Phylloquinone (vitamin K(1) ) biosynthesis in plants: two peroxisomal thioesterases of Lactobacillales origin hydrolyze 1,4-dihydroxy-2-naphthoyl-CoA.

It is not known how plants cleave the thioester bond of 1,4-dihydroxy-2-naphthoyl-CoA (DHNA-CoA), a necessary step to form the naphthoquinone ring of phylloquinone (vitamin K(1) ). In fact, only recently has the hydrolysis of DHNA-CoA been demonstrated to be enzyme driven in vivo, and the cognate thioesterase characterized in the cyanobacterium Synechocystis. With a few exceptions in certain prokaryotic (Sorangium and Opitutus) and eukaryotic (Cyanidium, Cyanidioschyzon and Paulinella) organisms, orthologs of DHNA-CoA thioesterase are missing outside of the cyanobacterial lineage. In this study, genomic approaches and functional complementation experiments identified two Arabidopsis genes encoding functional DHNA-CoA thioesterases. The deduced plant proteins display low percentages of identity with cyanobacterial DHNA-CoA thioesterases, and do not even share the same catalytic motif. GFP-fusion experiments demonstrated that the Arabidopsis proteins are targeted to peroxisomes, and subcellular fractionations of Arabidopsis leaves confirmed that DHNA-CoA thioesterase activity occurs in this organelle. In vitro assays with various aromatic and aliphatic acyl-CoA thioester substrates showed that the recombinant Arabidopsis enzymes preferentially hydrolyze DHNA-CoA. Cognate T-DNA knock-down lines display reduced DHNA-CoA thioesterase activity and phylloquinone content, establishing in vivo evidence that the Arabidopsis enzymes are involved in phylloquinone biosynthesis. Extraordinarily, structure-based phylogenies coupled to comparative genomics demonstrate that plant DHNA-CoA thioesterases originate from a horizontal gene transfer with a bacterial species of the Lactobacillales order.

Cloning, purification, crystallization and preliminary X-ray diffraction crystallographic study of acyl-protein thioesterase 1 from Saccharomyces cerevisiae.

Palmitoylation/depalmitoylation plays an important role in protein modification. yApt1 is the only enzyme in Saccharomyces cerevisiae that catalyses depalmitoylation. In the present study, recombinant full-length yApt1 was cloned, expressed, purified and crystallized. The crystals diffracted to 2.40 Šresolution and belonged to space group P4(2)2(1)2, with unit-cell parameters a = b = 146.43, c = 93.29 Å. A preliminary model of the three-dimensional structure has been built and further refinement is ongoing.

Distinct subcellular localization in the cytosol and apicoplast, unexpected dimerization and inhibition of Plasmodium falciparum glyoxalases.

The ubiquitous glyoxalase system removes methylglyoxal as a harmful by-product of glycolysis. Because malaria parasites have drastically increased glycolytic fluxes, they could be highly susceptible to the inhibition of this detoxification pathway. Here we analysed the intracellular localization, oligomerization and inhibition of the glyoxalases from Plasmodium falciparum. Glyoxalase I (GloI) and one of the two glyoxalases II (cGloII) were located in the cytosol of the blood stages. The second glyoxalase II (tGloII) was detected in the apicoplast pointing to alternative metabolic pathways. Using a variety of methods, cGloII was found to exist in a monomer-dimer equilibrium that might have been overlooked for homologues from other organisms and that could be of physiological importance. The compounds methyl-gerfelin and curcumin, which were previously shown to inhibit mammalian GloI, also inhibited P. falciparum GloI. Inhibition patterns were predominantly competitive but were complicated because of the two different active sites of the enzyme. This effect was neglected in previous inhibition studies of monomeric glyoxalases I, with consequences for the interpretation of inhibition constants. In summary, the present work reveals novel general glyoxalase properties that future research can build on and provides a significant advance in characterizing the glyoxalase system from P. falciparum.

Thioesterase activity and subcellular localization of acylprotein thioesterase 1/lysophospholipase 1.

Acylprotein thioesterase 1 (APT1), also known as lysophospholipase 1, is an important enzyme responsible for depalmitoylation of palmitoyl proteins. To clarify the substrate selectivity and the intracellular function of APT1, we performed kinetic analyses and competition assays using a recombinant human APT1 (hAPT1) and investigated the subcellular localization. For this purpose, an assay for thioesterase activity against a synthetic palmitoyl peptide using liquid chromatography/mass spectrometry was established. The thioesterase activity of hAPT1 was most active at neutral pH, and did not require Ca(2+) for its maximum activity. The K(M) values for thioesterase and lysophospholipase (against lysophosphatidylcholine) activities were 3.49 and 27.3 microM, and the V(max) values were 27.3 and 1.62 micromol/min/mg, respectively. Thus, hAPT1 revealed much higher thioesterase activity than lysophospholipase activity. One activity was competitively inhibited by another substrate in the presence of both substrates. Immunocytochemical and Western blot analyses revealed that endogenous and overexpressed hAPT1 were mainly localized in the cytosol, while some signals were detected in the plasma membrane, the nuclear membrane and ER in HEK293 cells. These results suggest that eliminating palmitoylated proteins and lysophospholipids from cytosol is one of the functions of hAPT1.

Deficiency of the INCL protein Ppt1 results in changes in ectopic F1-ATP synthase and altered cholesterol metabolism.

Infantile neuronal ceroid lipofuscinosis (INCL) is a severe neurodegenerative disease caused by deficiency of palmitoyl protein thioesterase 1 (PPT1). INCL results in dramatic loss of thalamocortical neurons, but the disease mechanism has remained elusive. In the present work we describe the first interaction partner of PPT1, the F(1)-complex of the mitochondrial ATP synthase, by co-purification and in vitro-binding assays. In addition to mitochondria, subunits of F(1)-complex have been reported to localize in the plasma membrane, and to be capable of acting as receptors for various ligands such as apolipoprotein A-1. We verified here the plasma membrane localization of F(1)-subunits on mouse primary neurons and fibroblasts by cell surface biotinylation and TIRF-microscopy. To gain further insight into the Ppt1-mediated properties of the F(1)-complex, we utilized the Ppt1-deficient Ppt1(Delta ex4) mice. While no changes in the mitochondrial function could be detected in the brain of the Ppt1(Delta ex4) mice, the levels of F(1)-subunits alpha and beta on the plasma membrane were specifically increased in the Ppt1(Delta ex4) neurons. Significant changes were also detected in the apolipoprotein A-I uptake by the Ppt1(Delta ex4) neurons and the serum lipid composition in the Ppt1(Delta ex4) mice. These data indicate neuron-specific changes for F(1)-complex in the Ppt1-deficient cells and give clues for a possible link between lipid metabolism and neurodegeneration in INCL.

An unusual thioesterase promotes isochromanone ring formation in ajudazol biosynthesis.

The ajudazols are antifungal secondary metabolites produced by a hybrid polyketide synthase (PKS)-nonribosomal peptide synthetase (NRPS) multienzyme "assembly line" in the myxobacterium Chondromyces crocatus Cm c5. The most striking structural feature of these compounds is an isochromanone ring system; such an aromatic moiety is only known from two other complex polyketides, the electron transport inhibitor stigmatellin and the polyether lasalocid. The cyclization and aromatization reactions in the stigmatellin pathway are presumed to be catalyzed by a cyclase domain located at the end of the PKS, while the origin of the lasalocid benzenoid ring remains obscure. Notably, the ajudazol biosynthetic machinery does not incorporate a terminal cyclase, but instead a variant thioesterase (TE) domain. Here we present detailed phylogenetic and sequence analysis, coupled with experiments both in vitro and in vivo, that suggest that this TE promotes formation of the isochromanone ring, a novel reaction for this type of domain. As the ajudazol TE has homologues in several other secondary-metabolite pathways, these results are likely to be generalizable.

The metal ion requirements of Arabidopsis thaliana Glx2-2 for catalytic activity.

In an effort to better understand the structure, metal content, the nature of the metal centers, and enzyme activity of Arabidopsis thaliana Glx2-2, the enzyme was overexpressed, purified, and characterized using metal analyses, kinetics, and UV-vis, EPR, and (1)H NMR spectroscopies. Glx2-2-containing fractions that were purple, yellow, or colorless were separated during purification, and the differently colored fractions were found to contain different amounts of Fe and Zn(II). Spectroscopic analyses of the discrete fractions provided evidence for Fe(II), Fe(III), Fe(III)-Zn(II), and antiferromagnetically coupled Fe(II)-Fe(III) centers distributed among the discrete Glx2-2-containing fractions. The individual steady-state kinetic constants varied among the fractionated species, depending on the number and type of metal ion present. Intriguingly, however, the catalytic efficiency constant, k(cat)/K(m), was invariant among the fractions. The value of k(cat)/K(m) governs the catalytic rate at low, physiological substrate concentrations. We suggest that the independence of k(cat)/K(m) on the precise makeup of the active-site metal center is evolutionarily related to the lack of selectivity for either Fe versus Zn(II) or Fe(II) versus Fe(III), in one or more metal binding sites.

Monitoring enzyme catalysis in the multimeric state: direct observation of Arthrobacter 4-hydroxybenzoyl-coenzyme A thioesterase catalytic complexes using time-resolved electrospray ionization mass spectrometry.

The ability to examine real-time reaction kinetics for multimeric enzymes in their native state may offer unique insights into understanding the catalytic mechanism and its interplay with three-dimensional structure. In this study, we have used a time-resolved electrospray mass spectrometry approach to probe the kinetic mechanism of 4-hydroxybenzoyl-coenzyme A (4-HBA-CoA) thioesterase from Arthrobacter sp. strain SU in the millisecond time domain. Intact tetrameric complexes of 4-HBA-CoA thioesterase with up to four natural substrate (4-HBA-CoA) molecules bound were detected at times as early as 6 ms using an online rapid-mixing device directly coupled to an electrospray ionization time-of-flight mass spectrometer. Species corresponding to the formation of a folded tetramer of the thioesterase at charge states 16+, 17+, 18+, and 19+ around m/z 3800 were observed and assigned as individual tetramers of thioesterase and noncovalent complexes of the tetramers with up to four substrate and/or product molecules. Real-time evaluation of the reaction kinetics was accomplished by monitoring change in peak intensity corresponding to the substrate and product complexes of the tetrameric protein. The mass spectral data suggest that product 4-HBA is released from the active site of the enzyme prior to the release of product CoA following catalytic turnover. This study demonstrates the utility of this technique to provide additional molecular details for an understanding of the individual enzyme states during the thioesterase catalysis and ability to observe real-time interactions between enzyme and substrates and/or products in the millisecond time range.

The neuron-specific protein PGP 9.5 is a ubiquitin carboxyl-terminal hydrolase.

A complementary DNA (cDNA) for ubiquitin carboxyl-terminal hydrolase isozyme L3 was cloned from human B cells. The cDNA encodes a protein of 230 amino acids with a molecular mass of 26.182 daltons. The human protein is very similar to the bovine homolog, with only three amino acids differing in over 100 residues compared. The amino acid sequence deduced from the cDNA was 54% identical to that of the neuron-specific protein PGP 9.5. Purification of bovine PGP 9.5 confirmed that it is also a ubiquitin carboxyl-terminal hydrolase. These results suggest that a family of such related proteins exists and that their expression is tissue-specific.

Benzoyl-coenzyme A thioesterase of Azoarcus evansii: properties and function.

The aerobic benzoate metabolism in Azoarcus evansii follows an unusual route. The intermediates of the pathway are processed as coenzyme A (CoA) thioesters and the cleavage of the aromatic ring is non-oxygenolytic. The enzymes of this pathway are encoded by the box gene cluster which harbors a gene, orf1, coding for a putative thioesterase. Benzoyl-CoA thioesterase activity (20 nmol min(-1) mg(-1) protein) was present in cells grown aerobically on benzoate, but was lacking in cells grown on other aromatic or aliphatic substrates under oxic or anoxic conditions. The gene was cloned and overexpressed in Escherichia coli to produce a C-terminal His-tag fusion protein. The recombinant enzyme was a homotetramer of 16 kDa subunits. It catalyzed not only the hydrolysis of benzoyl-CoA, but also of 2,3-dihydro-2,3-dihydroxybenzoyl-CoA, the second intermediate in the pathway. The enzyme exhibited higher activity with mono-substituted derivatives of benzoyl-CoA, showing highest activity with 4-hydroxybenzoyl-CoA. Di-substituted derivatives of benzoyl-CoA, phenylacetyl-CoA, and aliphatic CoA thioesters were not hydrolyzed but some acted as inhibitors. The thioesterase appears to protect the cell from CoA pool depletion. It may constitute the prototype of a new subfamily within the hotdog fold enzyme superfamily.

Characterization and cloning of a stearoyl/oleoyl specific fatty acyl-acyl carrier protein thioesterase from the seeds of Madhuca longifolia (latifolia).

Deposition of oleate, stearate and palmitate at the later stages of seed development in Mahua (Madhuca longifolia (latifolia)), a tropical non-conventional oil seed plant, has been found to be the characteristic feature of the regulatory mechanism that produces the saturated fatty acid rich Mahua seed fat (commonly known as Mowrah fat). Although, the content of palmitate has been observed to be higher than that of stearate at the initial stages of seed development, it goes down when the stearate and oleate contents consistently rise till maturity. The present study was undertaken in order to identify the kind of acyl-ACP thioesterase(s) that drives the characteristic composition of signature fatty acids (oleate 37%, palmitate 25%, stearate 23%, linoleate 12.5%) in its seed oil at maturity. The relative Fat activities in the crude protein extracts of the matured seeds towards three thioester substrates (oleoyl-, stearoyl- and palmitoyl-ACP) have been found to be present in the following respective ratio 100:31:8. Upon further purification of the crude extract, the search revealed the presence of two partially purified thioesterases: a long-chain oleoyl preferring house-keeping LC-Fat and a novel stearoyl-oleoyl preferring SO-Fat. The characteristic accumulation of oleate and linoleate in the M. latifolia seed fat is believed to be primarily due to the thioesterase activity of the LC-Fat or MlFatA. On the other hand, the SO-Fat showed almost equal substrate specificity towards stearoyl- and oleoyl-ACP, when its activity towards palmitoyl-ACP compared to stearoyl-ACP was only about 12%. An RT-PCR based technique for cloning of a DNA fragment from the mRNA pool of the developing seed followed by nucleotide sequencing resulted in the identification of a FatB type of thioesterase gene (MlFatB). This gene was found to exist as a single copy in the mother plant genome. Ectopic expression of this MlFatB gene product in E. coli strain fadD88 further proved that it induced a higher level of accumulation of both stearic and oleic acids when compared to the negative control line that did not contain this MlFatB gene. It also indicated that SO-Fat indeed is the product of the MlFatB gene present in the maturing seeds of M. latifolia in nature. Additionally, a predicted 3D-structure for MlFatB protein has been developed through use of bioinformatics tools.

Purification, crystallization and preliminary X-ray diffraction analysis of the glyoxalase II from Leishmania infantum.

In trypanosomatids, trypanothione replaces glutathione in all glutathione-dependent processes. Of the two enzymes involved in the glyoxalase pathway, glyoxalase I and glyoxalase II, the latter shows absolute specificity towards trypanothione thioester, making this enzyme an excellent model to understand the molecular basis of trypanothione binding. Cloned glyoxalase II from Leishmania infantum was overexpressed in Escherichia coli, purified and crystallized. Crystals belong to space group C222(1) (unit-cell parameters a = 65.6, b = 88.3, c = 85.2 angstroms) and diffract beyond 2.15 angstroms using synchrotron radiation. The structure was solved by molecular replacement using the human glyoxalase II structure as a search model. These results, together with future detailed kinetic characterization using lactoyltrypanothione, should shed light on the evolutionary selection of trypanothione instead of glutathione by trypanosomatids.

The role Acyl-CoA thioesterases play in mediating intracellular lipid metabolism.

Acyl-CoA thioesterases are a group of enzymes that catalyze the hydrolysis of acyl-CoAs to the free fatty acid and coenzyme A (CoASH), providing the potential to regulate intracellular levels of acyl-CoAs, free fatty acids and CoASH. These enzymes are localized in almost all cellular compartments such as endoplasmic reticulum, cytosol, mitochondria and peroxisomes. Acyl-CoA thioesterases are highly regulated by peroxisome proliferator-activated receptors (PPARs), and other nutritional factors, which has led to the conclusion that they are involved in lipid metabolism. Although the physiological functions for these enzymes are not yet fully understood, recent cloning and more in-depth characterization of acyl-CoA thioesterases has assisted in discussion of putative functions for specific enzymes. Here we review the acyl-CoA thioesterases characterized to date and also address the diverse putative functions for these enzymes, such as in ligand supply for nuclear receptors, and regulation and termination of fatty acid oxidation in mitochondria and peroxisomes.

Purification and characterization of two forms of glyoxalase II from the liver and brain of Wistar rats.

Glyoxalase II (S-(2-hydroxyacyl)glutathione hydrolase, EC 3.1.2.6) was purified to homogeneity and separated into two forms (alpha, pI = 8.0; beta, pI = 7.4) from both liver and brain of wistar rats by column isoelectric focusing. These forms were also found to have different electrophoretic mobilities. No significant differences were found between the alpha and beta forms from either source in the relative molecular mass (about 24,000) or in Km values using three substrates. The temperature-inactivation profiles were also similar, the two forms being stable up to 50 degrees C. Chemical modification studies with phenylglyoxal suggest that these enzyme forms probably contain arginine residues near the active site. Inactivation of alpha and beta forms by diethylpyrocarbonate and by photooxidation with methylene blue, and protection by S-D-mandeloylglutathione, a slowly reacting substrate, suggest the presence of histidine at the active site. The alpha and beta forms show different half-life values in inactivation by histidine reagents, which may be due to a difference in the active-site structures of these enzymes. The results probably indicate distinct structures (sequences) for alpha and beta forms.

Purification and characterization of mouse liver glyoxalase II.

Glyoxalase II (S2-hydroxyacylglutatione hydrolase, EC 3.1.2.6) was purified from Swiss mouse liver to homogeneity by a rapid, two-step affinity chromatographic scheme. Homogeneity was established by multiple electrophoretic determinations. The purified enzyme exhibited a specific activity of 920 I.U./mg protein and has a molecular weight of approx. 29 500 as estimated by SDS polyacrylamide gel electrophoresis. The enzyme is a basic protein with a pI of approx. 8.1. Mouse liver glyoxalase II is competitively inhibited by the substrate of glyoxalase I (the hemimercaptal of methylglyoxal and glutathione); the Ki is 0.3 mM. The Km for S-D-lactoylglutathione is 0.27 mM, and the enzyme has a turnover number of approx. 27 000 mumol substrate per min per mumol enzyme.

Demonstration of glyoxalase II in rat liver mitochondria. Partial purification and occurrence in multiple forms.

Glyoxalase II (S-(2-hydroxyacyl)glutathione hydrolase, EC 3.1.2.6), which has been regarded as a cytosolic enzyme, was also found in rat liver mitochondria. The mitochondrial fraction contained about 10-15% of the total glyoxalase II activity in liver. The actual existence of the specific mitochondrial glyoxalase II was verified by showing that all of the activity of the crude mitochondrial pellet was still present in purified mitochondria prepared in a Ficoll gradient. Subfractionation of the mitochondria by digitonin treatment showed that 56% of the activity resided in the mitochondrial matrix and 19% in the intermembrane space. Partial purification of the enzyme (420-fold) was also achieved. Statistically significant differences were found in the substrate specificities of the mitochondrial and the cytosolic glyoxalase II. Electrophoresis and isoelectric focusing of either the crude mitochondrial extract or of the purified mitochondrial glyoxalase II resolved the enzyme activity into five forms with the respective pI values of 8.1, 7.5, 7.0, 6.85 and 6.6. Three of these forms (pI values 7.0-6.6) were exclusively mitochondrial, with no counterpart in the cytosol. The relative molecular mass of the partially purified enzyme, as estimated by Superose 12 gel chromatography, was 21,000. These results give evidence for the presence of mitochondrial glyoxalase II which is different from the cytosolic enzymes in several characteristics.

Purificaton and characterization of S-formylglutathione hydrolase from a methanol-utilizing yeast, Kloeckera sp. No. 2201.

S-Fromylglutathione hydrolase (EC 3.1.2.12), a glutathione thiol esterase, was purified from a methanol-utilizing yeast, Kloeckera sp. No. 2201, to homogeneity as judged by polyacrylamide gel electrophoresis. The molecular weight of the native enzyme was determined to be 58 000 by gel filtration. The enzyme appeared to be composed of two identical subunits (Mr = 31 000). The apparent Km for S-formylglutathione was 0.077 mM. The optimum temperature was 50 degrees C and the optimum pH was 6.4-6.6. The enzyme was inhibited by several types of sulfhydryl reagents. The purified enzyme preparation contained no activity of formaldehyde dehydrogenase or of formate dehydrogenase. It is thought that three enzymes, formaldehyde dehydrogenase, S-formylglutathione hydrolase and formate dehydrogenase, participate in the oxidation of formaldehyde to CO2 in Kloeckera sp. No. 2201.

Purification and characterization of a long-chain acyl-CoA hydrolase from rat liver microsomes.

A long-chain acyl-CoA hydrolase from rat liver microsomes has been purified by solvent extraction and gel chromatography to homogeneity as judged by polyacrylamide gel electrophoresis in the presence and absence of sodium dodecyl sulfate. The enzyme was a monomer of molecular weight 59 000. In a sucrose gradient it sedimented at 4.3 S. The isoelectric point, pI was 6.9, and the Stokes radius was approx. 31 A. The enzyme hydrolyzed long-chain fatty acyl-CoA (C7--C18) with maximum activity for palmitoyl-CoA. Bovine serum albumin activation of the enzyme was related to the ratio acyl-CoA/bovine serum albumin, and at high ratios, acyl-CoA inhibited the enzyme activity. Disregarding the substrate inhibition, an apparent Km of 65 nmol/mg protein or 1-10(-7) M and a V of 750 nmol/mg protein per min were calculated. The enzyme was inhibited by p-hydroxymercuribenzoate and N-ethylmaleimide. Reactivation by means of dithiothreitol was not complete.

Purification and characterization of mitochondrial thioesterase from rabbit myocardium and its inhibition by palmitoyl carnitine.

Mitochondrial thioesterase from rabbit myocardium was purified to homogeneity by sequential ion exchange, hydroxylapatite, chromatofocusing and gel filtration chromatographies. The purified protein had an apparent molecular mass of 170 kDa. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and subsequent silver staining revealed a single band (Mr 43 000) demonstrating that the protein is a tetramer. The specific activity of the purified thioesterase for palmitoyl-CoA hydrolysis was 1.8 mumol/mg per min. Thioesterase activity was maximal at pH 8 and was activated by Mg2+ but inhibited by Ca2+. Pathophysiological concentrations of L-palmitoyl carnitine (20-400 microM) competitively inhibited enzymic activity. The purified enzyme was also inhibited by high concentrations of substrate (over 20 microM palmitoyl-CoA).

Liberation, purification, and properties of thioesterase component of pigeon liver fatty acid synthetase complex.

Proteolysis of pigeon liver fatty acid synthetase with elastase results in the quantitative cleavage of the thioesterase component from the enzyme complex. This thioesterase component is two or three times more active catalytically in the isolated state than in the native fatty acid synthetase, and its activity is not affected by the presence or absence of reducing thiols. The proteolytically cleaved thioesterase is separated from the core enzyme in one step by size-exclusion chromatography on a Sephadex G-75 column. The peptide obtained by gel permeation is homogeneous with respect to size and charge, as shown by polyacrylamide gel electrophoresis in the presence and absence of SDS. Size-exclusion chromatography on Bio-Gel A 0.5 m and Sephadex G-75 columns, sucrose density gradient ultracentrifugation, and N-terminal amino acid analysis also indicate that the proteolytically cleaved thioesterase is homogeneous. The sedimentation coefficient of the thioesterase is approximately 2.9 S. Proteolytic cleavage with elastase also quantitatively releases the [1,3-14C]- or [1,3-3H]diisopropylphosphofluoridate-labeled thioesterase component from the correspondingly labeled fatty acid synthetase. Binding studies with 14C- or 3H-labelled diisopropylphosphofluoridate and fatty acid synthetase show that 2 mol of the label are bound per mol of the enzyme when complete loss of fatty acid-synthesizing activity occurs. The molecular weight of the thioesterase component is estimated to be 36000 by size-exclusion chromatography, SDS-polyacrylamide gel electrophoresis and amino acid analysis.