Pesticide Biotransformation in Plants and Microorganisms - American

Chapter 8

Biodegradation of Pesticides Containing Carbon-to-Phosphorus Bond 1

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Downloaded by NORTH CAROLINA STATE UNIV on September 17, 2012 | http://pubs.acs.org Publication Date: December 1, 2000 | doi: 10.1021/bk-2001-0777.ch008

Pawel Kafarski , Barbara Lejczak , and Giuseppe Forlani 1

Institute of Organic Chemistry, Biochemistry and Biotechnology, Wroclaw University of Technology, Wybrzeze Wyspianskiego 27, 50-370 Wroclaw, Poland Department of Genetics and Microbiology, University of Pavia, via Ferrata 1, 27100 Pavia, Italy

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Phosphonates constitute a class of organic compounds containing a direct covalent carbon-to-phosphorus bond. They are in widespread use these days, mainly as insecticides, herbicides, antibiotics, lubricants or flame retardants. The fate of phosphonates in the environment attracts considerable attention especially if resistance of the stable C-P bond to chemical, photolytic and thermal cleavage is considered. Organophosphonates are generally considered to be non-persistent because a number of microorganisms provide suitable pathways for the biodegradation of these compounds. Thus, the ability to catabolize phosphonates is widesperad among bacteria and fungi. Metabolic pathways which have been characterized so far are reviewed in this presentation.

Organophosphonates are a group of both synthetic and biogenic compounds characterized by the presence of covalent carbon-to-phosphorus bond. The conversion of phosphonates to phosphate products by living systems, apart from being essential for the return of carbon-bound phosphorus to the phosphate metabolic pool, is of considerable practical importance since phosphonates have recently found extensive application. Compounds containing C-P bond occur in an increasing number of industrial, agricultural, medical and housecleaning products. As a consequence, thousands of tones of these xenobiotics are introduced annually into the environment (1). Intensive use of organophosphonate herbicides (glyphosate and glufosinate) and insecticides has raised an increasing concern due to their possible pollution of the environment and stimulated intensive studies on their biodegradation. Although the C-P bond is resistant to chemical degradation (hydrolytic, thermal or photochemical) (2) organophosphonates are generally considered to be non-persistent © 2001 American Chemical Society In Pesticide Biotransformation in Plants and Microorganisms; Hall, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2000.

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146 because a number of microorganisms possess pathways suitable for conversion to non toxic metabolites or complete mineralization of these compounds. Thus, the ability to catabolize phosphonates is widespread among bacteria, and many soilborne strains of Escherichia, Salmonella, Shigella, Klebsiella, Enterobacter, Serratia, Pseudomonas, Rhizobium, Agrobacterium, Bacillus, Arthrobacter and Kluyvera are able to grow on phosphonates as the sole source of phosphorus (for review see Refs. 3, 4 and 5). The history of studies on the biodegradation of phosphonates began with the reports of Zeleznick (6) and Mastalerz (7) on microbial strains capable of growth on various simple phosphonic acids as the sole source of phosphorus. The following thirty-five years of studies resulted in isolation of several hundred bacterial strains capable to split carbon-to-phosphorus bond. On the contrary, surprisingly little is known about the metabolism of these xenobiotics by fungi (8-12) although these organisms are supposedly responsible for the biodegradation of organophosphonates in soil. Even though many synthetic organophosphonates may be readily degraded in the environment by biotic transformations, our knowledge of their environmental fate remains limited (5,13).

Catabolism of 2-Aminoethanephosphonic Acid (Ciliatine) 2-Aminoethanephosphonic acid (I, A E P ) was isolated in 1959 from ciliated sheep rumen protozoa, and thus named ciliatine (14). Independently, it was isolated from lower marine animals (15). Ciliatine (I) is the simplest natural phosphonate and is also the most ubiquitous considering the high levels found in some organisms. Occurrence of A E P is well documented in Monera, Protista and animal kingdoms whereas its presence in plants and fungi has not been confirmed (16). Therefore, the fact that A E P acid may serve as the sole source of phosphorus for most of the examined microorganisms is not surprising and may be taken as an indication that it is degraded more readily than other phosphonates. The study of A E P utilization by soil microflora has provided useful insight to understand the molecular basis of the catabolism of C-P bond-containing xenobiotics. Ciliatine catabolism by many bacteria is a two-step pathway by which this amino phosphonate is ultimately converted to acetaldehyde and orthophosphate (17-19). The first step involves a transamination reaction in which the amino group of ciliatine is donated to pyruvate and phosphonoacetaldehyde (II) is produced. Phosphonoacetaldehyde is then cleaved by phosphonatase (phosphonoacetaldehyde hydrolase), which exhibits strong substrate specificity towards compound II. These two enzymes were isolated from various bacterial sources, mechanisms of their action were thoroughly studied, and the corresponding genes were characterized (20-28). Phosphonatase [EC 3.11.1.1] is perhaps the only well-characterized enzyme responsible for carbon-to-phosphorus bond cleavage (24-26). Formation of a protonated Schiff base, at a lysine in the active site of the enzyme and the carbonyl group of the substrate, facilitates cleavage of the C-P bond resulting in liberation of acetaldehyde and phosphite.

In Pesticide Biotransformation in Plants and Microorganisms; Hall, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2000.

147 POaHj


pyruvate

alanine

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p

.

Bacteria capable of metabolizing ciliatine may be divided into two distinct groups. In the first one, represented by Enterobacter aerogenes (5), Salmonella typhimurium (29) and Pseudomonas sp. (30), A E P utilization is regulated by inorganic phosphate, and occurs only when it is the sole source of phosphorus. The second group of bacteria, Pseudomonas putida (31), Pseudomonas fluorescens (30,33), Bacillus cereus (18) and a recently isolated strain of Streptomyces sp. (32), are not regulated by phosphate concentration and utilize ciliatine as a source of nitrogen, phosphorus and carbon, usually with excretion of inorganic phosphate into the culture media. 2-Oxoalkanephosphonates (III), which are compounds structurally related to phosphonoacetaldehyde, are also readily degraded by bacteria and fungi (8,9,33). However, they do not act as substrates or as inhibitors of phosphonatase (Lacoste A . M , University of Bordeaux, France, private communication). The mechanism of their degradation awaits elucidation.

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o II

III

Bacterial strains which degrade A E P to orthophosphate and ethylamine by C-P lyase(s) have also been identified (34,35). These enzymes catalyze direct cleavage of the carbon-to-phosphorus bond in a wide variety of structurally diverse organophosphonates by a free-radical mechanism that will be discussed later.

H

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I The degradation of A E P by Protista has been studied less extensively. Quite surprisingly, no release of inorganic phosphate from ciliatine, amino phosphonate endogenous to Tetrahymena thermophila, was ever directly observed in vivo in this thoroughly studied organism (36,37). Snail eggs contain virtually all stored phosphorus in the form of phosphonates, and during embryonic development the C-P bond is rapidly converted to inorganic phosphate (38-40). The mechanisms of this degradation, intermediates, and enzymes involved in this process are unknown.

In Pesticide Biotransformation in Plants and Microorganisms; Hall, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2000.

148


Catabolism of Other Naturally Occurring Organophosphonates The catabolism of other biogenic organophosphonates was studied only incidentally. 2-Amino-3-phosphonopropionic acid (phosphonoalanine, IV) seems to accompany ciliatine in some organisms, but its biological role is completely unknown. The decarboxylation of this amino acid may serve as an alternative pathway for ciliatine synthesis (41). Phosphonoalanine is also catabolized through a two-step pathway with initial conversion to phosphonopyruvate (V) with the release of inorganic phosphate. Most likely the cleavage of phosphonopyruvate is catalysed by phosphoenolpyruvate phosphomutase (P-C bond forming enzyme) or a related enzyme (42-45). Since phosphonoalanine is a biogenic organophosphonate and its formation is strictly bound to the metabolic pathway of ciliatine, this finding is not surprising. COOH

> —* °i —- M

H2N-

Pesticide Biotransformation in Plants and Microorganisms - American

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