Mechanism for Biotransformation of Nonylphenol Polyethoxylates to Xenoestrogens in Pseudomonas putida

ABSTRACT A strain of Pseudomonas putida isolated from activated sewage grew aerobically on the xenoestrogen precursor, nonylphenol polyethoxylate (NPEO x , where x is the number of ethoxylate units) as sole carbon source. Comparative growth yields on NPEOav6, NPEOav9, and NPEOav20 (mixtures with average ethoxylate numbers as indicated) were consistent with utilization of all but two ethoxylate units, and the final accumulating metabolite was identified by gas chromatography-mass spectroscopy as nonylphenol diethoxylate (NPEO2). There was no growth on nonylphenol or polyethylene glycols, and there was no evidence for production of carboxylic acid analogs of NPEO x . Biodegradation kinetics measured by high-pressure liquid chromatography (HPLC) for each component in NPEO x mixtures showed that biodegradation proceeded via successive exoscission of the ethoxylate chain and not by direct scission between the second and third ethoxylate residues. The NPEO x -degrading activity was inducible by substrate, and cell extracts of NPEOav9-induced cells were also active on the pure alcohol ethoxylate, dodecyl octaethoxylate (AEO8), producing sequentially, under either aerobic or anaerobic conditions, AEO7, AEO6, AEO5, etc., thus demonstrating that the pathway involved removal of single ethoxylate units. HPLC analysis of 2,4-dinitrophenylhydrazone derivatives revealed acetaldehyde (ethanal) as the sole aldehydic product from either NPEOav9 or AEO8 under either aerobic or anaerobic conditions. We propose a mechanism for biotransformation which involves an oxygen-independent hydroxyl shift from the terminal to the penultimate carbon of the terminal ethoxylate unit of NPEO x and dissociation of the resulting hemiacetal to release acetaldehyde and the next-lower homolog, NPEO x−1, which then undergoes further cycles of the same reaction until x = 2.

Special transport and neurological significance of two amino acids in a configuration conventionally designated as D.

We point out an ability of certain amino acids to be recognized at a biological receptor site as though their amino group bore, instead of an alpha relationship to a carboxylate group, a beta, gamma or delta relationship to the same or a second carboxylate group. For aspartate, the unbalanced position of its amino group between a pair of carboxylates allows its occasional biorecognition as a beta-rather than as an alpha-amino acid, whereas for proline and its homologs, their cyclic arrangement may allow the imino group, without its being replicated, to be sensed analogously as falling at either of two distances from the single carboxylate group. The greater separation might allow proline to be seen as biologically analogous to gamma-aminobutyric acid. This more remote positioning of the imino group would allow the D-form of both amino acids to present its amino group in the orientation characteristic of the natural L-form. The dual modes of recognition should accordingly be signalled by what appears to be low stereospecificity, actually due to a distinction in the enantiorecognition of the two isomers. Competing recognition for transport between their respective D- and L-forms, although it does not prove that phenomenon, has been shown for proline and, significantly, even more strongly for its lower homolog, 2-azetidine carboxylate. Such indications have so far revealed themselves rather inconspicuously for the central nervous system binding of proline, reviewed here as a possible feature of a role suspected for proline in neurotransmission.

A New Technique for Determining Chemical Structure by Gas Chromatography

A new technique that helps determine the chemical structure of organic compounds in microgram amounts utilizes a hot catalyst-containing tube that attaches to the injection port of a flameionization gas chromatograph. Hydrogen, the carrier gas, conducts the sample over the heated catalyst, and in this process saturates multiple bonds and removes halogen, oxygen, sulfur, and nitrogen atoms. The hydrocarbon products, which exit into the chromatographic column and separate in it, are the parent hydrocarbon and/or the next lower homolog. These data help determine the carbon skeleton of a wide variety of compounds, which may contain as many as 24 connected carbon atoms. The position of functional groups (e.g., ketones, alcohols, amines) may frequently be determined from the fragmentation pattern. A "neutral" catalyst composed of palladium on diatomaceous earth is the best catalyst found to date. Temperature programming of the analytical column has certain advantages in this procedure. Experimental data and discussion of different catalysts, flow rate, ring structures, operating parameters, comparison of this technique with pyrolysis and mass spectrometry, and limitations are included. Two apparatus designs permit collection of sufficient hydrocarbon product for characterization by another procedure. The technique is especially useful for identification of compounds available in only minute amounts, such as gas chromatographic fractions and degradation fragments of natural products.