Structural Remodelling of the Carbon-Phosphorus Enzymatic Machinery by a Dual ATP-Binding Cassette Module

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Phosphorus (P) is an essential macronutrient that can be extracted from the environment by several metabolic pathways. In E. coli, phosphate deprivation activates the the 14-cistron phn operon that encodes carbon-phosphorus (C-P) lyase. C-P lyase confers the ability to extract phosphorus from a wide range of phosphonate compounds containing the chemically highly stable C-P bond. 1 Phosphonates are widespread in nature and due to their similarity to phosphate esters, have important applications in agriculture and biomedicine, as pesticides (glyphosate, RoundUp) and antibiotics, respectively. C-P lyase catalyses a complex multi-step pathway that directly depends on 10 of the encoded proteins (PhnGHIJKLMNOP). Of these, the PhnJ subunit was shown to be responsible for cleavage of the C-P bond via a strict anaerobic glycyl radical mechanism that requires an iron-sulphur (Fe 4 S 4 ) cluster and S-adenosyl methionine (SAM) for radical activation. Surprisingly, however, this mechanism was not immediately compatible with the crystal structure of a PhnGHIJ C-P lyase core complex, which placed key residues at a significant distance, thus leaving a large gap in our understanding of the mechanism of phosphonate breakdown. 2,3 The phn operon also encodes two ATP-binding cassette (ABC) proteins with homology to the nucleotide-binding domains of ABC transporters, PhnK and PhnL, for which no function has been assigned. Here, we present four high-resolution (~2 Å), single-particle cryo-electron microscopy structures of 300+ kDa C-P lyase complexes in several key, functional states. Together, these structures reveal how the PhnGHIJ core complex interacts with a unique double dimer of PhnK and PhnL subunits, in which PhnK, in a tight ATP-bound conformation, bridges the core complex and PhnL. We also show using additional structures determined under ATP turnover conditions, that ATP hydrolysis in PhnK induces a dramatic remodelling of the core complex, leading to opening and large-scale movement of several subunits. Moreover, we show that ATP hydrolysis and binding of both ABC subunits is required for growth of E. coli on phosphonate. Finally, we demonstrate that C-P lyase in the closed state binds substrate at a metal-binding site located at the interface between the PhnI and PhnJ subunits, and that ATP hydrolysis leads to a reorganisation of this active site in a way likely contributes to substrate/product exchange. In summary, we provide several novel insights into the elusive process of phosphonate breakdown by C-P lyase in microorganisms and represent a solid structural basis for understanding the catalytic process. Our structural data also uncover a hitherto unknown configuration of ABCs in which two ABC dimers directly interact with each other that have broad implications for our understanding of the role of this module in biological systems. 1. Metcalf, W. W. & Wanner, B. L. Gene 129, 27-32, (1993). 2. Kamat, S. S., Williams, H. J., Dangott, L. J., Chakrabarti, M. & Raushel, F. M. Nature 497, 132-136, (2013). 3. Seweryn, P. et al. Nature 525, 68-72, (2015).

Original languageEnglish
JournalThe FASEB Journal
Publication statusPublished - May 2022


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