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BIOCHEMICAL AND GENETIC CHARACTERIZATION OF AN EARLY STEP IN A NOVEL PATHWAY FOR THE BIOSYNTHESIS OF AROMATIC AMINO ACIDS AND P-AMINOBENZOIC ACID IN THE ARCHAEON METHANOCOCCUS MARIPALUDIS
Citation:
PORAT, I., M. SIEPRAWSKA-LUPA, Q. TENG, F. J. BOHANON, R. H. WHITE, AND W. B. WHITMAN. BIOCHEMICAL AND GENETIC CHARACTERIZATION OF AN EARLY STEP IN A NOVEL PATHWAY FOR THE BIOSYNTHESIS OF AROMATIC AMINO ACIDS AND P-AMINOBENZOIC ACID IN THE ARCHAEON METHANOCOCCUS MARIPALUDIS. Molecular Microbiology. Blackwell Publishing Limited, Oxford, Uk, 62(4):1117-1131, (2006).
Impact/Purpose:
This task is divided into four major research areas: (1) Development of computational tools and databases for screening-level modeling of the environmental fate of organic chemicals; (2) Metabolism of xenobiotics: Enhancing the development of a metabolic simulator; (3) Metabonomics: The use of advanced analytical tools to identify toxicity pathways; and (4) Software infrastructure to support development and application of transformation/metabolic simulators.
For many chemicals, multiple transformation/metabolic pathways can exist. Consequently, transformation/metabolic simulators must utilize transformation rate data for prioritization of competing pathways. The prioritization process thus requires the integration of reliable rate data. When this data is absent, it is necessary to generate a database with metabolic and transformation rate constants based on: (1) experimentally measured values, including those requiring the use of advanced analytical techniques for measuring metabolic rate constants in vivo and in vitro; (2) rate constants derived from SPARC and mechanistic-based QSAR models; and (3) data mined from the literature and Program Office CBI. A long-term goal of this project is to build this database. This information will be used to enhance the predictive capabilities of the transformation/metabolic simulators. As indicated previously, exposure genomics, which provide early signs of chemical exposure based on changes in gene expression, will be used to guide chemical fate and metabolism studies. The incorporation of exposure genomics into fate studies will provide information concerning (1) the minimal concentrations at which biological events occur; and (2) the identification of biologically relevant chemicals(s) in mixtures.
The capability of categorizing chemicals and their metabolites based on toxicity pathway is imperative to the success of the CompTox Research Program. Metabonomics, which is the multi-parametric measurement of metabolites in living systems due to physiological stimuli and/or genetic modification, provides such a capability. The application of metabonomics to toxicity testing involves the elucidation of changes in metabolic patterns associated with chemical toxicity based on the measurement of component profiles in biofluids, and enables the generation of spectral profiles for a wide range of endogenous metabolites. Metabolic profiles can provide a measure of the real outcome of potential changes as the result of xenobiotic exposure.
Description:
Methanococcus maripaludis is a strictly anaerobic, methane-producing archaeon and facultative autotroph capable of biosynthesizing all the amino acids and vitamins required for growth. In this work, the novel 6-deoxy-5-ketofructose-1-phosphate (DKFP) pathway for the biosynthesis of aromatic amino acids (AroAAs) and p-aminobenzoic acid (PABA) was demonstrated in M. maripaludis. Moreover, PABA was shown to be derived from an early intermediate in AroAA biosynthesis and not from chorismate. Following metabolic labeling with [U13C]-acetate, the expected enrichments for phenylalanine and arylamine derived from PABA were observed. DKFP pathway activity was reduced following growth with aryl acids, an alternative source of the AroAAs. Lastly, a deletion mutant of aroA', which encodes the first step in the DKFP pathway, required AroAAs and PABA for growth. Complementation of the mutants by an aroA' expression vector restored the wild-type phenotype. In contrast, a deletion of aroB', which encodes the second step in the DKFP pathway, did not require AroAAs or PABA for growth. Presumably, methanococci contain an alternative activity for this step. These results identify the initial reactions of a new pathway for the biosynthesis of PABA in methanococci.