Time-resolved extrinsic fluorescence of aromatic-L-amino-acid decarboxylase

The coenzyme-linked fluorescence of aromatic-L-amino-acid decarboxylase decays non-exponentially. The decay of both native and NaBH4 reduced samples can only be fitted by two exponentials each roughly accounting for about half of the total fluorescence. Denaturation of the reduced protein with 8 M urea makes the fluorescence decay mono-exponential, like that observed for the reference compound pyridoxamine-5-phosphate. An extra pyridoxyl moiety can be bound to the enzyme after incubation with excess pyridoxal phosphate and reduction with NaBH4. This sample is almost twice as fluorescent and shows also two lifetimes. After denaturation only one fluorescence lifetime is observed. The presence of two non-equivalent pyridoxal sites in the native enzyme can be postulated. The heterogeneous decay behaviour of the pyridoxyl moiety in the enzyme together with the variability of lifetime shown, makes this fluorophore an even more interesting fluorescent probe for proteins.     

Purification and characterisation of acetolactate decarboxylase from Leuconostoc lactis NCW1

A two-step strategy involving DEAE-cellulose and POROS PI anion exchange chromatography has been developed for rapid purification of acetolactate decarboxylase (ALD) from Leuconostoc lactis NCW1. This results in 5333-fold purification with a yield of 30%. Purified ALD is a dimer of 49-kDa subunits, has a pH optimum of 6.0, a pI of 4.2 and its activity is independent of metals or branched chain amino acids. At the optimum pH, the K(m) for 2-acetolactate (ALA) was found to be 1.3 mM and the turnover number was 4000 min(-1). N-terminal sequence comparison with other ALDs showed little sequence conservation in this region. Purified ALD does not catalyse direct production of diacetyl from ALA, unlike the crude extract.     

Purification, characterisation and cDNA sequencing of pyruvate decarboxylase from Zygosaccharomyces bisporus

Cells of the wild-type yeast strain Zygosaccharomyces bisporus CBS 702 form alpha-hydroxy ketones from aromatic amino acid precursors during fermentation. Pyruvate decarboxylase (PDC, E.C. 4.1.1.1), the key enzyme of this biotransformation catalysing the non-oxidative decarboxylation of pyruvate and other 2-oxo-acids, was purified and characterised. The active enzyme is homotetrameric (alpha4) with a molecular mass of about 244 kDa. Activation of PDC by its substrate pyruvate results in a sigmoidal dependence of the reaction rate from substrate concentration (apparent Km value 1.73 mM; Hill coefficient 2.10). A cDNA library was screened using a PCR-based procedure, and a 1856 bp cDNA of PDC was identified and sequenced. The cDNA encodes a polypeptide of 563 amino acid residues (monomeric unit). Sequence alignments demonstrate high homologies (> 80%) to PDC genes from Saccharomyces cerevisiae, Kluyveromyces lactis and Kluyveromyces marxianus.     

Adrenergic enzymes in cultured mouse neuroblastoma: absence of detectable aromatic-L-amino-acid decarboxylase.

The relative activities of tyrosine hydroxylase, aromatic-l -amino-acid decarboxylase and dopamine beta-hydroxylase were established in a number of clones of neuroblastoma cells isolated from the uncloned mouse C-1300 tumor. One clone, NBD-2, was chosen for further analysis on the basis of its relatively high activities of tyrosine hydroxylase and dopamine beta-hydroxylase. The levels of these enzymes, and monoamine oxidase and catechol O-methyltransferase, were at least 20-80 fold lower in the neuroblastoma culture than in mouse superior cervical ganglion. More importantly, aromatic-l -amino-acid decarboxylase activity was not even detectable in any neuroblastoma clone examined. Based on the relative sensitivities of the tyrosine hydroxylase and aromatic-l -amino-acid decarboxylase assays and on the ratio of these two enzymes in the mouse ganglion, decarboxylase activity is more than 10 fold lower in the cultured cells than would be predicted on the basis of tyrosine hydroxylase activity. Dialysis and mixing studies with neuroblastoma extracts and partially purified aromatic-l -amino-acid decarboxylase did not reveal the presence of any endogenous inhibitors that could account for the low level of decarboxylase activity in the cultured cells. During growth of the neuroblastoma cells to confluency, only one enzyme, monoamine oxidase, exhibited an elevated specific activity on the basis of cell number. However, when based on the amount of protein, the specific activity of all measurable enzymes increased in culture-because cell protein decreased 5 fold during growth to confluency. These findings are discussed with respect to individual cell function.     

Purification and partial gene sequence of the tyrosine decarboxylase of Lactobacillus brevis IOEB 9809

Some lactic acid bacteria contain a tyrosine decarboxylase (TDC) which converts tyrosine to tyramine, a biogenic amine frequently encountered in fermented food and wine. Purification and microsequencing of the TDC of Lactobacillus brevis IOEB 9809 allowed us to determine a partial sequence of the TDC gene encoding 264 amino acids of the enzyme. Analysis of this protein sequence revealed typical features of pyridoxal phosphate-dependent amino acid decarboxylases while not any known decarboxylase was closely related to the TDC of L. brevis IOEB 9809. In addition, we could detect other L. brevis strains carrying a TDC gene in a rapid assay based on the polymerase chain reaction.     

Purification, characterisation and reconstitution of glutaconyl-CoA decarboxylase, a biotin-dependent sodium pump from anaerobic bacteria

Glutaconyl-CoA decarboxylase from Acidaminococcus fermentans is a biotin enzyme, which is integrated into membranes. It is activated by Triton X-100 and inhibited by avidin. The results obtained by a combination of both agents indicate that biotin and the substrate-binding site are located on the same side of the membrane. The decarboxylase was solubilized with Triton X-100 and purified by affinity chromatography on monomeric avidin-Sepharose. The enzyme is composed of three types of polypeptides: the group of alpha chains (Mr 120000-140000) containing the biotin, the beta chain (60000) and an apparently hydrophobic gamma chain (35000). Sodium ions specifically protected the latter chain from tryptic digestion. It was supposed, therefore, that this chain might function as the Na+ channel. The beta and gamma chains but not the alpha chain could be labelled by N-ethyl-[14C]maleimide. Similar decarboxylases but with much smaller biotin peptides (Mr 15000-20000) were isolated from Peptococcus aerogenes and Clostridium symbiosum. The decarboxylases from all three organisms could be reconstituted to active sodium pumps by incubation with phospholipid vesicles and octylglucoside followed by dilution. The Na+ uptake catalysed by the enzyme from A. fermentans was completely inhibited by monensin and activated twofold by valinomycin/K+ indicating an electrogenic Na+ pump. The coupling between Na+ transport and decarboxylation was not tight. During the reaction the ratio decreased from initially 1 to 0.2. The three organisms mentioned above and Clostridium tetanomorphum without glutaconyl-CoA decarboxylase are able to ferment glutamate and require 10 mM Na+ for rapid growth. There is no correlation between the concentration of monensin necessary to inhibit growth and the presence of decarboxylase in these organisms.     

Purification and properties of cis-aconitic decarboxylase

Summary Lyophilized and stored in a deep-freeze, the mycelial material was found to retain cis-aconitic decarboxylase activity unimpaired at the end of 2 months. Mycelia could be stored also in the frozen condition but after squeezing hard to remove as much of adherent water as possible. Extracts with maximum cis-aconitic decarboxylase activity were obtained when the frozen or better the lyophilized mycelia of Aspergillus terreus were ground in a mortar with phosphate buffer using pyrex glass powder as abrasive. Cis-aconitic decarboxylase was purified 25-fold by fractionation with ammonium sulfate, starting from extracts of the mycelia in phosphate buffer. The purified enzyme was considerably more stable than the crude extracts to storage and dialysis. The optimum pH was 5.8 using 0.2 m phosphate buffer; Km value was 5×10^-3 m at pH 5.8 and 37°C. EDTA and 8-hydroxyquinoline activated the enzyme; all metals tested inhibited the enzyme, Zn⁺⁺ and Cu⁺⁺ leading to complete inactivation. Fluoride, arsenite and azide also inhibited the enzyme activity.     

Purification and crystallization of benzoylformate decarboxylase.

A new large-scale purification method for benzoylformate decarboxylase from Pseudomonas putida has allowed us to undertake an X-ray crystallographic study of the enzyme. The previously observed instability of the enzyme was overcome by addition of 100 microM thiamine pyrophosphate to buffers used in the purification. The final enzyme preparation was more than 97% pure, as determined by denaturing gel electrophoresis and densitometry. The mobility of the enzyme on a gel filtration column indicates that it is a tetramer of 57-kDa subunits. Large, single crystals of benzoylformate decarboxylase were grown from solutions of buffered polyethylene glycol 400, pH 8.5. The crystals diffract to beyond 1.6 A resolution and are stable for days to X-ray radiation. Analysis of X-ray data from the crystals, along with the newly determined quaternary structure, identifies the space group as I222. The unit cell dimensions are a = 82 A, b = 97 A, c = 138 A. An average Vm value for the crystals is consistent with one subunit per asymmetric unit. The subunits of the tetramer must be arranged with tetrahedral 222 symmetry.     

Purification and characterisation of a lactococcal aminoacylase

The amd1-encoded aminoacylase from Lactococcus lactis MG1363 was cloned and overexpressed in Escherichia coli and purified. The assumed dimeric enzyme has a subunit molecular mass of about 42 kDa and contains 2.0+/-0.1 g-atoms of zinc and cobalt, in equimolar amounts, per subunit of Amd1. The enzyme was characterised with respect to substrate specificity, pH, temperature and metal dependence. Amd1 exhibited a broad activity range towards N-acetylated- l-amino acids with a strong preference towards those containing neutral aliphatic and aromatic side chains. It hydrolysed N-acetyl- l-alanine most efficiently, and exhibited temperature and pH optima of 30 degrees C and 7.0, respectively. The activity of Amd1 towards N-acetyl- l-alanine was enhanced by the divalent cation Co(2+), while Cd(2+ )inhibited activity. Interestingly, Amd1 was shown to catalyse the hydrolysis of several dipeptides at pH 7.0, although with reduced V(max) values as compared to hydrolysis of N-acetylated- l-amino acids. This characteristic has also biological significance since Amd1 was able to complement a growth deficiency in a L. lactis triple peptidase mutant.     

Purification and properties of diaminopimelate decarboxylase ofMicrococcus glutamicus

Diaminopimelate decarboxylase (EC 4.1.1.20) ofMicrococcus glutamicus ATCC 13059 was purified to homogeneity. The enzyme had an apparent molecular weight of 191,000 as determined by gel filtration on Sephadex G-200. At protein concentrations of 20 and 10 μg per ml and in the absence of pyridoxal-5′-phosphate, it dissociated into a species of molecular weight 94,000. The polypeptide chain molecular weight as determined by sodium dodecyl sulphate Polyacrylamide gel electrophoresis was 100,000. TheK _m formeso diaminopimelate was 0.5 mM and that for pyridoxal-5′-phosphate was 0.6 μI. Sulphydryl groups and pyridoxal-5′-phosphate were essential for activity and stability. The enzyme was inhibited significantly by L-lysine and DL-aspartic β-semialdehyde.     

Purification and properties of oxaloacetate decarboxylase fromCorynebacterium glutamicum

Oxaloacetate (OAA) decarboxylase (E.C. 4.1.1.3) was isolated fromCorynebacterium glutamicum. In five steps the enzyme was purified 300-fold to apparent homogeneity. The molecular mass estimated by gel filtration was 118 ± 6 kDa. SDS-PAGE showed a single subunit of 31.7 KDa, indicating an ₄ subunit structure for the native enzyme. The enzyme catalyzed the decarboxylation of OAA to pyruvate and CO₂, but no other -ketoacids were used as substrate. The cation Mn²⁺ was required for full activity, but could be substituted by Mg²⁺, Co²⁺, Ni²⁺ and Ca²⁺. Monovalent ions like Na⁺, K⁺ or NH ₄ ⁺ were not required for activity. The enzyme was inhibited by Cu²⁺, Zn²⁺, ADP, coenzyme A and succinate. Avidin did not inhibit the enzyme activity, indicating that biotin is not involved in decarboxylation of OAA. Analysis of the kinetic properties revealed a K _m for OAA of 2.1 mM and a K _m of 1.2 mM for Mn²⁺. The V _max was 158 µmol of OAA converted per min per mg of protein, which corresponds to an apparent k _cat of 311 s^–1.Abbreviations OAA oxaloacetate - LDH lactate dehydrogenase     

Purification and properties of Selenomonas ruminantium lysine decarboxylase

Selenomonas ruminantium, a strictly anaerobic, gram-negative bacterium isolated from sheep rumen, contains lysine decarboxylase (Y. Kamio et al., J. Bacteriol. 145:122-128, 1981). This report describes the synthesis, purification, and characterization of the enzyme. Lysine decarboxylase was synthesized in cells grown in chemically defined medium without lysine. The enzyme was purified approximately 1,800-fold to electrophoretic homogeneity. The native enzyme of approximate molecular weight 88,000 consisted of two identical subunits, each with a molecular weight of 44,000. Several properties of the enzyme were determined and compared with those of the lysine decarboxylases from Escherichia coli and Bacterium cadaverisis.     

Purification and characterization of rat-liver 3,4-dihydroxyphenylalanine decarboxylase

A simple and rapid procedure, which takes advantage of the effectiveness of conventional and HPLC hydrophobic interaction, for the isolation of highly purified rat liver 3,4-dihydroxyphenylalanine decarboxylase is described in detail. Some of its structural and functional properties are reported and discussed in comparison with those of pig kidney 3,4-dihydroxyphenylalanine decarboxylase.     

Purification and characterization of bovine hepatic uroporphyrinogen decarboxylase

Uroporphyrinogen decarboxylase (EC 4.1.1.37) has been purified to homogeneity from bovine liver by using isoelectric and salt precipitations, followed by chromatography on DEAE-cellulose, phenyl-Sepharose, hydroxylapatite, and Sephacryl S-200. The purified enzyme is a monomer with an Mr approximately 57 000 and an isoelectric point at pH 4.6. Enzyme activity is optimal in buffers having an ionic strength of approximately 0.1 M and a pH of 6.8. The purified enzyme has a specific activity (expressed as the disappearance of uroporphyrinogen I) of 936 nmol X h-1 X (mg of protein)-1. The purified enzyme catalyzes all four decarboxylation reactions in the conversion of uroporphyrinogen I or III to the corresponding coproporphyrinogen. The rate-limiting step in the physiologically significant conversion of uroporphyrinogen III to coproporphyrinogen III is the decarboxylation of heptacarboxylate III. Kinetic data suggest that the enzyme has at least two noninteracting active sites. At least one sulfhydryl group is required for catalytic activity. The enzyme is inhibited by sulfhydryl-specific reagents and by divalent metal ions including Fe2+, Co2+, Cu2+, Zn2+, and Pb2+. The pattern of accumulation of intermediate (hepta-, hexa-, and pentacarboxylate porphyrinogens) and final (coproporphyrinogen) decarboxylation products is affected by the ratio of substrate (uroporphyrinogen I or III) concentration to enzyme concentration. Under physiologic conditions where the uroporphyrinogen to enzyme ratio is low, the substrate is nearly quantitatively decarboxylated, and the major product is coproporphyrinogen. If the ratio of uroporphyrinogen to enzyme is high, intermediates accumulate, and heptacarboxylate porphyrinogen becomes the major decarboxylation product.(ABSTRACT TRUNCATED AT 250 WORDS)     

Purification and characterization of a new sodium-transport decarboxylase. Methylmalonyl-CoA decarboxylase from Veillonella alcalescens

Upon resolution of the particulate cell fraction of Veillonella alcalescens by gel chromatography, membranes and ribosomes were clearly resolved. Methylmalonyl-CoA decarboxylase was bound to the membranes and not to ribosomes as reported earlier. Membrane vesicles containing methylmalonyl-CoA decarboxylase were prepared by disrupting V. alcalescens cells with a French pressure chamber. About 64% of the decarboxylase was oriented in these vesicles with the substrate binding site facing to the outside. The vesicles performed a rapid accumulation of Na+ ions in response to the decarboxylation of methylmalonyl-CoA. Decarboxylation and transport were highly uncoupled. The efficiency of the transport was considerably increased if methylmalonyl-CoA decarboxylation was retarded by using a low temperature or by slowly generating the substrate enzymically from propionyl-CoA. Under optimized conditions Na+ was concentrated inside the inverted vesicles eight-times higher than in the incubation medium. Methylmalonyl-CoA decarboxylase was solubilized from the membranes with Triton X-100 and purified about 20-fold by affinity chromatography on monomeric avidin-Sepharose columns. The decarboxylase was specifically activated by Na+ ions (apparent Km approximately equal to 0.6 mM). Whereas (S)-methylmalonyl-CoA was the superior substrate (apparent Km approximately equal to 7 microM), malonyl-CoA was also decarboxylated (apparent Km approximately equal to 35 microM). The decarboxylation of methylmalonyl-CoA yielded CO2 and not HCO-3 as the primary reaction product. Analysis of the purified enzyme by dodecylsulfate gel electrophoresis indicated the presence of four different polypeptides alpha, beta, gamma, delta with Mr 60 000, 33 000, 18 5000 and 14 000. The latter of these polypeptides was clearly visible only after silver staining but not after staining with Coomassie brilliant blue. A low molecular weight polypeptide with similar staining properties was also found in oxaloacetate decarboxylase. Methylmalonyl-CoA decarboxylase contained about 1 mol covalently bound biotin per 125 500 g protein which was localized exclusively in the gamma-subunit. This subunit therefore represents the biotin carboxyl carrier protein of methylmalonyl-CoA decarboxylase. A new very sensitive method for the detection of biotin-containing proteins is described.     

Purification of L-glutamate decarboxylase from monkey brain

Glutamate decarboxylase (GAD) is an enzyme that synthesizes gamma-aminobutyrate (GABA), a major inhibitory neurotransmitter in the central nervous system. Post-translational modification of GAD, such as N-terminal blockage, phosphorylation-dephosphorylation, and palmitoylation, is an important factor in the biological activity of GAD. In order to address the significance of post-translational events on GAD, we thought it crucial to obtain a non-recombinant form of GAD. In this study, we attempted to isolate GAD protein from the monkey brain, a model animal close to the human that has not been studied. Monkey brain was homogenized, fractionated with ammonium sulphate, and applied to a series of chromatographic steps, including hydrophobic, ion-exchange, and gel filtration. Purified GAD showed a single band on SDS-PAGE, and the enzyme was found to have a molecular weight of 61,000 and exhibited 1,100 nmol/min/mg of specific activity. It had an optimal pH of 7 and optimal thermal stability at 40 degrees C.     

Purification of L-glutamate decarboxylase by affinity chromatography

L-Glutamate decarboxylase (L-glutamate 1-carboxy-lyase, EC 4.1.1.15) from rat brain synaptosomal extract was partially purified by affinity chromatography. On further purification by DEAE-Sephadex A 50 and Sephadex G-200, L-glutamate decarboxylase was purified to greater extent. It was found that a single affinity chromatography by appropriate elution gave a highly purified protein giving a single band of high specific activity on polyacrylamide gradient gel slab electrophoresis with minimal contamination. Substrate specificity of the purified enzyme was modified in the presence of 6-azauracil or phenylalanine resulting in decreased specificity to L-glutamate and increased specificity to L-aspartate.