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

Takeshi Kanamori , Norihisa Kanou , Shingo Kusakabe , Haruyuki Atomi , Tadayuki Imanaka
DOI: http://dx.doi.org/10.1016/j.femsle.2005.02.023 61-65 First published online: 1 April 2005

Abstract

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.

Key words
  • Allophanate hydrolase
  • Urea amidolyase
  • Urea carboxylase
  • Urea metabolism
  • Nitrogen assimilation

1 Introduction

The biological conversion of urea to ammonia and carbon dioxide has been reported from various organisms. Two distinct enzymes, urease and urea amidolyase, are known to degrade urea (Fig. 1) [1,2]. Urease, which catalyzes the ATP-independent hydrolysis of urea, has been characterized in plants, fungi, and bacteria, and a wealth of information on its characteristics, physiological functions, and three-dimensional structures have been obtained [37]. On the other hand, some yeast strains and green algae that lack urease have been known to utilize urea as a nitrogen source through the function of urea amidolyase [8,9]. Urea amidolyase has been found to consist of two distinct enzymatic activities, urea carboxylase activity and allophanate hydrolase activity [10]. Urea carboxylase, a member of the biotin-dependent carboxylases, catalyzes the ATP-dependent carboxylation of urea to allophanate. Allophanate is subsequently hydrolyzed to ammonia and carbon dioxide by the function of allophanate hydrolase. Studies on urea amidolyase from yeast have revealed that the yeast allophanate hydrolase is fused with urea carboxylase on a single polypeptide [1113]. Urea amidolyase activity in green microalgae is the result of two separate enzymes, urea carboxylase and allophanate hydrolase [14]. Although allophanate hydrolase has been purified and enzymatically characterized from Chlamydomonas reinhardtii [15], urea carboxylase has not yet been purified from algae.

1

Reactions catalyzed by urease and urea amidolyase. The two reactions catalyzed by urea carboxylase and allophanate hydrolase in the urea amidolyase reaction are individually shown [1].

In Bacteria, the orthologues of the urea carboxylase domain or the allophanate hydrolase domain of yeast enzymes have been found to be widely distributed among several subdomains of Bacteria as separate genes [1]. Martinez et al. [16] identified the first bacterial allophanate hydrolase gene, atzF, from Pseudomonas sp. strain ADP as a metabolic gene for atrazine degradation. Although the protein product of the gene actually exhibited allophanate hydrolase activity, its involvement in urea degradation remains unclear.

The first prokaryotic urea carboxylase was previously identified and biochemically characterized from a novel α-Proteobacterium, Oleomonas sagaranensis [1]. O. sagaranensis urea carboxylase displayed carboxylase activities towards urea, acetamide, and formamide. Although comparison of the kcat/Km values indicated that urea was the most preferred substrate among them, this substrate specificity raised the possibility that O. sagaranensis urea carboxylase might participate in the assimilation of acetamide or formamide because these compounds served as a sole nitrogen source for O. sagaranensis. Furthermore, though O. sagaranensis urea carboxylase was a monofunctional enzyme without an allophanate hydrolase domain, a putative gene for allophanate hydrolase was found adjacent to the O. sagaranensis urea carboxylase gene, initiating 15 bp downstream. The deduced primary structure of the putative allophanate hydrolase exhibited 40% of identity to that of the allophanate hydrolase domain of Saccharomyces cerevisiae urea amidolyase (residues 1–622) and 50% identity to AtzF of Pseudomonas sp. strain ADP.

In this report, a detailed biochemical examination of bacterial allophanate hydrolase from O. sagaranensis was performed, clarifying the substrate specificity of the novel urea carboxylase/allophanate hydrolase system in bacterial nitrogen assimilation.

2 Materials and methods

2.1 Bacterial strains, plasmids, and DNA analyses

Oleomonas sagaranensis strain HD-1 was isolated from an oil field in Sagara, Shizuoka, Japan [17]. Escherichia coli strain DH5α and pUC118 were used for DNA manipulation, and E. coli strain BL21(DE3) and pET21a were used for gene expression. Methods for DNA manipulation, gene isolation and sequencing analyses have been described previously [1].

2.2 Gene expression and purification of recombinant proteins

Overproduction and purification of O. sagaranensis urea carboxylase have been described elsewhere [1]. For allophanate hydrolase, an expression vector was constructed according to the methods for urea carboxylase [1] using a pair of primers as follows: 5′-AAAAACATATGACGCTGCCCAAGATGTTGACCATCG-3′ and 5′-AAAAAGGATCCGATCCCCAGAGATTCACTTGGCAGCGCA-3′ (underlining indicates the NdeI and BamHI sites). The obtained vector, pET-ahy, was introduced into E. coli BL21(DE3) and the transformant was grown until the turbidity reached OD660= 0.4 in Luria–Bertani (LB) medium containing 100 μg ampicillin ml−1 at 37 °C. Expression of the allophanate hydrolase gene was induced by adding 0.1 mM isopropyl-β-D-thiogalactopyranoside, and the culture was incubated for another 24 h at 17 °C.

Unless otherwise mentioned, all purification steps were performed at 4 °C. The cells were harvested, washed, and suspended in 50 mM Tris–HCl buffer (pH 8.0) (buffer A). The cells were sonicated on ice and centrifuged (20,000g, 30 min), followed by ultracentrifugation (100,000g, 30 min) to remove cell debris. The supernatant was brought to 30–40% saturation with saturated (NH4)2SO4 solution on ice. The precipitated proteins were recovered by centrifugation (20,000g, 15 min) and resuspended in a minimal volume of buffer A, and dialyzed against 2 l of buffer A twice. The desalted protein solution was applied to a Resource Q column (Amersham Biosciences) equilibrated with buffer A. Proteins were eluted with an increasing linear gradient of 0–400 mM NaCl. Fractions with activity were concentrated 5-fold and applied to a gel filtration column (Superdex 200 HR 10/30; Amersham Biosciences) equilibrated with 100 mM HEPES buffer (pH 7.5) containing 150 mM NaCl. The apparent homogeneity of the purified protein was confirmed by sodium dodecylsulfate–polyacrylamide gel electrophoresis [18,19] and Coomassie Brilliant Blue staining [18]. The native molecular mass of allophanate hydrolase was determined by gel filtration chromatography with a Superdex 200 HR 10/30 column as described elsewhere [1].

2.3 Preparation of potassium allophanate

Potassium allophanate was synthesized by saponification of ethyl allophanate (TCI, Tokyo, Japan) with potassium hydroxide [16]. The actual amount of allophanate produced was determined as follows. Synthesized allophanate was added into reaction mixtures (1 ml) at calculated concentrations of 0 (control reaction), 1, 2, 3, or 4 mM, that included 100 mM HEPES (pH 7.5) and 3.0 U of purified O. sagaranensis allophanate hydrolase. The reaction mixtures were incubated at 37 °C until allophanate was completely converted (15 min), and the amounts of ammonia produced were determined using NH3 Kit (Wako, Osaka, Japan). The actual amount was estimated as 65.8% of the calculated value, and all concentrations of allophanate mentioned below represent the actual values.

The amount of contaminant urea in the preparation of synthesized allophanate was also quantified by adding urease (0.2 U) from sword bean (Urea N B Kit, Wako) into the reaction mixture described above. The amount of additional ammonia produced with the addition of urease was measured, and as a result, the amount of urea was determined to be 3.0% of the calculated concentration of allophanate. As addition of urea at a concentration of 2.0 or 4.0 mM into the reaction mixture along with 2.0 mM of allophanate did not lead to enhancement or inhibition of O. sagaranensis allophanate hydrolase activity (data not shown), this amount of urea is negligible under the conditions used in this study.

2.4 Ammonia releasing assay

The amide bond hydrolyzing activity of allophanate hydrolase towards urea, acetamide, formamide, acetoacetamide, allophanate, biuret, diacetamide, or malonamide, was determined spectrophotometrically in a coupled assay with glutamate dehydrogenase [20]. The reaction mixture was buffered with 100 mM HEPES (pH 7.5). When allophanate was used as a substrate, it was added into the reaction mixture at a concentration of 2.0 mM. Other substrates were examined at concentrations of 20 mM. The oxidation of NADH linked with the ammonia generation was determined at 340 nm at 25 °C. One unit of activity was defined as hydrolysis of 1 μmol of a substrate per min. Values in a control reaction without O. sagaranensis allophanate hydrolase were subtracted.

Ammonia-generating activity of the combination of O. sagaranensis urea carboxylase and allophanate hydrolase from urea, acetamide, formamide was determined using the spectrophotometric assay coupled with glutamate dehydrogenase [1]. Urea, acetamide, or formamide was added at a final concentration of 50, 250, or 100 mM, respectively. O. sagaranensis urea carboxylase and/or allophanate hydrolase were added at a final concentration of 5.5 and 131 mU ml−1, respectively. The oxidation of NADH was monitored as described above.

3 Results

3.1 Primary structure of O. sagaranensis allophanate hydrolase

The O. sagaranensis allophanate hydrolase gene was composed of 1800 bp, corresponding to a protein of 600 amino acid residues and a molecular weight of 61,999. Allophanate hydrolase has been reported as a member of amidase signature protein family [16]. Sequence comparison with several members of the family indicated that catalytic residues involved in the Ser–cisSer–Lys catalytic triad of amidase signature family [21], Ser177, Ser153, and Lys79, were conserved in O. sagaranensis allophanate hydrolase.

3.2 Gene expression and purification of recombinant O. sagaranensis allophanate hydrolase

In order to carry out a detailed biochemical examination of a bacterial allophanate hydrolase, overexpression of the O. sagaranensis allophanate hydrolase gene was performed. Through three purification steps, the recombinant O. sagaranensis allophanate hydrolase was purified 2.8-fold with a yield of 32.8%. The specific activity of the purified enzyme towards 2.0 mM of allophanate was 108U mg−1. The molecular mass of purified O. sagaranensis allophanate hydrolase was estimated as approximately 138 kDa, indicating that the recombinant protein was a dimer.

3.3 Substrate specificity and kinetic examination of O. sagaranensis allophanate hydrolase

The substrate specificity of O. sagaranensis allophanate hydrolase was performed using compounds described in Materials and methods as a substrate. No activity was detected with urea, acetamide, formamide, acetoacetamide, biuret, diacetamide, or malonamide. As allophanate was the only substrate to be catalyzed by the enzyme, the kinetic analysis of the allophanate hydrolase reaction with allophanate was carried out. The reaction followed Michaelis–Menten kinetics. The Vmax value of O. sagaranensis allophanate hydrolase for allophanate was calculated as 110 ± 1 U mg−1 protein and the Km value was 42.0 ± 1.9 μM.

3.4 Urea degrading activity of O. sagaranensis urea carboxylase/allophanate hydrolase system

As some yeast strains and green algae that lack urease have been known to hydrolyze urea to ammonia and carbon dioxide by the reaction catalyzed by urea amidolyase, the ammonia-generating activity of the O. sagaranensis urea carboxylase/allophanate hydrolase system from urea was examined. When O. sagaranensis urea carboxylase or allophanate hydrolase was mixed with urea individually, no activity was observed. However, addition of O. sagaranensis allophanate hydrolase along with urea carboxylase led to the production of a significant amount of ammonia (10.2 U mg−1 of urea carboxylase). The apparent ammonia-generating rate of O. sagaranensis urea carboxylase/allophanate hydrolase system was almost equivalent to that of the carboxylation of urea, indicating that there is no significant loss of the intermediate, allophanate, between the reactions of urea carboxylase and allophanate hydrolase.

The previously determined substrate specificity of O. sagaranensis urea carboxylase [1] suggested that acetamide and formamide might also be substrates of the O. sagaranensis urea carboxylase/allophanate hydrolase system. The predicted carboxylated products of acetamide and formamide, N-carboxyacetamide and N-carboxyformamide, have not been examined in the experiments described above. Therefore, the ATP-dependent ammonia-releasing activity of the system with 250 mM acetamide or 100 mM formamide was examined. However, no activities were observed with acetamide or formamide. Since acetamide and formamide scarcely affect the allophanate hydrolyzing activity of O. sagaranensis allophanate hydrolase at the concentrations examined, the lack of the activity for acetamide and formamide were not due to inhibition or inactivation of O. sagaranensis allophanate hydrolase by acetamide or formamide.

Acknowledgments

4 Discussion

Although the kinetic parameters of O. sagaranensis allophanate hydrolase were determined, only a few examples for comparison have been reported from other known allophanate hydrolase and urea amidolyase enzymes. The Km value of O. sagaranensis allophanate hydrolase towards allophanate was over 10 times lower than those of the purified allophanate hydrolase from C. reinhardtii [15] and the partially purified urea amidolyase from Pichia jadinii [22]. Although the Vmax value towards allophanate of a purified enzyme has not been obtained from any other organism, comparison of the purification tables suggested that the catalytic activity of O. sagaranensis allophanate hydrolase was much higher than those of other known enzymes.

Further examination for the substrate specificity of O. sagaranensis allophanate hydrolase revealed that O. sagaranensis allophanate hydrolase specifically hydrolyzed allophanate among substrates examined. Maitz et al. [15] described that the structures of substrates in which a keto and a carboxyl group were separated by one atom were important in the recognition of substrates by allophanate hydrolase. Indeed, all of the substrates examined, other than allophanate, did not contain this structure supposed to be recognized by allophanate hydrolase. Allophanate contains three C–N bonds, and the specific C–N bond that is hydrolyzed by allophanate hydrolase is still unclear. Contrary to our expectations, the experiments for the substrate specificity did not provide information on the cleavage site because of the strict substrate specificity of O. sagaranensis allophanate hydrolase. On the other hand, the comparison of the primary structure of O. sagaranensis allophanate hydrolase with other amidase enzymes indicated that O. sagaranensis allophanate hydrolase was a member of the amidase signature family. As all known members of this enzyme family catalyze the hydrolysis of the terminal amide bond [21,23], O. sagaranensis allophanate hydrolase can also be supposed to hydrolyze the terminal C–N bond, followed by the spontaneous hydrolysis of the product (N-carboxylcarbamate) in water.

Ammonia-generating activity of the urea carboxylase/allophanate hydrolase system towards urea suggested that these two enzymes might form a complex to directly transfer allophanate generated in the urea carboxylase reaction to allophanate hydrolase. If so, one would expect a difference in the elution profiles in gel filtration chromatography for either enzyme in the presence of the other. Complex formation can also be expected to affect the reaction kinetics of the individual enzymes. Further, mixing O. sagaranensis urea carboxylase, allophanate hydrolase and the avidin affinity resin that specifically binds biotinylated proteins might result in co-precipitation of both enzymes. We carried out all of the above experiments, but none of the results suggested complex formation (data not shown). This suggested that O. sagaranensis urea carboxylase does not interact with allophanate hydrolase, or the interaction between them was too weak or unstable to detect. This is similar to the case of the algal enzymes [14].

Our results on the substrate specificity of the urea carboxylase/allophanate hydrolase system of O. sagaranensis indicated that the system is specific for urea. On the other hand, the O. sagaranensis cells displayed not only urea amidolyase activity but also an ATP-independent ammonia-generating activity from urea that might correspond to the urease activity. The analyses on the relationship between the culture conditions and the levels of activities of urea carboxylase, allophanate hydrolase and putative urease in the O. sagaranensis cells may lead to new information on the roles of these enzymes in the urea decomposition in the prokaryotic cells.

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