A radiochemical assay for beta-ureidopropionase using radiolabeled N-carbamyl-beta-alanine obtained via hydrolysis of [2-(14)C]5, 6-dihydrouracil.

A radiochemical assay was developed to measure the activity of beta-ureidopropionase in human liver homogenates which is based on the detection of the reaction product (14)CO(2) by liquid scintillation counting. Radiolabeled N-carbamyl-beta-alanine was prepared within 15 min by a simple hydrolysis of [2-(14)C]5, 6-dihydrouracil under alkaline conditions at 37 degrees C. The enzymatic reaction proved to be linear with time up to at least 3.5 h and protein concentrations up to at least 1 mg/ml. Human beta-ureidopropionase obeyed Michaelis-Menten kinetics with an apparent Km for N-carbamyl-beta-alanine of 15.5 +/- 1.9 microM. The assay proved to be very accurate and sensitive with an intraassay coefficient of variation of 2% and a detection limit of 28 pmol for the product CO(2).     

cDNA cloning, genomic structure and chromosomal localization of the human BUP-1 gene encoding beta-ureidopropionase.

A full-length cDNA clone encoding human beta-ureidopropionase was isolated. A 1152-nucleotide open reading frame which corresponds to a protein of 384 amino acids with a calculated molecular weight of 43? omitted?158 Da, surrounded by a 5'-untranslated region of 61 nucleotides and a 3'-untranslated region of 277 nucleotides was identified. The protein showed 91% similarity with the translation product of the rat beta-ureidopropionase cDNA. Expression of the human cDNA in an Escherichia coli and eukaryotic COS-7 expression system revealed a very high beta-ureidopropionase enzymatic activity, thus confirming the identity of the cDNA. Since human EST libraries from brain, liver, kidney and heart contained partial beta-ureidopropionase cDNAs, the enzyme seems to be expressed in these tissues, in agreement with the expression profile of this enzyme in rat. Using the human cDNA as a probe a genomic P1 clone could be isolated containing the complete human beta-ureidopropionase gene. The gene consist of 11 exons spanning approximately 20 kB of genomic DNA. Fluorescence in situ hydridization localized the human beta-ureidopropionase gene to 22q11.2.     

Kinetic study on the reaction mechanism of pantothenase: existence of an acyl-enzyme intermediate and role of general acid catalysis.

A kinetic study was performed on the reaction mechanism of pantothenase (EC 3.5.1.22) catalyzed hydrolysis of the pantothenic acid. A nonlinear progress curve is derived if the reaction occurs at low buffer concentrations. The nonlinearity is due to partial reversibility of the reaction; an acylenzyme (pantoyl-enzyme) is formed during the reaction, and beta-alanine, the other end product, is able to react with the acyl-enzyme and return back to pantothenate. The dependence of the beta-alanine return reaction on buffer concentration and on pH suggests a general acid catalysis during the reaction. A reaction mechanism is suggested, in which the -NH3+ form of beta-alanine participates in the return reaction, and the deacylation of the acyl-enzyme is acid catalyzed.     

Beta-alanine transport in synaptic plasma membrane vesicles from rat brain. Efflux, exchange and stoichiometry

The efflux and exchange of beta-alanine were studied in synaptic plasma membrane vesicles from rat brain. The mechanism of beta-alanine translocation has been probed by comparing the ion dependence of net efflux to that of exchange. Dilution-induced efflux requires the simultaneous presence of internal sodium and chloride ions while influx is dependent on the presence of these two ions on the outside [Zafra, F., Aragón, M. C., Valdivieso, F. and Giménez, C. (1984) Neurochem Res. 9, 695-707]. These data show that the release of beta-alanine occurs via the carrier system and that it is cotransported with sodium and chloride ions. beta-Alanine efflux from the membrane vesicles is stimulated by external beta-alanine. This exchange does not require external sodium and chloride but it is dependent on the external concentration of beta-alanine. Half-maximal stimulation is obtained at a beta-alanine concentration similar to the Km for beta-alanine influx. Results of the direct measurements of the coupling of sodium and chloride to the transport of beta-alanine by using a kinetic approach allow us to propose a stoichiometry for the translocation cycle catalyzed by the beta-alanine transporter of three sodium ions and one chloride ion per beta-alanine zwitterion. To account for all the observed effects of external ions, beta-alanine concentrations and membrane potential on beta-alanine influx and efflux, a kinetic model of the Na+/Cl-/beta-alanine cotransport system is discussed.     

Crystal structure of the pantothenate synthetase from Mycobacterium tuberculosis, snapshots of the enzyme in action

Pantothenate synthetase (PS) from Mycobacterium tuberculosis represents a potential target for antituberculosis drugs. PS catalyzes the ATP-dependent condensation of pantoate and beta-alanine to form pantothenate. Previously, we determined the crystal structure of PS from M. tuberculosis and its complexes with AMPCPP, pantoate, and pantoyl adenylate. Here, we describe the crystal structure of this enzyme complexed with AMP and its last substrate, beta-alanine, and show that the phosphate group of AMP serves as an anchor for the binding of beta-alanine. This structure confirms that binding of beta-alanine in the active site cavity can occur only after formation of the pantoyl adenylate intermediate. A new crystal form was also obtained; it displays the flexible wall of the active site cavity in a conformation incapable of binding pantoate. Soaking of this crystal form with ATP and pantoate gives a fully occupied complex of PS with ATP. Crystal structures of these complexes with substrates, the reaction intermediate, and the reaction product AMP provide a step-by-step view of the PS-catalyzed reaction. A detailed reaction mechanism and its implications for inhibitor design are discussed.     

Isolation and Properties of a β-Alanine Transaminaseless Mutant of Pseudomonas fluorescens

beta-Alanine catabolism in Pseudomonas fluorescens is initiated by the enzyme beta-alanine transaminase. We have isolated mutants which fail to produce this enzyme and thus cannot grow on beta-alanine as the sole nitrogen source. The accumulation of beta-alanine-1-(14)C has been studied in one of these mutants, strain 67, and in the wild type. In the mutant, beta-alanine remains in a stable intracellular pool, whereas in the wild type conversion of beta-alanine to an intermediate, presumably malonate semialdehyde, and to CO(2) can be detected. The membrane transport system for beta-alanine can be conveniently studied in this transaminaseless mutant.     

Pyrimidine-degrading enzymes. Purification and properties of beta-ureidopropionase of Euglena gracilis.

In photoorganotrophically grown, mid-log phase cells of Euglena gracilis, enzymes of pyrimidine degradation including uracil reductase, dihydrouracil dehydrogenase, dihydropyrimidinase, and beta-ureidopropionase, were detected in a crude extract. beta-Ureidopropionase (N-carbamoyl-beta-alanine amidohydrolase, EC 3.5.1.6) was purified 100-fold by heat treatment, ammonium sulphate fractionation and chromatography using Sepharose 6B and DEAE-Sephadex A-25. The enzyme follows Michaelis-Menten kinetics (Km of beta-ureidopropionase for beta-ureidopropionate 3.8 . 10(-5) M, Hill coefficient n = 1). Other enzyme properties are: pH optimum 6.25, temperature optimum 60 degrees C, stimulation by Mg2+, inhibition by Cu2+, Mr approximately 1.5--2 . 10(6). beta-Ureidoisobutyrate, the intermediate of thymine degradation, and beta-ureidopropionate are competing substrates of beta-ureidopropionase (Ki = Km of beta-ureidopropionase for beta-ureidoisobutyrate 1.8 . 10(-5) M). Structural analogues of beta-ureidopropionate, isobutyrate and propionate are competitive inhibitors (Ki of beta-ureidopropionase 0.3 and 0.16 mM, respectively). There were no indications of regulatory function of beta-ureidopropionase in pyrimidine degradation.     

beta-Amino acid transport across the renal brush-border membrane is coupled to both Na and Cl

Sodium-dependent beta-alanine uptake into dog renal brush-border membrane vesicles was studied. Kinetic analysis indicated a single transport system, highly specific for beta-amino acids, with Km = 35 microM at 100 mM NaCl. Sodium-dependent beta-alanine transport was markedly anion-dependent, being highest in the presence of chloride (Cl greater than Br greater than SCN greater than NO3 approximately I greater than F) and virtually nonexistent in the presence of gluconate and other nonphysiological chloride substitutes. In addition, it was observed that beta-alanine uptake could be driven against a concentration gradient by a chloride gradient. Similar results were found for sodium. Taken together, these observations provide strong evidence that beta-alanine transport across the renal brush-border membrane is coupled to both sodium and chloride. Studies of the dependence of beta-alanine flux on chloride and sodium concentrations indicated that one chloride ion and multiple sodium ions were involved in the beta-alanine transport event. beta-Alanine flux on chloride found to involve the net transfer of positive charge, consistent with these stoichiometric assignments. The hallucinogen harmaline inhibited beta-alanine uptake in a 1:1 fashion, presumably by acting at a single site on the transport molecule. The ability of harmaline to inhibit beta-alanine uptake was decreased when the chloride concentration was lowered but was unchanged when the sodium concentration was decreased. These results indicate that harmaline does not compete with sodium for a binding site on the carrier as has been suggested for other sodium-coupled transport systems, and that instead, chloride may be required for harmaline binding to the beta-alanine transporter.     

Beta-alanine transaminase activity in black and suppressor of black mutations of Drosophila melanogaster.

An assay for beta-alanine transaminase activity in extracts of Drosophila melanogaster has been developed. By use of this assay, the levels of beta-alanine transaminase activity in several strains of flies has been examined as a function of developmental age. The black mutation shows elevated levels of activity compared to wild type, while suppressor of black strains show decreased levels compared to wild type.     

Genetic analysis of the first 4 patients with beta-ureidopropionase deficiency

beta-Ureidopropionase is the third enzyme of the pyrimidine degradation pathway and it catalyses the irreversible hydrolysis of N-carbamyl-ss-aminoisobutyric acid or N-carbamyl-ss-alanine to beta-aminoisobutyric acid or ss-alanine, ammonia, and CO2. Analysis of the beta-ureidopropionase gene (UPB1) of the first 4 patients presenting with a complete enzyme deficiency, revealed the presence of 2 splice-site mutations (IVS1-2A>G and IVS8-1G>A) and one missense mutation (A85E). RT-PCR analysis of the complete beta-ureidopropionase cDNA suggested that both splice-site mutations lead to a variety of alternative splice variants, with deletions of a single or several exons. The alanine at position 85 was not conserved in other eukaryotic beta-ureidopropionase protein sequences.     

Amidohydrolases of the reductive pyrimidine catabolic pathway purification, characterization, structure, reaction mechanisms and enzyme deficiency

In the reductive pyrimidine catabolic pathway uracil and thymine are converted to beta-alanine and beta-aminoisobutyrate. The amidohydrolases of this pathway are responsible for both the ring opening of dihydrouracil and dihydrothymine (dihydropyrimidine amidohydrolase) and the hydrolysis of N-carbamyl-beta-alanine and N-carbamyl-beta-aminoisobutyrate (beta-alanine synthase). The review summarizes what is known about the properties, kinetic parameters, three-dimensional structures and reaction mechanisms of these proteins. The two amidohydrolases of the reductive pyrimidine catabolic pathway have unrelated folds, with dihydropyrimidine amidohydrolase belonging to the amidohydrolase superfamily while the beta-alanine synthase from higher eukaryotes belongs to the nitrilase superfamily. beta-Alanine synthase from Saccharomyces kluyveri is an exception to the rule and belongs to the Acyl/M20 family.     

Beta-alanine transport in the isolated hepatocytes of the elasmobranch Raja erinacea

Cells were isolated from the liver of the skate and the uptake of beta-alanine followed using [14C]-beta-alanine. The isolated hepatocytes showed good viability, were found to accumulate beta-alanine from the incubation medium, and did so in a manner indicating a transport system involving a saturable carrier. The data for the rate of beta-alanine uptake suggest that this may be a rate-limiting step in the oxidation of the amino acid by the liver. Experiments indicated that the transport system could distinguish beta-alanine from certain structurally similar molecules (L-alanine and taurine, but not gamma-amino butyrate). Cells isolated from fish adapted to a diluted environment (50% seawater) showed no significant change in the uptake rate. However, evidence indicates that, over the range of beta-alanine concentrations occurring in the fish, the uptake rate would be acutely sensitive to small changes in the concentration in the blood, thus forming a self-regulating system for the metabolism of beta-alanine.     

Ebony,a novel nonribosomal peptide synthetase for beta-alanine conjugation with biogenic amines in Drosophila

Using Ebony protein either expressed in Escherichia coli or in Schneider S2 cells, we provide evidence for its substrate specificity and reaction mechanism. Ebony activates beta-alanine to aminoacyladenylate by an adenylation domain and covalently attaches it as a thioester to a thiolation domain in a nonribosomal peptide synthetase (NRPS) related mechanism. In a second reaction, biogenic amines act as external nucleophiles on beta-alanyl-S-pantetheine-Ebony, thereby releasing in a fast reaction the dipeptide (peptidoamine) in a process that is novel in higher eucaryotes. Therefore, we define Ebony as a beta-alanyl-biogenic amine synthetase. Insight into the reaction mechanism stems from mutational analysis of an invariant serine that disclosed Ebony as a multienzyme with functional analogy to the starting modules of NRPSs. In light of a putative biogenic amine-deactivating capacity, Ebony function in the nervous system must be reconsidered. We propose that in the Drosophila eye Ebony is involved in the transmission process by inactivation of histamine through beta-alanyl conjugation.     

Pantothenases from pseudomonads produce either pantoyl lactone or pantoic acid.

A pseudomonad was separated having a pantothenase that produced beta-alanine and pantoic acid. The pantothenase from an old strain, Pseudomonas fluorescens UK-1, was shown to produce beta-alanine and pantoyl lactone. The pantoic acid-producing pantothenase was characterized and compared with the pantothenase from the strain UK-1. The Mr was 240,000; it apparently consists of four subunits. The Km value for pantothenate is 3 mM. The enzymic activity is affected by an ionizable group of pK 8.4, the enzyme is active at higher pH, and V but not Km is affected by pH. This pantothenase is not inhibited by di-isopropyl phosphorofluoridate or phenylmethanesulphonyl fluoride, unlike the enzyme from the strain UK-1. Both pantothenases are inhibited by m-aminophenylboronic acid, oxalate, oxaloacetate and Cl- ions. The pantoic acid-producing pantothenase is inhibited also by SO4(2-) ions. The strong inhibitions by many compounds make this pantothenase unsuitable for the assay of pantothenic acid.     

Two beta-alanyl-CoA:ammonia lyases in Clostridium propionicum

The fermentation of beta-alanine by Clostridium propionicum proceeds via activation to the CoA-thiol ester, followed by deamination to acryloyl-CoA, which is also an intermediate in the fermentation of l-alanine. By shifting the organism from the carbon and energy source alpha-alanine to beta-alanine, the enzyme beta-alanyl-CoA:ammonia lyase is induced 300-fold (approximately 30% of the soluble protein). The low basal lyase activity is encoded by the acl1 gene, whereas the almost identical acl2 gene (six amino acid substitutions) is responsible for the high activity after growth on beta-alanine. The deduced beta-alanyl-CoA:ammonia lyase proteins are related to putative beta-aminobutyryl-CoA ammonia lyases involved in lysine fermentation and found in the genomes of several anaerobic bacteria. beta-Alanyl-CoA:ammonia lyase 2 was purified to homogeneity and characterized as a heteropentamer composed of 16 kDa subunits. The apparent K(m) value for acryloyl-CoA was measured as 23 +/- 4 microm, independent of the concentration of the second substrate ammonia; k(cat)/K(m) was calculated as 10(7) m(-1) x s(-1). The apparent K(m) for ammonia was much higher, 70 +/- 5 mm at 150 microm acryloyl-CoA with a much lower k(cat)/K(m) of 4 x 10(3) m(-1) x s(-1). In the reverse reaction, a K(m) of 210 +/- 30 microM was obtained for beta-alanyl-CoA. The elimination of ammonia was inhibited by 70% at 100 mm ammonium chloride. The content of beta-alanyl-CoA:ammonia lyase in beta-alanine grown cells is about 100 times higher than that required to sustain the growth rate of the organism. It is therefore suggested that the enzyme is needed to bind acryloyl-CoA, in order to keep the toxic free form at a very low level. A formula was derived for the calculation of isomerization equilibra between L-alanine/beta-alanine or D-lactate/3-hydroxypropionate.     

Reverse structure of carnosine-induced sedative and hypnotic effects in the chick under acute stress

In the central nervous system, beta-alanine is thought to act as an inhibitory neurotransmitter, but the role or precise mechanism of beta-alanine in the brain has not been clearly defined. beta-Alanine is found in high levels in the chicken brain as a component of the dipeptides carnosine (beta-alanyl-L-histidine) and anserine, or as a free amino acid. We focused on the position of beta-alanine, i.e., at the carboxyl terminus. In Experiment 1, the central effects of glycyl-beta-alanine, L-histidyl-beta-alanine and L-valyl-beta-alanine were compared with a saline control in chicks. L-Histidyl-beta-alanine significantly induced sedative and hypnotic effects. In Experiment 2, the effects of carnosine, its reverse (L-histidyl-beta-alanine), and their combination were investigated. Central carnosine-induced hyperactivity while reverse carnosine-induced hypoactivity, and the behaviors were intermediate following the combination of the two peptides. Finally, the central effect of reverse carnosine was compared with beta-alanine alone and L-seryl-beta-alanine in Experiment 3. Reverse carnosine showed similar effects to beta-alanine. In conclusion, L-histidyl-beta-alanine not only has the reverse structure of carnosine, but also reverse function. Thus, we propose to name reverse carnosine (L-histidyl-beta-alanine) rev-carnosine.     

Two new reactive targets of 2,5-hexanedione in vitro – beta-alanine and glycine

Summary. In this study, we found that two amino acids reacted with 2,5-hexanedione to form new reaction products in vitro, respectively. In the reaction of beta-alanine and 2,5-hexanedione, a reaction product was obtained and analyses of obtained results showed it was 3-(2,5-dimethyl-1H-pyrrol-1-yl)propanoic acid; in the reaction of glycine and 2,5-hexanedione, a reaction product was also obtained and analyses showed it was (2,5-dimethyl-1H-pyrrol-1-yl)acetic acid. Two reaction products were found to be oxidized easily; in addition, the latter was more easily to be oxidized than the former in the air. Our discoveries demonstrated that reactions between amino acids and 2,5-hexanedione could exist possibly in vitro. At present, it is clear that 2,5-hexanedione causes either axon atrophy or swelling, but the underlying molecular mechanism is still unclear. Since both beta-alanine and glycine are considered as neurotransmitter in the central nervous system, the reaction products remain to be identified in vivo.     

Neutral amino acid transport in Pseudomonas fluorescens

Membrane transport of beta-alanine, l-alanine, and l-proline was studied in a beta-alanine transaminaseless mutant (strain 67) of Pseudomonas fluorescens. In this mutant beta-alanine is metabolically inert, and it was therefore possible to demonstrate active transport of this substrate in the absence of intracellular catabolism. The permease which catalyzes the uptake of beta-alanine also transports l-proline and l-alanine. This common transport system was distinguished from permeases which transport only l-alanine and only l-proline by competition studies in strain 67 and by studies of transport specificity in a permeaseless mutant (strain 67/4MTR).     

Beta-alanine synthesis in Escherichia coli.

The enzyme, aspartate 1-decarboxylase (L-aspartate 1-carboxy-lyase; EC 4.1.1.15), that catalyzes the reaction aspartate leads to beta-alanine + CO2 was found in extracts of Escherichia coli. panD mutants of E. coli are defective in beta-alanine biosynthesis and lack aspartate 1-decarboxylase. Therefore, the enzyme functions in the biosynthesis of the beta-alanine moiety of pantothenate. The genetic lesion in these mutants is closely linked to the other pantothenate (pan) loci of E. coli K-12.