinases. The obtained results clearly indicate that cyclic AMP-independent yeast protein kinases might be involved in the modification in vivo of some ribosomal proteins, in particular of the strongly acidic proteins of 60S ribosome subunit.     

Biochemical characterization of the minimal polyketide synthase domains in the lovastatin nonaketide synthase LovB

The biosynthesis of lovastatin in Aspergillus terreus requires two megasynthases. The lovastatin nonaketide synthase, LovB, synthesizes the intermediate dihydromonacolin L using nine malonyl-coenzyme A molecules, and is a reducing, iterative type I polyketide synthase. The iterative type I polyketide synthase is mechanistically different from bacterial type I polyketide synthases and animal fatty acid synthases. We have cloned the minimal polyketide synthase domains of LovB as standalone proteins and assayed their activities and substrate specificities. The didomain proteins ketosynthase-malonyl-coenzyme A:acyl carrier protein acyltransferase (KS-MAT) and acyl carrier protein-condensation (ACP-CON) domain were expressed solubly in Escherichia coli. The monodomains MAT, ACP and CON were also obtained as soluble proteins. The MAT domain can be readily labeled by [1,2-(14)C]malonyl-coenzyme A and can transfer the acyl group to both the cognate LovB ACP and heterologous ACPs from bacterial type I and type II polyketide synthases. Using the LovB ACP-CON didomain as an acyl acceptor, LovB MAT transferred malonyl and acetyl groups with k(cat)/K(m) values of 0.62 min(-1).mum(-1) and 0.032 min(-1).mum(-1), respectively. The LovB MAT domain was able to substitute the Streptomyces coelicolor FabD in supporting product turnover in a bacterial type II minimal polyketide synthase assay. The activity of the KS domain was assayed independently using a KS-MAT (S656A) mutant in which the MAT domain was inactivated. The KS domain displayed no activity towards acetyl groups, but was able to recognize malonyl groups in the absence of cerulenin. The relevance of these finding to the priming mechanism of fungal polyketide synthase is discussed.     

Purification and characterization of neutral trehalase from the yeast ABYS1 mutant

Neutral trehalase was purified from stationary yeast ABYS1 mutant cells deficient in the vacuolar proteinases A and B and the carboxypeptidases Y and S. The purified electrophoretically homogeneous preparation of phosphorylated neutral trehalase exhibited a molecular mass of 160,000 Da on nondenaturing gel electrophoresis and of 80,000 Da on sodium dodecyl sulfate-gel electrophoresis. Maximal activity (114 mumol of trehalose min-1 x mg-1 at 37 degrees C) was observed at pH 6.8-7.0. The apparent Km for trehalose was 34.5 mM. Among seven oligosaccharides studied, the enzyme formed glucose only from trehalose. Neutral trehalase is located in the cytosol. A polyclonal rabbit antiserum raised against neutral trehalase precipitates the enzyme in the presence of protein A. The antiserum does not react with acid trehalase. Dephosphorylation by alkaline phosphatase from Escherichia coli of the active phosphorylated enzyme is accompanied by greater than or equal to 90% inactivation. Rephosphorylation by incubation with the catalytic subunit of beef heart protein kinase is accompanied by reactivation and incorporation of 0.85 mol of phosphate/mol subunit (80,000 Da). The phosphorylated amino acid residue was identified as phosphoserine.