User:Jonathan Cline/Notebook/Melaminometer/Specific Metabolic Activity

Primary Metabolic Pathways

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Comparison of the reactions catalyzed by melamine deaminase (TriA) from Pseudomonas sp. strain NRRL B-12227 (A) and AtzA from Pseudomonas sp. strain ADP (B).

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Time course of melamine and ammeline deamination by cell extracts prepared from the TriA clone E. coli(pJS3). Error bars represent the standard error of the mean; n = 2.

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Pathway of melamine metabolism by Pseudomonas strain A and presumptive pathway of melamine metabolism by K. terragena.

Note: path is not to urea, it is to allophanate; see below.

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Growth of K. terragena with melamine as the sole N source.

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[MetaCyc Pathway: melamine degradation]

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Catabolism of the s-triazines ammelide and cyanuric acid in Pseudomonas sp. strain NRRLB-12228. Pseudomonas sp. strain NRRLB-12228 does not metabolize melamine but uses the same pathway to metabolize ammeline.

Research

• [Full Text] The degradative pathway of the s-triazine melamine. The steps to ring cleavage. Biochem J. 1982 Dec 15;208(3):679-84.
• [Full Text] Cloning and analysis of s-triazine catabolic genes from Pseudomonas sp. strain NRRLB-12227. J Bacteriol. 1991 Feb;173(3):1215-22.
• [Full Text] Cloning and comparison of the DNA encoding ammelide aminohydrolase and cyanuric acid amidohydrolase from three s-triazine-degrading bacterial strains. J Bacteriol. 1991 Feb;173(3):1363-6.

The enzymes are encoded by the genes trzB, trzC, trzD for metabolizing ammeline to biuret. Enzymes: B, ammeline aminohydrolase; C, ammelide aminohydrolase; D,and cyanuric acid amidohydrolase.

• [Full Text] Metabolism of Melamine by Klebsiella terragena. APPLIED AND ENVIRONMENTAL MICROBIOLOGY, July 1997, p. 2832¿2835 Vol. 63, No. 7
• [Full Text] Gene sequence and properties of an s-triazine ring-cleavage enzyme from Pseudomonas sp. strain NRRLB-12227. Appl Environ Microbiol. 1999 Aug;65(8):3512-7.
• [Full Text] Melamine deaminase and atrazine chlorohydrolase: 98 percent identical but functionally different. J Bacteriol. 2001 Apr;183(8):2405-10. PMID: 11274097

The gene encoding melamine deaminase (TriA) from Pseudomonas sp. strain NRRL B-12227 was identified, cloned into Escherichia coli, sequenced, and expressed for in vitro study of enzyme activity. Melamine deaminase displaced two of the three amino groups from melamine, producing ammeline and ammelide as sequential products. [...] Remarkably, melamine deaminase is 98% identical to the enzyme atrazine chlorohydrolase (AtzA) from Pseudomonas sp. strain ADP. [...] Melamine (2,4,6-triamino-1,3,5-triazine), a related s-triazine that predates the use of atrazine (29), is also metabolized by soil bacteria. [...] The triA gene was cloned into the EcoRI and HindIII sites of pUC18, and the resulting plasmid, pJS3, was transformed into Maximum Efficient E. coli DH5alpha (Gibco BRL, Gaithersburg, Md.). The melamine degradation phenotype of transformed cells was confirmed by incubating cell extracts with melamine, followed by analysis using high-pressure liquid chromatography (HPLC) [...] The triA sequence has been entered into GenBank under accession number AF312304. [...] Sequence analysis indicated a single open reading frame 1,425 nucleotides in length.

• [Full Text] Regulation of the Pseudomonas sp. strain ADP cyanuric acid degradation operon. J Bacteriol. 2005 Jan;187(1):155-67.

Expression of the cyanuric acid degradation operon atzDEF is specifically induced by the substrate of the pathway, cyanuric acid. The product of the orf99 gene, renamed here atzR, is required for cyanuric acid-mediated activation (Fig. 7). AtzR is homologous to a ubiquitous family of proteins designated LysR-type transcriptional regulators (LTTRs). Like most LTTRs (42), AtzR activates the expression of divergently transcribed genes (the atzDEF operon) in the presence of a small inducing molecule (cyanuric acid) and represses its own synthesis (Fig. 7).

Pseudomonas sp. strain ADP is not amenable to genetic studies. Conjugation and electrotransformation occur with low efficiency, making insertion mutagenesis impracticable, and plasmids are often unstable or severely retard growth under selective conditions (V. García-González and F. Govantes, unpublished observations). To avoid these inconveniences, we sought to reproduce the regulation of the atzDEF operon in a heterologous background. P. putida KT2440 was chosen for this purpose because it is a widely used model Pseudomonas strain for which a variety of genetic tools are available (2, 43) and its genome sequence was recently made available (37).

• [Full Text] Allophanate hydrolase, not urease, functions in bacterial cyanuric acid metabolism. Appl Environ Microbiol. 2005 Aug;71(8):4437-45.

Growth substrates containing an s-triazine ring are typically metabolized by bacteria to liberate 3 mol of ammonia via the intermediate cyanuric acid.

Previous studies concluded that the metabolism of s-triazine ring compounds, such as melamine and atrazine, proceeds through cyanuric acid, biuret, and urea, with the last metabolic step carried out via the enzyme urease (1, 3, 4, 10, 24, 36). The present study provides evidence that allophanate, not urea, is the last intermediate in this pathway and that allophanate hydrolase, not urease, is the enzyme involved.

• Degradation of cyanuric acid in soil by Pseudomonas sp. NRRL B-12227 using bioremediation with self-immobilization system. J Biosci Bioeng. 2006 Sep;102(3):206-9.