Structural Superimposition

Abstact

Arginine metabolism in plants is targeted by phytopathogens such as Pseudomonas syringae strains, which produce toxins that affect enzymes of plant arginine metabolism, causing devastating infections in crops. In this work, the structural and functional properties of plant N-acetylornithine glutamate acetyltransferase (NAOGAT), the key deacetylating enzyme of the cyclic ornithine pathway and the target of mangotoxin, were investigated. We determined the structures of rice and maize NAOGATs to near-atomic resolution. Plant NAOGATs demonstrate a characteristic ArgJ-like N-terminal nucleophile fold, undergoing autoproteolytic maturation into alpha and beta subunits, generating an active-site threonine nucleophile at the N-terminus of the beta-chain. The enzyme catalyses acetyl transfer via a bi-bi ping-pong mechanism in a bidirectional manner, although the acetylornithine-to-acetylglutamate conversion is preferred. NAOGAT undergoes minimal structural rearrangements during the reaction, however threonine acetylation causes pronounced changes in electrostatic potential around the active site which is also exhibited by the increased NAOGAT stability in the thermal shift assay. We did not detect clear evidence of NAOGAT acetylation by acetylCoA in our structural and biophysical data, which supports the specialisation of plant NAOGATs towards acetylornithine-glutamate transacetylation. Finally, predictive bioinformatic analysis of the P. syringae mangotoxin-producing operon, in the context of NAOGAT architecture, provided a basis for potential structural features of mangotoxin.