Biochemical characterization of mutant propionyl CoA carboxylases from two minor genetic complementation groups

We have biochemically characterized several parameters of propionyl CoA carboxylase (PCC) activity in fibroblast extracts from PCC-deficient patients belonging to the two minor genetic complementation groups, pcc B and pcc BC. Comparison of PCCs from these groups with those of the two major complementation groups, pcc A and pcc C, has demonstrated that PCCs from both the pcc B and pcc BC groups closely resemble each other as well as PCC from the pcc C group. These results further support the hypothesis that the pcc B and pcc BC lines are interallelic with respect to pcc C and consequently that the structural mutations in the PCCs from these groups involve the same subunit.     

Kinetic analysis of genetic complementation in heterokaryons of propionyl CoA carboxylase-deficient human fibroblasts.

We studied genetic complementation of propionyl CoA carboxylase (PCC) deficiency in cultures of polyethylene glycol (PEG)-induced heterokaryons, using mutant fibroblast lines assigned to five mutant classes, designated bio, pcc A, pcc B, pcc C, and pcc BC. By measuring PCC activity directly in extracts of fused cells or indirectly in intact cells by [1-14C]propionate utilization, we confirmed the nonlinear nature of the PCC deficiency complementation map described by Gravel et al. [1]. When we studied the kinetics of complementation, we detected three distinct patterns using the [1-14C]propionate utilization assay. When either pcc A or pcc C lines were fused to bio cells, 14C-fixation increased to half of the maximally restored values within 4 hrs. In pcc A x pcc C crosses or in pcc A x pcc B crosses, however, complementation was much slower. In fusions between pcc B and pcc C cells, a third pattern was elicited; complementation was incomplete, maximum restoration of PCC activity begin less than 20% of that observed in other complementing crosses. From these data and previous biochemical evidence, we suggest (1) that the bio and pcc mutations affect different genes; (2) that complementation between pcc A and either pcc B, pcc C, or pcc BC lines is intergenic and involves subunit exchange and synthesis of new PCC molecules; and (3) that complementation between pcc B and pcc C mutants is interallelic.     

Absence of cross-reacting material in isolated propionyl CoA carboxylase deficiency: nature of residual carboxylating activity.

Fibroblast extracts and fetal liver homogenates from patients with propionic acidemia due to inherited deficiency of propionyl CoA carboxylase (PCC) were analyzed for the presence of immunologically cross-reactive PCC protein. Using several rabbit antisera raised against homogeneous human liver PCC, homogeneous pig heart PCC, or the individual non-identical subunits of the human liver enzyme, we found no detectable cross-reacting material by direct or competitive immunotitration in several cell lines from patients in either major complementation group (pcc A; pcc C) with isolated PCC deficiency. In contrast, cells of a patient from the bio complementation group contained normal amounts of immunoreactive PCC. Further analysis of the pcc A and pcc C mutants revealed that their residual propionyl CoA carboxylating activity varied greatly depending on the concentration of extract or homogenate protein used in the PCC assay. When propionyl CoA carboxylation was assayed at high protein concentration in a fetal liver homogenate from a pcc C patient, the apparent PCC activity was comparable to that found in normal human fetal liver. Significantly, the specific activity in the mutant, but not in the control, extract declined steeply as protein concentration was lowered, and this loss could not be prevented by adding PCC substrates, bovine serum albumin, glycerol, or 2-mercaptoethanol. Moreover, detailed analyses of immunotitration curves of control fibroblasts extracts showed that fresh extracts contained an amount of nonimmunotitratable carboxylating activity corresponding to the residual activity present in fresh extracts of mutant cell lines. We conclude that the residual propionyl CoA carboxylating activity found in isolated PCC deficiency represents another carboxylase that can utilize propionyl CoA as a substrate rather than a mutant form of PCC with markedly different immunochemical and physicochemical properties.     

Evidence for two genetic complementation groups in pyruvate carboxylase-deficient human fibroblast cell lines

We have examined genetic complementation in pyruvate carboxylase deficiency by comparing the enzyme activity in polyethylene glycol-induced heterokaryons with that in unfused mixtures of fibroblasts from three affected children. Complementation, manifested as a three- to sevenfold increase in pyruvate carboxylase activity, was observed in fusions between a biotin-responsive multiple carboxylase (pyruvate carboxylase, propionyl CoA carboxylase, and -methylcrotonyl CoA carboxylase) deficient fibroblast line and two other lines deficient only in pyruvate carboxylase activity. Kinetic analysis of complementing pyruvate carboxylase deficient lines, measured by the rate of restoration of enzyme activity as a function of time, revealed that maximum restoration was achieved within 10–24 hr after fusion. This profile is similar to those observed for fusions between the multiple carboxylase deficient line and two lines deficient in propionyl CoA carboxylase activity that are known to represent different gene mutations. Although the patients with pyruvate carboxylase deficiency had similar clinical findings, our studies indicate that pyruvate carboxylase deficiency is genetically heterogeneous, with at least two distinct, probably intergenic, complementation groups.This work was supported by an NIH research grant (AM 25675) and an A. D. Williams research grant (6-48360). B. Wolf is the recipient of an NIH Research Career Development Award (AM 00677) and is aided by a Basil O'Connor Starter Research Grant from The National Foundation-March of Dimes (5-263). G. Feldman is the recipient of an NIH predoctoral training grant (GM 07492). This article is No. 100 from the Department of Human Genetics at the Medical College of Virginia.     

Investigating the role of native propionyl‐CoA and methylmalonyl‐CoA metabolism on heterologous polyketide production in Escherichia coli

6‐Deoxyerythronolide B (6dEB) is the macrocyclic aglycone precursor of the antibiotic natural product erythromycin. Heterologous production of 6dEB in Escherichia coli was accomplished, in part, by designed over‐expression of a native prpE gene (encoding a propionyl‐CoA synthetase) and heterologous pcc genes (encoding a propionyl‐CoA carboxylase) to supply the needed propionyl‐CoA and (2S)‐methylmalonyl‐CoA biosynthetic substrates. Separate E. coli metabolism includes three enzymes, Sbm (a methylmalonyl‐CoA mutase), YgfG (a methylmalonyl‐CoA decarboxylase), and YgfH (a propionyl‐CoA:succinate CoA transferase), also involved in propionyl‐CoA and methylmalonyl‐CoA metabolism. In this study, the sbm, ygfG, and ygfH genes were individually deleted and over‐expressed to investigate their effect on heterologous 6dEB production. Our results indicate that the deletion and over‐expression of sbm did not influence 6dEB production; ygfG over‐expression reduced 6dEB production by fourfold while ygfH deletion increased 6dEB titers from 65 to 129 mg/L in shake flask experiments. It was also found that native E. coli metabolism could support 6dEB biosynthesis in the absence of exogenous propionate and the substrate provision pcc genes. Lastly, the effect of the ygfH deletion was tested in batch bioreactor cultures in which 6dEB titers improved from 206 to 527 mg/L. Biotechnol. Bioeng. 2010; 105: 567–573. © 2009 Wiley Periodicals, Inc.     

Biochemical characterization of propionyl CoA carboxylase deficiency: Heterogeneity within a single genetic complementation group

Liver tissues and fibroblasts from patients with propionic acidemia assigned to the pcc BC genetic complementation group have previously been shown to contain normal or near-normal quantities of structurally altered propionyl CoA carboxylases (PCC). Biochemical comparisons of PCCs from extracts of three livers and one placenta belonging to the pcc BC complementation group revealed that the K _m values for the enzyme's major substrates, propionyl CoA, bicarbonate, and ATP, and its monovalent activator, potassium, were similar to those of normal PCC. PCC in extracts of one of the livers, however, had an altered isoelectric point (pI = 5.4) compared to that of PCC from normal and other PCC-deficient tissues (pK = 4.6–4.7). Thermostability in the presence of sucrose or ATP differed among several of the mutant PCCs, including the PCC with an altered pI, and from that of normal PCC. To confirm these results and to determine whether valid inferences may be derived from comparisons of mutant and normal PCC in crude extracts, PCC was purified from normal liver and from one of the PCC-deficient livers. The biochemical parameters of the purified carboxylases were similar to those observed in liver extracts. These studies further-more confirmed that, whether purified or in extracts, PCC from the pcc BC group reflects structural mutations. Nevertheless, the abnormal enzyme structure appears to have no corresponding effect on the clinical features of the disorder in various affected individuals. Moreover, there is biochemical heterogeneity within the pcc BC complementation group that probably represents different interallelic gene mutations.This work was supported by NIH Research Grants Am 25675 and AM 26127. B. Wolf is the recipient of NIH Research Career Development Award AM 00677 and is aided by Basil O'Connor Starter Research Grant 5-263 from The National Foundation-March of Dimes. This article is No. 131 from the Department of Human Genetics at the Medical College of Virginia.     

Biochemical characterization of biotin-responsive multiple carboxylase deficiency: heterogeneity within the bio genetic complementation group

Three biotin-dependent enzymes, pyruvate carboxylase (PC), propionyl CoA carboxylase (PCC), and beta-methylcrotonyl CoA carboxylase (beta MCC), were biochemically characterized in fibroblasts from two patients with neonatal multiple carboxylase deficiency. Genetic complementation analyses indicated that both cell lines, designated lines 1 and 2, were deficient in the various carboxylase activities and belonged to the bio complementation group. The activities of the three carboxylases became normal when line 2 cells were incubated in medium supplemented with biotin (1 mg/l) for 24 hrs, whereas 4-6 days were required to achieve maximum activities of PC, PCC, and beta MCC (57%, 46%, and 29% of mean normal enzyme activity, respectively) in line 1 cells incubated in medium containing up to 10 mg/1 biotin. Furthermore, PC activity in line 2 continued to increase under apparent gluconeogenic conditions in culture, but not in line 1. Thermostability studies suggested that biotin stabilizes PC and beta MCC in both cell lines. PC in line 1 cells incubated with or without biotin was less stable than that in normal or line 2 cells, and the less than normal increase of enzyme activities in line 1, especially that of PC, may represent incomplete biotination. These results indicate that there is biochemical heterogeneity within the bio complementation group. Immunotitration with antibodies prepared against purified pig heart PCC demonstrated normal quantities of cross-reacting material in both lines and no differences in the amount of this material after incubation with supplemental biotin, despite the seven- to 20-fold increase in PCC activity. Thus, the increase in carboxylase activity in both bio lines appears to represent activation of rpe-existing apocarboxylase rather than de novo enzyme synthesis. The primary defect in this form of multiple carboxylase deficiency may be in a common holocarboxylase synthetase or in biotin transport. If the defect is in the synthetase, the differences noted between the two bio lines could be explained by a difference in the enzyme's Km for biotin.     

Evaluation of biotin responsiveness in cultured fibroblasts from patients with propionic acidemia: Absence of response by structurally altered carboxylases

We have demonstrated that, although propionyl CoA carboxylase (PCC) activity is deficient in fibroblast extracts from PCC-deficient patients belonging to the two major and two minor genetic complementation groups, the activity of another biotin-dependent carboxylase, -methylcrotonyl CoA carboxylase (MCC), is normal. Moreover, MCC activity is stimulated when the fibroblasts are cultured in high concentrations of biotin, in the same way that it is in normal fibroblasts, whereas the depressed PCC activity remains essentially unchanged. Because these results are parallel with the in vivo failure of high-dose biotin to stimulate PCC activity in peripheral blood leukocytes, we conclude that the biotin responsiveness of PCC in cultured fibroblasts from patients with PCC deficiency may be used to predict or confirm biotin responsiveness in vivo.     

Complementation in somatic hybrids indicates four types of nitrate reductase deficient lines in Nicotiana plumbaginifolia

Summary Allelism of nine nitrate reductase deficient (NR^–) Nicotiana plumbaginifolia cell lines was tested by complementation after protoplast fusion. Complementation was recognized by the appearance of somatic hybrid colonies growing on a selective NH₄ ⁺/NO₃ ^– medium which cannot support the growth of NR^– lines. All five apoenzyme defective (NA) lines were non-complementing and therefore allelic. The apoenzyme and the cofactor defective (NX) lines were complementing, as expected, and gave somatic hybrids with restored nitrate reductase activity. The four cofactor defective lines were found to belong to three complementation groups (NX1 and NX9; NX21; NX24). Two of these (NX21 and NX24) are of new types which have not been previously described in flowering plants. In the somatic hybrids restoration of NR activity was accompanied by the restoration of plant regeneration ability. On leave from: Instituto di Mutagenesi e Differenziamento CNR, Via Svezia, 10, 56100, Pisa, Italy     

Biochemical evidence for diverse etiologies in biotin-responsive multiple carboxylase deficiency

Biotin-responsive multiple carboxylase deficiency can be categorized by clinical criteria into a neonatal-onset disorder and a distinct syndrome of infantile onset. Pedigrees in each instance are consistent with autosomal recessive inheritance. For a neonatal-onset proband, the sensitivity to relative biotin deprivation and the rapid clinical response to biotin supplementation are reflected by in vitro studies. Specific activities of biotin-dependent pyruvate carboxylase, propionyl CoA carboxylase, and 3-methylcrotonyl CoA carboxylase are 0.8 to 16% of mean control values after growth of fibroblasts in intermediate and very low biotin concentrations. Following relative biotin depletion, pyruvate carboxylase activity returns to normal after only 14 hr of growth in biotin-supplemented medium. In contrast, carboxylase activities in fibroblasts of an infantile-onset proband remain normal at very low biotin concentrations, even when avidin is added to the growth medium. The clinical heterogeneity, taken together with the distinct responses of cultured skin fibroblasts to biotin deprivation in vitro, probably reflect fundamentally different etiologies for the two categories of biotin-responsive multiple carboxylase deficiency.This work was supported by USPHS Grants GM28838 and AM25884.     

Hydrogen-stimulated CO2 fixation and coordinate induction of hydrogenase and ribulosebiphosphate carboxylase in a H2-uptake positive strain of Rhizobium japonicum

H₂-uptake positive strains (122 DES and SR) and H₂-uptake negative strains SR2 and SR3 of Rhizobium japonicum were examined for ribulosebisphosphate (RuBP) carboxylase and H₂-uptake activities during growth conditions which induced formation of the hydrogenase system. The rate of ¹⁴CO₂ uptake by hydrogenase-derepressed cells was about 6-times greater in the presence than in the absence of H₂. RuBP carboxylase activity was observed in free-living R. japonicum strains 122 DES or SR only when the cells were derepressed for their hydrogenase system. Hydrogenase and RuBP carboxylase activities were coordinately induced by H₂ and both were repressed by added succinate. Hydrogenase-negative mutant strains SR2 and SR3 derived from R. japonicum SR showed no detecyable RuBP carboxylase activities under hydrogenase derepression conditions. No detectable RuBP carboxylase was observed in bacteroids formed by H₂-uptake positive strains R. japonicum 122 DES or SR. Propionyl CoA carboxylase activity was consistently observed in extracts of cells from free-living cultures of R. japonicum but activity was not appreciably influenced by the addition of H₂. Neither phosphoenolpyruvate carboxylase nor phosphoenolpyruvate carboxykinase activity was detected in extracts of R. japonicum.Abbreviations RuBP Ribulose 1,5-bisphosphate - (Na₂EDTA) (Ethylenedinitrilo)-tetraacetic acid, disodium salt - (propionyl CoA) Propionyl coenzyme A - (PEP) Phosphoenolpyruvate - (GSH) Reduced glutathione - (Tricine) N-tris(hydroxymethyl)-methylglycine     

Triheptanoin protects against status epilepticus‐induced hippocampal mitochondrial dysfunctions,oxidative stress and neuronal degeneration

Triheptanoin, the triglyceride of heptanoate, is anaplerotic (refills deficient tricarboxylic acid cycle intermediates) via the propionyl‐CoA carboxylase pathway. It has been shown to be neuroprotective and anticonvulsant in several models of neurological disorders. Here, we investigated the effects of triheptanoin against changes of hippocampal mitochondrial functions, oxidative stress and cell death induced by pilocarpine‐induced status epilepticus (SE ) in mice. Ten days of triheptanoin pre‐treatment did not protect against SE , but it preserved hippocampal mitochondrial functions including state 2, state 3 ADP , state 3 uncoupled respiration, respiration linked to ATP synthesis along with the activities of pyruvate dehydrogenase complex and oxoglutarate dehydrogenase complex 24 h post‐SE . Triheptanoin prevented the SE ‐induced reductions of hippocampal mitochondrial superoxide dismutase activity and plasma antioxidant status as well as lipid peroxidation. It also reduced neuronal degeneration in hippocampal CA 1 and CA 3 regions 3 days after SE . In addition, heptanoate significantly reduced hydrogen peroxide‐induced cell death in cultured neurons. In situ hybridization localized the enzymes of the propionyl‐CoA carboxylase pathway, specifically Pcc α, Pcc β and methylmalonyl‐CoA mutase to adult mouse hippocampal pyramidal neurons and dentate granule cells, indicating that anaplerosis may occur in neurons. In conclusion, triheptanoin appears to have anaplerotic and antioxidant effects which contribute to its neuroprotective properties.

     

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.     

Acetyl-CoA-carboxylase activity in normally developing wheat leaves

In order to investigate the role of acetyl CoA carboxylase (ACC) in the regulation of fatty-acid biosynthesis in chloroplasts, the activities and relative amounts of the enzyme have been measured in the tissue of wheat (Triticum aestivum L.) leaves undergoing development and cellular differentiation. The total activity in the first leaves of 5- to 7-d-old plants was similar but decreased to less than half in 9-d-old plants. The activity of ACC in the cells of the first leaf of 7-d-old plants doubled when cell age increased from 24 to 48 h, remained relatively constant for a further 24 h and then declined. The amount of ACC in cells increased 15-fold during the first 36 h of cell enlargement. Cells more than 36 h old contained about two-thirds the maximum amount of ACC found in younger cells. The most rapid phase of fatty-acyl accumulation in lipids was in cells aged between 60 and 84 h. Tenfold changes in the activity of ACC were observed when the assay conditions with respect to ATP, ADP, Mg²⁺ and pH were changed to correspond to the physiological conditions in chloroplasts during light/dark transitions. This observation and the magnitude of the changes in the optimum activity and amount of ACC in leaf cells undergoing development are consistent with a role for ACC in the regulation of the flow of carbon from acetyl CoA to fatty acids in chloroplasts.Abbreviation ACC acetyl CoA carboxylase     

Modulation in vivo by Nitrate Salts of the Activity and Properties of Phosphoenolpyruvate Carboxylase in Leaves of Alternanthera pungens (C4 plant) and A. sessilis (C3 species)

Feeding K⁺ or Na⁺ nitrate salts in vivo enhanced the activity of phosphoenolpyruvate carboxylase (PEPC) in the leaf extracts of Alternanthera pungens (C₄ plant) and A. sessilis (C₃ species). The increase was more pronounced in A. pungens than in A. sessilis. Chloride salts increased the PEPC activity only marginally. However, the sulfate salts were either not effective or inhibitory. Feeding nitrate modulated the regulatory properties of PEPC in A. pungens, resulting in increased K_I (malate) and decreased K_A (glucose-6-P). The sensitivity of PEPC to malate, which gives a measure of phosphorylation status of the enzyme, indicated that feeding leaves with NO₃ ^– enhanced the phosphorylation status of the enzyme. The reduction in PEPC activity due to cycloheximide treatment suggested that increased synthesis of PEPC protein kinase may be one of the reasons for the enhancement in PEPC activity, after the nitrate feeding. We suggest that nitrate salts could be used as a tool to modulate and analyze the properties of PEPC in C₃ and C₄ plants.     

Inborn errors of cobalamin metabolism: Effect of cobalamin supplementation in culture on methylmalonyl CoA mutase activity in normal and mutant human fibroblasts

We have examined the effect of addition of hydroxocobalamin to growth medium on the activity of the adenosylcobalamin-requiring enzyme methylmalonyl CoA mutase in normal human fibroblasts and in mutant human fibroblasts derived from patients with inherited methylmalonicacidemia. The mutant cell lines were assigned to four distinct genetic complementation groups (cbl A, cbl B, cbl C, and cbl D), each deficient in some step in the synthesis of adenosylcobalamin from hydroxocobalamin. After control cells were grown in cobalamin-supplemented medium, mutase holoenzyme activity increased markedly in a time- and concentration-dependent fashion. Growth in cobalamin-supplemented medium had no effect on mutase activity in some mutant lines belonging to the cbl B group, while activity increased severalfold in other cbl B mutants and in all cbl A, cbl C, and cbl D mutants examined, although mutase activity was still <10% of control. Comparison of mutase holoenzyme activity and total propionate pathway activity suggests that enhancement of mutase activity in mutant cells after cobalamin supplementation to values 5–10% of control may be sufficient to overcome the inherited metabolic block and to restore total pathway activity to normal.This work was supported in part by a research grant from the National Institutes of Health (AM 12579). H. F. W. is a recipient of a traineeship from the National Institutes of Health (T01-GM02299).     

Metabolism of dimethylsulphoniopropionate by Ruegeria pomeroyi DSS‐3

Ruegeria pomeroyi DSS‐3 possesses two general pathways for metabolism of dimethylsulphoniopropionate (DMSP), an osmolyte of algae and abundant carbon source for marine bacteria. In the DMSP cleavage pathway, acrylate is transformed into acryloyl‐CoA by propionate‐CoA ligase (SPO2934) and other unidentified acyl‐CoA ligases. Acryloyl‐CoA is then reduced to propionyl‐CoA by AcuI or SPO1914. Acryloyl‐CoA is also rapidly hydrated to 3‐hydroxypropionyl‐CoA by acryloyl‐CoA hydratase (SPO0147). A SPO1914 mutant was unable to grow on acrylate as the sole carbon source, supporting its role in this pathway. Similarly, growth on methylmercaptopropionate, the first intermediate of the DMSP demethylation pathway, was severely inhibited by a mutation in the gene encoding crotonyl‐CoA carboxylase/reductase, demonstrating that acetate produced by this pathway was metabolized by the ethylmalonyl‐CoA pathway. Amino acids and nucleosides from cells grown on ¹³C‐enriched DMSP possessed labelling patterns that were consistent with carbon from DMSP being metabolized by both the ethylmalonyl‐CoA and acrylate pathways as well as a role for pyruvate dehydrogenase. This latter conclusion was supported by the phenotype of a pdh mutant, which grew poorly on electron‐rich substrates. Additionally, label from [¹³C‐methyl] DMSP only appeared in carbons derived from methyl‐tetrahydrofolate, and there was no evidence for a serine cycle of C‐1 assimilation.     

LC-MS/MS quantification of short-chain acyl-CoA's in Escherichia coli demonstrates versatile propionyl-CoA synthetase substrate specificity

Aims: This paper utilized quantitative LC‐MS/MS to profile the short‐chain acyl‐CoA levels of several strains of Escherichia coli engineered for heterologous polyketide production. To further compare and potentially expand the levels of available acyl‐CoA molecules, a propionyl‐CoA synthetase gene from Ralstonia solanacearum (prpE‐RS) was synthesized and expressed in the engineered strain BAP1. Methods and Results: Upon feeding propionate, the engineered E. coli strains had increased the levels of both propionyl‐ and methylmalonyl‐CoA of 6‐ to 30‐fold and 3·7‐ to 6·8‐fold, respectively. Expression of prpE‐RS resulted in no significant increases in acetyl‐, butyryl‐ and propionyl‐CoA when fed the corresponding substrates (sodium acetate, butyrate or propionate). More interesting, however, were the results from strain BAP1 engineered for native prpE overexpression, which indicated increases in the same range of acyl‐CoA formation. Conclusions: The increased acyl‐CoA levels across the strains profiled in this study reflect the genetic modifications implemented for improved polyketide production and also indicate flexibility of the native PrpE. Significance and Impact of the Study: The results provide direct evidence of enhanced acyl‐CoA levels correlating to those strains engineered for polyketide biosynthesis. This information and the inherent flexibility of the native PrpE enzyme support future efforts to characterize, engineer and extend acyl‐CoA precursor supply for additional heterologous biosynthetic attempts.