The Unusual Enzymes of Phosphonate Metabolism

The submitted project deals with the biodegradation of phosphonates. The biological importance of this class of compounds was long time underestimated. Thus, we only know relatively few details about the enzymatic processes involved in their metabolism. What we know by now is that phosphonate biochemistry is a treasure trove of unusual enzymology, where already numerous novel enzymatic transformations have been discovered. Still the known pathways leading to their breakdown are limited. Due to the natural abundance of phosphonates it is very likely that new metabolic pathways will be discovered in the near future. Whereas higher organisms such as mammals cannot metabolise phosphonates, a variety of microorganisms can. Among those we can also find several pathogens synthesising and degrading phosphonates. This fact makes them ideal candidates for drug design and already led to the development of varied phosphonic acid based crop protection products and pharmaceuticals. Thus a fundamental understanding of the processes involved in the biosynthesis and biodegradation of these compounds is of crucial interest. This will help to understand resistance mechanisms and allow designing improved drugs. Furthermore the influence of phosphonates in the global phosphorus cycle was discovered only recently. Breakdown of phosphonates was shown to be involved in marine methane production and therefore also influences the global climate. In the current project we want to contribute to the elucidation of the biodegradation pathways of fosfomycin, phosphaisoserine and 2-aminoethylphosphonic acid (2-AEP). These three compounds were chosen due to their significance and natural abundance. We will synthesise potential metabolites of the biodegradation of those compounds. These will be tested with the corresponding enzymes/cell cultures in cooperation with the groups of Prof. John Quinn (Belfast), Prof. David Zechel (Ontario)and Dr. Sascha Martens (Vienna). We plan to study the recently discovered PhnY/PhnZ enzyme system which is degrading 2-AEP and phosphaisoserine, respectively, and to further characterise its substrate specifity. We will also continue the work of former group members concerning the biodegradation of fosfomycin. Further we want to study the stereochemistry of a crucial protonation step in the biodegradation of 2-AEP by phosphonoacetaldehyde hydrolase using chirally labelled 2-AEP analogues. In a last part of the project we will deal with a newly isolated bacterial strain, which is able to use phosphaisoserine as sole phosphorus and carbon source.

Phosphorus is an essential and often growth-limiting, nutrient for all plants and animals. Especially marine ecosystems are often depleted in phosphate, which is the main phosphorus source for all higher organisms. Thus, a variety of microorganisms has developed strategies to use so-called phosphonates as alternative phosphorus source. Phosphonates are organic phosphorus compounds containing a direct, covalent phosphorus-carbon bond. For a long time, mechanistic details of phosphonate use by microorganisms were regarded as topics of limited scientific interest as they were assumed to be an evolutionary relic of low relevance. Nowadays we know that about 40% of all marine, bacterial genomes encode pathways for phosphonate breakdown. Thus, these compounds have a high environmental impact. High total quantities of phosphonates are released into the environment and their degradation is linked to the global phosphorus cycle. It was only found recently that phosphonate metabolism can lead to the formation of high amounts of methane in the upper, oxygen rich ocean zones and is thus climate relevant. Despite this knowledge about the relevance of phosphonates , the precise mechanisms leading to their breakdown is often missing. During the course of this FWF funded project, we studied several completely new aspects of phosphonate breakdown and could characterise a couple of new cleavage routes for the first time. Thus, we highly contributed to a better general understanding of phosphonate biochemistry. The project dealt with aspects of the metabolism of phosphonates of industrial relevance, like the clinically used antibiotic fosfomycin and of less known compounds alike. For example, we elucidated the three-dimensional structure of the recently discovered natural product hydroxynitrilaphos. A special focus was however on the discovery and study of new, up-to-now unknown degradation pathways for phosphonates. Together with our collaborators in Italy, Canada, Northern Ireland and the USA, we were able to show that there are other, yet unknown degradation pathways for phosphonate cleavage and characterised them for the first time.