Stereochemistry of the reaction catalysed by 2‐aminoethylphosphonate aminotransferase

Abstract

(R)-and (S)-2-amino[2-D,]ethylphosphonic acids ([2-D1]AEP) were synthesised to investigate the stereochemistry of the reaction catalysed by 2-aminoethylphosphonate aminotransferase from Pseudomonas aeruginosa. This enzyme catalyses the transfer of the amino group of AEP to pyruvate to produce 2-phosphonoacetaldehyde and alanine. The enzymic reaction proceeding through the abstraction of a proton from the Schiff-base complex formed between the enzyme-bound pyridoxal 5'-phosphate, and the substrate, was carried out in an aqueous buffer at pH 8.5; it was followed by high-field 'H-NMR measurements (SOOMHz, H,O) on an AMX 500 Bruker spectrometer. The spectra, recorded with chiral (R)-or (S)-[2-D1]AEP, both showed the methylenic signal (3.0 ppm), whereas (S)-[2-D1]AEP gave the additional aldehydic signal (CHO, 9.6 ppm). These data clearly show that AEP-aminotransferase catalyses the abstraction of the pro-S hydrogen atom at the prochiral C2 carbon of AEP. Furthermore, careful timing of NMR measurements over a 2-hour period allows us to show the occurrence of an isotopic effect.2-Aminoethylphosphonic acid (AEP) is a naturally occurring compound discovered in Protozoa collected from sheep mmen, then found in a variety of microorganisms and marine animals [l]. The ability to cleave the C-P bond of AEP is widespread among microorganisms, but not among plants and animals. The catabolism of AEP by Pseudomonas aeruginosa involves a double-step pathway, allowing this molecule to be a source of nitrogen, carbon and phosphorus for this bacterium [2]. The first step of this pathway implies an inducible specific 2-aminoethylphosphonate aminotransferase, requiring pyridoxal 5'-phosphate (pyridoxal-P), which we have purified and characterized [3]. This enzyme catalyses the transfer of the amino group of AEP to pyruvate to produce 2-phosphonoacetaldehyde and alanine according to Scheme 1.The aminotransferase-catalysed reaction proceeds through the abstraction of a proton from the Schiff-base complex formed between the enzyme-bound pyridoxal-P and the substrate [4]. Thus, w-aminotransferases, unlike a-aminotransferases, exhibit two stereochemical possibilities for the proton abstraction.

Discovery of a New, Recurrent Enzyme in Bacterial Phosphonate Degradation: (R)-1-Hydroxy-2-aminoethylphosphonate Ammonia-lyase

“…These experiments showed that R- HAEP is efficiently processed by the enzyme, whereas S -HAEP is completely unreactive. When the reaction between PbfA and R -HAEP was monitored by 1 H NMR, consumption of this aminophosphonate was observed, with the simultaneous appearance of a compound showing the 1 H NMR signature of PAA (t at 9.60 ppm, J = 4.25 Hz; dd at 2.90–2.94 ppm, J = 19.90 and 4.25 Hz) 32 , 33 ( Figure 4 ).…”

Biosynthesis of Phosphonic and Phosphinic Acid Natural Products

“…At present, aminotransferases involved in the anabolism of AEP have not been characterized biochemically, but several enzymes catalyzing the same reaction in organisms that use AEP as a source of nutrients have been studied (29-31). These proteins from Salmonella enterica (31) and Pseudomonas aeruginosa (32) utilize pyridoxal 5′-diphosphate (PLP) as cofactor and pyruvate as ammonium acceptor.…”

Molecular cloning, mapping, and regulation of Pho regulon genes for phosphonate breakdown by the phosphonatase pathway of Salmonella typhimurium LT2

“…AEPn is converted to phosphonoacetaldehyde by an AEPnspecific transaminase, and phosphonoacetaldehyde is subsequently converted to acetaldehyde and P i by phosphonoacetaldehyde hydrolase (trivial name, phosphonatase). Enzymes carrying out both of these steps have been characterized for Bacillus cereus (26) and Pseudomonas aeruginosa (12,23). Uptake systems specific for AEPn have also been demonstrated for these bacteria (22,39), consistent with breakdown occurring in the cytoplasm.…”