An enzymatic pathway for the biosynthesis of the formylhydroxyornithine required for rhodochelin iron coordination

Rhodochelin, a mixed catecholate-hydroxamate type siderophore isolated from Rhodococcus jostii RHA1, holds two L-δ-N-formyl-δ-N-hydroxyornithine (L-fhOrn) moieties essential for proper iron coordination. Previously, bioinformatic and genetic analysis proposed rmo and rft as the genes required for the tailoring of the L-ornithine (L-Orn) precursor [Bosello, M. (2011) J. Am. Chem. Soc.133, 4587-4595]. In order to investigate if both Rmo and Rft constitute a pathway for L-fhOrn biosynthesis, the enzymes were heterologously produced and assayed in vitro. In the presence of molecular oxygen, NADPH and FAD, Rmo monooxygenase was able to convert L-Orn into L-δ-N-hydroxyornithine (L-hOrn). As confirmed in a coupled reaction assay, this hydroxylated intermediate serves as a substrate for the subsequent N(10)-formyl-tetrahydrofolate-dependent (N(10)-fH(4)F) Rtf-catalyzed formylation reaction, establishing a route for the L-fhOrn biosynthesis, prior to its incorporation by the NRPS assembly line. It is of particular interest that a major improvement to this study has been reached with the use of an alternative approach to the chemoenzymatic FolD-dependent N(10)-fH(4)F conversion, also rescuing the previously inactive CchA, the Rft-homologue in coelichelin assembly line [Buchenau, B. (2004) Arch. Microbiol.182, 313-325; Pohlmann, V. (2008) Org. Biomol. Chem.6, 1843-1848].     

Radiochemical malonyl-CoA decarboxylase assay: activity and subcellular distribution in heart and skeletal muscle

Malonyl-CoA decarboxylase is the main route for the disposal of malonyl-CoA, the key metabolite in the regulation of mitochondrial fatty acid oxidation. We have developed a simple and sensitive radiochemical assay to determine malonyl-CoA decarboxylase activity. The decarboxylation of [2-14C]malonyl-CoA produces [2-14C]acetyl-CoA, which is converted to [2-14C]acetylcarnitine in the presence of excess L-carnitine and carnitine acetyltransferase. The positively charged radiolabeled product, acetylcarnitine, is separated from negatively charged excess radiolabeled substrate and the radioactivity measured by scintillation counting. Measurement of malonyl-CoA decarboxylase activities with this method gives values comparable to those obtained with assays currently in use, but has the advantage of being simpler and less labor intensive. We have applied this assay to rat skeletal muscle of different fiber-type composition and to rat heart. Malonyl-CoA decarboxylase activity (mU/g wet wt) correlates with the oxidative capacity of the muscles, being lowest in type IIb fibers (42.7 +/- 3.0) and highest in heart (1071.4 +/- 260), with intermediate activity in type IIa fibers (150.7 +/- 4.3) and type I fibers (107.8 +/- 7.6). Studies on subcellular distribution of malonyl-CoA decarboxylase activity in rat heart and rat skeletal muscle show that approximately 50 and 65% is localized to mitochondria, while 50 and 35% of the activity is extramitochondrial.     

Functions of Malonate Decarboxylase Subunits from Pseudomonas putida

Malonate decarboxylase from Pseudomonas putida is composed of five subunits, α, β, γ, δ, and ε. Two subunits, δ and ε, have been identified as an acyl-carrier protein (ACP) and malonyl-CoA:ACP transacylase, respectively. Functions of the other three subunits have not been identified, because recombinant subunits expressed in Escherichia coli formed inclusion bodies. To resolve this problem, we used a coexpression system with GroEL/ES from E. coli, and obtained active recombinant subunits. Enzymatic analysis of the purified recombinant subunits showed that the α subunit was an acetyl-S-ACP:malonate ACP transferase and that the βγ-subunit complex was a malonyl-S-ACP decarboxylase.     

MCD encodes peroxisomal and cytoplasmic forms of malonyl-CoA decarboxylase and is mutated in malonyl-CoA decarboxylase deficiency.

Malonyl-CoA decarboxylase (MCD) catalyzes the proton-consuming conversion of malonyl-CoA to acetyl-CoA and CO(2). Although defects in MCD activity are associated with malonyl-CoA decarboxylase deficiency, a lethal disorder characterized by cardiomyopathy and developmental delay, the metabolic role of this enzyme in mammals is unknown. A computer-based search for novel peroxisomal proteins led to the identification of a candidate gene for human MCD, which encodes a protein with a canonical type-1 peroxisomal targeting signal of serine-lysine-leucine(COOH). We observed that recombinant MCD protein has high intrinsic malonyl-CoA decarboxylase activity and that a malonyl-CoA decarboxylase-deficient patient has a severe mutation in the MCD gene (c.947-948delTT), confirming that this gene encodes human MCD. Subcellular fractionation experiments revealed that MCD resides in both the cytoplasm and peroxisomes. Cytoplasmic MCD is positioned to play a role in the regulation of cytoplasmic malonyl-CoA abundance and, thus, of mitochondrial fatty acid uptake and oxidation. This hypothesis is supported by the fact that malonyl-CoA decarboxylase-deficient patients display a number of phenotypes that are reminiscent of mitochondrial fatty acid oxidation disorders. Additional support for this hypothesis comes from our observation that MCD mRNA is most abundant in cardiac and skeletal muscles, tissues in which cytoplasmic malonyl-CoA is a potent inhibitor of mitochondrial fatty acid oxidation and which derive significant amounts of energy from fatty acid oxidation. As for the role of peroxisomal MCD, we propose that this enzyme may be involved in degrading intraperoxisomal malonyl-CoA, which is generated by the peroxisomal beta-oxidation of odd chain-length dicarboxylic fatty acids.     

Ethylmalonyl-CoA decarboxylase, a new enzyme involved in metabolite proofreading

A limited number of enzymes are known that play a role analogous to DNA proofreading by eliminating non-classical metabolites formed by side activities of enzymes of intermediary metabolism. Because few such "metabolite proofreading enzymes" are known, our purpose was to search for an enzyme able to degrade ethylmalonyl-CoA, a potentially toxic metabolite formed at a low rate from butyryl-CoA by acetyl-CoA carboxylase and propionyl-CoA carboxylase, two major enzymes of lipid metabolism. We show that mammalian tissues contain a previously unknown enzyme that decarboxylates ethylmalonyl-CoA and, at lower rates, methylmalonyl-CoA but that does not act on malonyl-CoA. Ethylmalonyl-CoA decarboxylase is particularly abundant in brown adipose tissue, liver, and kidney in mice, and is essentially cytosolic. Because Escherichia coli methylmalonyl-CoA decarboxylase belongs to the family of enoyl-CoA hydratase (ECH), we searched mammalian databases for proteins of uncharacterized function belonging to the ECH family. Combining this database search approach with sequencing data obtained on a partially purified enzyme preparation, we identified ethylmalonyl-CoA decarboxylase as ECHDC1. We confirmed this identification by showing that recombinant mouse ECHDC1 has a substantial ethylmalonyl-CoA decarboxylase activity and a lower methylmalonyl-CoA decarboxylase activity but no malonyl-CoA decarboxylase or enoyl-CoA hydratase activity. Furthermore, ECHDC1-specific siRNAs decreased the ethylmalonyl-CoA decarboxylase activity in human cells and increased the formation of ethylmalonate, most particularly in cells incubated with butyrate. These findings indicate that ethylmalonyl-CoA decarboxylase may correct a side activity of acetyl-CoA carboxylase and suggest that its mutation may be involved in the development of certain forms of ethylmalonic aciduria.     

Effect of DL-alpha-hydrazino-delta-aminovaleric acid, an inhibitor of ornithine decarboxylase, on polyamine metabolism in isoproterenol-stimulated mouse parotid glands.

The effects of DL-alpha-hydrazino-delta-aminovaleric acid (DL-HAVA) on polyamine metabolism in isoproterenol(IPR)-stimulated mouse parotid glands were investigated both in vitro and in vivo. Using partially enzyme preparations, it was found that DL-HAVA strongly inhibited ornithine decarboxylase (EC 4.1.1.17) by competing with L-ornithine. Other enzymes metabolizing ornithine and pyridoxal phosphate-dependent enzymes were at least 2-3 orders of magnitude less sensitive to DL-HAVA than ornithine decarboxylase. Administration of DL-HAVA greatly depressed the increases in both the putrescine level and putrescine formation from L-ornithine induced by IPR in the mouse parotid glands. Under the same conditions, the stimulation of DNA synthesis and subsequent cell proliferation in the glands were also suppressed. However, the IPR-dependent increases in S-adenosyl-L-methionine decarboxylase (EC 4.1.1.50) activity, synthesis and the tissue concentration of spermidine, and RNA synthesis in the parotid glands were not affected appreciably by DL-HAVA. The inhibition of DNA synthesis by DL-HAVA was effectively prevented by putrescine, but not by spermidine or 1,7-diaminoheptane, given at the same time when DL-HAVA inhibited stimulation of putrescine formation by IPR. From these results, it is proposed that putrescine is involved in cell proliferation besides being a precursor of spermidine. The effects of methylglyoxal bis(guanylhydrazone) (MGBG), an inhibitor of S-adenosyl-L-methionine decarboxylase, on the metabolism of polyamines and nucleic acids in growing parotid glands were also examined.     

Biosynthesis of inducible L-ornithine decarboxylase by bacterial of the Vibrionaceae family

The biosynthesis of L-ornithine decarboxylase was investigated in 73 strains of the Vibrionaceae family. V. harveyi 1175 was found to be most active. The maximum accumulation of the enzyme was observed after 4-hour cultivation at pH 5.5 and 33 degrees in the presence of 0.8% L-ornithine HCl used as an inducer without aeration. Under these conditions L-ornithine decarboxylase activity was 3.87 units/mg dry cells.     

Developmental expression of peptidylglycine alpha-amidating monooxygenase (PAM) in primary cultures of neonatal rat cardiocytes: a model for studying regulation of PAM expression in the rat heart.

Primary cultures of neonatal rat atrial and ventricular cardiomyocytes were used to investigate the expression of peptidylglycine alpha-amidating monooxygenase (PAM), a bifunctional enzyme required for the production of alpha-amidated neuroendocrine peptides. The use of assays for the individual enzymes, peptidylglycine alpha-amidating monooxygenase (PHM) and peptidyl-alpha-hydroxyglycine alpha-amidating lyase (PAL), demonstrated that the levels of expression observed in vitro approximated those observed in vivo. Both in vivo and in vitro, atrial and ventricular PAL activity greatly exceeded PHM activity. Atrial and ventricular cardiomyocytes secreted PHM and PAL activity at a constant rate throughout the culture period. Immunofluorescence studies localized PAM proteins to the perinuclear region, with intense punctate staining. Both in vivo and in vitro, PAM mRNAs encoding integral membrane proteins predominated throughout the neonatal period, with PAM-1 mRNA becoming more prevalent after the first week in culture. Although PAM-2 mRNA decreased in prevalence in vivo at the time when PAM-1 expression increased, levels of PAM-2 mRNA remained elevated throughout 2 weeks in vitro. Western blot analysis demonstrated intact PAM-1 and PAM-2 proteins in atrial cultures, with the prevalence of PAM-1 increasing in older cultures. Atrial cardiomyocytes secreted only bifunctional PAM proteins. Many of the features of PAM expression, processing, and storage that are unique to cardiomyocytes as opposed to endocrine cells are faithfully replicated by primary atrial and ventricular cultures.     

Putrescine-sensitive (artifactual) and insensitive (biosynthetic) S-adenosyl-L-methionine decarboxylase activities of Lathyrus sativus seedlings.

The crude extracts of 3-day-old etiolated seedlings of Lathyrus sativus contained two S-adenosyl-L-methionine decarboxylase activities. The artifactual putrescine-dependent activity was due to the H2O2 generated by diamine oxidase (EC 1.4.3.6) of this plant system and was inhibited by catalase. This observation was confirmed by using an electrophoretically and immunologically homogeneous preparation of L. sativus diamine oxidase. In the presence of putrescine, diamine oxidase, in addition to S-adenosylmethionine, decarboxylated L-lysine, L-arginine, L-ornithine, L-methionine and L-glutamic acid to varying degrees. The decarboxylation was not metal-ion dependent. The biosynthetic S-adenosylmethionine decarboxylase (EC 4.1.1.21) was detected after removing diamine oxidase specifically from the crude extracts by employing an immunoaffinity column. This Mg2+-dependent decarboxylase was not stimulated by putrescine or inhibited by catalase. The enzyme activity was inhibited by semicarbazide, 4-bromo-3-hydroxybenzoylamine dihydrogen phosphate and methylglyoxal-bis (guanylhydrazone). It was largely localized in the shoots of the etiolated seedlings and was purified 40-fold by employing a p-hydroxymercuribenzoate/AH-Sepharose affinity column, which also separated the decarboxylase activity from spermidine synthase.     

Malonyl-CoA decarboxylase is not a substrate of AMP-activated protein kinase in rat fast-twitch skeletal muscle or an islet cell line.

The AMP-activated protein kinase (AMPK) plays an important role in fuel metabolism in exercising skeletal muscle and possibly in the islet cell with respect to insulin secretion. Some of these effects are due to AMPK-mediated regulation of cellular malonyl-CoA content, ascribed to the ability of AMPK to phosphorylate and inactivate acetyl-CoA carboxylase (ACC), reducing malonyl-CoA formation. It has been suggested that AMPK may also regulate malonyl-CoA content by activation of malonyl-CoA decarboxylase (MCD). We have investigated the potential regulation of MCD by AMPK in exercising skeletal muscle, in an islet cell line, and in vitro. Three rat fast-twitch muscle types were studied using two different contraction methods or after exposure to the AMPK activator AICAR. Although all muscle treatments resulted in activation of AMPK and phosphorylation of ACC, no stimulus had any effect on MCD activity. In 832/13 INS-1 rat islet cells, two treatments that result in the activation of AMPK, namely low glucose and AICAR, also had no discernable effect on MCD activity. Last, AMPK did not phosphorylate in vitro either recombinant MCD or MCD immunoprecipitated from skeletal muscle or heart. We conclude that MCD is not a substrate for AMPK in fast-twitch muscle or the 832/13 INS-1 islet cell line and that the principal mechanism by which AMPK regulates malonyl-CoA content is through its regulation of ACC.     

Identification of the amino acid residues conferring substrate specificity upon Selenomonas ruminantium lysine decarboxylase

Lysine decarboxylase (LDC, EC 4.1.1.18) from Selenomonas ruminantium has decarboxylating activities towards both L-lysine and L-ornithine with similar K(m) and Vmax. Here, we identified four amino acid residues that confer substrate specificity upon S. ruminantium LDC and that are located in its catalytic domain. We have succeeded in converting S. ruminantium LDC to an enzyme with a preference in decarboxylating activity for L-ornithine when the four-residue of LDC were replaced by the corresponding residues of mouse ornithine decarboxylase (EC 4.1.1.17).     

NXP-1, a human protein related to Rad21/Scc1/Mcd1, is a component of the nuclear matrix

Nuclear matrix is a complex intranuclear network supposed to be involved in the various nuclear functions. In order to identify the nuclear matrix proteins, we isolated a cDNA clone from a human placenta cDNA library. This clone was partially represented a known cDNA clone HA1237. HA1237 encoded a 631-amino-acid peptide, which we designated NXP-1. NXP-1 was related to yeast Rad21/Scc1/Mcd1, Xenopus XRAD21, and mouse PW29, and identical with HR21spA isolated from a human testis cDNA library. We developed a polyclonal antibody to the purified NXP-1 bacterially expressed as a fusion protein with GST. Western blot analysis with anti-NXP-1 polyclonal antibody showed nuclear matrix localization of NXP-1 in HeLa cells. Indirect immunofluorescence staining also showed nuclear and nuclear matrix localization of the NXP-1. Results of in vitro binding assays employing nuclear matrix preparations indicated that the N-terminal region (16-128 amino acid) of NXP-1 has an important role in nuclear matrix distribution.     

TetX is a flavin-dependent monooxygenase conferring resistance to tetracycline antibiotics

The tetracycline antibiotics block microbial translation and constitute an important group of antimicrobial agents that find broad clinical utility. Resistance to this class of antibiotics is primarily the result of active efflux or ribosomal protection; however, a novel mechanism of resistance has been reported to be oxygen-dependent destruction of the drugs catalyzed by the enzyme TetX. Paradoxically, the tetX genes have been identified on transposable elements found in anaerobic bacteria of the genus Bacteroides. Overexpression of recombinant TetX in Escherichia coli followed by protein purification revealed a stoichiometric complex with flavin adenine dinucleotide. Reconstitution of in vitro enzyme activity demonstrated a broad tetracycline antibiotic spectrum and a requirement for molecular oxygen and NADPH in antibiotic degradation. The tetracycline products of TetX activity were unstable at neutral pH, but mass spectral and NMR characterization under acidic conditions supported initial monohydroxylation at position 11a followed by intramolecular cyclization and non-enzymatic breakdown to other undefined products. TetX is therefore a FAD-dependent monooxygenase. The enzyme not only catalyzed efficient degradation of a broad range of tetracycline analogues but also conferred resistance to these antibiotics in vivo. This is the first molecular characterization of an antibiotic-inactivating monooxygenase, the origins of which may lie in environmental bacteria.     

Functions of malonate decarboxylase subunits from Pseudomonas putida

Malonate decarboxylase from Pseudomonasputida is composed of five subunits, alpha, beta, gamma, delta, and epsilon. Two subunits, delta and epsilon, have been identified as an acyl-carrier protein (ACP) and malonyl-CoA:ACP transacylase, respectively. Functions of the other three subunits have not been identified, because recombinant subunits expressed in Escherichia coi formed inclusion bodies. To resolve this problem, we used a coexpression system with GroEL/ES from E. coli, and obtained active recombinant subunits. Enzymatic analysis of the purified recombinant subunits showed that the alpha subunit was an acetyl-S-ACP:malonate ACP transferase and that the betagamma-subunit complex was a malonyl-S-ACP decarboxylase.     

Arginine biosynthesis in Neisseria gonorrhoeae: enzymes catalyzing the formation of ornithine and citrulline.

Many of the Neisseria gonorrhoeae strains isolated from patients require arginine for growth in a defined medium. As a basis for genetic studies of these Arg- strains, we examined two biosynthetic enzymes of Arg+ (nonrequiring) gonococci. Cell-free extracts contained (i) glutamate acetyltransferase, which catalyzes the formation of L-ornithine from alpha-N-acetyl-L-ornithine, and (ii) ornithine transcaramylase, which catalyzes the reaction between L-ornithine and carbamyl phosphate, yielding L-citrulline. Arg- strains were unable to utilze alpha-N-acetyl-L-ornithine for growth lacked significant activity of glutamate acetyltransferase, and activity was gained by Arg+ clones derived by DNA-mediated transformation. Some of the Arg- patient isolates were unable to use either alpha-N-acetyl-L-ornithine or L-ornithine in place of arginine, and two separate steps of genetic transformation were required to yield Arg+ cells. Extracts of these doubly auxotrophic cells lacked glutamate acetyltransferase activity, but, unexpectedly, they displayed normal ornithine transcarbamylase activity. This finding illustrates the importance of identifying the products specified by arg loci during genetic studies of arginine auxotrophy.     

Dual sites of occurrence of malonyl-CoA decarboxylase and their possible functional significance in avian tissues.

1. Uropygial glands of domestic goose and mallard which synthesize methyl-branched fatty acids, contain large quantities of cytosolic malonyl-CoA decarboxylase and a small quantity of mitochondrial enzyme. 2. Uropygial glands of chicken and the liver of geese which generate little methyl-branched acids, contain only small quantities of malonyl-CoA decarboxylase and in such cases the enzyme is in the mitochondria. 3. The mitochondrial decarboxylase from the uropygial gland and liver of goose is immunologically similar to the cytosolic decarboxylase of the uropygial gland. 4. The mitochondrial enzyme probably protects the mitochondrial enzymes which are susceptible to inhibition by malonyl-CoA, whereas the cytosolic enzyme promotes the synthesis of methyl-branched acids.     

Characterization of ornithine decarboxylase of tobacco cells and tomato ovaries.

Some characteristics of L-ornithine decarboxylase of tomato ovaries and tobacco cells are described. The enzyme has a pH optimum of 8.0. It requires pyridoxal phosphate and thiol reagent (dithiothreitol) for activity. It is specific for L-ornithine and has an apparent Km of 1.4 X 10-4 M. It has an apparent molecular weight of 107000. Putrescine inhibited the activity in vitro. Spermidine and spermine also inhibit the enzyme, but less effectively. It is concluded that the enzyme is similar to that of mammalian origin and likewise fulfils a function related to cell proliferation.     

A role for hypothalamic malonyl-CoA in the control of food intake

The cellular level of malonyl-CoA, an intermediate in fatty acid biosynthesis, depends on its rate of synthesis catalyzed by acetyl-CoA carboxylase relative to its rate of utilization and degradation catalyzed by fatty acid synthase and malonyl-CoA decarboxylase, respectively. Recent evidence suggests that hypothalamic malonyl-CoA functions in the regulation of feeding behavior by altering the expression of key orexigenic and anorexigenic neuropeptides. Here we report that 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR), a 5'-AMP kinase activator, rapidly lowers malonyl-CoA both in GT1-7 hypothalamic neurons and in the hypothalami of mice. These effects correlate closely with the phosphorylation of acetyl-CoA carboxylase, an established target of AMP kinase. Intracerebroventricular (i.c.v.) administration of AICAR rapidly lowers hypothalamic [malonyl-CoA] and increases food intake. Expression of an adenoviral cytosolic malonyl-CoA decarboxylase vector (Ad-cMCD) in hypothalamic GT1-7 cells decreases malonyl-CoA. When delivered by bilateral stereotaxic injection into the ventral hypothalamus (encompassing the arcuate nucleus) of mice, Ad-cMCD increases food intake and body weight. Ad-MCD delivered into the ventral hypothalamus also reverses the rapid suppression of food intake caused by i.c.v.-administered C75, a fatty acid synthase inhibitor that increases hypothalamic [malonyl-CoA]. Taken together these findings implicate malonyl-CoA in the hypothalamic regulation of feeding behavior.