Interaction of formycin A-5'-triphosphate with pyruvate carboxylase

Formycin triphosphate (FTP), a fluorescent analogue of ATP, is a competitive inhibitor of chicken liver pyruvate carboxylase with respect to ATP. The chicken liver enzyme is unable to utilise FTP as a substrate at a measureable rate, but FTP is a poor substrate for the sheep liver enzyme. When FTP binds to the enzyme, its fluorescence is enhanced and in this way the formation of enzyme-FTP complexes can be monitored. Using this property of FTP, the effect of Mg2+ and acetyl-CoA on the binding of nucleoside triphosphates to the chicken liver enzyme was examined. Mg2+ was found to enhance the binding of FTP whilst acetyl-CoA reduced the fluorescence intensity of a mixture of Mg2+, enzyme and FTP. Most probably, this was caused by a conformational change in the enzyme which changed the environment of the fluorophore.     

Conserved Glu40 and Glu433 of the biotin carboxylase domain of yeast pyruvate carboxylase I isoenzyme are essential for the association of tetramers

The native form of pyruvate carboxylase is an alpha4 tetramer but the tetramerisation domain of each subunit is currently unknown. To identify this domain we co-expressed yeast pyruvate carboxylase 1 isozyme (Pyc1) with an N-terminal myc tag, together with constructs encoding either the biotin carboxylase (BC) domain or the transcarboxylase-biotin carboxyl carrier domain (TC-BCC), each with an N-terminal 9-histidine tag. From tag-affinity chromatography experiments, the subunit contacts within the tetramer were identified to be primarily located in the 55 kDa BC domain. From modelling studies based on known structures of biotin carboxylase domains and subunits we have predicted that Arg36 and Glu433 and Glu40 and Lys426, respectively, are involved pairwise in subunit interactions and are located on opposing subunits in the putative subunit interface of Pyc1. Co-expression of mutant forms with wild type Pyc1 showed that the R36E mutation had no effect on the interaction of these subunits with those of wild type Pyc1, while the E40R, E433R and R36E:E433R mutations caused severe loss of interaction with wild type Pyc1. Ultracentrifugal analysis of these mutants when expressed and purified separately indicated that the predominant form of E40R, E433R and R36R:E433R mutants is the monomer, and that their specific activities are less than 2% of the wild type. Studies on the association state and specific activity of the R36E mutant at different concentrations showed it to be much more susceptible to tetramer dissociation and inactivation than the wild type. Our results suggest that Glu40 and Glu433 play essential roles in subunit interactions.     

Mitochondrial location of pyruvate carboxylase in Aspergillus niger

Pyruvate carboxylase has been found in the mitochondrial fraction of two strains of Aspergillus niger along with the marker enzymes of citrate synthase and cytochrome c oxidase. The location of pyruvate carboxylase in A. nidulans was, however, confirmed to be in the cytosolic fraction. The enzyme from the former sources was dependent upon the presence of acetyl-CoA for full activity; the enzyme from A. nidulans was unaffected by the presence or absence of acetyl-CoA.     

By-product formation during exposure of respiring Saccharomyces cerevisiae cultures to excess glucose is not caused by a limited capacity of pyruvate carboxylase

Upon exposure to excess glucose, respiring cultures of Saccharomyces cerevisiae produce substantial amounts of ethanol and acetate. A possible role of a limited anaplerotic capacity in this process was investigated by overexpressing pyruvate carboxylase and by replacing it with a heterologous enzyme (Escherichia coli phosphoenolpyruvate carboxylase). Compared to the wild-type, neither the pyruvate carboxylase (Pyc)-overexpressing nor the transgenic strain exhibited reduced by-product formation after glucose pulses to aerobic glucose-limited chemostat cultures. An increased intracellular malate concentration was observed in the two engineered strains. It is concluded that by-product formation in S. cerevisiae is not caused by a limited anaplerotic capacity.     

Molecular domain structure of porcine vinculin and metavinculin

Summary Metavinculin is a higher molecular weight variant of vinculin expressed only in cardiac and smooth muscle. Using microsequencing methods on the intact molecules and their proteolytic subfragments we have been able to map the common and different parts of these closely related proteins. Both vinculin and metavinculin, from mammals and birds exhibit a relatively protease resistant 90 kD core fragment. N-terminal sequencing analysis of the avian and mammalian core fragments as well as of major core subfragments obtained by extended proteolysis placed the core domain at the N-terminus of the intact molecules and revealed identity between metavinculin and vinculin as well as between species. Limited chymotryptic digestion of porcine vinculin and metavinculin yielded a common 16 kD fragment which could be placed at the C-terminus of the cDNA sequence derived from chick fibroblast vinculin (G. J.Price, P.Jones, M. D.Davison, R.Bendori, S.Griffiths, B.Patel, B.Geiger and D. R.Critchley 1988, in press). From additional sequence data the metavinculin specific fragment could be placed at the metavinculin C-terminus. Using a polyclonal antibody specific for porcine metavinculin a peptide unique to metavinculin could be identified. Direct sequencing of this, as well as of related, overlapping fragments, purified by reversed phase HPLC revealed a 68 amino acid insert in procine metavinculin, between the core fragment and the C-terminal piece, common to vinculin and metavinculin. The domain organizazions of vinculin and metavinculin and their possible functional implications are discussed.Abbreviations SDS sodium dodecyl sulfate - EDTA ethylendinitrilotetra acetic acid - HPLC high pressure liquid chromatography     

Changes in catalytic activity and association state of pyruvate carboxylase which are dependent on enzyme concentration

The specific activity of chicken liver pyruvate carboxylase has been shown to decrease with decreasing enzyme concentration, even at 100 microM, which is close to the estimated physiological concentration. The kinetics of the loss of enzyme specific activity following dilution were biphasic. Incubation of dilution-inactivated enzyme with ATP, acetyl CoA, Mg2+ + ATP or, to a lesser degree, with Mg2+ alone resulted in a high degree of reactivation, while no reactivation occurred in the presence of pyruvate. The association state of the enzyme before, during, and after dilution inactivation has been assessed by gel filtration chromatography. These studies indicate that on dilution, there is dissociation of the catalytically active tetrameric enzyme species into inactive dimers. Reactivation of the enzyme resulted in reassociation of enzymic dimers into tetramers. The enzyme was shown to form high molecular weight aggregates at high enzyme concentrations.     

Expression of human pyruvate carboxylase in insect cells using the baculovirus system

Constructs containing cDNA encoding human pyruvate carboxylase (PC) with and without a hexahistidine (6x His) tag at the N-terminal of the mature enzyme have been cloned under the control of the polyhedrin promoter. These two constructs were co-transfected with the baculovirus genome into Sf9 cells to produce recombinant baculoviruses harbouring human PC cDNA. The expression of human PC under the control of the polyhedrin promoter was found to be at its highest level at 4 days post-infection. The expressed material accounted for up to 70% of total cellular protein with 5% of this expressed material being found in the soluble fraction. The recombinant human 6x His-PC isolated with a purity of approximately 50% using a Ni-NTA agarose column was found to have the specific activity of 7U/mg, which was similar to that produced from a 293T stable line [Biochem. Biophys. Res. Commun. 266 (1999) 512]. This is the first report of a heterologous expression system for recombinant human PC.     

Molecular evolution of phosphoeno/pyruvate carboxylase

Phosphoenolpyruvate carboxylase is an enzyme involved in a wide variety of important metabolic pathways of plants such as anaplerotic reactions and C4 and CAM photosynthetic pathways. The accumulation of molecular sequence data of phosphoenolpyruvate carboxylases has enabled us to investigate the function and molecular evolution of the enzymes by computer-assisted sequence comparison. Here we report the results of sequence comparison of phosphoenolpyruvate carboxylases: (1) Phosphoenofpyruvate carboxylases were classified into four groups; a subgroup of bacterial enzymes and three subgroups of plants enzymes. (2) The divergence time of the monocot enzymes involved in the C₄ pathways was roughly estimated to be 150—300 million years. On the other hand, the phylogenetic tree of the enzymes suggested that those for the dicot enzymes involved in the C₄ and CAM pathways might be close to the divergence time between the monocots and the dicots. (3) The evolutionary positions of the enzymes prevalent in roots or root nodules were identified. (4) Although sorghum and maize contained at least three genes for the enzymes in their genomes, the rates of amino acid substitution of the enzymes were different from gene to gene. The difference could not be explained by either lineage effects nor bias in base contents.     

Sequence and domain structure of yeast pyruvate carboxylase

The nucleotide sequence of the yeast pyruvate carboxylase gene has been determined from a cloned fragment of yeast genomic DNA. The deduced translation product codes for a polypeptide of 1178 amino acids, having a calculated molecular weight of 130,100. The protein shows strong sequence homology to specific regions of other biotin carboxylases, lipoamide transferases, and carbamyl phosphate synthetases. The homologous regions suggest the presence of three subsites in the enzyme: a biotin attachment site, a keto acid-binding site, and an ATP-binding site. Partial proteolysis with a variety of proteases under nondenaturing conditions indicates the presence of structural domains corresponding to these subsites.     

Estimation of domain length of chicken erythrocyte chromatin

Chromatin of chicken erythrocyte nuclei was extracted by digestion with micrococcal nuclease. The length distribution of the soluble chromatin was determined by gel electrophoresis and electron microscopy. These results were fitted with a theoretical distribution which was an outcome of the domain model proposed by Igo-Kemenes and Zachau (Igo-Kemenes, T. and H.G. Zachau (1977) Cold Spring Harbour Symp. Quant. Biol. 42, 109–118). A domain length of 45 kbp was obtained.     

Increased expression of pyruvate carboxylase and biotin protein ligase increases lysine production in a biotin prototrophic Corynebacterium glutamicum strain

Corynebacterium glutamicum, a Gram‐positive bacterium used for the production of various biochemicals, is naturally a biotin auxotroph. We introduced the biotin genes from Bacillus subtilis on a plasmid, pBIO, into a lysine‐producing derivative (termed AHP‐3) that has been described previously, and achieved biotin prototrophy. We found that AHP‐3, containing pBIO, was able to produce lysine in a medium lacking biotin and that the lysine yield on glucose was similar to what is obtained when using a medium containing biotin. However, there was a decrease in specific growth rate of 20% when the strain was cultivated without biotin, indicating a suboptimal intracellular concentration of biotin. In an attempt to locate the potential bottleneck, we added pimelic acid, an early biotin precursor, and found that growth rate could be restored fully, which demonstrates that the bottleneck is in pimeloyl‐CoA (or pimeloyl‐Acyl Carrier Protein [ACP]) formation. Pyruvate carboxylase (pycA), a biotin‐dependent enzyme needed for lysine biosynthesis and biotin ligase (birA), which is responsible for attaching biotin to pyruvate carboxylase, were overexpressed by replacing the native promoters with the strong superoxide dismutase (sod) promoter, to see whether growth could be restored. Neither pycA nor birA overexpression, whether alone or in combination, had an effect on specific growth rate, but they did have a positive effect on lysine yield, which increased by 55% in the strain overexpressing both enzymes.     

Tissue manganese levels and liver pyruvate carboxylase activity in magnesium-deficient rats

To investigate the manganese status in magnesium deficiency, 40 male Wistar rats, 3 wk old, were divided into two groups and fed a magnesium deficient diet or a normal synthetic diet for 2 wk. Dietary magnesium depletion decreased magnesium levels in brain, spinal cord, lung, spleen, kidney, testis, bone, blood, and plasma, while it elevated the magnesium level in liver. In magnesium-depleted rats, calcium concentration was increased in lung, liver, spleen, kidney, and testis, while it was decreased in tibia. In magnesium-depleted rats, manganese concentration was decreased in plasma and all tissues except adrenal glands and blood. Dietary magnesium depletion diminished pyruvate carboxylase (EC 6.4.1.1) activity in the crude mitochondrial fraction of liver. Positive correlation was found between the liver manganese concentration and the pyruvate carboxylase activity. In the magnesium-depleted rats, glucose was decreased while plasma lipids (triglycerides, phospholipids, and total cholesterol) were increased. These results suggest that dietary magnesium deficiency changes manganese metabolism in rats.     

The transcarboxylase domain of pyruvate carboxylase is essential for assembly of the peroxisomal flavoenzyme alcohol oxidase

Pyruvate carboxylase (Pyc1p) has multiple functions in methylotrophic yeast species. Besides its function as an enzyme, Pyc1p is required for assembly of peroxisomal alcohol oxidase (AO). Hence, Pyc1p-deficient cells share aspartate auxotrophy (Asp-) with a defect in growth on methanol as sole carbon source (Mut-). To identify regions in Hansenula polymorpha Pyc1p that are required for the function of HpPyc1p in AO assembly, a series of random mutations was generated in the HpPYC1 gene by transposon mutagenesis. Upon introduction of 18 mutant genes into the H. polymorpha PYC1 deletion strain (pyc1), four different phenotypes were obtained, namely Asp- Mut-, Asp- Mut+, Asp+ Mut-, and Asp+ Mut+. One mutant showed an Asp+ Mut- phenotype. This mutant produced HpPyc1p containing a pentapeptide insertion in the region that links the conserved N-terminal biotin carboxylation domain (BC) with the central transcarboxylation (TC) domain. Three mutants that were Asp- Mut- contained insertions in the TC domain, suggesting that this domain is important for both functions of Pyc1p. Analysis of a series of constructed C-terminal and N-terminal truncated versions of HpPyc1p showed that the TC domain of Pyc1p, including the region linking this domain to the BC domain, is essential for AO assembly.     

Molecular cloning and expression of two chicken invariant chain isoforms produced by alternative splicing

The biosynthesis of distinct forms of the invariant chain (Ii) protein from a unique gene as the result of differential splicing patterns has been observed in humans and mice. However, there have been no reports on the existence of Ii isoforms in avian species. In the present study, we identified two chicken Ii cDNAs by RT-PCR and RACE, and examined the Ii gene copy number, mRNA expression and protein expression by Southern blotting, Northern blotting and immunofluorescence confocal microscopy, respectively. One of the Ii cDNAs, named Ii-1, was 1,151 bp in length, and had an open reading frame (ORF) of 672 nucleotides, in agreement with a previously identified chicken Ii sequence; the other, named Ii-2, was 1,337 bp long and had an ORF of 861 nucleotides. Southern blotting confirmed that these cDNAs were derived from a single copy gene. Northern blotting performed with total RNA from various tissues of 6-week-old chickens revealed high levels of Ii-1 and Ii-2 mRNA expression in the spleen and bursa of Fabricius, and low levels of Ii-1 expression in the thymus, heart and liver, while Ii-2 was not expressed in these tissues. High levels of expression of both Ii isoforms were detected in the spleen and bursa of Fabricius during late embryogenesis. Immunofluorescence staining showed that Ii proteins were expressed in the cell membranes of the splenocytes. These data suggest that chicken Ii exists in two isoforms resulting from alternative splicing, and is strongly expressed in the major immune organs.     

Molecular cloning and sequence analysis of a chicken ornithine decarboxylase cDNA

A chicken embryo cDNA library was screened with a mouse probe for ornithine decarboxylase (ODC) and 14 positively hybridizing clones isolated. The longest of these (1.7 kb) was sub-cloned and sequenced. It is estimated that the clone comprises approximately 98% of the coding region for chicken ODC. The DNA sequence shows 78% identity with the human ODC cDNA sequence and the deduced amino acid sequence is almost 90% homologous to mouse and human. Both the peptide and cDNA sequences show interesting potential regulatory features which are discussed here.     

DNA sequences in chromosomes 11 and VII code for pyruvate carboxylase isoenzymes in Saccharomyces cerevisiae: analysis of pyruvate carboxylase-deficient strains

Summary A gene encoding pyruvate carboxylase has previously been isolated from Saccharomyces cerevisiae. We have isolated a second gene, PYC2, from the same organism also encoding a pyruvate carboxylase. The gene PYC2 is situated on the right arm of chromosome II between the DUR 1, 2 markers and the telomere. We localized the previously isolated gene, which we designate PYC1, to chromosome VII. Disruption of either of the genes did not produce marked changes in the phenotype. However, simultaneous disruption of both genes resulted in inability to grow on glucose as sole carbon source, unless aspartate was added to the medium. This indicates that in wild-type yeast there is no bypass for the reaction catalysed by pyruvate carboxylase. The coding regions of both genes exhibit a homology of 90% at the amino acid level and 85% at the nucleotide level. No appreciable homology was found in the corresponding flanking regions. No differences in the K _m values for ATP or pyruvate were observed between the enzymes obtained from strains carrying inactive, disrupted versions of one or other of the genes.A preliminary report of this work was presented at the 15th International Conference on Yeast Genetics and Molecular Biology, The Hague, Netherlands. Abstract appeared in Yeast 6, S-240 (1990)