Uncovering the function of novel proteins in the microbial degradation of phosphonates

Phosphonates, characterized by their stable C-P bond, represent a significant pool of organophosphorus compounds in both natural environments and anthropogenic systems. They serve as alternative phosphorus, carbon, and nitrogen sources for many microorganisms, thus playing an important role in nutrient cycling. The environmental importance of these C-P-containing compounds has raised interest in reaching a more complete understanding of the microbial degradation of phosphonates. In particular, microorganisms possess specialized enzymes for C-P degradation that occur in different pathways. These can be divided into the broad-specificity pathway characterized by the C-P lyase complex, capable of degrading a wide range of phosphonates through a radical mechanism, and the substrate-specific pathways, in which the C-P bond is oxidized or hydrolyzed. Among the latter, the pathways for aminoethyl-phosphonate (AEPn) degradation are the most abundant. In this work, we explored the gene clusters for AEPn hydrolysis to identify new genes of unknown function associated with these clusters. In particular, we found new enzymes that increase the utility of the substrate-specific PhnWX and PhnWYA pathways for AEPn hydrolysis. This work was encouraged by the previous discovery of the novel accessory enzyme, PbfA whose gene was recurrently found grouped with the phnWX or phnWAY operons. PbfA is a lyase that catalyzes an elimination reaction on the R-hydroxy-aminoethylphosphonate (R-HAEPn), yielding phosphonoacetaldehyde and ammonia.1,2 Therefore, we performed further genomic analyses that showed the presence of new sets of genes associated with the phnWX and phnWAY clusters. Three groups of genes encode proteins annotated as FAD-dependent oxidoreductases belonging to the D-amino oxidase family (DAO - Pfam01266), and we named the three genes PbfB, PbfC, and PbfD. Another group is constituted of genes for proteins annotated as NAD-dependent D-hydroxy acid dehydrogenases (Pfam02826), which we named PbfF. Functional analyses revealed that the PbfB, PbfC, and PbfD enzymes catalyze an oxidative deamination reaction on the monomethyl-aminoethyl phosphonate (M1AEPn) yielding phosphonoacetaldehyde (PnAA) and methylamine. On the other hand, we found that PbfF is a NAD-dependent racemase acting on the R- and S- enantiomers of HAEPn. These accessory enzymes can, therefore, expand the utility of the well-known hydrolytic pathways for AEPn degradation by channeling other phosphonates into these routes, thus expanding the repertoire of phosphonate compounds that microorganisms can process. Finally, this thesis also describes an initial characterization of PbtR, a transcriptional regulator belonging to the LysR family, whose gene was found to be associated with the cluster for AEPn hydrolysis in Azospirillum sp. B510. In particular, we found that PbtR can bind to the non-coding region containing the putative promoter region of the AEPn degradation cluster, thus suggesting that it may be involved in the Pho-independent transcriptional regulation of this cluster.