Enzymatic characterization of a prokaryotic urea carboxylase

We identified the first prokaryotic urea carboxylase (UCA) from a member of the alpha subclass of the class Proteobacteria, Oleomonas sagaranensis. This enzyme (O. sagaranensis Uca) was composed of 1,171 amino acids, and its N-terminal region resembled the biotin carboxylase domains of various biotin-dependent carboxylases. The C-terminal region of the enzyme harbored the Met-Lys-Met motif found in biotin carboxyl carrier proteins. The primary structure of the enzyme was 45% identical to that of the urea carboxylase domain of urea amidolyase from Saccharomyces cerevisiae. O. sagaranensis Uca did not harbor the allophanate hydrolase domain found in the yeast enzyme, but a separate gene with structural similarity was found to be adjacent to the uca gene. Purified recombinant O. sagaranensis Uca displayed ATP-dependent carboxylase activity towards urea (V(max) = 21.2 micro mol mg(-1) min(-1)) but not towards acetyl coenzyme A (acetyl-CoA) and propionyl-CoA, indicating that the gene encoded a bona fide UCA and not an acetyl-CoA or propionyl-CoA carboxylase. The enzyme also exhibited high levels of activity towards acetamide and formamide. Kinetic parameters of the enzyme reaction were determined with ATP, urea, acetamide, and formamide. O. sagaranensis could grow on urea, acetamide, and formamide as sole nitrogen sources; moreover, ATP-dependent urea-degrading activity was found in cells grown with urea but not in cells grown with ammonia. The results suggest that the UCA of this organism may be involved in the assimilation of these compounds as nitrogen sources. Furthermore, orthologues of the O. sagaranensis uca gene were found to be widely distributed among BACTERIA: This implies that there are two systems of urea degradation in Bacteria, a pathway catalyzed by the previously described ureases and the UCA-allophanate hydrolase pathway identified in this study.     

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.     

Fluoroacetamide resistance mutations in Chlamydomonas reinhardtii

Acetamide, a nitrogen and carbon source for Chlamydomonas reinhardtii, is hydrolyzed by acetamidase to ammonium and acetate. It also induces urea pathway activities. Fluoroacetamide (F-acetamide) is toxic to wild-type through conversion to F-citrate, a respiratory inhibitor. Resistant mutants were selected on plates of F-acetamide plus urea. When tested on acetamide plates two mutant classes were obtained, acm⁺ (utilized acetamide as sole N source) and acm^-. All acm⁺ isolates had acetamidase activity and were obligate phototrophs (i.e. dark-diers). Acm^- isolates had either normal urea assimilation (ure⁺) or lacked all urea pathway activities, namely transport, urea carboxylase and allophanate hydrolase (ure^-). Inheritance patterns for both types indicated single nuclear gene mutations. The acm^- ure⁺ type presumably resulted from a defective acetamidase gene, and the acm^- ure^- strains might be regulatory gene mutants. Temperature conditional F-acetamide tolerant mutants were also obtained. Acetamidase extracted from one such strain was more thermolabile than the wild-type enzyme, indicating a mutation in the coding region. The hypothesis that acetamidase is involved in urea assimilation was not supported by the genetic and biochemical evidence.Abbreviations F-acetamide fluoroacetamide - F-acetate fluoroacetate - TAP tris-acetate-phosphate medium - CDB Chlamydomonas dilution buffer - TCA trichloroacetic acid - AH allophanate hydrolase - UC urea carboxylase - PAR photosynthetically active radiation - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea     

Purification and properties of the allophanate hydrolase from Chlamydomonas reinhardii

Allophanate hydrolase was purified to homogeneity from extracts of Chlamydomonas reinhardii grown phototrophically using urea as sole source of nitrogen. The following sequence of steps comprised the purification procedure: (1) protamine sulfate precipitation; (2) ammonium sulfate fractionation; (3) poly(ethylene glycol) fractionation; (4) batch-wise DEAE-cellulose adsorption; (5) Sepharose 6-B gel filtration; (6) hydroxyapatite chromatography. This procedure yielded an allophanate hydrolase preparation which was homogenous as judged by polyacrylamide gel electrophoresis. The molecular weight, as determined by gradient polyacrylamide electrophoresis and gel filtration, was 110 000 and 100 000, respectively. The pH optimum of this enzyme was approximately 9.0, while the K_m for allophanate was 0.55 mM. Allophanate hydrolase was sensitive to N-ethylmaleimide but was protected from this inhibition by allophanate. Malonic acid, oxaloacetic acid, and acetoacetic acid were inhibitory to allophanate hydrolysis.     

Structure and Function of Allophanate Hydrolase

Allophanate hydrolase converts allophanate to ammonium and carbon dioxide. It is conserved in many organisms and is essential for their utilization of urea as a nitrogen source. It also has important functions in a newly discovered eukaryotic pyrimidine nucleic acid precursor degradation pathway, the yeast-hypha transition that several pathogens utilize to escape the host defense, and an s-triazine herbicide degradation pathway recently emerged in many soil bacteria. We have determined the crystal structure of the Kluyveromyces lactis allophanate hydrolase. Together with structure-directed functional studies, we demonstrate that its N and C domains catalyze a two-step reaction and contribute to maintaining a dimeric form of the enzyme required for their optimal activities. Our studies also provide molecular insights into their catalytic mechanism. Interestingly, we found that the C domain probably catalyzes a novel form of decarboxylation reaction that might expand the knowledge of this common reaction in biological systems.     

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.     

Purification and properties of the urea amidolyase from Candida utilis.

Urea amidolyase was purified to homogeneity from extracts of Candida utilis. The purification involves protamine sulfate precipitation, ammonium sulfate precipitation, polyethylene glycol precipitation, Sepharose 6B gel filtration, DEAE-cellulose column chromatography, and hydroxylapatite column chromatography. The final preparation is pure as judged by disc-gel electrophoresis. The molecular weight of urea amidolyase, as determined by gel filtration and disc-gel electrophoresis, is between 500,000 and 520,000. Treatment with sodium dodecyl sulfate results in two peptides with molecular weights of 70,000 and 170,000. The urea carboxylase and allophanate hydrolase activities of urea amidolyase may be distinguished from one another on the basis of (a) the effect of the stabilizers, urea and glycerol, (b) the effect of storage pH on activity, and (c) selective inhibition by sulfhydryl reagents.     

Urea carboxylase and allophanate hydrolase are components of a multifunctional protein in yeast

Saccharomyces cerevisiae can use urea as sole nitrogen source by degrading it in two steps (urea carboxylase and allophanate hydrolase) to ammonia and carbon dioxide. We previously demonstrated that: 1) the enzymatic functions required for degradation are encoded in two tightly linked genetic loci and 2) pleiotropic mutations each resulting in the loss of both activities are found in both loci. These and other observations led to the hypothesis that urea degradation might be catalyzed by a multifunctional polypeptide. Waheed and Castric (1977) J. Biol. Chem. 252, 1628-1632), on the other hand, purified urea amidolyase from Candida utilis and reported it to be a tetramer composed of nonidentical 70- and 170-kilodalton subunits. To resolve the differing views of urea amidolyase structure, we purified the protein using rapid methods designed to avoid proteolytic cleavage. Application of these methods resulted in the isolation of a single, inducible and repressible, 204-kilodalton species. We observed no evidence for the existence of nonidentical subunits. A similar inducible, high molecular weight species was also detected in C. utilis. These biochemical results support our earlier hypothesis that urea degradation is carried out in yeast by an inducible and repressible protein composed of identical, multifunctional subunits.     

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.     

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.     

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.     

Metabolic control of urea catabolism in Chlamydomonas reinhardi and Chlorella pyrenoidosa.

In the unicellular green alga Chlamydomonas reinhardi (strain y-1), synthesis of the enzymes required for urea hydrolysis is under substrate induction control by urea and under end product repression control by ammonia. Hydrolysis of urea if effected by the sequential action of the discrete enzymes urea carboxylase and allophanate lyase, collectively called urea amidolyase. The carboxylase converts urea to allophanate in a reaction requiring biotin, adenosine 5'-triphosphate, and Mg2+. The lyase hydrolzyes allophanate to ammonium ions and bicarbonate. Neither activity is present in more than trace amounts when cultures are grown with ammonia or urea plus ammonia, or when they are starved for nitrogen for 8 h. Urea in the absence of ammonia induces both activities 10 to 100 times the basal levels. Addition of ammonia to an induced culture causes complete cessation of carboxylase accumulation and an 80% depression of lyase accumulation. Ammonia does not reduce urea uptake by repressed cells, so it does not prevent induction by the mechanism of inducer exclusion. The unicellular green alga Chlorella pyrenoidosa (strain 3 Emerson) also has discrete carboxylase and lyase enzymes, but only the carboxylase exhibits metabolic control.     

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.     

X-Ray Structure of the Amidase Domain of AtzF,the Allophanate Hydrolase from the Cyanuric Acid-Mineralizing Multienzyme Complex

The activity of the allophanate hydrolase from Pseudomonas sp. strain ADP, AtzF, provides the final hydrolytic step for the mineralization of s-triazines, such as atrazine and cyanuric acid. Indeed, the action of AtzF provides metabolic access to two of the three nitrogens in each triazine ring. The X-ray structure of the N-terminal amidase domain of AtzF reveals that it is highly homologous to allophanate hydrolases involved in a different catabolic process in other organisms (i.e., the mineralization of urea). The smaller C-terminal domain does not appear to have a physiologically relevant catalytic function, as reported for the allophanate hydrolase of Kluyveromyces lactis, when purified enzyme was tested in vitro. However, the C-terminal domain does have a function in coordinating the quaternary structure of AtzF. Interestingly, we also show that AtzF forms a large, ca. 660-kDa, multienzyme complex with AtzD and AtzE that is capable of mineralizing cyanuric acid. The function of this complex may be to channel substrates from one active site to the next, effectively protecting unstable metabolites, such as allophanate, from solvent-mediated decarboxylation to a dead-end metabolic product.     

Sequence of molecular events involved in induction of allophanate hydrolase.

Addition of urea to an uninduced culture of Saccharomyces at 22 C results in appearance of allophanate hydrolase activity after a lag of 12 min. We have previously demonstrated that both ribonucleic acid (RNA) and protein synthesis are needed for this induction to occur. To elucidate the time intervals occupied by known processes involved in induction, temperature-sensitive mutants defective in messenger RNA transport from nucleus to cytoplasm (rna1) and in protein synthesis initiation (prt1) were employed along with an RNA polymerase inhibitor in experiments that measure cumulative synthetic capacity to produce allophanate hydrolase. These measurements identify the time within the lag period at which each of the above processes is completed. We observed that RNA synthesis, rna1 gene product function, and protein synthesis initiation are completed at 1 to 1.5, 4, and 9 to 10 min, respectively.     

Glyceraldehyde-3-phosphate ferredoxin oxidoreductase (GAPOR) and nonphosphorylating glyceraldehyde-3-phosphate dehydrogenase (GAPN), key enzymes of the respective modified Embden-Meyerhof pathways in the hyperthermophilic crenarchaeota Pyrobaculum aerophilum and Aeropyrum pernix

The growth of Pyrobaculum aerophilum on yeast extract and nitrate was stimulated by the addition of maltose. Extracts of maltose/yeast extract/nitrate-grown cells contained all enzyme activities of a modified Embden-Meyerhof (EM) pathway, including ATP-dependent glucokinase, phosphoglucose isomerase, ATP-dependent 6-phosphofructokinase, fructose-1,6-phosphate aldolase, triose-phosphate isomerase, GAPOR, phosphoglycerate mutase, enolase and pyruvate kinase. The activity of GAPOR was stimulated about fourfold by maltose, indicating a role in sugar degradation. GAPOR was purified 200-fold to homogeneity and characterized as a 67 kDa monomeric, extremely thermostable protein. The enzyme showed high specificity for glyceraldehyde-3-phosphate and did not use glyceraldehyde, acetaldehyde or formaldehyde as substrates. By matrix-assisted laser desorption/ionization-time of flight analysis of the purified enzyme, ORF PA1029 was identified as a coding gene, gapor, in the sequenced genome of Pyrobaculum aerophilum. The data indicate that the (micro)aerophilic Pyrobaculum aerophilum contains a functional GAPOR as part of a modified EM pathway. Cells of the strictly aerobic crenarchaeon Aeropyrum pernix also contain enzyme activities of a modified EM pathway similar to that of Pyrobaculum aerophilum, except that a GAPN activity replaces GAPOR activity.     

Overexpression and characterization of a novel chitinase gene from a marine bacterium Pseudomonas sp. BK1

The chitinase A (ChiA)-coding gene of Pseudomonas sp. BK1, which was isolated from a marine red alga Porphyra dentata, was cloned and expressed in Escherichia coli. The structural gene consists of 1602 bp encoding a protein of 534 amino acids, with a predicted molecular weight of 55,370 Da. The deduced amino acid sequence of ChiA showed low identity (less than 32%) with other bacterial chitinases. The ChiA was composed of multiple domains, unlike the arrangement of domains in other bacterial chitinases. Recombinant ChiA overproduced as inclusion bodies was solubilized in the presence of 8 M urea, purified in a urea-denatured form and re-folded by removing urea. The purified enzyme showed maximum activity at pH 5.0 and 40 degrees C. It exhibited high activity towards glycol chitosan and glycol chitin, and lower activity towards colloidal chitin. The enzyme hydrolyzed the oligosaccharides from (GlcNAc)4 to (GlcNAc)6, but not GlcNAc to (GlcNAc)3. The results suggest that the ChiA is a novel enzyme, with different domain structure and action mode from bacterial family 18 chitinases.