Malonyl-CoA decarboxylase from Streptomyces erythreus: purification, properties, and possible role in the production of erythromycin

Malonyl-CoA decarboxylase was purified (800-fold) from an erythromycin-producing strain of Streptomyces erythreus using DEAE-cellulose, Sephadex G-100, SP-Sephadex, and gel filtration with Sephadex G-75. The molecular weight of the native enzyme was 93,000 as determined by gel filtration and the subunit molecular weight was 45,000 as estimated by sodium dodecyl sulfate-polyacrylamide electrophoresis, suggesting an alpha 2 subunit composition for the native enzyme. Evidence is presented that during the purification procedure and storage a proteolytic cleavage occurred resulting in the formation of 30- and 15-kDa peptides. The enzyme showed a pH optimum of about 5.0 whereas the vertebrate enzyme showed an optimum at alkaline pH. The enzyme decarboxylated malonyl-CoA with a Km of 143 microM and V of 250 nmol min-1 mg-1. For the decarboxylation of methylmalonyl-CoA this enzyme showed the opposite stereospecificity to that shown by vertebrate enzyme; the (R) isomer was decarboxylated at 3% of the rate observed with malonyl-CoA while the (S) isomer was not a substrate. Neither avidin nor biotin affected the rate of malonyl-CoA decarboxylation, suggesting that biotin is not involved in catalysis. Acetyl-CoA and free CoA were found to be competitive inhibitors. Propionyl-CoA, butyryl-CoA, succinyl-CoA, and methylmalonyl-CoA showed little inhibition, and neither thiol-directed reagents nor chelating agents inhibited the enzyme. High ionic strength and sulfate ions caused reversible inhibition of the enzymatic activity. Under two different cultural conditions the time course of appearance of malonyl-CoA decarboxylase was determined by measuring the enzyme activity and the level of enzyme protein by an immunological method using rabbit antibodies prepared against the enzyme. In both cases the increase and decrease in the decarboxylase correlated with the rate of production of erythromycin, suggesting a possible role for this enzyme in the antibiotic production.     

Malonyl-CoA decarboxylase from Mycobacterium tuberculosis and Pseudomonas fluorescens.

Malonyl-CoA decarboxylase was partially purified (nearly 1000-fold) from Mycobacterium tuberculosis H37Ra by ammonium sulfate precipitation, gel filtration with Sepharose 6B, and chromatography on DEAE Sephacel, carboxymethyl-Sephadex, and NADP-agarose. Polyacrylamide gel electrophoresis showed a major band (60–70%), which contained the enzymatic activity, and a minor band which had no decarboxylase activity. The molecular weight of the enzyme was 44,000, and the PI and pH optimum were 6.7 and 5.5, respectively. The enzyme showed a typical Michaelis-Menten substrate saturation, with an apparent K_m and V of 0.2 mm and 3.85 μmol/min/mg, respectively. It catalyzed decarboxylation of methylmalonyl-CoA only at 5% of the rate observed with malonyl-CoA, whereas malonic acid and succinyl-CoA were not decarboxylated. Antibodies prepared against malonyl-CoA decarboxylase from the uropygial glands of goose and rat liver mitochondria did not inhibit the bacterial enzyme. Avidin did not inhibit the enzyme suggesting that biotin was not involved in the reaction. Thiol-directed reagents inhibited the enzyme as did CoA, acetyl-CoA, propionyl-CoA, methylmalonyl-CoA, and succinyl-CoA. Malonyl-CoA decarboxylase was also partially purified from malonate-grown Pseudomonas fluorescens. The molecular weight of this enzyme was 56,000 and the pH optimum and apparent K_m were 5.5 and 1 mm, respectively. Unlike the mycobacterial enzyme, this enzyme was insensitive to p-hydroxymercuribenzoate, acetyl-CoA, and propionyl-CoA, and it was less sensitive to inhibition by succinyl-CoA and CoA than the mycobacterial enzyme. The size and properties of the two bacterial enzymes suggest that these are quite unlike the mammalian and avian enzymes and that they constitute a different class of malonyl-CoA decarboxylases.     

Purification and properties of malonyl-CoA decarboxylase from rat liver mitochondria and its immunological comparison with the enzymes from rat brain, heart, and mammary gland.

Malonyl-CoA decarboxylase (EC 4.1.1.9) was found to be localized in the mitochondria in rat liver. Low ionic strength (10 mm Na phosphate) buffer extracted the bulk (>85%) of the enzyme from the mitochondria. From this extract the enzyme was purified over 2,000-fold using a combination of (NH₄)₂SO₄ precipitation, gel filtration with Sepharose 4B and Sephadex G-150, ion exchange chromatography with QAE-Sephadex and CM-Sephadex, and finally chromatography on NADP-agarose. The purified enzyme, which had a specific activity of about 16 μmol/min/mg, appeared to be electrophoretically homogeneous and had a molecular weight of 160,000. The decarboxylase had a broad pH optimum between 8.5 and 10.0 and showed a typical Michaelis-Menten substrate saturation pattern from which K_m and V were calculated to be 54 μm and 18.8 μmol/min/mg, respectively. This enzyme decarboxylated neither malonic acid nor methylmalonyl-CoA and was severely inhibited by thiol-directed reagents such as p-hydroxymercuribenzoate and N-ethylmaleimide but not by iodoacetamide. Acetyl-CoA, propionyl-CoA, and methylmalonyl-CoA also inhibited the enzyme. The purified decarboxylase was immunogenic in rabbits and Ouchterlony double diffusion analysis revealed a single precipitant line with the purified enzyme. The IgG fraction isolated from the antiserum inhibited the enzyme from not only liver mitochondria but also the mammary gland, heart, and kidney of the rat. However, malonyl-CoA decarboxylase from rat brain mitochondria was not inhibited by the antibody. Malonyl-CoA decarboxylase purified from the uropygial gland of a domestic goose neither cross reacted nor was it inhibited by the antiserum prepared against the rat liver mitochondrial enzyme and the antibody against the goose enzyme neither cross-reacted nor inhibited the enzyme from the rat. It is proposed that a role for mitochondrial malonyl-CoA decarboxylase is to decarboxylate malonyl-CoA generated by propionyl-CoA carboxylase and thus protect mitochondrial enzymes susceptible to inhibition by malonyl-CoA.     

Life by a new decarboxylation-dependent energy conservation mechanism with Na as coupling ion

We report here a new mode of ATP synthesis in living cells. The anaerobic bacterium Propionigenium modestum gains its total energy for growth from the conversion of succinate to propionate according to: succinate + H₂O → propionate + HCO₃^- (G^o' = -20.6 kJ/mol). The small free energy change of this reaction does not allow a substrate-linked phosphorylation mechanism, and no electron transport phosphorylation takes place. Succinate was degraded by cell-free extracts to propionate and CO₂ via succinyl-CoA, methyl-malonyl-CoA and propionyl-CoA. This pathway involves a membrane-bound methylmalonyl-CoA decarboxylase which couples the exergonic decarboxylation with a Na⁺ ion transport across the membrane. The organism also contained a membrane-bound ATPase which was specifically activated by Na⁺ ions and catalyzed and transport of Na⁺ ions into inverted bacterial vesicles upon ATP hydrolysis. The transport was abolished by monensin but not by the uncoupler carbonylcyanide-p-trifluoromethoxy phenylhydrazone. Isolated membrane vesicles catalyzed the synthesis of ATP from ADP and inorganic phosphate when malonyl-CoA was decarboxylated and malonyl-CoA synthesis from acetyl-CoA when ATP was hydrolyzed. These syntheses were sensitive to monensin which indicates that Na⁺ functions as the coupling ion. We conclude from these results that ATP synthesis in P. modestum is driven by a Na⁺ ion gradient which is generated upon decarboxylation of methylmalonyl-CoA.     

Purification and characterization of a new sodium-transport decarboxylase. Methylmalonyl-CoA decarboxylase from Veillonella alcalescens

Upon resolution of the particulate cell fraction of Veillonella alcalescens by gel chromatography, membranes and ribosomes were clearly resolved. Methylmalonyl-CoA decarboxylase was bound to the membranes and not to ribosomes as reported earlier. Membrane vesicles containing methylmalonyl-CoA decarboxylase were prepared by disrupting V. alcalescens cells with a French pressure chamber. About 64% of the decarboxylase was oriented in these vesicles with the substrate binding site facing to the outside. The vesicles performed a rapid accumulation of Na+ ions in response to the decarboxylation of methylmalonyl-CoA. Decarboxylation and transport were highly uncoupled. The efficiency of the transport was considerably increased if methylmalonyl-CoA decarboxylation was retarded by using a low temperature or by slowly generating the substrate enzymically from propionyl-CoA. Under optimized conditions Na+ was concentrated inside the inverted vesicles eight-times higher than in the incubation medium. Methylmalonyl-CoA decarboxylase was solubilized from the membranes with Triton X-100 and purified about 20-fold by affinity chromatography on monomeric avidin-Sepharose columns. The decarboxylase was specifically activated by Na+ ions (apparent Km approximately equal to 0.6 mM). Whereas (S)-methylmalonyl-CoA was the superior substrate (apparent Km approximately equal to 7 microM), malonyl-CoA was also decarboxylated (apparent Km approximately equal to 35 microM). The decarboxylation of methylmalonyl-CoA yielded CO2 and not HCO-3 as the primary reaction product. Analysis of the purified enzyme by dodecylsulfate gel electrophoresis indicated the presence of four different polypeptides alpha, beta, gamma, delta with Mr 60 000, 33 000, 18 5000 and 14 000. The latter of these polypeptides was clearly visible only after silver staining but not after staining with Coomassie brilliant blue. A low molecular weight polypeptide with similar staining properties was also found in oxaloacetate decarboxylase. Methylmalonyl-CoA decarboxylase contained about 1 mol covalently bound biotin per 125 500 g protein which was localized exclusively in the gamma-subunit. This subunit therefore represents the biotin carboxyl carrier protein of methylmalonyl-CoA decarboxylase. A new very sensitive method for the detection of biotin-containing proteins is described.     

Anaerobic degradation of malonatevia malonyl-CoA bySporomusa malonica,Klebsiella oxytoca,andRhodobacter capsulatus

Anaerobic decarboxylation of malonate to acetate was studied withSporomusa malonica, Klebsiella oxytoca, andRhodobacter capsulatus. WhereasS. malonica could grow with malonate as sole substrate (Y=2.0 g·mol^–1), malonate decarboxylation byK. oxytoca was coupled with anaerobic growth only in the presence of a cosubstrate, e.g. sucrose or yeast extract (Y _s =1.1–1.8 g·mol malonate^–1).R. capsulatus used malonate anaerobically only in the light, and growth yields with acetate and malonate were identical. Malonate decarboxylation in cell-free extracts of all three bacteria was stimulated by catalytic amounts of malonyl-CoA, acetyl-CoA, or Coenzyme A plus ATP, indicating that actually malonyl-CoA was the substrate of decarboxylation. Less than 5% of malonyl-CoA decarboxylase activity was found associated with the cytoplasmic membrane. Avidin (except forK. oxytoca) and hydroxylamine inhibited the enzyme completely, EDTA inhibited partially. InS. malonica andK. oxytoca, malonyl-CoA decarboxylase was active only after growth with malonate; malonyl-CoA: acetate CoA transferase was found as well. These results indicate that malonate fermentation by these bacteria proceedsvia malonyl-CoA mediated by a CoA transferase and that subsequent decarboxylation to acetyl-CoA is catalyzed, at least withS. malonica andR. capsulatus, by a biotin enzyme.Abbreviations CoASH Coenzyme A - EDTA ethylenediamine tetraacetate     

Malonyl-CoA decarboxylase from the uropygial gland of waterfowl: purification, properties, immunological comparison, and role in regulating the synthesis of multimethyl-branched fatty acids.

Analysis of the acyl portion of the wax from the uropygial gland of muscovy duck, wood duck, (Cairininae subfamily) and Canadian goose (Anserinae) by combined gas-liquid chromatography and mass spectrometry showed that 2,4,6-trimethyloctanoic acid and 2,4,6-trimethylnonanoic acid were the major (~100%) components. Similar analyses of the wax from the glands of mallard and Peking duck (Anatinae) showed that 2- and 4-mono-methylhexanoic acids predominated (>75%) with no multimethyl-branched acids. The uropygial glands of the former group contained 20 to 100 times as much malonyl-CoA decarboxylase activity as those of the latter group. These results strongly support the hypothesis that this decarboxylase, by causing specific decarboxylation of malonyl-CoA, makes available only methylmalonyl-CoA for fatty acid synthesis, and thus causes the production of multimethyl-branched acids. Malonyl-CoA decarboxylase was purified to apparent homogeniety in 30% yield from the uropygial glands of muscovy and wood ducks. Properties of the enzyme from the ducks, such as S_20.w (7.8 S), molecular weight (190,000) subunit composition (4 × 47,000), amino acid composition, strict substrate specificity, pH optimum (~9.0), K_m (~33 μm), V (~80 μmol/min/mg), and inhibition by SH-directed reagents were similar to those observed with the decarboxylase from the domestic goose. Antiserum prepared against the goose enzyme cross-reacted with and inhibited the decarboxylase from the four genera of ducks and Canadian goose. Ouchterlony double-diffusion analyses showed fusion of precipitant lines with the enzyme from muscovy, wood duck, and Canadian goose, whereas spurs were observed with the enzymes from mallard and Peking ducks. Immunoelectrophoresis showed that the decarboxylases from muscovy and wood ducks were similar and that they were different from the enzyme from the domestic goose. It appears that during evolution, the subfamilies (Anserinae and Cairininae) which synthesize multimethyl-branched acids acquired the ability to produce a high level of malonyl-CoA decarboxylase, an enzyme which is also present in low levels in other organisms.     

Synthesis of multimethyl-branched fatty acids by avian and mammalian fatty acid synthetase and its regulation by malonyl-CoA decarboxylase in the uropygial gland

Fatty acid synthetase, partially purified by gel filtration with Sepharose 4B from goose liver, showed the same relative rate of incorporation of methylmalonyl-CoA (compared to malonyl-CoA) as that observed with the purified fatty acid synthetase from the uropygial gland. In the presence of acetyl-CoA, methylmalonyl-CoA was incorporated mainly into 2,4,6,8-tetramethyldecanoic acid and 2,4,6,8,10-pentamethyl-dodecanoic acid by the enzyme from both sources. Methylmalonyl-CoA was a competitive inhibitor with respect to malonyl-CoA for the enzyme from the gland just as previously observed for fatty acid synthetase from other animals. Furthermore, rabbit antiserum prepared against the gland enzyme cross-reacted with the liver enzyme, and Ouchterlony double-diffusion analyses showed complete fusion of the immunoprecipitant lines. The antiserum inhibited both the synthesis of n-fatty acids and branched fatty acids catalyzed by the synthetase from both liver and the uropygial gland. These results suggest that the synthetases from the two tissues are identical and that branched and n-fatty acids are synthesized by the same enzyme. Immunological examination of the 105,000g supernatant prepared from a variety of organs from the goose showed that only the uropygial gland contained a protein which cross-reacted with the antiserum prepared against malonyl-CoA decarboxylase purified from the gland. Thus, it is concluded that the reason for the synthesis of multimethyl-branched fatty acids by the fatty acid synthetase in the gland is that in this organ the tissue-specific and substrate-specific decarboxylase makes only methylmalonyl-CoA available to the synthetase. Fatty acid synthetase, partially purified from the mammary gland and the liver of rats, also catalyzed incorporation of [methyl-¹⁴C]methylmalonyl-CoA into 2,4,6,8-tetramethyldecanoic acid and 2,4,6,8-tetramethylundecanoic acid with acetyl-CoA and propionyl-CoA, respectively, as the primers. Evidence is also presented that fatty acids containing straight and branched regions can be generated by the fatty acid synthetase from the rat and goose, from methylmalonyl-CoA in the presence of malonyl-CoA or other precursors of n-fatty acids. These results provide support for the hypothesis that, under the pathological conditions which result in accumulation of methylmalonyl-CoA, abnormal branched acids can be generated by the fatty acid synthetase.     

Decarboxylation of homoarginine and lysine by an enzyme from Lathyrus sativus seedlings

Homoarginine decarboxylase has been purified ca 110-fold from Lathyrus sativus seedlings and resolved from arginine decarboxylase by DEAE-Sephadex column chromatography. The enzyme was less active than arginine decarboxylase and was highly labile. This preparation decarboxylated l-lysine in addition to L-homoarginine. The purified enzyme preparation had an absolute requirement for exogenous Mn²⁺ or Fe²⁺ for both the enzyme activities. The pH and temperature optima for decarboxylation of both homoarginine and lysine were the same viz. 8·4 and 41° respectively. The K_m value l-homoarginine was 3·33 mM and for l-lysine was 0·88 mM. Arginine and homoarginine decarboxylases appear to be different and separable entities having different physico-chemical characteristics, despite the fact that their respective guanido amino acid substrates undergo similar metabolic conversion to guanido- and diamines in this plant system.     

Decarboxylation of malonyl-CoA and biosynthesis of mevalonic acid in rat liver]

Biosynthesis of mevalonic acid (MVA), total formation of 14CO2 from [1,3-14C]malonyl-CoA and the activity of malonyl-CoA decarboxylase in subcellular fractions of rat liver were studied. The dependence of the rate of MVA biosynthesis on malonyl-CoA concentration was found to be linear both in 140,000 g supernatant and solubilized microsomal fractions. It was shown that in a composite system (140,000 g supernatant fraction added to washed microsomes, 10 : 1) the optimal concentration ratio for the substrates of MVA biosynthesis (malonyl-CoA and acetyl-CoA) is 1 to 2. In the absence of acetyl-CoA decarboxylation of [1,3-14C]malonyl-CoA was prevalent. In all subcellular fractions studied decarboxylation of [1,3-14C]malonyl-CoA prevailed over its incorporation into MVA, total non-saponified lipid fraction and fatty acids. The degree of malonyl-CoA, decarboxylation was not correlated with the rate of its incorporation into MVA, i. e. the increase in the 14CO2 formation was not accompanied by stimulation of [1,3-14C]malonyl-CoA incorporation either into MVA or into total non-saponified lipid fractions. The incorporation of [1-14C]acetyl-CoA into MVA under the same conditions was considerably lower than that of [1,3-14C]malonyl-CoA. In all subcellular fractions under study the activity of malonyl-CoA decarboxylase was found. The experimental data suggest that a remarkable part of malonyl-CoA is incorporated into MVA without preliminary decarboxylation. A possible role of malonyl-CoA decarboxylase as an enzyme which protects the cell against accumulation of malonyl-CoA and its immediate metabolites -- malonate and methylmalonyl-CoA is disucssed.     

An investigation into the role of malonyl-coenzyme A in isoprenoid biosynthesis

1. [¹⁴C]Malonyl-CoA was incorporated into isoprenoids by cell-free yeast preparations, by preparations from pigeon and rat liver, and by Hevea brasiliensis latex. 2. In agreement with previous reports the incorporation of acetyl-CoA into isoprenoids was not inhibited by avidin and was not stimulated by HCO₃⁻. In a cell-free yeast preparation addition of HCO₃⁻ stimulated the formation of fatty acids from acetyl-CoA and decreased the incorporation into unsaponifiable lipids. 3. The labelling patterns of β-hydroxy-β-methylglutaryl-CoA formed from [2-¹⁴C]- and [1,3-¹⁴C]-malonyl-CoA in rat and pigeon liver preparations were those that would be expected if malonyl-CoA underwent decarboxylation to acetyl-CoA before incorporation. 4. The labelling pattern of ergosterol formed by cell-free yeast preparations from [2-¹⁴C]malonyl-CoA was also consistent with decarboxylation of malonyl-CoA before incorporation. 5. The incorporation of [2-¹⁴C]malonyl-CoA into mevalonate by rat liver preparations was related to the malonyl-CoA decarboxylase activity present in the preparation.     

The malonate decarboxylase enzyme system of Malonomonas rubra: evidence for the cytoplasmic location of the biotin-containing component

Malonate decarboxylase of Malonomonas rubra is a complex enzyme system involving cytoplasmic and membrane-bound components. One of these is a biotin-containing protein of M_r 120'000, the location of which in the cytoplasm was deduced from the following criteria: (i) If the cytoplasm was incubated with avidin and the malonate decarboxylase subsequently completed with the membrane fraction the decarboxylase activity was abolished. The corresponding incubation of the membrane with avidin, however, was without effect. (ii) Western blot analysis identified the single biotin-containing polypeptide of M_r 120'000 within the cytoplasm. (iii) Transmission electron micrographs of immuno-gold labeled M. rubra cells clearly showed the location of the biotinyl protein within the cytoplasm, whereas the same procedure with Propionigenium modestum cells indicated the location of the biotin enzyme methylmalonyl-CoA decarboxylase in the cell membrane. The biotin-containing protein of the M. rubra malonate decarboxylase enzyme system was not retained by monomeric avidin-Sepharose columns but could be isolated with this column in a catalytically inactive form in the presence of detergents. If the high binding affinity of tetrameric avidin towards biotin was reduced by destructing part of the tryptophan residues by irradiation or oxidation with periodate, the inhibition of malonate decarboxylase by the modified avidin was partially reversed with an excess of biotin. Attempts to purify the biotin protein in its catalytically active state using modified avidin columns were without success.     

Lipid biosynthesis in sebaceous glands: regulation of the synthesis of n- and branched fatty acids by malonyl-coenzyme A decarboxylase.

Crude cell-free extracts isolated from the uropygial glands of goose catalyzed the carboxylation of propionyl-CoA but not acetyl-CoA. However, a partially purified preparation catalyzed the carboxylation of both substrates and the characteristics of this carboxylase were similar to those reported for chicken liver carboxylase. The Km and Vmax for the carboxylation of either acetyl-CoA or propionyl-CoA were 1.5 times 10- minus-5 M and 0.8 mumol per min per mg, respectively. In the crude extracts an inhibitor of the acetyl-CoA carboxylase activity was detected. The inhibitor was partially purified and identified as a protein that catalyzed the rapid decarboxylation of malonyl-CoA. This enzyme was avidin-insenitive and highly specific for malonyl-CoA with very low rates of decarboxylation for methylmalonyl-CoA and malonic acid. Vmax and Km for malonyl-CoA decarboxylation, at the pH optimum of 9.5, were 12.5 mumol per min per mg and 8 times 10- minus-4 M, respectively. The relative activities of the acetyl-CoA carboxylase and malonyl-CoA decarboxylase were about 4 mumol per min per gland and 70 mumoles per min per gland, respectively. Therefore acetyl-CoA and methylmalonyl-CoA should be the major primer and elongating agent, respectively, present in the gland. The major fatty acid formed from these precursors by the fatty acid synthetase of the gland would be 2,4,6,8-tetramethyl-decanoic acid which is known to be the major fatty acid of the gland (Buckner, J. S. and Kolattukudy, P. E. (1975), Biochemistry, following paper). Therefore it is concluded that the malonyl-CoA decarboxylase controls fatty acid synthesis in this gland.     

Sodium-dependent succinate decarboxylation by a new anaerobic bacterium belonging to the genus Peptostreptococcus

An anaerobic bacterium was isolated from a polluted sediment, with succinate and yeast extract as carbon and energy sources. The new strain was Gram-positive, the cells were coccal shaped, the mol% G+C content of the genomic DNA was 29, and the peptidoglycan was of the L-ornithine-D-glutamic acid type. Comparative sequence analysis of the 16S rRNA gene showed the new strain to belong to the genus Peptostreptococcus. Succinate, fumarate, pyruvate, 3-hydroxybutyrate and lysine supported growth. Succinate was degraded to propionate and presumably CO₂, with a stoichiometric cell yield. Key enzymes of the methylmalonyl-CoA decarboxylase pathway were present. The methylmalonyl-CoA decarboxylase activity was avidin-sensitive and sodium dependent, and about 5 mM Na⁺ was required for maximal activity. Whole cells, however, required at least 50 mM sodium for maximal succinate decarboxylation activity and to support the maximum growth rate. Sodium-dependent energy conservation coupled to succinate decarboxylation is shown for the first time to occur in a bacterium belonging to the group of Gram-positive bacteria containing the peptostreptococci and their relatives.     

Purification and properties of myo-inositol-1-phosphate dehydrogenase from germinating mung bean seeds

A novel enzyme, myo-inositol-1-phosphate dehydrogenase, which catalyzes the conversion of myo-inositol 1-phosphate to ribulose 5-phosphate has been purified 84-fold from mung bean seedling employing several common techniques. The molecular weight of this purified enzyme has been recorded as 88,500 by Sephadex G-200 column chromatography, and in sodium dodecyl sulfate-polyacrylamide gel electrophoresis one protein band containing three subunits of M_r 32,000 each was discernible. K_m values for NAD⁺ and myo-inositol 1-phosphate have been recorded as 2.8 × 10^?4 and 5.0 × 10^?4m, respectively. Production of NADH in myo-inositol-1-phosphate dehydrogenase reaction has also been evidenced by measurement of NADH fluorescence. Dehydrogenation and decarboxylation of myo-inositol 1-phosphate are mediated by the same enzyme. In fact, the rate of dehydrogenation corroborates with that of decarboxylation. Stoichiometry of this reaction suggests that for the production of 1 mol of ribulose 5-phosphate 2 mol of NAD⁺ are reduced.     

Developmental changes in malonate-related enzymes of rat brain.

Mitochondria and high-speed supernatant were prepared from rat brain homogenates at 0–50 days of age. The development of malonyl-CoA synthetase, malonyl-CoA decarboxylase, coenzyme A-transferases and acetyl-CoA hydrolase was examined and compared to de novo fatty acid biosynthesis. The specific activity of malonyl-CoA synthetase rose steeply between 6 and 10 days, and this sudden increase coincided with peak specific activity of fatty acid synthetase. Similarly, malonate activation by coenzyme A-transfer from succinyl-CoA increased rapidly at the same time. Transfer of the coenzyme A moiety from acetoacetyl-CoA was only minimal during this period. Brain mitochondria had active malonyl-CoA decarboxylase which showed an almost linear increase of specific activity between 0 and 50 days. Acetyl-CoA resulting from malonyl-CoA decarboxylation underwent enzymatic hydrolysis to acetate and free coenzyme A. Only traces of acetoacetate were recovered. In mitochondria, acetyl-CoA hydrolase increased progressively whereas the cytosolic enzyme had high specific activity at birth which declined slowly during maturation.     

Isolation and Characterization of Acyl Coenzyme A Carboxylases from Mycobacterium tuberculosis and Mycobacterium bovis, Which Produce Multiple Methyl-Branched Mycocerosic Acids

Mycobacterium tuberculosis H37Ra and M. bovis BCG produce multiple methyl-branched fatty acids called mycocerosic acids, presumably from methyl-malonyl coenzyme A (CoA). An acyl-CoA carboxylase was isolated from these organisms at a 30 to 50% yield by a purification procedure involving ammonium sulfate fractionation, gel filtration, and affinity chromatography with a monomeric avidin–Sepharose 4B-CL gel with d-biotin as the eluant. Sodium dodecyl sulfate electrophoresis and avidin binding indicate that each enzyme is probably composed of two dissimilar subunits with a covalently bound biotin in the larger subunit. The enzyme preparations from H37Ra and BCG had specific activities of 2.1 and 5.5 μmol min⁻¹ mg⁻¹, respectively, when propionyl-CoA was the substrate. The enzymes from the two species displayed striking similarities in their kinetic parameters. They showed maximal activity at pH 8.0 when propionyl-CoA was the substrate, but displayed a relatively broad pH-activity profile when acetyl-CoA was the substrate. With both substrates, potassium phosphate buffer gave maximal activity. Apparent K_m values for propionyl-CoA, ATP, Mg²⁺, and NaHCO₃ were 70 μM, 100 μM, 5.4 mM, and 2.2 mM, respectively. The enzyme also carboxylated acetyl-CoA and butyryl-CoA, and high-performance liquid chromatography showed the expected products of carboxylation. However, with these substrates, the K_m was higher and the V_max was lower than those of propionyl-CoA. The enzyme was shown to be stereospecific, synthesizing exclusively (S)-methylmalonyl-CoA from propionyl-CoA. No other acyl-CoA carboxylase was observed during the purification procedure, indicating that the present carboxylase may provide malonyl-CoA for the synthesis of n-fatty acids as well as methylmalonyl-CoA for the synthesis of mycocerosic acids.     

Purification and characterization of 1-aminocyclopropane-1-carboxylate N-malonyltransferase from etiolated mung bean hypocotyls

1-Aminocyclopropane-1-carboxylate (ACC) N-malonyltransferase converts ACC, an immediate precursor of ethylene, to the presumably inactive product malonyl-ACC (MACC). This enzyme plays a role in ethylene production by reducing the level of free ACC in plant tissue. In this study, ACC N-malonyltransferase was purified 3660-fold from etiolated mung bean (Vigna radiata) hypocotyls, with a 6% overall recovery. The final specific activity was about 83,000 nmol of MACC formed mg⁻¹ protein h⁻¹. The five-step purification protocol consisted of polyethylene glycol fractionation, Cibacron blue 3GA-agarose chromatography using salt gradient elution, Sephadex G-100 gel filtration, MonoQ anion-exchange chromatography, and Cibacron blue 3GA-agarose chromatography using malonyl-CoA plus ACC for elution. The molecular mass of the native enzyme determined by Sephadex G-100 chromatography was 50 ± 3 kD. Protein from the final purification step showed one major band at 55 kD after sodium dodecyl sulfate polyacrylamide gel electrophoresis, indicating that ACC N-malonyltransferase is a monomer. The mung bean ACC N-malonyltransferase has a pH optimum of 8.0, an apparent K_m of 0.5 mm for ACC and 0.2 mm for malonyl-coenzyme A, and an Arrhenius activation energy of 70.29 kJ mol⁻¹ degree⁻¹.     

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.