The relative activities of PEP carboxylase and RuDP carboxylase in blue-green algae

The activity of phosphoenolpyruvate (PEP) carboxylase in preparations obtained by lysis of spheroplasts of three species of blue-green algae was found to be 1.5- to 5-fold higher at 20°C than that of RuDP carboxylase. This level of enzyme activity suggests that these algae may have the ability to fix carbon dioxide by a C₄ pathway.     

Propionyl-CoA carboxylase in Nocardia mediterranei

Abstract The propionyl-CoA carboxylase activity of crude extracts of Nocardia mediterranei fractionated by ammonium sulfate (40–60%) is described. Such an enzyme may play an important role in the biosynthesis of rifamycin as well as in the degradation of various amino acids. The effect of different compounds on the enzymatic reaction have been investigated. Product P8/1-OG and citrate inhibited the enzyme. An activation by the amino acids isoleucine, methionine, threonine and valine was found. The results do not exclude that the same enzyme is responsible for the carboxylation of acetyl-CoA and propionyl-CoA.     

Wheat acetyl-CoA carboxylase

The acetyl-CoA carboxylase present in both wheat germ and total wheat leaf protein contains ca. 220 kDa subunits. It is the major biotin-dependent carboxylase present in wheat chloroplasts. Active acetyl-CoA carboxylase purified from wheat germ is a homodimer with an apparent molecular mass of ca. 500 kDa. The enzyme from wheat germ or from wheat chloroplasts is sensitive to the herbicide haloxyfop at micromolar levels. The incorporation of ¹⁴C-acetate into fatty acids in freshly cut wheat seedling leaves provides a convenient in vivo assay for both acetyl-CoA carboxylase and haloxyfop.     

Plants contain multiple biotin enzymes: discovery of 3-methylcrotonyl-CoA carboxylase, propionyl-CoA carboxylase and pyruvate carboxylase in the plant kingdom

Acetyl-CoA carboxylase is the sole biotin enzyme previously reported in plants. Western analysis with 125I-streptavidin of proteins extracted from carrot somatic embryos visualized six biotin-containing polypeptides, the relative molecular masses of which are 210,000, 140,000, 73,000, 50,000, 39,000, and 34,000. This multiplicity of the biotin-containing polypeptides can be partly explained by the discovery of 3-methylcrotonyl-CoA carboxylase, propionyl-CoA carboxylase, and pyruvate carboxylase in extracts of somatic carrot embryos, biotin enzymes previously unknown in the plant kingdom. These biotin enzymes seem to be widely distributed in the plant kingdom.     

Multiple carboxylase deficiency

1. The multiple carboxylase deficiencies are inborn errors in the metabolism of biotin in which there is defective activity of propionyl CoA carboxylase, 3-methylcrotonyl CoA carboxylase and pyruvate carboxylase. 2. Two distinct disorders have been described. 3. In one the fundamental defect is in the enzyme holocarboxylase synthetase which catalyzes the molecular activation of the apocarboxylase proteins. 4. In the other the fundamental defect is in biotinidase which catalyzes the reutilization of biotin and may be involved in its digestion and intestinal absorption.     

Changes in phosphoenolpyruvate carboxylase and ribulosebisphosphate carboxylase activities in tobacco plants infected with tobacco mosaic virus

Considerable changes in the activities of phosphoenolpyruvate carboxylase and ribulosebisphosphate carboxylase were found inNicotiana tabacum cv. Sarasun plants infected with TMV. Ribulosebisphosphate carboxylase is inhibited at the time of maximum TMV reproduction, but its decreased activity is at the same time partly compensated by phosphoenol-pyruvate carboxylase in the shoots of infected plants. The pattern of activity of this enzyme nearly exactly reflects the pattern of reproduction of the tobacco mosaic virus.     

Acetyl-coenzyme A carboxylase in maize leaves

Purified chloroplasts from mesophyll and bundle sheath cells of maize leaves have been shown to be the location of acetyl-CoA carboxylase. In disrupted chloroplasts the enzyme was recovered in the stromal fraction, along with protein-bound biotin; acetyl-CoA carboxylase activity did not require a membrane component. Mg²⁺ and ATP are required for activity and sulfhydryl protecting agents enhance stability of the enzyme. Acetyl-CoA carboxylase activity was independent of leaf development in cell-free extracts of maize. Comparison of acetyl-CoA carboxylase activity with [¹⁴C]acetate incorporation into lipids, in isolated chloroplasts from developing leaves of maize, indicate that acetyl-CoA carboxylase is not limiting fatty acid synthesis.     

Essential arginine residues in the active sites of propionyl CoA carboxylase and beta-methylcrotonyl CoA carboxylase

At least one arginine residue is essential for substrate binding in or near the active sites of propionyl CoA carboxylase (PCC) and beta-methylcrotonyl CoA carboxylase (beta MCC) in cultured human fibroblasts. This conclusion is based on studies of enzyme inhibition by phenylglyoxal, a reagent which specifically modifies arginine residues. Human fibroblast PCC both in extracts and in a 20-fold purified preparation was nearly completely protected from phenylglyoxal inhibition following incubation with propionyl CoA or ATP. It appears that a phosphate group from either ATP or the CoA moiety of propionyl CoA reacts with the essential arginine residue(s). beta MCC which was similarly inhibited by phenylglyoxal was protected by beta-methylcrotonyl CoA and ATP. Thus phenylglyoxal may be used to label specific arginine residues within the active sites of previously sequenced carboxylases.     

Isoenzymes of vitamin-K-dependent carboxylase

Vitamin-K-dependent carboxylase was prepared from bovine liver, kidney, lung and testis and it was checked that these systems obeyed the laws of normal enzyme kinetics. Four carboxylatable substrates were obtained from different sources and the apparent Michaelis constants of the various carboxylases for these four substrates were measured. From the results thus obtained we concluded that carboxylase is a group name for a number of isoenzymes which are present in hepatic as well as in various non-hepatic tissues.     

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.     

Sequence homology around the biotin-binding site of human propionyl-CoA carboxylase and pyruvate carboxylase

Biotin-dependent carboxylases require covalently bound biotin for enzymatic activity. The biotin is attached through a lysine residue, which in a number of bacterial, avian, and mammalian carboxylases, is found within the conserved sequence Ala-Met-Lys-Met. We have determined the partial nucleotide sequence of cDNA clones for human propionyl-CoA carboxylase and pyruvate carboxylase. The predicted amino acid sequence of both these proteins contains the conserved tetrapeptide 35 residues from the carboxy terminus. In addition, both proteins contain the tripeptide, Pro-Met-Pro, 26 residues toward the amino terminus from the biotin attachment site. The overall amino acid homology through this region is 43%. Similar findings have been made for the biotin-containing polypeptides of transcarboxylase of Propionibacterium shermanii and acetyl-CoA carboxylase of Escherichia coli (W. L. Maloy, B. U. Bowien, G. K. Zwolinski, K. G. Kumar, and H. G. Wood (1979) J. Biol. Chem. 254, 11615-11622). The implications of this sequence conservation with regard to the function and evolution of biotin-dependent carboxylases is discussed. We propose that the 60 amino acids surrounding the biotin site are bounded by a proline "hinge" and the carboxy terminus has remained conserved as a result of constraints imposed by biotinylation of the enzyme.     

Ribulose bisphosphate carboxylase activity in halophilic Archaebacteria

Among the several strains of halobacteria grown heterotrophically, ribulose bisphosphate carboxylase activity was detected in those which accumulate poly (-hydroxybutyrate), viz. Haloferax mediterranei, Haloferax volcanii and Halobacterium marismortui. In H. mediterranei, the activity was present in cell extracts prepared after growth on a variety of carbohydrates. The ribulose bisphosphate carboxylase activity in H. mediterranei was inhibited by carboxyarabinitol bisphosphate, and the enzyme cross-reacted with antibodies raised against the spinach enzyme. CO₂ fixation by cell extract was stimulated by the addition of ATP and NADH. Preliminary data suggested that hydrogen could be a possible reductant.Abbreviations RuBP ribulose bisphosphate - Ru5P ribulose 5-phosphate - R5P ribose 5-phosphate - CABP carboxyarabinitol bisphosphate - PHB poly (-hydroxybutyrate) - DTT dithiothreitol