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

Abstract

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.

FIG. 1.

Proposed metabolic pathways for the degradation of cynauric acid in bacteria. The lower pathway is that found in Pseudomonas sp. strain ADP and operates via enzymes encoded by the atzDEF operon.

FIG. 2.

Instability of synthetic allophanate under differing pH and buffer conditions. Buffers used were 10 (▿), 50 (○), or 100 (▾) mM sodium phosphate buffer, pH 8.0, or 100 mM sodium phosphate buffer, pH 7.3 (•). Allophanate solutions were sampled at the indicated time points and analyzed by HPLC using conditions under which the remaining allophanate is stable.

FIG. 3.

Incubation of [UL-¹⁴C]cyanuric acid with cell extracts from Pseudomonas sp. strain ADP that had been grown on cyanuric acid. The reaction mixtures were sampled at 0, 1, 2, 3.5, and 7.5 h and analyzed by HPLC for cyanuric acid and allophanate. Solid bars, cyanuric acid; hatched bars, allophanate.

FIG. 4.

Kinetic course of [UL-¹⁴C]cyanuric acid metabolism by Pseudomonas huttiensis NRRLB-12228 and radiometric detection of metabolites separated by HPLC. (A) HPLC radiochromatogram at one time point, with each bar showing the amount of radioactivity collected in that fraction. Positions of elution for standards of urea, biuret, bicarbonate, allophanate, and cyanuric acid are shown by the arrows. (B) Concentrations of cyanuric acid, biuret, allophanate, and bicarbonate shown as a function of time as determined by multiple HPLC analyses as illustrated in panel A.

FIG. 5.

Urease and allophanate hydrolase activity levels in crude protein extracts from (A) Pseudomonas huttiensis NRRLB-12228 and (B) Enterobacter cloacae strain 99, each measured after growth on ammonia, urea, or cyanuric acid as the sole nitrogen source. Black bars, urease activity; gray bars, allophanate hydrolase activity.

FIG. 6.

Dendrogram of protein sequence relatedness comparing allophanate hydrolase homologs in all bacteria indicated. Boxed entries are found in bacteria that contain a urea carboxylase or s-triazine biodegradation genes, consistent with an assignment as an allophanate hydrolase. Those strains highlighted in gray have been shown in this study to specifically function as allophanate hydrolases involved in cyanuric acid metabolism. The protein sequences used for the dendrogram are as follows: Pseudomonas sp. strain ADP AtzF (NP_862539), R. pickettii strain D (this study), A. radiobacter J14a (this study), Enterobacter cloacae (AAK11683), Oleomonas sagaranensis (BAD16655), Novosphingobium aromaticivorans (ZP_00304984), Caulobacter crescentus (NP_420637), Gloeobacter violaceus (NP_923907), Magnetospirillum magnetotacticum (ZP_00208237), Rhodopseudomonas palustris (CAE26847), Bradyrhizobium japonicum (NP_772446), Mesorhizobium loti (NP_107459.1), Rhizobium sp. strain NGR234 (AAQ87317.1), Wolinella succinogenes (NP_907309), Microbulbifer degradans (ZP_00314767), Pseudomonas fluorescens (ZP_00266691), Burkholderia cepacia (ZP_00211646), Dechloromonas aromatica (ZP_00152874), Ralstonia eutropha (ZP_00202612), Rubrivivax gelatinosus (ZP_00241447), Xanthomonas axonopodis (NP_644621), Erwinia carotovora (YP_050236), Burkholderia fungorum (ZP_00282169), Pseudomonas syringae (NP_791188), Saccharomyces cerevisiae (AAC41643), Streptomyces avermitilis (NP_827873), and Kineococcus radiotolerans (ZP_00198103.1).

FIG. 7.

Theoretical pathways of amide bond hydrolysis from biuret. Only the upper pathway is experimentally verified. Urea derived from cyanuric acid metabolism arises from allophanate decarboxylation (downward arrow).