Bacterial ammeline metabolism via guanine deaminase

Abstract

Melamine toxicity in mammals has been attributed to the blockage of kidney tubules by insoluble complexes of melamine with cyanuric acid or uric acid. Bacteria metabolize melamine via three consecutive deamination reactions to generate cyanuric acid. The second deamination reaction, in which ammeline is the substrate, is common to many bacteria, but the genes and enzymes responsible have not been previously identified. Here, we combined bioinformatics and experimental data to identify guanine deaminase as the enzyme responsible for this biotransformation. The ammeline degradation phenotype was demonstrated in wild-type Escherichia coli and Pseudomonas strains, including E. coli K12 and Pseudomonas putida KT2440. Bioinformatics analysis of these and other genomes led to the hypothesis that the ammeline deaminating enzyme was guanine deaminase. An E. coli guanine deaminase deletion mutant was deficient in ammeline deaminase activity, supporting the role of guanine deaminase in this reaction. Two guanine deaminases from disparate sources (Bradyrhizobium japonicum USDA 110 and Homo sapiens) that had available X-ray structures were purified to homogeneity and shown to catalyze ammeline deamination at rates sufficient to support bacterial growth on ammeline as a sole nitrogen source. In silico models of guanine deaminase active sites showed that ammeline could bind to guanine deaminase in a similar orientation to guanine, with a favorable docking score. Other members of the amidohydrolase superfamily that are not guanine deaminases were assayed in vitro, and none had substantial ammeline deaminase activity. The present study indicated that widespread guanine deaminases have a promiscuous activity allowing them to catalyze a key reaction in the bacterial transformation of melamine to cyanuric acid and potentially contribute to the toxicity of melamine.

FIG. 1.

The known metabolic pathway in bacteria for transforming melamine to cyanuric acid.

FIG. 2.

A clustering network diagram of a subset of the amidohydrolase superfamily, representing enzymes tested in this study for activity with melamine, ammeline, and ammelide. The node colors represent different functions, as indicated on the figure. The distance between nodes represents the closeness of sequence relatedness. Adda, adenosine deaminases; Cretdeim, creatinine deaminase; Cyda, cytosine deaminases; Daa, d-aminoacylases; Guda, guanine deaminases; Gudabra, guanine deaminase from B. japonicum USDA 110; GudaEcol, guanine deaminase from E. coli strain K-12 MG16655; GudaHu, guanine deaminase from humans.

FIG. 3.

Gene regions of the annotated guanine deaminase genes in P. putida KT2440 (gad) (A) and E. coli K-12 strain MG1655 (guaD) (B).

FIG. 4.

HPLC traces of E. coli wild type and a GuaD guanine deaminase mutant after incubation for 14 h with ammeline. The small amount of material eluting at 2.9 min with the guanine deaminase mutant is also present in a control incubation without cells.

FIG. 5.

Crystal structure and docking analysis of guanine deaminases. (A) Overlay of X-ray structure backbones of guanine deaminases from B. japonicum USDA 110 (PDB 2ood; yellow), human (PDB 2uz9; blue), and C. acetobutylicum (PDB 2i9u; purple). (B) Catalytically significant residues in the guanine deaminase active sites. The spheres represent the active-site zinc atoms. (C) Docking results of guanine (purple) and ammeline (cyan) tetrahedral intermediates within B. japonicum USDA 110. Residues that interact with both substrates include Arg206, Gln84, and Glu237.

FIG. 6.

Comparative tautomeric structures for ammeline and guanine and their reaction products, ammelide and xanthine, respectively. The ammeline (15) and guanine (10) tautomers shown are the predominant forms in aqueous solution at neutral pH.