Phosphonic acids, or phosphonates, are a class of organophosphorus compounds characterized by a chemically very stable carbon-phosphorus (C-P) bond. In organisms, the stability of these compounds is exploited to cover a wide range of biological functions. Moreover, many bacteria have acquired the ability to degrade and use phosphonates as a source of C, N and P. Based on current knowledge, the catabolism of phosphonates can be subdivided into three groups of enzymatic pathways: C-P hydrolase, C-P oxidase and C-P lyase. With the exception of C-P lyase, which is as flexible as it is complex, most of them appear to be specialized pathways for the degradation of a specific compound. In this work, we explored through bioinformatics analysis the genomic contexts of genes encoding for C-P hydrolase pathways involved in 2-aminoethylphosphonic acid (2-AEP) degradation, in order to identify additional novel enzymes functionally related with these pathways and characterized their activity and role. Specifically, we detected four genes frequently associated with the hydrolytic phnW-phnX and phnW-phnA-phnY clusters: one automatically annotated as encoding for a PLP-dependent 4-aminobutyrate transaminase (GABA-T) (PF00202) and three, others that, although different from each other, are annotated as hypothetical FAD-dependent oxidoreductases (PF01266); we labeled them as PbfA, PbfB, PbfC and PbfD, respectively. We report here that the PLP-dependent enzyme PbfA from the marine bacterium Vibrio splendidus catalyzes an elimination reaction on the natural compound R-1-hydroxy-2-aminoethylphosphonate (R-OH-AEP), yielding ammonia and phosphonoacetaldehyde, and that the FAD-dependent enzyme PbfC from the rhizobacterium Azospirillum lipoferum catalyzes an oxidation reaction on N-monomethyl-2-aminoethylphosphonate (MMAEP), yielding methylamine and phosphonoacetaldehyde. Although these two aminophosphonates are structurally close to 2-AEP, they cannot be processed properly by the PhnW-PhnX (or PhnW-PhnY-PhnA) pathway. However, the scope of these specialized pathways is extended by the accessory enzymes PbfA and PbfC, which channel phosphonoacetaldehyde into the main pipeline. In addition to filling in some missing pieces of the phosphonate metabolism puzzle, our results contribute to the advancement in the field of phosphonate catabolism by emphasizing that such accessory enzymes increase the utility of the common (but very specific) hydrolytic pathways for 2-AEP degradation.
Gli acidi fosfonici, o fosfonati, sono una classe di composti organofosforici caratterizzati da un legame carbonio-fosforo (C-P) chimicamente molto stabile. Negli organismi, la stabilità di questi composti viene sfruttata per coprire un’incredibile gamma di attività biologiche. Tuttavia, molti batteri hanno sviluppato anche la capacità di degradarli e utilizzarli come fonte di carbonio, azoto e fosforo, rivelando un loro ruolo significativo nei cicli biogeochimici di questi elementi acquisendo importanza ecologica. Sulla base delle conoscenze attuali, il catabolismo dei fosfonati può essere suddiviso in tre gruppi di vie enzimatiche: la via idrolitica, la via ossidativa e la via radicalica (C-P liasi). Con l’eccezione della C-P liasi, che è tanto promiscua quanto complessa, le altre sono vie specializzate per la degradazione di un solo composto. In questo lavoro, abbiamo esplorato attraverso l’analisi bioinformatica i contesti genomici dei geni che codificano per le vie idrolitiche coinvolte nella degradazione dell’acido 2-aminoetilfosfonico (2-AEP; ciliatina), con lo scopo di trovare geni frequentemente associati ad essi codificanti per proteine ancora non caratterizzate e di identificarne la loro funzione e ruolo. In particolare, abbiamo individuato quattro geni spesso associati ai cluster phnW-phnX e phnW-phnA-phnY; uno annotato automaticamente come codificante per la 4-aminobutirrato transaminasi PLP-dipendente (GABA-T) (Pfam00202) e tre, che sebbene diversi tra loro, sono annotati come ipotetica ossidoreduttasi FAD-dipendente (Pfam01266); li abbiamo chiamati PbfA, PbfB, PbfC e PbfD, rispettivamente. In questo lavoro riportiamo che l’enzima PLP-dipendente PbfA, del batterio marino Vibrio splendidus, catalizza una reazione di eliminazione sul composto naturale R-1-idrossi-2-aminoetilfosfonato (R-OH-AEP), producendo ammoniaca e fosfonoacetaldeide, e che l’enzima FAD-dipendente PbfC, del rizobatterio Azospirillum lipoferum, catalizza una reazione di ossidazione sull’ N-monometil-2-aminoetilfosfonato (MMAEP), producendo metilammina e fosfonoacetaldeide. Anche se l’ R-OH-AEP e MMAEP sono analoghi strutturali del 2-AEP, essi non possono essere processati correttamente dal percorso PhnW-PhnX (o PhnW-PhnY-PhnA). Tuttavia, la portata di queste vie specializzate è estesa dagli enzimi accessori PbfA e PbfC, che incanalano la fosfonoacetaldeide nella via principale. I risultati ottenuti in questo lavoro contribuiscono all’avanzamento nel campo del catabolismo dei fosfonati ad oggi ancora frammentato e incompleto, sottolineando che tali enzimi accessori aumentano l’utilità della comune (ma molto specifica) via idrolitica per la degradazione del 2-AEP.
Identificazione e caratterizzazione di nuovi enzimi coinvolti nel catabolismo batterico dei fosfonati
Erika, Zangelmi
2022
Abstract
Phosphonic acids, or phosphonates, are a class of organophosphorus compounds characterized by a chemically very stable carbon-phosphorus (C-P) bond. In organisms, the stability of these compounds is exploited to cover a wide range of biological functions. Moreover, many bacteria have acquired the ability to degrade and use phosphonates as a source of C, N and P. Based on current knowledge, the catabolism of phosphonates can be subdivided into three groups of enzymatic pathways: C-P hydrolase, C-P oxidase and C-P lyase. With the exception of C-P lyase, which is as flexible as it is complex, most of them appear to be specialized pathways for the degradation of a specific compound. In this work, we explored through bioinformatics analysis the genomic contexts of genes encoding for C-P hydrolase pathways involved in 2-aminoethylphosphonic acid (2-AEP) degradation, in order to identify additional novel enzymes functionally related with these pathways and characterized their activity and role. Specifically, we detected four genes frequently associated with the hydrolytic phnW-phnX and phnW-phnA-phnY clusters: one automatically annotated as encoding for a PLP-dependent 4-aminobutyrate transaminase (GABA-T) (PF00202) and three, others that, although different from each other, are annotated as hypothetical FAD-dependent oxidoreductases (PF01266); we labeled them as PbfA, PbfB, PbfC and PbfD, respectively. We report here that the PLP-dependent enzyme PbfA from the marine bacterium Vibrio splendidus catalyzes an elimination reaction on the natural compound R-1-hydroxy-2-aminoethylphosphonate (R-OH-AEP), yielding ammonia and phosphonoacetaldehyde, and that the FAD-dependent enzyme PbfC from the rhizobacterium Azospirillum lipoferum catalyzes an oxidation reaction on N-monomethyl-2-aminoethylphosphonate (MMAEP), yielding methylamine and phosphonoacetaldehyde. Although these two aminophosphonates are structurally close to 2-AEP, they cannot be processed properly by the PhnW-PhnX (or PhnW-PhnY-PhnA) pathway. However, the scope of these specialized pathways is extended by the accessory enzymes PbfA and PbfC, which channel phosphonoacetaldehyde into the main pipeline. In addition to filling in some missing pieces of the phosphonate metabolism puzzle, our results contribute to the advancement in the field of phosphonate catabolism by emphasizing that such accessory enzymes increase the utility of the common (but very specific) hydrolytic pathways for 2-AEP degradation.File | Dimensione | Formato | |
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https://hdl.handle.net/20.500.14242/193323
URN:NBN:IT:UNIPR-193323