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.

Biodegradation of melamine and its hydroxy derivatives by a bacterial consortium containing a novel Nocardioides species

“…The co-culture also appeared to accelerate ammeline degradation. Seffernick et al (2010) demonstrated that guanine deaminase in B. japonicum USDA 110 also showed considerable activity to catalyze ammeline deamination compared with AtzB and related s-triazine hydrolases which show almost no activity. The enzyme responsible for the ammeline deamination in strain CDB21 is currently unknown.…”

Chiral biomarkers and microfossils in carbonaceous meteorites

“…At the time it was thought that these compounds had no possible relationship to biology. However, in 2010 Seffernick et al 35 described the metabolic pathways that use the guanine deaminase enzyme to allow ammeline to act as an intermediate in the bacterial metabolism of melamine.) Hyatsu et al 36 presented a detailed review of nitrogen compounds in carbonaceous meteorites.…”

Plasmid Localization and Organization of Melamine Degradation Genes in Rhodococcus sp. Strain Mel

“…The trzA expression strain was supplemented with 0.2 mM ZnSO 4 , and chaperones GroEL and GroES were induced from pAG with 0.5% L-arabinose for 1 h prior to induction of trzA with 1.0 mM isopropyl-␤-Dthiogalactopyranoside (IPTG) for 19 h. The synthetic putative CDase was induced with 0.1 mM IPTG for 20 h at 16°C. Cells were harvested and broken, and proteins were purified as previously described (28). High-purity fractions (determined by SDS-PAGE) were pooled and dialyzed.…”

“…High-purity fractions (determined by SDS-PAGE) were pooled and dialyzed. Activity was measured as ammonia release from 1.0 mM melamine or 0.3 mM ammeline or ammelide by 1.0 to 10.0 g protein using the Berthelot method (22,28).…”

“…While some of the melamine pathway genes were characterized in molecular genetic studies by Eaton and Karns (10,11), not all genes in the pathway were identified. For example, an ammeline (4,6-diamino-2-hydroxy-1,3,5-triazine)-deaminating enzyme was not identified until recently and was shown in some organisms to be a guanine deaminase that "moonlights" as an ammeline deaminase (28). Similarly, the last step was initially thought to be catalyzed by urease, but more recently allophanate hydrolase was found to be the terminal enzyme releasing the last two equivalents of ammonia from allophanate (carboxyurea) (4) (Fig.…”