Genes for two subunits of acetyl coenzyme A carboxylase of Anabaena sp. strain PCC 7120: biotin carboxylase and biotin carboxyl carrier protein.

Genes for two subunits of acetyl-coenzyme A carboxylase, biotin carboxylase and biotin carboxyl carrier protein, have been cloned from Anabaena sp. strain PCC 7120. The two proteins are 181 and 447 amino acids long and show 40 and 57% identity to the corresponding Escherichia coli proteins, respectively. The sequence of the biotinylation site in Anabaena sp. strain PCC 7120 is MetLysLeu, not the MetLysMet found in other sequences of biotin-dependent carboxylases. The amino acid sequence of biotin carboxylase is also very similar (32 to 47% identity) to the sequence of the biotin carboxylase domain of other biotin-dependent carboxylases. Genes for these two subunits of acetyl-coenzyme A carboxylase are not linked in Anabaena sp. strain PCC 7120, contrary to the situation in E. coli, in which they are in one operon.     

Multiple acyl-coenzyme A carboxylases in Pseudomonas citronellolis.

Pseudomonas citronellolis was shown to contain four different acyl-coenzyme A carboxylases, including acetyl-, propionyl-, 3-methylcrotonyl-, and geranyl-CoA carboxylases, when grown on the appropriate carbon sources. Acetyl-CoA carboxylase activity in crude extracts was stimulated approximately 40-fold by inclusion of 0.4-0.5 M ammonium sulfate in the assay. Unexpectedly high levels of propionyl-CoA carboxylase activity, also stimulated by ammonium sulfate, were found in acetate-grown cells. That these acetyl- and propionyl-CoA carboxylase activities were due to different enzymes was shown by their resolution during purification by a procedure that stabilized acetyl-CoA carboxylase as a complex and separated propionyl-CoA carboxylase into two required protein fractions. Propionate- or valine-grown cells contained a propionyl-CoA carboxylase activity that was strongly inhibited by ammonium sulfate in the assay, and which may represent an inducible form of the enzyme. Geranyl- and 3-methylcrotonyl-CoA carboxylases that catalyze the carboxylation of the 3-methyl groups of homologous acyl-CoA acceptors, were induced by growth on the monoterpenes, citronellic or geranoic acid; only 3-methylcrotonyl-CoA carboxylase was induced by growth on leucine or isovaleric acid. Induction of either carboxylase was associated with the appearance of similar high-molecular-weight, biotin-containing proteins as measured by gel filtration. These two carboxylases are probably distinct enzymes since 3-methyl-crotonyl-CoA carboxylase from isovalerate-grown cells does not carboxylate geranyl-CoA, while geranyl-CoA carboxylase will carboxylate both acyl-CoA homologues. P. citronellolis appears to be a useful system for studying the structural aspects of pairs of homologous acyl-CoA carboxylases.     

Biosynthesis of Leaf Alcohol: Kinetic Study of Lipoxygenase Activation Process Caused by Hydroperoxylinoleic Acid

Quaternary structure of ribulose-1, 5-bisphosphate (RuP₂) carboxylase from the autotrophically grown cells of blue-green alga, Anabaena cylindrica, was studied. Sedimentation coefficient (s_{20, w}) of the enzyme was determined to be 18.3 S by the sucrose density gradient centrifugation. The molecular weight was estimated to be 5.0 × 10⁵ by the Sepharose 4B gel filtration technique. The purification of the enzyme from the algal cells was undertaken by means of sucrose density gradient centrifugation and DEAE-Sephadex A–50 ion-exchange column chromatography, and the structural make-up of the enzyme containing two subunits, A (M. W., 5.2 × 10⁴) and B (M. W., 1.2 × 10⁴) was established by the Na-dodecylsulfate polyacrylamide gel electrophoresis experiment. Structural similarity of the algal RuP₂carboxylase with the spinach enzyme was further demonstrated by the Ouchterlony double immunodiffusion experiment.     

Ribulose Diphosphate Carboxylase Synthesis in Euglena: III. Serological Relationships of the Intact Enzyme and its Subunits

Ribulose 1,5-diphosphate carboxylase was isolated from Euglena gracilis Klebs strain Z Pringsheim, Chlorella fusca var. vacuolata, and Chlamydobotrys stellata, and the subunits from each enzyme were separated and purified by gel filtration on Sephadex G-200 in the presence of sodium dodecyl sulfate. Rabbit antibody was elicited against purified Euglena ribulose 1,5-diphosphate carboxylase whole enzyme and the isolated large and small subunits. Euglena ribulose 1,5-diphosphate carboxylase showed partial immunological identity on Ouchterlony gels with the Chlorella and Chlamydobotrys carboxylases. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of immunoprecipitates between antibody to the Euglena large subunit and the isolated large subunits of the Chlorella and Chlamydobotrys enzymes showed this was due to determinants on the large subunit. There was no serological affinity between the small subunits of the Euglena, Chlorella, and Chlamydobotrys carboxylases, and NH₂-terminal amino acid analyses provided further evidence of variability in the structure of the small subunits.     

Purification and Characterization of Acetyl-CoA Carboxylase from the Diatom Cyclotella cryptica

Acetyl-CoA carboxylase from the diatom Cyclotella cryptica has been purified to near homogeneity by the use of ammonium sulfate fractionation, gel filtration chromatography, and affinity chromatography with monomeric avidin-agarose. The specific activity of the final preparation was as high as 14.6 micromoles malonyl-CoA formed per milligram protein per minute, indicating a 600-fold purification. Native acetyl-CoA carboxylase has a molecular weight of approximately 740 kilodaltons and appears to be composed of four identical biotin-containing subunits. The enzyme has maximal activity at pH 8.2, but enzyme stability is greater at pH 6.5. K_m values for MgATP, acetyl-CoA, and HCO₃- were determined to be 65, 233, and 750 micromolar, respectively. The purified enzyme is strongly inhibited by palmitoyl-CoA, and is inhibited to a lesser extent by malonyl-CoA, ADP, and phosphate. Pyruvate stimulates enzymatic activity to a slight extent. Acetyl-CoA carboxylase from Cyclotella cryptica is not inhibited by cyclohexanedione or aryloxyphenoxypropionic acid herbicides as strongly as monocot acetyl-CoA carboxylases; 50% and 0% inhibition was observed in the presence of 23 micromolar clethodim and 100 micromolar haloxyfop, respectively.     

Nitrate-dependent degradation of acetone by Alicycliphilus and Paracoccus strains and comparison of acetone carboxylase enzymes

A novel acetone-degrading, nitrate-reducing bacterium, strain KN Bun08, was isolated from an enrichment culture with butanone and nitrate as the sole sources of carbon and energy. The cells were motile short rods, 0.5 to 1 by 1 to 2 μm in size, which gave Gram-positive staining results in the exponential growth phase and Gram-negative staining results in the stationary-growth phase. Based on 16S rRNA gene sequence analysis, the isolate was assigned to the genus Alicycliphilus. Besides butanone and acetone, the strain used numerous fatty acids as substrates. An ATP-dependent acetone-carboxylating enzyme was enriched from cell extracts of this bacterium and of Alicycliphilus denitrificans K601(T) by two subsequent DEAE Sepharose column procedures. For comparison, acetone carboxylases were enriched from two additional nitrate-reducing bacterial species, Paracoccus denitrificans and P. pantotrophus. The products of the carboxylase reaction were acetoacetate and AMP rather than ADP. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of cell extracts and of the various enzyme preparations revealed bands corresponding to molecular masses of 85, 78, and 20 kDa, suggesting similarities to the acetone carboxylase enzymes described in detail for the aerobic bacterium Xanthobacter autotrophicus strain Py2 (85.3, 78.3, and 19.6 kDa) and the phototrophic bacterium Rhodobacter capsulatus. Protein bands were excised and compared by mass spectrometry with those of acetone carboxylases of aerobic bacteria. The results document the finding that the nitrate-reducing bacteria studied here use acetone-carboxylating enzymes similar to those of aerobic and phototrophic bacteria.     

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.     

Pyruvate carboxylase from Pseudomonas citronellolis: shape of the enzyme, and localization of its prosthetic biotin group by electron microscopic affinity labeling

Pseudomonas citronellolis is known to contain a pyruvate carboxylase with an alpha 4 beta 4 composition. All the other pyruvate carboxylases investigated so far are made up of four seemingly identical subunits. Nevertheless, this exceptional pyruvate carboxylase exhibits a size and overall shape similar to other pyruvate carboxylases. Electron microscopic affinity labeling with avidin revealed that the prosthetic biotin groups (one per alpha beta unit, i.e. four per enzyme particle) are located close to the inter-unit junctions of pairs of alpha beta units making up the enzyme. This position of the prosthetic biotin groups is very similar to the location of the biotin in the other carboxylases.     

C-phycocyanin and allophycocyanin in two species of blue-green algae

1. The biliproteins C-phycocyanin and allophycocyanin were purified from the blue-green alga Anabaena variabilis by ammonium sulphate fractionation and gel filtration. 2. An assay procedure that enabled the proportion of the two pigments, present as a mixture, to be determined was devised by using the data provided by spectrophotometric analysis of the purified biliproteins. 3. The degree of association and relative proportions of the two pigments were analysed by the application of this procedure to the components separated by thin-layer gel filtration. 4. The C-phycocyanin/allophycocyanin ratio remained essentially constant in algal extracts prepared at various stages throughout the growth cycle or after growth under conditions of reduced illumination. 5. The behaviour of the C-phycocyanin aggregate species from Anacystis nidulans suggested that they were of appreciably lower molecular weight than those observed in extracts of Anabaena variabilis.     

A multienzyme complex for CO2 fixation.

Acetyl-coenzyme A carboxylase from Euglena gracilis strain Z was isolated as a component of a multienzyme complex which includes phosphoenolpyruvate carboxylase and malate dehydrogenase. The multienzyme complex was shown to exist in crude extracts and was purified to a homogeneous protein with a molecular weight of 360,000 by gel filtration. The ratio of the activities of the constituent enzymes was acetyl-CoA carboxylase:phosphoenolpyruvate carboxylase:malate dehydrogenase, 1:25:500. The complex is proposed to operate in conjunction with malic enzyme, which is present in Euglena, to facilitate the formation of substrates, malonyl-CoA, and NADPH, for fatty acid biosynthesis. The interaction of the enzymes may represent a means of control of acetyl-CoA carboxylase activity in organisms which do not possess an enzyme subject to allosteric regulation. The acetyl-CoA carboxylase activity from Euglena is unaffected by citrate and isocitrate.     

Characterization of a bifunctional archaeal acyl coenzyme A carboxylase

Acyl coenzyme A carboxylase (acyl-CoA carboxylase) was purified from Acidianus brierleyi. The purified enzyme showed a unique subunit structure (three subunits with apparent molecular masses of 62, 59, and 20 kDa) and a molecular mass of approximately 540 kDa, indicating an alpha(4)beta(4)gamma(4) subunit structure. The optimum temperature for the enzyme was 60 to 70 degrees C, and the optimum pH was around 6.4 to 6.9. Interestingly, the purified enzyme also had propionyl-CoA carboxylase activity. The apparent K(m) for acetyl-CoA was 0.17 +/- 0.03 mM, with a V(max) of 43.3 +/- 2.8 U mg(-1), and the K(m) for propionyl-CoA was 0.10 +/- 0.008 mM, with a V(max) of 40.8 +/- 1.0 U mg(-1). This result showed that A. brierleyi acyl-CoA carboxylase is a bifunctional enzyme in the modified 3-hydroxypropionate cycle. Both enzymatic activities were inhibited by malonyl-CoA, methymalonyl-CoA, succinyl-CoA, or CoA but not by palmitoyl-CoA. The gene encoding acyl-CoA carboxylase was cloned and characterized. Homology searches of the deduced amino acid sequences of the 62-, 59-, and 20-kDa subunits indicated the presence of functional domains for carboxyltransferase, biotin carboxylase, and biotin carboxyl carrier protein, respectively. Amino acid sequence alignment of acetyl-CoA carboxylases revealed that archaeal acyl-CoA carboxylases are closer to those of Bacteria than to those of Eucarya. The substrate-binding motifs of the enzymes are highly conserved among the three domains. The ATP-binding residues were found in the biotin carboxylase subunit, whereas the conserved biotin-binding site was located on the biotin carboxyl carrier protein. The acyl-CoA-binding site and the carboxybiotin-binding site were found in the carboxyltransferase subunit.     

Improved purification and further characterization of acetyl-CoA carboxylase from cultured cells of parsley (Petroselinum hortense)

Acetyl-CoA carboxylase from irradiated cell-suspension cultures of parsley (Petroselinum hortense) has been purified to apparent homogeneity. The procedure included affinity chromatography of the enzyme on avidinmonomer--Sepharose 4B. Molecular weights of about 420000 for the native enzyme and about 220000 for the enzyme subunit were determined respectively by gel filtration or sucrose-density-gradient sedimentation and by electrophoresis in the presence of dodecyl sulfate. The purified enzyme showed an isoelectric point of 5. The enzyme carboxylated the straight-chain acyl-CoA esters of acetate, propionate, and butyrate at decreasing rates in this order. The catalytic efficiency of the carboxylase was highest when ATP existed largely as MgATP2- complex. At the optimum pH of 8 the apparent Km values for the substrates were: acetyl-CoA, 0.15 mmol/1; bicarbonate, 1 mmol/1; MgATP2-, 0.07 mmol/1. The carboxylase was inhibited by greater than 50 mmol/l NaCl, KCl, or Tris/HCl buffer. The putative allosteric activator, citrate, stimulated the enzyme only slightly at concentrations below 2 mmol/l, but strongly inhibited the carboxylase at higher concentrations. The results of these studies demonstrate that several properties of the light-inducible acetyl-CoA carboxylase of parsley cells, an enzyme of the flavonoid pathway, are remarkably similar to those of acetyl-CoA carboxylases from a variety of other organisms.     

Purification and characterization of the thermostable ribulose-1,5-bisphosphate carboxylase/oxygenase from the thermophilic purple bacterium Chromatium tepidum

The Calvin cycle enzyme ribulose-bisphosphate carboxylase/oxygenase has been purified and characterized from the thermophilic and obligately anaerobic purple sulfur bacterium, Chromatium tepidum. The enzyme is an L8S8 carboxylase with a molecular mass near 550 kDa. No evidence for a second form of the enzyme lacking small subunits was obtained. C. tepidum ribulose-bisphosphate carboxylase/oxygenase was stable to heating to temperatures of 60 degrees C and could be readily purified in an active form at room temperature. Both carboxylase and oxygenase activities of this enzyme were Mg2+-dependent and carboxylase activity was sensitive to the effector 6-phosphogluconic acid. The Km for ribulose bisphosphate for the carboxylase activity of the C. tepidum enzyme was substantially higher than that observed in mesophilic Calvin cycle autotrophs. Amino acid composition and immunological analyses of C. tepidum and Chromatium vinosum ribulose-bisphosphate carboxylases showed the enzymes to be highly related despite significant differences in heat stability. It is hypothesized that thermal stability of C. tepidum ribulose-bisphosphate carboxylase/oxygenase is due to differences in primary structure affecting folding patterns in both the large and small subunits and is clearly not the result of any unique quaternary structure of the thermostable enzyme.     

Carboxylase genes of Sulfolobus metallicus

Carbon dioxide limitation of Sulfolobus metallicus resulted in increased cellular concentrations of polypeptides that were predicted to be biotin carboxylase and biotin carboxyl-carrier-protein components of a protein complex. These polypeptides were coeluted from a native polyacrylamide gel and were estimated at 19 and 59 kDa after separation by denaturing gel electrophoresis. Their encoding genes were identified, sequenced and shown to code for polypeptides of 18,580 and 58,235 Da with similarities to biotin carboxyl carrier proteins and biotin carboxylases, respectively. The genes overlapped at the second of two stop codons that terminated the carboxylase gene. A third gene occurred on the opposite strand, 293 bp upstream of the biotin carboxylase gene. Its deduced amino acid sequence was similar to those of carboxyl transferase subunits of carboxylase enzymes, in particular to those of the propionyl-CoA carboxylases. It is proposed that the three described genes could encode the key enzyme complex responsible for carbon dioxide fixation during autotrophic growth of the thermoacidophilic archaea. Received: 24 February 1999 / Accepted: 30 July 1999     

Purification and Characterization of 3-Methylcrotonyl-Coenzyme A Carboxylase from Higher Plant Mitochondria

3-Methylcrotonyl-coenzyme A (CoA) carboxylase was purified to homogeneity from pea (Pisum sativum L.) leaf and potato (Solanum tuberosum L.) tuber mitochondria. The native enzyme has an apparent molecular weight of 530,000 in pea leaf and 500,000 in potato tuber as measured by gel filtration. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate disclosed two nonidentical subunits. The larger subunit (B subunit) is biotinylated and has an apparent molecular weight of 76,000 in pea leaf and 74,000 in potato tuber. The smaller subunit (A subunit) is biotin free and has an apparent molecular weight of 54,000 in pea leaf and 53,000 in potato tuber. The biotin content of the enzyme is 1 mol/133,000 g of protein and 1 mol/128,000 g of protein in pea leaf and potato tuber, respectively. These values are consistent with an A4B4 tetrameric structure for the native enzyme. Maximal 3-methylcrotonyl-CoA carboxylase activity was found at pH 8 to 8.3 and at 35 to 38[deg]C in the presence of Mg2+. Kinetic constants (apparent Km values) for the enzyme substrates 3-methylcrotonyl-CoA, ATP, and HCO3- were: 0.1 mM, 0.1 mM, and 0.9 mM, respectively, for pea leaf 3-methylcrotonyl-CoA carboxylase and 0.1 mM, 0.07 mM, and 0.34 mM, respectively, for potato tuber 3-methylcrotonyl-CoA carboxylase. A steady-state kinetic analysis of the carboxylase-catalyzed carboxylation of 3-methylcrotonyl-CoA gave rise to parallel line patterns in double reciprocal plots of initial velocity with the substrate pairs 3-methylcrotonyl-CoA plus ATP and 3-methylcrotonyl-CoA plus HCO3- and an intersecting line pattern with the substrate pair HCO3- plus ATP. It was concluded that the kinetic mechanism involves a double displacement. Purified 3-methylcrotonyl-CoA carboxylase was inhibited by end products of the reaction catalyzed, namely ADP and orthophosphate, and by 3-hydroxy-3-methylglutaryl-CoA. Finally, as for the 3-methylcrotonyl-CoA carboxylases from mammalian and bacterial sources, plant 3-methylcrotonyl-CoA carboxylase was sensitive to sulfhydryl and arginyl reagents.     

Comparison of acetyl-CoA carboxylases from parsley cell cultures and wheat germ

The activity of acetyl-CoA carboxylase of suspension-cultured cells of parsley (Petroselinum hortense Hoffm.) is greatly stimulated by light soon after transferring cells to new culture medium. Parsley acetyl-CoA carboxylase has been purified from frozen cells by treatment of the crude protein extract with Dowex 1 × 2 and polyethyleneimine, precipitation with (NH₄)₂SO₄, chromatography on DEAE-cellulose and blue Sepharose CL-6B, and gel filtration on Sepharose 6B. A recovery of about 8% has been achieved with a 300-fold increase in specific activity. Wheat germ acetyl-CoA carboxylase has been purified 2180-fold by a similar procedure. The two carboxylases have the following characteristics: Molecular weights of 840,000 for the parsley carboxylase and 700,000 for the wheat germ carboxylase have been estimated from the elution volumes of a calibrated Sepharose 6B column. Analysis by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate showed that the carboxylases from parsley and wheat each are composed of one large subunit (M_r = 210,000 and 240,000, respectively) and possibly one smaller polypeptide component (M_r = 105,000 and 98,000, respectively). Avidin-binding experiments demonstrated that the 240,000 — M_r component of wheat germ carboxylase is the biotin-containing subunit of this enzyme. No isoenzymes of the parsley carboxylase could be demonstrated.     

Biochemical relationship of phosphoenolpyruvate carboxylases (PEPCs) from thermophilic archaea

An archaeal phosphoenolpyruvate carboxylase (PEPC) was purified from an acidophilic extreme thermophile, Sulfolobus acidocaldarius. The native enzyme was a homotetramer of 260±20 kDa molecular mass composed of 60±5 kDa subunits. The enzyme appeared to have a temperature optimum of 90°C and a pH optimum of 8.0. The activity of S. acidocaldarius phosphoenolpyruvate carboxylase was inhibited by l-aspartate and l-malate, but not enhanced by any metabolites. In comparison to the enzymatic and molecular properties of all other phosphoenolpyruvate carboxylases including another archaeal entity from the hyperthermophilic methanogen Methanothermus sociabilis, the archaeal phosphoenolpyruvate carboxylases were quite different from bacterial and eucaryal counterparts, and their small size and the lack of positively allosteric regulation were likely to be peculiar to the enzyme of the domain Archaea.     

Biotin subunits of acetyl CoA carboxylase and pyruvate carboxylase from a thermophilic Bacillus.

Two biotin-containing polypeptides of molecular weights 140,000 and 22,000 have been identified by gel electrophoresis in a sodium dodecyl sulfate-denatured extract of cells of a thermophilic Bacillus. These polypeptides can be separated from each other by either gel filtration through Sepharose 6B or affinity chromatography on a Sepharose-avidin column. The larger polypeptide is renatured under appropriate conditions to yield enzymically active pyruvate carboxylase. Enzyme reconstitution experiments show that the smaller polypeptide is a component of acetyl CoA carboxylase. The biotin subunits of these two carboxylases are thus distinct from, and dissimilar to, each other. The demonstration that a pyruvate carboxylase-deficient mutant of the Bacillus contains the smaller, but not the larger, polypeptide corroborates this conclusion.     

Acetone and butanone metabolism of the denitrifying bacterium "Aromatoleum aromaticum" demonstrates novel biochemical properties of an ATP-dependent aliphatic ketone carboxylase

The anaerobic and aerobic metabolism of acetone and butanone in the betaproteobacterium "Aromatoleum aromaticum" is initiated by their ATP-dependent carboxylation to acetoacetate and 3-oxopentanoic acid, respectively. Both reactions are catalyzed by the same enzyme, acetone carboxylase, which was purified and characterized. Acetone carboxylase is highly induced under growth on acetone or butanone and accounts for at least 5.5% of total cell protein. The enzyme consists of three subunits of 85, 75, and 20 kDa, respectively, in a (αβγ)(2) composition and contains 1 Zn and 2 Fe per heterohexamer but no organic cofactors. Chromatographic analysis of the ATP hydrolysis products indicated that ATP was exclusively cleaved to AMP and 2 P(i). The stoichiometry was determined to be 2 ATP consumed per acetone carboxylated. Purified acetone carboxylase from A. aromaticum catalyzes the carboxylation of acetone and butanone as the only substrates. However, the enzyme shows induced (uncoupled) ATPase activity with many other substrates that were not carboxylated. Acetone carboxylase is a member of a protein family that also contains acetone carboxylases of various other organisms, acetophenone carboxylase of A. aromaticum, and ATP-dependent hydantoinases/oxoprolinases. While the members of this family share several characteristic features, they differ with respect to the products of ATP hydrolysis, subunit composition, and metal content.     

N-terminal amino acid sequence of persimmon fruit beta-galactosidase.

beta-Galactosidase (EC 3.2.1.23) from persimmon fruit was purified 114-fold with a 15% yield using Sephadex G-100 gel filtration, CM-Sephadex ion exchange, and Sephacryl S-200 gel filtration chromatography, with subsequent electroelution from nondenaturing polyacrylamide gel electrophoresis (PAGE) gels. The estimated molecular mass of the native beta-galactosidase by Sephacryl S-200 was 118 kD. After sodium dodecyl sulfate-PAGE of the enzyme electroeluted from native gels, two subunits with estimated molecular masses of 34 and 44 kD were observed, suggesting that the native enzyme was an aggregate of several subunits. Amino acid composition and N-terminal amino acid sequences of the two major subunits were different.