Urea amidolyase of Candida utilis. Characterization of the urea cleavage reactions.

Evidence is presented that the enzymes catalyzing the three reactions involved in urea cleavage in Candida utilis, biotin carboxylation, urea carboxylation, and allophanate hydrolysis occur as a complex of enzymes. The allophanate-hydrolyzing activity could not be separated from the urea-cleaving activity using common methods of protein purification. Further, urea cleavage and allophanate hydrolysis activities are induced coordinately in cells grown on various nitrogen sources. The reactions involved in urea cleavage can be distinguished from one another on the basis of their sensitivities to (a) heat, (b) pH, and (c) chemical inhibitors. Evidence is presented for the product of the first reaction in urea cleavage, biotin carboxylation. Production of carboxylated enzyme is ATP dependent and avidin sensitive. Carboxylated enzyme is not observed in the presence of 1 mM urea.     

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.     

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.     

The urea amidolyase (DUR1,2) gene of Saccharomyces cerevisiae.

The DNA sequence of the urea amidolyase (DUR1,2) gene from S. cerevisiae has been determined. The polypeptide structure deduced from the DNA sequence contains 1,835 amino acid residues and possesses a calculated weight of 201,665 daltons which favorably correlates with that predicted from compositional analysis of purified protein (1,881 amino acid residues and a molecular weight of 203,900). The C-terminal 57 residues of the polypeptide exhibit significant homology with similarly situated sequences found in five other biotin carboxylases whose primary structures have been determined or deduced from protein and DNA sequence data, respectively. Major S1 nuclease protection fragments derived from DUR1,2 RNA-DNA hybrids exhibit apparent termini at positions -140 and -141 upstream of the coding region. The termini of minor protection fragments also occur at eleven other positions as well.     

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.     

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.     

Urease and urea amidolyase: Determination of activity in liverworts

Using highly sensitive techniques, we have investigated urea degradation in the liverworts and found that they have high urease but no detectable urea amidolyase activity.     

Cloning and nucleotide sequence of the urea amidolyase gene from Candida utilis

Urea amidolyase (EC 3.5.1.45) is an important multi-functional enzyme for the degradation of urea. The urea amidolyase gene from Candida utilis CA(u)-37 (DUR1,2^c) was cloned by plaque hybridization, and the nucleotide sequences of DUR1, 2^c and its flanking regions were determined. DUR1, 2^c was found to be composed of 5,490 base pairs and 1,830 amino acid residues. Using Edman degradation of the purified enzyme, it was revealed that the amino-terminal residue (methionine) was processed for maturation. A TATA-box like sequence was found 112 bases upstream from the translation start site (ATG). The site of the poly (A) tail was found 54 bases downstream from the translation stop site (TGA), since cDNA of DUR1, 2^c was synthesized from mRNA and sequenced. The nucleotide sequences of the urea amidolyase gene from Saccharomyces cerevisiae and DUR1, 2^c were very similar to each other (65.3%), as were the deduced amino acid sequences (67.2%). The molecular weight of DUR1, 2^c was calculated to be 200,700. This value corresponded to the result obtained from SDS-polyacrylamide gel electrophoresis of the purified enzyme. The enzyme functions in a dimeric form. Three important regions were found in the amino acid sequence of urea amidolyase through the homology search. It was predicted that each region was equivalent to the active site of allophanate hydrolase, that of urea carboxylase, and the biotin-binding site. This was verified by deletion analysis of the DUR1, 2^c gene in S. cerevisiae. The function of the upstream region of the C. utilis gene is also discussed.     

Single‐particle analysis of urea amidolyase reveals its molecular mechanism

Urea amidolyase (UA), a bifunctional enzyme that is widely distributed in bacteria, fungi, algae, and plants, plays a pivotal role in the recycling of nitrogen in the biosphere. Its substrate urea is ultimately converted to ammonium, via successive catalysis at the C‐terminal urea carboxylase (UC) domain and followed by the N‐terminal allophanate hydrolyse (AH) domain. Although our previous studies have shown that Kluyveromyces lactis UA (KlUA) functions efficiently as a homodimer, the architecture of the full‐length enzyme remains unresolved. Thus how the biotin carboxyl carrier protein (BCCP) domain is transferred within the UC domain remains unclear. Here we report the structures of full‐length KlUA in its homodimer form in three different functional states by negatively‐stained single‐particle electron microscopy. We report here that the ADP‐bound structure with or without urea shows two possible locations of BCCP with preferred asymmetry, and that when BCCP is attached to the carboxyl transferase domain of one monomer, it is attached to the biotin carboxylase domain in the second domain. Based on this observation, we propose a BCCP‐swinging model for biotin‐dependent carboxylation mechanism of this enzyme.     

The regulation of urea amidolyase of Saccharomyces cerevisiae: mating type influence on a constitutivity mutation acting in cis.

Summary Constitutivity for the synthesis of the urea amidolyase bienzymatic complex is obtained by dur0^hmutations located in the regulatory genetic region adjacent to the dur1, dur2 gene cluster. The dur0^hmutations act only in cis and are a new case of cis effect strongly cancelled in /a diploid, similar to cargA ⁺0^hmutation shown previously to lead to arginase constitutivity. Illegitimate diploids do not show such a cancellation of constitutivity.The constitutivity produced by dur0^hmutation comprises the process of induction and the release of the glutamine effect. It does not impair the NH ₄ ⁺ effect.     

Reductive cleavage of S-adenosylmethionine by biotin synthase from Escherichia coli

Biotin synthase (BioB) catalyzes the insertion of a sulfur atom between the C6 and C9 carbons of dethiobiotin. Reconstituted BioB from Escherichia coli contains a [4Fe-4S](2+/1+) cluster thought to be involved in the reduction and cleavage of S-adenosylmethionine (AdoMet), generating methionine and the reactive 5'-deoxyadenosyl radical responsible for dethiobiotin H-abstraction. Using EPR and M?ssbauer spectroscopy as well as methionine quantitation we demonstrate that the reduced S = 1/2 [4Fe-4S](1+) cluster is indeed capable of injecting one electron into AdoMet, generating one equivalent of both methionine and S = 0 [4Fe-4S](2+) cluster. Dethiobiotin is not required for the reaction. Using site-directed mutagenesis we show also that, among the eight cysteines of BioB, only three (Cys-53, Cys-57, Cys-60) are essential for AdoMet reductive cleavage, suggesting that these cysteines are involved in chelation of the [4Fe-4S](2+/1+) cluster.     

Urea transport in the toad bladder; Coupling of urea flows

Summary Urea transport across amphibian membranes is influenced by interactions with the membrane, the solvent and other solutes. One case of solute interaction, that in which the two species are chemically identical, is investigated here. Because of the effects of hypertonic urea on permeability, the demonstration of interaction required consideration of the ratior of bidirectional tracer permeabilities. Mucosal-to-serosal (MS) and serosal-to-mucosal (MS) tracer urea fluxes were determined in paired toad urinary bladders, in the absence and presence of abundant urea. In the control state,r was 1.0. Addition of 0.3m urea toM increasedr, and toS decreasedr. These results indicate coupling of abundant and tracer urea flows (isotope interaction), probably occurring in specialized regions. The effects persisted after the addition of antidiuretic hormone, despite the opposing influence of osmotic water flow. Quantitatively different effects of mucosal and serosal hypertonicity, both with and without antidiuretic hormone, are explicable in terms of heterogeneous parallel and series permeability barriers.     

Functional involvement of G8 in the hairpin ribozyme cleavage mechanism.

The catalytic determinants for the cleavage and ligation reactions mediated by the hairpin ribozyme are integral to the polyribonucleotide chain. We describe experiments that place G8, a critical guanosine, at the active site, and point to an essential role in catalysis. Cross-linking and modeling show that formation of a catalytic complex is accompanied by a conformational change in which N1 and O6 of G8 become closely apposed to the scissile phosphodiester. UV cross-linking, hydroxyl-radical footprinting and native gel electrophoresis indicate that G8 variants inhibit the reaction at a step following domain association, and that the tertiary structure of the inactive complex is not measurably altered. Rate-pH profiles and fluorescence spectroscopy show that protonation at the N1 position of G8 is required for catalysis, and that modification of O6 can inhibit the reaction. Kinetic solvent isotope analysis suggests that two protons are transferred during the rate-limiting step, consistent with rate-limiting cleavage chemistry involving concerted deprotonation of the attacking 2'-OH and protonation of the 5'-O leaving group. We propose mechanistic models that are consistent with these data, including some that invoke a novel keto-enol tautomerization.     

Biochemical characterization of the Arabidopsis biotin synthase reaction. The importance of mitochondria in biotin synthesis.

Biotin synthase, encoded by the bio2 gene in Arabidopsis, catalyzes the final step in the biotin biosynthetic pathway. The development of radiochemical and biological detection methods allowed the first detection and accurate quantification of a plant biotin synthase activity, using protein extracts from bacteria overexpressing the Arabidopsis Bio2 protein. Under optimized conditions, the turnover number of the reaction was >2 h(-1) with this in vitro system. Purified Bio2 protein was not efficient by itself in supporting biotin synthesis. However, heterologous interactions between the plant Bio2 protein and bacterial accessory proteins yielded a functional biotin synthase complex. Biotin synthase in this heterologous system obeyed Michaelis-Menten kinetics with respect to dethiobiotin (K(m) = 30 microM) and exhibited a kinetic cooperativity with respect to S-adenosyl-methionine (Hill coefficient = 1.9; K(0.5) = 39 microM), an obligatory cofactor of the reaction. In vitro inhibition of biotin synthase activity by acidomycin, a structural analog of biotin, showed that biotin synthase reaction was the specific target of this inhibitor of biotin synthesis. It is important that combination experiments using purified Bio2 protein and extracts from pea (Pisum sativum) leaf or potato (Solanum tuberosum) organelles showed that only mitochondrial fractions could elicit biotin formation in the plant-reconstituted system. Our data demonstrated that one or more unidentified factors from mitochondrial matrix (pea and potato) and from mitochondrial membranes (pea), in addition to the Bio2 protein, are obligatory for the conversion of dethiobiotin to biotin, highlighting the importance of mitochondria in plant biotin synthesis.