Allophanate hydrolase of Oleomonas sagaranensis involved in an ATP-dependent degradation pathway specific to urea

The first prokaryotic urea carboxylase has previously been purified and characterized from Oleomonas sagaranensis. As the results indicated the presence of an ATP-dependent urea degradation pathway in Bacteria, the characterization of the second component of this pathway, allophanate hydrolase, was carried out. The gene encoding allophanate hydrolase was found adjacent to the urea carboxylase gene. The purified, recombinant enzyme exhibited ammonia-generating activity towards allophanate, and, together with urea carboxylase, efficiently produced ammonia from urea in an ATP-dependent manner. The substrate specificity of the enzyme was strict, and analogs of allophanate were not hydrolyzed. Moreover, although the urea carboxylase exhibited carboxylase activity towards urea, acetamide, and formamide, ammonia-releasing activity of the two enzymes combined was detected only towards urea, indicating that the pathway was specific for urea degradation.     

The induction of allophanate lyase during the vegetative cell cycle in light-synchronized cultures of Chlamydomonas reinhardi.

Allophanate lyase can be induced by urea or acetamide 20-40-fold within 4 h in NH4 + -deprived cultures of Chlamydomonas reinhardi. In light-synchronized cultures, allophanate lyase induction appeared to be limited to the light phase of the cell cycle, provided that culture samples were induced under ongoing illumination conditions (i.e. light induction of light phase cells and dark induction of dark phase cells). However, when culture samples were induced under constant light conditions this cell cycle pattern was abolished. Light was found to be required for allophanate lyase induction and this was shown to be due, in part, to the light requirement for inducer uptake. The relationship between allophanate lyase induction and gametogenesis is discussed.     

The induction of allophanate lyase during the vegetative cell cycle in light-synchronized cultures of Chlamydomonas reinhardi

Allophanate lyase can be induced by urea or acetamied 20–40-fold within 4 h in NH₄⁺-deprived cultures of Chlamydomonas reinhardi. In light-synchronized cultures, allophanate lyase induction appeared to be limited to the light phase of the cell cycle, provided that culture samples were induced under ongoing illumination conditions (i.e. light induction of light phase cells and dark induction of dark phase cells). However, when culture samples were induced under constant light conditions this cell cycle pattern was abolished. Light was found to be required for allophanate lyase induction and this was shown to be due, in part, to light requirement for inducer uptake. The relationship between allophanate lyase induction and gametogenesis is discussed.     

Induction of the allantoin degradative enzymes by allophanic acid, the last intermediate of the pathway

$\text{Saccharomyces cerevisiae}$ can utilize allantoin as a sole nitrogen source by degrading it in five steps to ammonia, “CO₂”, and glyoxylate. We have previously shown that allophanic acid is the inducer of the urea carboxylase: allophanate hydrolase multienzyme complex. Since these enzymes catalyse the last two steps of allantoin degradation, experiments were performed to determine if allophanate was also the inducer of any other enzymes in the pathway. Our data demonstrate that allophanate induces synthesis of at least five of the seven purine degradative enzymes.     

Oxaluric Acid: a Non-Metabolizable Inducer of the Allantoin Degradative Enzymes in Saccharomyces cerevisiae

Saccharomyces cerevisiae degrades allantoin in five steps to ammonia, CO(2), and glyoxylate. Previously we demonstrated that allophanic acid, the last intermediate of the pathway, was required for induction of all five degradative enzymes. The data presented here indicate that oxaluric acid, an allophanate analogue, is capable of serving as a non-metabolizable inducer. Oxaluric acid brings about a high level of induction even in strains lacking urea carboxylase. Induction observed with parabanic acid was found to result from its spontaneous breakdown to oxaluric acid.     

Post-translational processing of urea amidolyase in Saccharomyces cerevisiae.

Urea amidolyase catalyzes the two reactions (urea carboxylase and a allophanate hydrolase) associated with urea degradation in Saccharomyces cerevisiae. Past work has shown that both reactions are catalyzed by a 204-kilodalton, multifunctional protein. In view of these observations, it was surprising to find that on induction at 22 degrees C, approximately 2 to 6 min elapsed between the appearance of allophanate hydrolase and urea carboxylase activities. In search of an explanation for this apparent paradox, we determined whether or not a detectable period of time elapsed between the appearance of allophanate hydrolase activity and activation of the urea carboxylase domain by the addition of biotin. We found that a significant portion of the protein produced immediately after the onset of induction lacked the prosthetic group. A steady-state level of biotin-free enzyme was reached 16 min after induction and persisted indefinitely thereafter. These data are consistent with the suggestion that sequential induction of allophanate hydrolase and urea carboxylase activities results from the time required to covalently bind biotin to the latter domain of the protein.     

Allophanate hydrolase, not urease, functions in bacterial cyanuric acid metabolism

Growth substrates containing an s-triazine ring are typically metabolized by bacteria to liberate 3 mol of ammonia via the intermediate cyanuric acid. Over a 25-year period, a number of original research papers and reviews have stated that cyanuric acid is metabolized in two steps to the 2-nitrogen intermediate urea. In the present study, allophanate, not urea, was shown to be the 2-nitrogen intermediate in cyanuric acid metabolism in all the bacteria examined. Six different experimental results supported this conclusion: (i) synthetic allophanate was shown to readily decarboxylate to form urea under acidic extraction and chromatography conditions used in previous studies; (ii) alkaline extraction methods were used to stabilize and detect allophanate in bacteria actively metabolizing cyanuric acid; (iii) the kinetic course of allophanate formation and disappearance was consistent with its being an intermediate in cyanuric acid metabolism, and no urea was observed in those experiments; (iv) protein extracts from cells grown on cyanuric acid contained allophanate hydrolase activity; (v) genes encoding the enzymes AtzE and AtzF, which produce and hydrolyze allophanate, respectively, were found in several cyanuric acid-metabolizing bacteria; and (vi) TrzF, an AtzF homolog found in Enterobacter cloacae strain 99, was cloned, expressed in Escherichia coli, and shown to have allophanate hydrolase activity. In addition, we have observed that there are a large number of genes homologous to atzF and trzF distributed in phylogenetically distinct bacteria. In total, the data indicate that s-triazine metabolism in a broad class of bacteria proceeds through allophanate via allophanate hydrolase, rather than through urea using urease.     

Structural Analysis of the dur Loci in S. CEREVISIAE: Two Domains of a Single Multifunctional Gene

In Saccharomyces cerevisiae, the degradation of urea to carbon dioxide and ammonia is catalyzed by urea carboxylase and allophanate hydrolase. The loci coding for these enzymes (dur1 and dur2) are very tightly linked on the right arm of chromosome II between pet11 and met8. Pleiotropic mutations that fail to complement mutations in either of the dur loci were found to be predominantly located in or near the dur2 locus. We interpret these data as suggesting that the two dur loci might in reality be domains of a single gene that codes for a multifunctional polypeptide. In view of this conclusion, we have renamed the dur loci as the dur1,2 locus.     

Metabolite compartmentation in Saccharomyces cerevisiae.

Uninduced cultures of Saccharomyces cerevisiae exhibit high basal levels of allantoinase, allantoicase, and ureidoglycolate hydrolase, the enzymes responsible for degrading allantoin to urea. As a result, these activities increase only 4- to 8-fold upon induction, whereas the urea-degrading enzymes, urea carboxylase and allophanate hydrolase, have very low basal levels and routinely increase 30-fold on induction. Differences in the inducibility of these five enzymes were somewhat surprising because they are all part of the same pathway and have the same inducer, allophanate. Our current studies reconcile these observations. S. cerevisiae normally contained up to 1 mM allantoin sequestered in a cellular organelle, most likely the vacuole. Separation of the large amounts of allantoin and the enzymes that degrade it provide the cell with an efficient nitrogen reserve. On starvation, sequestered allantoin likely becomes accessible to these degradative enzymes. Because they are already present at high levels, the fact that their inducer is considerably removed from the input allantoin is of little consequence. This suggests that at times metabolite compartmentation may play an equal role with enzyme induction in the regulation of allantoin metabolism. Metabolism of arginine, another sequestered metabolite, must be controlled both by induction of arginase and compartmentation because arginine serves both as a reserve nitrogen source and a precursor of protein synthesis. The latter function precludes the existence of high basal levels of arginase.     

Purification and Characterization of TrzF: Biuret Hydrolysis by Allophanate Hydrolase Supports Growth

TrzF, the allophanate hydrolase from Enterobacter cloacae strain 99, was cloned, overexpressed in the presence of a chaperone protein, and purified to homogeneity. Native TrzF had a subunit molecular weight of 65,401 and a subunit stoichiometry of α₂ and did not contain significant levels of metals. TrzF showed time-dependent inhibition by phenyl phosphorodiamidate and is a member of the amidase signature protein family. TrzF was highly active in the hydrolysis of allophanate but was not active with urea, despite having been previously considered a urea amidolyase. TrzF showed lower activity with malonamate, malonamide, and biuret. The allophanate hydrolase from Pseudomonas sp. strain ADP, AtzF, was also shown to hydrolyze biuret slowly. Since biuret and allophanate are consecutive metabolites in cyanuric acid metabolism, the low level of biuret hydrolase activity can have physiological significance. A recombinant Escherichia coli strain containing atzD, encoding cyanuric acid hydrolase that produces biuret, and atzF grew slowly on cyanuric acid as a source of nitrogen. The amount of growth produced was consistent with the liberation of 3 mol of ammonia from cyanuric acid. In vitro, TrzF was shown to hydrolyze biuret to liberate 3 mol of ammonia. The biuret hydrolyzing activity of TrzF might also be physiologically relevant in native strains. E. cloacae strain 99 grows on cyanuric acid with a significant accumulation of biuret.     

Purification and characterization of TrzF: biuret hydrolysis by allophanate hydrolase supports growth

TrzF, the allophanate hydrolase from Enterobacter cloacae strain 99, was cloned, overexpressed in the presence of a chaperone protein, and purified to homogeneity. Native TrzF had a subunit molecular weight of 65,401 and a subunit stoichiometry of alpha(2) and did not contain significant levels of metals. TrzF showed time-dependent inhibition by phenyl phosphorodiamidate and is a member of the amidase signature protein family. TrzF was highly active in the hydrolysis of allophanate but was not active with urea, despite having been previously considered a urea amidolyase. TrzF showed lower activity with malonamate, malonamide, and biuret. The allophanate hydrolase from Pseudomonas sp. strain ADP, AtzF, was also shown to hydrolyze biuret slowly. Since biuret and allophanate are consecutive metabolites in cyanuric acid metabolism, the low level of biuret hydrolase activity can have physiological significance. A recombinant Escherichia coli strain containing atzD, encoding cyanuric acid hydrolase that produces biuret, and atzF grew slowly on cyanuric acid as a source of nitrogen. The amount of growth produced was consistent with the liberation of 3 mol of ammonia from cyanuric acid. In vitro, TrzF was shown to hydrolyze biuret to liberate 3 mol of ammonia. The biuret hydrolyzing activity of TrzF might also be physiologically relevant in native strains. E. cloacae strain 99 grows on cyanuric acid with a significant accumulation of biuret.     

Ribulose bisphosphate carboxylase from a mutant strain of Chlamydomonas reinhardii deficient in chloroplast ribosomes

The ac-20 mutant strain of the unicellular green alga, Chlamydomonas reinhardii, lacks both chloroplast ribosomes and ribulose bisphosphate carboxylase activity when grown on organic medium. Under these conditions, the cells do not posses pools of either the large or small subunit of this enzyme. When transferred to inorganic medium, the carboxylase activity recovers. During this recovery, de novo synthesis of both subunits occurs. Synthesis of both subunits is inhibited by chloramphenicol even when possible free subunit pools rather than just the subunits incorporated into whole enzyme are examined.Abbreviations RubP ribulose bisphosphate - CAP D-threochloramphenicol - CHI cycloheximide - PPO 2,5-diphenyloxazole - POPOP 1,4-bis[2(5-phenyloxazolyl)]-benzene - SDS sodium dodecyl sulfate     

The urea carboxylase and allophanate hydrolase activities of urea amidolyase are functionally independent

Urea amidolyase (UAL) is a multifunctional biotin‐dependent enzyme that contributes to both bacterial and fungal pathogenicity by catalyzing the ATP‐dependent cleavage of urea into ammonia and CO₂. UAL is comprised of two enzymatic components: urea carboxylase (UC) and allophanate hydrolase (AH). These enzyme activities are encoded on separate but proximally related genes in prokaryotes while, in most fungi, they are encoded by a single gene that produces a fusion enzyme on a single polypeptide chain. It is unclear whether the UC and AH activities are connected through substrate channeling or other forms of direct communication. Here, we use multiple biochemical approaches to demonstrate that there is no substrate channeling or interdomain/intersubunit communication between UC and AH. Neither stable nor transient interactions can be detected between prokaryotic UC and AH and the catalytic efficiencies of UC and AH are independent of one another. Furthermore, an artificial fusion of UC and AH does not significantly alter the AH enzyme activity or catalytic efficiency. These results support the surprising functional independence of AH from UC in both the prokaryotic and fungal UAL enzymes and serve as an important reminder that the evolution of multifunctional enzymes through gene fusion events does not always correlate with enhanced catalytic function.     

Molecular evolution of urea amidolyase and urea carboxylase in fungi

Background  

Urea amidolyase breaks down urea into ammonia and carbon dioxide in a two-step process, while another enzyme, urease, does this in a one step-process. Urea amidolyase has been found only in some fungal species among eukaryotes. It contains two major domains: the amidase and urea carboxylase domains. A shorter form of urea amidolyase is known as urea carboxylase and has no amidase domain. Eukaryotic urea carboxylase has been found only in several fungal species and green algae. In order to elucidate the evolutionary origin of urea amidolyase and urea carboxylase, we studied the distribution of urea amidolyase, urea carboxylase, as well as other proteins including urease, across kingdoms.     

Role of ureogenesis in tackling problems of ammonia toxicity during exposure to higher ambient ammonia in the air-breathing walking catfish<Emphasis Type="Italic">clarias batrachus</Emphasis>

In the present study, the possible role of ureogenesis to avoid the accumulation of toxic ammonia to a lethal level under hyper-ammonia stress was tested in the air-breathing walking catfishClarias batrachus by exposing the fish at 25 mM NH₄Cl for 7 days. Excretion of ammonia by the NH₄Cl-exposed fish was totally suppressed, which was accompanied by significant accumulation of ammonia in different body tissues. The walking catfish, which is otherwise predominantly ammoniotelic, turned totally towards ureotelism from ammoniotelism with a 5-to 6-fold increase of urea-N excretion during exposure to higher ambient ammonia. Stimulation of ureogenesis was accompanied with significant increase of some of the key urea cycle enzymes such as carbamyl phosphate synthetase (urea cycle-related), argininosuccinate synthetase and argininosuccinate lyase both in hepatic and non-hepatic tissues. Due to this unique physiological strategy of turning towards ureotelism from ammoniotelism via the induced urea cycle, this air-breathing catfish is able to survive in very high ambient ammonia, which they face in certain seasons of the year in the natural habitat.     

不同氮源对蛋白核小球藻生长、色素和中性脂肪积累的影响

单细胞微藻在生长发育过程中,所积累的中性脂肪具有潜在的生物燃料价值。不同氮源对藻类的生长具有显著影响。研究了氨态氮、尿素氮和硝酸态氮对蛋白核小球藻生长、色素和中性脂肪积累的影响。结果显示,不同氮源对培养液pH有显著影响,以氨态氮为氮源导致培养液pH降低,而硝酸态氮导致培养液pH升高,培养液pH的波动可被添加的Hepes所稳定,并促进以氨态氮为氮源的蛋白核小球藻的生长。尿素氮对蛋白核小球藻生长、色素积累的效果优于氨态氮和硝酸态氮,硝酸态氮在中性脂肪积累上优于尿素氮和氨态氮,添加Hepes对氮饥饿后蛋白核小球藻的中性脂肪积累无显著影响。     

Defence responses of chilli fruits to Colletotrichum capsici and Alternaria alternata

The induction of defence compounds and enzymes involved in the phenylpropanoid pathway were studied in the ripe and green chilli fruits inoculated with Colletotrichum capsici and Alternaria alternata. Total phenols and the activity of phenylalanine ammonia lyase (PAL), peroxidase (PO), polyphenol oxidase (PPO) and catalase (CAT) increased in the inoculated ripe and green chilli fruits compared to the corresponding healthy fruits. Total phenols and the activities of the enzymes were at the maximum 2–3 d after inoculation and thereafter declined sharply in ripe chilli fruits, whereas slowly in green chilli fruits. In comparison with ripe chilli fruits, green chilli fruits were more resistant as they showed higher accumulation of total phenols and also higher activities of enzymes.     

The induction of the CO2-concentrating mechanism is correlated with the formation of the starch sheath around the pyrenoid of Chlamydomonas reinhardtii

The pyrenoid is a prominent proteinaceous structure found in the stroma of the chloroplast in unicellular eukaryotic algae, most multicellular algae, and some hornworts. The pyrenoid contains the enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase and is sometimes surrounded by a carbohydrate sheath. We have observed in the unicellular green alga Chlamydomonas reinhardtii Dangeard that the pyrenoid starch sheath is formed rapidly in response to a decrease in the CO₂ concentration in the environment. This formation of the starch sheath occurs coincidentally with the induction of the CO₂-concentrating mechanism. Pyrenoid starch-sheath formation is partly inhibited by the presence of acetate in the growth medium under light and low-CO₂ conditions. These growth conditions also partly inhibit the induction of the CO₂-concentrating mechanism. When cells are grown with acetate in the dark, the CO₂-concentrating mechanism is not induced and the pyrenoid starch sheath is not formed even though there is a large accumulation of starch in the chloroplast stroma. These observations indicate that pyrenoid starch-sheath formation correlates with induction of the CO₂-concentrating mechanism under low-CO₂ conditions. We suggest that this ultrastructural reorganization under lowCO₂ conditions plays a role in the CO₂-concentrating mechanism C. reinhardtii as well as in other eukaryotic algae.     

UV-A/B radiation rapidly activates photoprotective mechanisms in Chlamydomonas reinhardtii

Conversion of light energy into chemical energy through photosynthesis in the chloroplasts of photosynthetic organisms is essential for photoautotrophic growth, and non-photochemical quenching (NPQ) of excess light energy prevents the generation of reactive oxygen species and maintains efficient photosynthesis under high light. In the unicellular green alga Chlamydomonas reinhardtii, NPQ is activated as a photoprotective mechanism through wavelength-specific light signaling pathways mediated by the phototropin (blue light) and ultra-violet (UV) light photoreceptors, but the biological significance of photoprotection activation by light with different qualities remains poorly understood. Here, we demonstrate that NPQ-dependent photoprotection is activated more rapidly by UV than by visible light. We found that induction of gene expression and protein accumulation related to photoprotection was significantly faster and greater in magnitude under UV treatment compared with that under blue- or red-light treatment. Furthermore, the action spectrum of UV-dependent induction of photoprotective factors implied that C. reinhardtii senses relatively long-wavelength UV (including UV-A/B), whereas the model dicot plant Arabidopsis (Arabidopsis thaliana) preferentially senses relatively short-wavelength UV (mainly UV-B/C) for induction of photoprotective responses. Therefore, we hypothesize that C. reinhardtii developed a UV response distinct from that of land plants.

In contrast to land plants, which sense short-wave UV light, the unicellular green alga Chlamydomonas reinhardtii senses long-wavelength UV light for photoprotective responses.