The Neighboring Subunit Is Engaged to Stabilize the Substrate in the Active Site of Plant Arginases

Abstract

Arginine acts as a precursor of polyamines in plants in two known pathways, agmatine and ornithine routes. It is decarboxylated to agmatine by arginine decarboxylase, and then transformed to putrescine by the consecutive action of agmatine iminohydrolase and Ncarbamoylputrescine amidohydrolase. Alternatively, it can be hydrolyzed to ornithine by arginase and then decarboxylated by ornithine decarboxylase to putrescine. Some plants lack a functional ornithine pathway, but all have one or two arginases that can have dual cellular localization, in mitochondria and plastids. It was recently shown that arginases from Arabidopsis thaliana and soybean act also as agmatinases, thus they can produce putrescine directly from agmatine. Therefore, arginase (together with arginine decarboxylase) can complement putrescine production in plastids, providing a third polyamine biosynthesis pathway in plants. Phylogenetic analysis suggests that arginases, highly conserved in the plant kingdom, create the only group of enzymes recognized in the family of ureohydrolases in plants. Arginases are metalloenzymes with binuclear manganese cluster in the active site. In this work, two arginases from A. thaliana and Medicago truncatula are structurally characterized and their binding properties are discussed. Crystal structures with bound ornithine show that plant hexameric arginases engage a long loop from the neighboring subunit to stabilize a-amino and carboxyl groups of the ligand. This unique ligand binding mode is unobserved in arginases from other domains of life. Structural analysis shows that substrate binding by residues from two neighboring subunits might also characterize some prokaryotic agmatinases. This feature of plant arginases is most likely the determinant of their ability to recognize not only arginine but also agmatine as their substrates, thus, to act as arginase and agmatinase.

Crystal Structure of Escherichia coli Agmatinase: Catalytic Mechanism and Residues Relevant for Substrate Specificity

“…Finally, putrescine and urea would leave the active site randomly, in agreement with the Uni-bi rapid equilibrium random kinetic mechanism reported for EcAGM [19]. Interestingly, His163 is conserved in other putative agmatinases (i.e., BtAGM and TvAGM), even is conserved in mammalian and plant arginases [31]. However, the only exception is the agmatinase from D. radiodurans, in whose position an asparagine is found, but since there are no kinetic characterizations of DrAGM, it is not possible to know the kinetic characteristics of this enzyme.…”

“…For the structure 7LOL (Figure 2B), the hexamer has a total buried surface of 23,560 Å2, and a ∆Gdiss of 58.8 Kcal/mol, consistent with the oligomeric association observed in DrAGM PISA analysis of EcAGM structures indicates that the hexamer is the most stable oligomeric state for both structures 7LOL and 7LOX (Figure 2B and Supplementary Figure S2), presenting as monomer and trimer in the asymmetric unit, respectively (rmsdCα = 0.47 Å for monomers of 7LOL and 7LOX). The hexamer as oligomeric state tallies as is described to other bacterial agmatinases and arginases from bacteria and plants [21,31,32]. For the structure 7LOL (Figure 2B), the hexamer has a total buried surface of 23,560 Å2, and a ∆G diss of 58.8 Kcal/mol, consistent with the oligomeric association observed in DrAGM and other prokaryotic ureohydrolases [28,32].…”

“…PISA analysis of EcAGM structures indicates that the hexamer is the most stable oligomeric state for both structures 7LOL and 7LOX (Figure 2B and Supplementary Figure S2), presenting as monomer and trimer in the asymmetric unit, respectively (rmsdCα = 0.47 Å for monomers of 7LOL and 7LOX). The hexamer as oligomeric state tallies as is described to other bacterial agmatinases and arginases from bacteria and plants [21,31,32]. For the structure 7LOL (Figure 2B), the hexamer has a total buried surface of 23,560 Å2, and a ∆Gdiss of 58.8 Kcal/mol, consistent with the oligomeric association observed in DrAGM PISA analysis of EcAGM structures indicates that the hexamer is the most stable oligomeric state for both structures 7LOL and 7LOX (Figure 2B and Supplementary Figure S2), presenting as monomer and trimer in the asymmetric unit, respectively (rmsdCα = 0.47 Å for monomers of 7LOL and 7LOX).…”

“…Interestingly, neighboring monomer residues' contribution to the formation of agmatine binding pocket appears not to be fully conserved in agmatinases, neither is a widely reported characteristic of ureohydrolases. It has recently been reported that plant's arginases (Arabidopsis thaliana and Medicago truncatula), residues of the equivalent α2-η2 loop of the neighboring subunit, participate in binding of the substrate arginine [31].…”

Compartmentation of Putrescine Synthesis in Plants

“…In A. thaliana , ADC2 and arginase/agmatinase ARGAH2 are localized to the chloroplast (Patel et al, 2017). All plant arginase/agmatinase enzymes are very structurally conserved and have both arginase and agmatinase activity (Patel et al,2017; Sekula 2020). A unique feature of plant ARGAHs is that they form hexameric complexes, and substrate binding of ornithine or agmatine is stabilized by a loop from the neighboring subunit (Sekula 2020).…”