Solving the Conundrum: Widespread Proteins Annotated for Urea Metabolism in Bacteria Are Carboxyguanidine Deiminases Mediating Nitrogen Assimilation from Guanidine.

Free guanidine is increasingly recognized as a relevant molecule in biological systems. Recently, it was reported that urea carboxylase acts preferentially on guanidine, and consequently, it was considered to participate directly in guanidine biodegradation. Urea carboxylase combines with allophanate hydrolase to comprise the activity of urea amidolyase, an enzyme predominantly found in bacteria and fungi that catalyzes the carboxylation and subsequent hydrolysis of urea to ammonia and carbon dioxide. Here, we demonstrate that urea carboxylase and allophanate hydrolase from Pseudomonas syringae are insufficient to catalyze the decomposition of guanidine. Rather, guanidine is decomposed to ammonia through the combined activities of urea carboxylase, allophanate hydrolase, and two additional proteins of the DUF1989 protein family, expansively annotated as urea carboxylase-associated family proteins. These proteins comprise the subunits of a heterodimeric carboxyguanidine deiminase (CgdAB), which hydrolyzes carboxyguanidine to N-carboxyurea (allophanate). The genes encoding CgdAB colocalize with genes encoding urea carboxylase and allophanate hydrolase. However, 25% of urea carboxylase genes, including all fungal urea amidolyases, do not colocalize with cgdAB. This subset of urea carboxylases correlates with a notable Asp to Asn mutation in the carboxyltransferase active site. Consistent with this observation, we demonstrate that fungal urea amidolyase retains a strong substrate preference for urea. The combined activities of urea carboxylase, carboxyguanidine deiminase and allophanate hydrolase represent a newly recognized pathway for the biodegradation of guanidine. These findings reinforce the relevance of guanidine as a biological metabolite and reveal a broadly distributed group of enzymes that act on guanidine in bacteria.

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.

Crystal structure analysis of c4763, a uropathogenic Escherichia coli-specific protein.

Urinary-tract infections (UTIs), which are some of the most common infectious diseases in humans, can cause sepsis and death without proper treatment. Therefore, it is necessary to understand their pathogenicity for proper diagnosis and therapeutics. Uropathogenic Escherichia coli, the major causative agents of UTIs, contain several genes that are absent in nonpathogenic strains and are therefore considered to be relevant to UTI pathogenicity. c4763 is one of the uropathogenic E. coli-specific proteins, but its function is unknown. To investigate the function of c4763 and its possible role in UTI pathogenicity, its crystal structure was determined at a resolution of 1.45 Šby a multiple-wavelength anomalous diffraction method. c4763 is a homodimer with 129 residues in one subunit that contains a GGCT-like domain with five α-helices and seven ß-strands. c4763 shows structural similarity to the C-terminal domain of allophanate hydrolase from Kluyveromyces lactis, which is involved in the degradation of urea. These results suggest that c4763 might be involved in the utilization of urea, which is necessary for bacterial survival in the urinary tract. Further biochemical and physiological investigation will elucidate its functional relevance in UTIs.

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.

Crystallization and preliminary X-ray diffraction analysis of the amidase domain of allophanate hydrolase from Pseudomonas sp. strain ADP.

The allophanate hydrolase from Pseudomonas sp. strain ADP was expressed and purified, and a tryptic digest fragment was subsequently identified, expressed and purified. This 50 kDa construct retained amidase activity and was crystallized. The crystals diffracted to 2.5 Šresolution and adopted space group P21, with unit-cell parameters a = 82.4, b = 179.2, c = 112.6 Å, ß = 106.6°.

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.

The structure of allophanate hydrolase from Granulibacter bethesdensis provides insights into substrate specificity in the amidase signature family.

Allophanate hydrolase (AH) catalyzes the hydrolysis of allophanate, an intermediate in atrazine degradation and urea catabolism pathways, to NH(3) and CO(2). AH belongs to the amidase signature family, which is characterized by a conserved block of 130 amino acids rich in Gly and Ser and a Ser-cis-Ser-Lys catalytic triad. In this study, the first structures of AH from Granulibacter bethesdensis were determined, with and without the substrate analogue malonate, to 2.2 and 2.8 Å, respectively. The structures confirm the identity of the catalytic triad residues and reveal an altered dimerization interface that is not conserved in the amidase signature family. The structures also provide insights into previously unrecognized substrate specificity determinants in AH. Two residues, Tyr(299) and Arg(307), are within hydrogen bonding distance of a carboxylate moiety of malonate. Both Tyr(299) and Arg(307) were mutated, and the resulting modified enzymes revealed >3 order of magnitude reductions in both catalytic efficiency and substrate stringency. It is proposed that Tyr(299) and Arg(307) serve to anchor and orient the substrate for attack by the catalytic nucleophile, Ser(172). The structure further suggests the presence of a unique C-terminal domain in AH. While this domain is conserved, it does not contribute to catalysis or to the structural integrity of the core domain, suggesting that it may play a role in mediating transient and specific interactions with the urea carboxylase component of urea amidolyase. Analysis of the AH active site architecture offers new insights into common determinants of catalysis and specificity among divergent members of the amidase signature family.

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.