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.     

Organization and nucleotide sequences of the genes encoding the biotin carboxyl carrier protein and biotin carboxylase protein of Pseudomonas aeruginosa acetyl coenzyme A carboxylase.

The genetic organization of the Pseudomonas aeruginosa acetyl coenzyme A carboxylase (ACC) was investigated by cloning and characterizing a P. aeruginosa DNA fragment that complements an Escherichia coli strain with a conditional lethal mutation affecting the biotin carboxyl carrier protein (BCCP) subunit of ACC. DNA sequencing and RNA blot hybridization studies indicated that the P. aeruginosa accB (fabE) homolog, which encodes BCCP, is part of a 2-gene operon that includes accC (fabG), the structural gene for the biotin carboxylase subunit of ACC. P. aeruginosa homologs of the E. coli accA and accD, encoding the alpha and beta subunits of the ACC carboxyltransferase, were identified by hybridization of P. aeruginosa genomic DNA with the E. coli accA and accD. Data are presented which suggest that P. aeruginosa accA and accD homologs are not located either immediately upstream or downstream of the P. aeruginosa accBC operon. In contrast to E. coli, where BCCP is the only biotinylated protein, P. aeruginosa was found to contain at least three biotinylated proteins.     

The primary structure of the subunits of carbon monoxide dehydrogenase/acetyl-CoA synthase from Clostridium thermoaceticum.

CO dehydrogenase/acetyl-coenzyme A synthase (CODH) is the central enzyme in the pathway of acetyl-coenzyme A biosynthesis in Clostridium thermoaceticum. It catalyzes the interconversion of CO and CO2 and the synthesis of acetyl-coenzyme A from the methylated corrinoid/iron sulfur protein, CO, and coenzyme A. It is a nickel-iron-sulfur protein and contains two subunits in the form (alpha beta)3. Reported here is the cloning and sequencing of the genes for both subunits of CODH. The gene for the alpha subunit codes for a protein with 729 amino acids and a molecular weight of 81,730, and the beta gene for a protein with 674 amino acids and a molecular weight of 72,928. The alpha subunit follows the beta subunit by 23 bases and the genes for both subunits are preceded by a sequence which is similar to the Shine-Dalgarno sequence of Escherichia coli. No significant amino acid sequence homology has been found to any known sequence. Labeling CODH with 2,4-dinitrophenylsulfenyl chloride and isolating labeled peptide fragments demonstrated that a tryptophan, residue 418 of the alpha subunit, is protected by coenzyme A and thus may be considered a potential part of the coenzyme A site.     

Recombinant carboxyltransferase responsive to redox of pea plastidic acetyl-CoA carboxylase

Acetyl-CoA carboxylase regulates the rate of fatty acid synthesis. This enzyme in plants is localized in plastids and is believed to be composed of biotin carboxyl carrier protein, biotin carboxylase, and carboxyltransferase made up of alpha and beta polypeptides, although the enzyme has not been purified yet. Accumulated evidence shows that pea plastidic acetyl-CoA carboxylase is activated by light and the activation is caused by light-dependent reduction of carboxyltransferase, but not of biotin carboxylase, via a redox cascade. To understand the reductive activation of carboxyltransferase at the molecular level here, we obtained the active enzyme composed of decahistidine-tagged (His tag) alpha and beta polypeptides through the expression of the pea plastidic carboxyltransferase gene in Escherichia coli. Gel filtration showed that the molecular size of the recombinant carboxyltransferase is in agreement with that of partially purified carboxyltransferase from pea chloroplasts. The catalytic activity of the recombinant enzyme was similar to that of native carboxyltransferase. These results indicate that the molecular structure and conformation of recombinant carboxyltransferase resemble those of its native counterpart and that native carboxyltransferase is indeed composed of alpha and beta polypeptides. This recombinant enzyme was activated by dithiothreitol, a known reductant of S-S bonds, with a profile similar to that of its native counterpart. The recombinant enzyme was activated by reduced thioredoxin-f, a signal transducer of redox potential in chloroplasts under irradiation. Thus, this enzyme was redox-regulated, like that of the native carboxyltransferase.     

Cloning, expression, and functional characterization of the beta regulatory subunit of human methionine adenosyltransferase (MAT II)

MAT II, the extrahepatic form of methionine adenosyltransferase (MAT), consists of catalytic alpha(2)/alpha(2') subunits and a noncatalytic beta subunit, believed to have a regulatory function. The full-length cDNA that encodes the beta subunit of human MAT II was cloned and found to encode for a 334-amino acid protein with a calculated molecular weight of 37,552. Analysis of sequence homology showed similarity with bacterial enzymes that catalyze the reduction of TDP-linked sugars. The beta subunit cDNA was cloned into the pQE-30 expression vector, and the recombinant His tagged protein, which was expressed in Escherichia coli, was recognized by antibodies to the human MAT II, to synthetic peptides copying the sequence of native beta subunit protein, and to the rbeta protein. There is no cross-reactivity between the MAT II alpha(2) or beta subunits. None of the anti-beta subunit antibodies reacted with protein extracts of E. coli host cells, suggesting that these bacteria have no beta subunit protein. Interestingly, the rbeta subunit associated with E. coli as well as human MAT alpha subunits. This association changed the kinetic properties of both enzymes and lowered the K(m) of MAT for L-methionine. Together, the data show that we have cloned and expressed the human MAT II beta subunit and confirmed its long suspected regulatory function. This knowledge affords a molecular means by which MAT activity and consequently the levels of AdoMet may be modulated in mammalian cells.     

Isolation and sequencing of cDNA clones encoding alpha and beta subunits of Drosophila melanogaster casein kinase II.

Cloned cDNAs encoding both subunits of Drosophila melanogaster casein kinase II have been isolated by immunological screening of lambda gt11 expression libraries, and the complete amino acid sequence of both polypeptides has been deduced by DNA sequencing. The alpha cDNA contained an open reading frame of 336 amino acid residues, yielding a predicted molecular weight for the alpha polypeptide of 39,833. The alpha sequence contained the expected semi-invariant residues present in the catalytic domain of previously sequenced protein kinases, confirming that it is the catalytic subunit of the enzyme. Pairwise homology comparisons between the alpha sequence and the sequences of a variety of vertebrate protein kinase suggested that casein kinase II is a distantly related member of the protein kinase family. The beta subunit was derived from an open reading frame of 215 amino acid residues and was predicted to have a molecular weight of 24,700. The beta subunit exhibited no extensive homology to other proteins whose sequences are currently known.     

The gene encoding the biotin carboxylase subunit of Escherichia coli acetyl-CoA carboxylase.

We report the molecular cloning and DNA sequence of the gene encoding the biotin carboxylase subunit of Escherichia coli acetyl-CoA carboxylase. The biotin carboxylase gene encodes a protein of 449 residues that is strikingly similar to amino-terminal segments of two biotin-dependent carboxylase proteins, yeast pyruvate carboxylase and the alpha-subunit of rat propionyl-CoA carboxylase. The deduced biotin carboxylase sequence contains a consensus ATP binding site and a cysteine-containing sequence preserved in all sequenced bicarbonate-dependent biotin carboxylases that may play a key catalytic role. The gene encoding the biotin carboxyl carrier protein (BCCP) subunit of acetyl-CoA carboxylase is located upstream of the biotin carboxylase gene and the two genes are cotranscribed. As previously reported by others, the BCCP sequence encoded a protein of 16,688 molecular mass. However, this value is much smaller than that (22,500 daltons) obtained by analysis of the protein. Amino-terminal amino acid sequencing of the purified BCCP protein confirmed the deduced amino acid sequence indicating that BCCP is a protein of atypical physical properties. Northern and primer extension analyses demonstrate that BCCP and biotin carboxylase are transcribed as a single mRNA species that contains an unusually long untranslated leader preceding the BCCP gene. We have also determined the mutational alteration in a previously isolated acetyl-CoA carboxylase (fabE) mutant and show the lesion maps within the BCCP gene and results in a BCCP species defective in acceptance of biotin. Translational fusions of the carboxyl-terminal 110 or 84 (but not 76) amino acids of BCCP to beta-galactosidase resulted in biotinated beta-galactosidase molecules and production of one such fusion was shown to result in derepression of the biotin biosynthetic operon.     

Distinct structural features of the alpha and beta subunits of nitrogenase molybdenum-iron protein of Clostridium pasteurianum: an analysis of amino acid sequences

Nitrogenase is composed of two separately purified proteins, a molybdenum-iron (MoFe) protein and an iron (Fe) protein. Structural genes (nifD and nifK) encoding alpha and beta subunits of the MoFe protein of Clostridium pasteurianum (Cp) have been cloned and sequenced. The deduced amino acid sequences were analyzed for structures that could be related to the unique properties of the Cp protein, particularly its low capacity to form an active enzyme with a heterologous Fe protein. Cp nifK is located immediately downstream from Cp nifD, with the start codon of nifK overlapping by one base with the stop codon of nifD. An open reading frame following nifK was identified as nifE. The amino acid sequence deduced from nifK encompasses the partial amino acid sequences previously reported from the isolated beta subunit. Cp nifK encodes a polypeptide of 458 amino acid residues (Mr 50 115) whose amino-terminal region is about 50 residues shorter than the otherwise conserved corresponding polypeptides from four other organisms. In contrast, Cp alpha subunit (nifD product) contains an additional stretch of 50 amino acid residues in the 380-430 region, which is unique to the Cp protein. It therefore appears that the combined size of the alpha and beta subunits could be important to nitrogenase function. An analysis of the predicted secondary structure from the amino acid sequence of each subunit from three species (C. pasteurianum, Azotobacter vinelandii, and Rhizobium japonicum) further revealed structural features, including regions adjacent to some of the conserved cysteine residues, differentiating the Cp MoFe protein from others. These different regions may be further tested for correlation with distinct properties of Cp nitrogenase.     

Cloning and sequence analysis of the genes encoding the alpha and beta subunits of the E1 component of the pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus

A 4175-bp EcoRI fragment of DNA that encodes the alpha and beta chains of the pyruvate dehydrogenase (lipoamide) component (E1) of the pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus has been cloned in Escherichia coli. Its nucleotide sequence was determined. Open reading frames (pdhA, pdhB) corresponding to the E1 alpha subunit (368 amino acids, Mr 41,312, without the initiating methionine residue) and E1 beta subunit (324 amino acids, Mr 35,306, without the initiating methionine residue) were identified and confirmed with the aid of amino acid sequences determined directly from the purified polypeptide chains. The E1 beta gene begins just 3 bp downstream from the E1 alpha stop codon. It is followed, after a longer gap of 73 bp, by the start of another but incomplete open reading frame that, on the basis of its known amino acid sequence, encodes the dihydrolipoyl acetyltransferase (E2) component of the complex. All three genes are preceded by potential ribosome-binding sites and the gene cluster is located immediately downstream from a region of DNA showing numerous possible promoter sequences. The E1 alpha and E1 beta subunits of the B. stearothermophilus pyruvate dehydrogenase complex exhibit substantial sequence similarity with the E1 alpha and E1 beta subunits of pyruvate and branched-chain 2-oxo-acid dehydrogenase complexes from mammalian mitochondria and Pseudomonas putida. In particular, the E1 alpha chain contains the highly conserved sequence motif that has been found in all enzymes utilizing thiamin diphosphate as cofactor.     

Amino acid sequence of the alpha and beta subunits of Methanosarcina barkeri ATPase deduced from cloned genes. Similarity to subunits of eukaryotic vacuolar and F0F1-ATPases

The atpA and atpB genes coding for the alpha and beta subunits, respectively, of membrane ATPase were cloned from a methanogen Methanosarcina barkeri, and the amino acid sequences of the two subunits were deduced from the nucleotide sequences. The methanogenic alpha (578 amino acid residues) and beta (459 amino acid residues) subunits were highly homologous to the large and small subunits, respectively, of vacuolar H+-ATPases; 52% of the residues of the methanogenic alpha subunit were identical with those of the large subunit of vacuolar enzyme of carrot or Neurospora crassa, respectively, and 59, 60, and 59% of the residues of the methanogenic beta subunit were identical with those of the small subunits of N. crassa, Arabidopsis thaliana, and Sacharomyces cerevisiae, respectively. The methanogenic subunits were also highly homologous to the corresponding subunits of Sulfolobus acidocaldarius ATPase. The methanogenic alpha and beta subunits showed 22 and 24% identities with the beta and the alpha subunits of Escherichia coli F1, respectively. Furthermore, important amino acid residues identified genetically in the E. coli enzyme were conserved in the methanogenic enzyme. This sequence conservation suggests that vacuolar, F1, methanogenic, and S. acidocaldarius ATPases were derived from a common ancestral enzyme.     

Molecular cloning of cDNA encoding the C subunit of H(+)-ATPase from bovine chromaffin granules

A cDNA encoding subunit C of the V-ATPase from bovine chromaffin granules was cloned and sequenced. The gene encodes a hydrophilic protein of 382 amino acids with a calculated molecular weight of 43,989. Hydropathy plots revealed no apparent transmembrane segments and a rather high helix content was detected. A cDNA encoding most of the C subunit of the V-ATPase of human brain was also cloned and sequenced. The deduced amino acid sequence of this gene is almost identical to the bovine polypeptide with only one change of tyrosine 336 that was replaced by histidine in the human gene. Two polypeptide fragments derived from subunit E of V-ATPase from chromaffin granules were sequenced and found to be identical to the predicted amino acid sequence of this subunit from bovine kidney. These observations support the idea that the amino acid sequences of corresponding subunits from different V-ATPases are highly conserved. Unlike the A and B subunits of V-ATPases, that are homologous to the beta and alpha subunits of F-ATPases, subunits C and E showed no homology with analogous subunits of the F-ATPase family. It is proposed that the addition of the C and gamma subunits to the respective V- and F-ATPases during evolution defined them as two separate families of H(+)-ATPases.     

Structure and ligand specificity of the Drosophila melanogaster insulin receptor.

The insulin-binding and protein tyrosine kinase subunits of the Drosophila melanogaster insulin receptor homolog have been identified and characterized by using antipeptide antibodies elicited to the deduced amino acid sequence of the alpha and beta subunits of the human insulin receptor. In D. melanogaster embryos and cell lines, the insulin receptor contains insulin-binding alpha subunits of 110 or 120 kilodaltons (kDa), a 95-kDa beta subunit that is phosphorylated on tyrosine in response to insulin in intact cells and in vitro, and a 170-kDa protein that may be an incompletely processed receptor. All of the components are synthesized from a proreceptor, joined by disulfide bonds, and exposed on the cell surface. The beta subunit is recognized by an antipeptide antibody elicited to amino acids 1142 to 1162 of the human insulin proreceptor, and the alpha subunit is recognized by an antipeptide antibody elicited to amino acids 702 to 723 of the human proreceptor. Of the polypeptide ligands tested, only insulin reacts with the D. melanogaster receptor. Insulinlike growth factors type I and II, epidermal growth factor, and the silkworm insulinlike prothoracicotropic hormone are unable to stimulate autophosphorylation. Thus despite the evolutionary divergence of vertebrates and invertebrates, the essential features of the structure and intrinsic functions of the insulin receptor have been remarkably conserved.     

Kinetic and structural analysis of a new group of Acyl-CoA carboxylases found in Streptomyces coelicolor A3(2)

Two acyl-CoA carboxylases from Streptomyces coelicolor have been successfully reconstituted from their purified components. Both complexes shared the same biotinylated alpha subunit, AccA2. The beta and the epsilon subunits were specific from each of the complexes; thus, for the propionyl-CoA carboxylase complex the beta and epsilon components are PccB and PccE, whereas for the acetyl-CoA carboxylase complex the components are AccB and AccE. The two complexes showed very low activity in the absence of the corresponding epsilon subunits; addition of PccE or AccE dramatically increased the specific activity of the enzymes. The kinetic properties of the two acyl-CoA carboxylases showed a clear difference in their substrate specificity. The acetyl-CoA carboxylase was able to carboxylate acetyl-, propionyl-, or butyryl-CoA with approximately the same specificity. The propionyl-CoA carboxylase could not recognize acetyl-CoA as a substrate, whereas the specificity constant for propionyl-CoA was 2-fold higher than for butyryl-CoA. For both enzymes the epsilon subunits were found to specifically interact with their carboxyltransferase component forming a beta-epsilon subcomplex; this appears to facilitate the further interaction of these subunits with the alpha component. The epsilon subunit has been found genetically linked to several carboxyltransferases of different Streptomyces species; we propose that this subunit reflects a distinctive characteristic of a new group of acyl-CoA carboxylases.     

Purification, cDNA cloning and Northern-blot analysis of mitochondrial chaperonin 60 from pumpkin cotyledons.

Two different cDNA clones, pMCPN60-1 and pMCPN60-2, encoding the mitochondrial homologues of chaperonin 60 (Cpn60) were isolated from a cDNA library of germinating pumpkin cotyledons by use of mixtures of synthetic oligonucleotides based on the N-terminal amino acid sequence of the protein. Determination of the complete nucleotide sequences of the two cDNA revealed that pMCPN60-1 and pMCPN60-2 each contain one open reading frame that encodes a protein of 575 amino acids with molecular masses of 61052 Da and 61127 Da, respectively. The deduced amino acid sequences of the two polypeptides include a 32-residue N-terminal putative mitochondrial presequence attached to the mature polypeptides, and they are 95.3% identical. From a comparison of deduced amino acid sequences with other Cpn60, it appears that the mature polypeptides of pumpkin mitochondrial Cpn60 are 44-59% identical to the other Cpn60, namely, GroEL of Escherichia coli, the 60-kDa heat-shock protein (Hsp60) of mitochondria in the yeast Saccharomyces cerevisiae, P1 protein of mammalian mitochondria and the Ribulose-1,5-bisphosphate carboxylase/oxygenase subunit-binding proteins alpha and beta of plastids in higher plants. Genomic Southern-blot analysis identified at least two copies of the gene for mitochondrial Cpn60 in the pumpkin genome. The levels of mRNA for mitochondrial Cpn60 in cotyledons, hooks and hypocotyls of pumpkin seedlings increased in response to heat stress, as deduced from Northern-blot analysis, indicating that pumpkin mitochondrial Cpn60 is a heat-induced stress protein.     

The nucleotide sequence of the luxA and luxB genes of Xenorhabdus luminescens HM and a comparison of the amino acid sequences of luciferases from four species of bioluminescent bacteria

The luxA and luxB genes of bioluminescent bacteria encode the alpha and beta subunits of luciferase, respectively. Sequences of the luxA and luxB genes of Xenorhabdus luminescens, the only terrestrial bioluminescent bacterium known, were determined and the amino acid sequence of luciferase deduced. The alpha subunit was found to contain 360 amino acids and has a calculated molecular weight of 41,005 Da, while the beta subunit contains 327 amino acids and has a calculated molecular weight of 37,684 Da. Alignment of this luciferase with the luciferases of three marine bacteria showed 196 (or 55%) conserved residues in the alpha subunit and 114 (or 35%) conserved residues in the beta subunit. The highest degree of homology between any two species was between the luciferases of X. luminescens and Vibrio harveyi with 84% identity in the alpha subunits and 59% identity in the beta subunits.     

Primary structure of the monomer of the 12S subunit of transcarboxylase as deduced from DNA and characterization of the product expressed in Escherichia coli.

Transcarboxylase from Propionibacterium shermanii is a complex biotin-containing enzyme composed of 30 polypeptides of three different types: a hexameric central 12S subunit to which 6 outer 5S subunits are attached through 12 1.3S biotinyl subunits. The enzyme catalyzes a two-step reaction in which methylmalonyl coenzyme A and pyruvate serve as substrates to form propionyl coenzyme A (propionyl-CoA) and oxalacetate, the 12S subunit specifically catalyzing one of the two reactions. We report here the cloning, sequencing, and expression of the 12S subunit. The gene was identified by matching amino acid sequences derived from isolated authentic 12S peptides with the deduced sequence of an open reading frame present in a cloned P. shermanii genomic fragment known to contain the gene encoding the 1.3S biotinyl subunit. The cloned 12S gene encodes a protein of 604 amino acids and of M(r) 65,545. The deduced sequence shows regions of extensive homology with the beta subunit of mammalian propionyl-CoA carboxylase as well as regions of homology with acetyl-CoA carboxylase from several species. Two genomic fragments were subcloned into pUC19 in an orientation such that the 12S open reading frame could be expressed from the lac promoter of the vector. Crude extracts prepared from these cells contained an immunoreactive band on Western blots (immunoblots) which comigrated with authentic 12S. The Escherichia coli-expressed 12S was purified to apparent homogeneity by a three-step procedure and compared with authentic 12S from P. shermanii. Their quaternary structures were identical by electron microscopy, and the E. coli 12S preparation was fully active in the reactions catalyzed by this subunit. We conclude that we have cloned, sequenced, and expressed the 12S subunit which exists in a hexameric active form in E.coli.     

Molecular cloning of the cDNA encoding the third polypeptide (gamma) of brain calmodulin-dependent protein kinase II

cDNA clones for three distinct types of rat brain calmodulin-dependent protein kinase II have been isolated. Two of them were identified as cDNA clones for the alpha and beta subunits of this kinase. The other showed a nucleotide sequence similar but, not identical, to that encoding either the alpha or beta subunit. The cDNA sequence encoded a polypeptide, designated gamma, consisting of 527 amino acid residues with a molecular weight of 59,038. The deduced amino acid sequence of gamma was 84 and 87% homologous to those of alpha and beta, respectively. Higher homologies of the sequences were found in the amino-terminal halves of the three species, alpha, beta, and gamma. RNA blot analysis revealed that the mRNAs for alpha, beta, and gamma were expressed in rat brain with different regional specificities.     

Similarity of the E1 subunits of branched-chain-oxoacid dehydrogenase from Pseudomonas putida to the corresponding subunits of mammalian branched-chain-oxoacid and pyruvate dehydrogenases

The genes encoding proteins responsible for activity of the E1 component of branched-chain-oxoacid dehydrogenase of Pseudomonas putida have been subcloned and the nucleotide sequence of this region determined. Open reading frames encoding E1 alpha (bkdA1, 1233 bp) and E1 beta (bkdA2, 1020 bp) were identified with the aid of the N-terminal sequence of the purified subunits. The Mr of E1 alpha was 45,158 and of E1 beta was 37,007, both calculated without N-terminal methionine. The deduced amino acid sequences of E1 alpha and E1 beta had no similarity to the published sequences of the E1 subunits of pyruvate and 2-oxoglutarate dehydrogenases of Escherichia coli. However, there was substantial similarity between the E1 alpha subunits of Pseudomonas and rat liver branched-chain-oxoacid dehydrogenases. In particular, the region of the E1 alpha subunit of the mammalian branched-chain-oxoacid dehydrogenase which is phosphorylated, was found to be highly conserved in the Pseudomonas E1 alpha subunit. There was also considerable similarity between the E1 beta subunits of Pseudomonas branched-chain-oxoacid dehydrogenase and human pyruvate dehydrogenase.     

Isolation and preliminary characterization of the subunits of the terminal component of naphthalene dioxygenase from Pseudomonas putida NCIB 9816-4.

The terminal oxygenase component (ISPNAP) of naphthalene dioxygenase from Pseudomonas putida NCIB 9816-4 was purified to homogeneity. The protein contained approximately 4 g-atoms each of iron and acid-labile sulfide per mol of ISPNAP, and enzyme activity was stimulated significantly by addition of exogenous iron. The large (alpha) and small (beta) subunits of ISPNAP were isolated by two different procedures. The NH2-terminal amino acid sequences of the alpha and beta subunits were identical to the deduced amino acid sequences reported for the ndoB and ndoC genes from P. putida NCIB 9816 and almost identical to the NH2-terminal amino acid sequences determined for the large and small subunits of ISPNAP from P. putida G7. Gel filtration in the presence of 6 M urea gave an alpha subunit with an absorption maximum at 325 nm and broad absorption between 420 and 450 nm. The alpha subunit contained approximately 2 g-atoms each of iron and acid-labile sulfide per mol of the subunit. The beta subunit did not contain iron or acid-labile sulfide. These results, taken in conjunction with the deduced amino acid sequences of the large subunits from several iron-sulfur oxygenases, indicate that each alpha subunit of ISPNAP contains a Rieske [2Fe-2S] center.     

G protein-effector coupling: interactions of recombinant inhibitory gamma subunit with transducin and phosphodiesterase

A bacterial expression vector for the inhibitory gamma subunit of retinal rod phosphodiesterase has been constructed by inserting a mouse gamma cDNA into pUC19. Escherichia coli 222 transformed with this plasmid produces a 12-kDa recombinant protein consisting of 18 additional amino acids attached to the amino terminus of gamma. The fusion protein, designated beta-gal-gamma, has been refolded into an active form in formic acid and partially purified by gel filtration chromatography. Despite a large extended sequence at the amino terminus, beta-gal-gamma is able to inhibit the activity of trypsin-activated phosphodiesterase, bind tightly to the catalytic alpha beta subunits, and interact with the alpha subunit of transducin in a nucleotide-dependent manner. The availability of large quantities of active bacterial gamma, together with the ability to change its primary structure by site-directed mutagenesis, promises to provide considerable new information on the interaction between transducin and phosphodiesterase, as well as insights into the molecular mechanism of G protein-effector coupling.