Abstract
Patatin is a non-specific plant lipase and the eponymous member of a broad class of serine hydrolases termed the patatin-like phospholipase domain containing proteins (PNPLAs). Certain PNPLA family members can be inhibited by organophosphorus (OP) compounds. Currently, no structural data are available on the modes of interaction between the PNPLAs and OP compounds or their native substrates. To this end, we present the crystal structure of patatin-17 (pat17) in its native state as well as following inhibition with methyl arachidonyl fluorophosphonate (MAFP) and inhibition/aging with diisopropylphosphorofluoridate (DFP). The native pat17 structure revealed the existence of two portals (portal1 and portal2) that lead to its active-site chamber. The DFP-inhibited enzyme underwent the aging process with the negatively charged phosphoryl oxygen, resulting from the loss of an isopropyl group, being within hydrogen-binding distance to the oxyanion hole. The MAFP-inhibited pat17 structure showed that MAFP did not age following its interaction with the nucleophilic serine residue (Ser77) of pat17 since its O-methyl group was intact. The MAFP moiety is oriented with its phosphoryl oxygen in close proximity to the oxyanion hole of pat17 and its O-methyl group located farther away from the oxyanion hole of pat17 relative to the DFP-bound state. The orientation of the alkoxy oxygens within the two OP compounds suggests a role for the oxyanion hole in stabilizing the emerging negative charge on the oxygen during the aging reaction. The arachidonic acid side chain of MAFP could be contained within portals 1 or 2. Comparisons of pat17 in the native, inhibited, and aged states showed no significant global conformational changes with respect to their Cα backbones, consistent with observations from other α/β hydrolases such as group VIIA phospholipase A2.
Highlights
Patatin is a soluble 40 kDa non-specific plant lipase that is expressed extensively in the tubers of potatoes [1,2] and other members of the nightshade family [3]
Given the lack of an experimentally derived structure for the interaction of a PNPLA with inhibitors or a native lipid substrate, we explored whether interaction of patatin with OP compounds could serve as a model to better understand the structural basis for the interaction of PNPLAs with their native substrates as well as the transition states present during substrate hydrolysis
While the selenomethionine-derivatized structure of native pat17 has been published [6], there are currently no structures available for the native protein as well as the modes of inhibition and aging of an OP compound on patatin. To address these gaps in knowledge, we present a high-resolution crystal structure of pat17 in its native state (PDB ID 4PK9) as well as pat17 structures inhibited by methyl arachidonyl fluorophosphonate (MAFP; PDB ID 4PKB) and aged by DFP (PDB ID 4PKA), a known neuropathic OP compound
Summary
Patatin is a soluble 40 kDa non-specific plant lipase that is expressed extensively in the tubers of potatoes [1,2] and other members of the nightshade family [3]. The crystal structure of a recombinant patatin isoform, patatin-17 (pat17), derived from the tubers of heartleaf nightshade (Solanum cardiophyllum), has shown this protein to have a modified a/b hydrolase fold containing a central 6stranded b-sheet sandwiched by 9 a-helices along with a catalytic center composed of a Ser77-Asp215 dyad [6]. This fold and active site configuration is reminiscent of the human cytosolic phospholipase A2 family of enzymes [7], but it is distinct from the traditional Ser-His-Asp/Glu catalytic triads found in serine proteases and esterases such as chymotrypsin and acetylcholinesterase (AChE), respectively [8,9]. One member of the PNPLA family, PNPLA6, which is better known
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