Structural Investigations of N-carbamoylputrescine Amidohydrolase from Medicago truncatula: Insights into the Ultimate Step of Putrescine Biosynthesis in Plants

Abstract

Putrescine, 1,4-diaminobutane, is an intermediate in the biosynthesis of more complexed polyamines, spermidine and spermine. Unlike other eukaryotes, plants have evolved a multistep pathway for putrescine biosynthesis that utilizes arginine. In the final reaction, N-carbamoylputrescine is hydrolyzed to putrescine by N-carbamoylputrescine amidohydrolase (CPA, EC 3.5.1.53). During the hydrolysis, consecutive nucleophilic attacks on the substrate by Cys158 and water lead to formation of putrescine and two by-products, ammonia and carbon dioxide. CPA from the model legume plant, Medicago truncatula (MtCPA), was investigated in this work. Four crystal structures were determined: the wild-type MtCPA in complex with the reaction intermediate, N-(dihydroxymethyl)putrescine as well as with cadaverine, which is a longer analog of putrescine; and also structures of MtCPA-C158S mutant unliganded and with putrescine. MtCPA assembles into octamers, which resemble an incomplete left-handed helical twist. The active site of MtCPA is funnel-like shaped, and its entrance is walled with a contribution of the neighboring protein subunits. Deep inside the catalytic cavity, Glu48, Lys121, and Cys158 form the catalytic triad. In this studies, we have highlighted the key residues, highly conserved among the plant kingdom, responsible for the activity and selectivity of MtCPA toward N-carbamoylputrescine. Moreover, since, according to previous reports, a close MtCPA relative from Arabidopsis thaliana, along with several other nitrilase-like proteins, are subjected to allosteric regulation by substrates, we have used the structural information to indicate a putative secondary binding site. Based on the docking experiment, we postulate that this site is adjacent to the entrance to the catalytic pocket.

Structural Analysis of Phosphoserine Aminotransferase (Isoform 1) From Arabidopsis thaliana– the Enzyme Involved in the Phosphorylated Pathway of Serine Biosynthesis

“…At PSAT1 was cloned and purified using a modified protocol recently applied for the production of Medicago truncatula N-carbamoylputrescine amidohydrolase ( Sekula et al, 2016 ) and thermospermine synthase ( Sekula and Dauter, 2018 ). Isolation of total RNA from leaves of A. thaliana was performed with RNeasy Plant Mini Kit (Qiagen).…”

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

“…The first reaction is catalyzed by agmatine iminohydrolase, the enzyme built of two subunits characterized by an αββαβ five-bladed propeller fold ( Sekula and Dauter, 2019 ). The second step is carried out by N-carbamoylputrescine amidohydrolase, which in plants forms characteristic octamers with four pairs of subunits arranged helically ( Sekula et al., 2016 ). An alternative route for putrescine production is the ornithine pathway.…”

On methylene‐bridged cysteine and lysine residues in proteins

“…Interestingly, even though the electron density maps clearly indicate the presence of CH 2  group, it was neglected in all cases, except for Mt HPP. It is also of note that an analogous, unintended, carbon‐mediated junction of Cys Sγ with a primary amine was observed in case of N ‐carbamoylputrescine amidohydrolase from M. truncatula . In this example, the amine group was not a part of a lysine residue but of 1,4‐diaminobutane (putrescine) which apparently reacted with carbon dioxide, absorbed by the crystallization solution from air.…”

“…It is also of note that an analogous, unintended, carbon-mediated junction of Cys Sg with a primary amine was observed in case of N-carbamoylputrescine amidohydrolase from M. truncatula. 5 In this example, the amine group was not a part of a lysine residue but of 1,4-diaminobutane (putrescine) which apparently reacted with carbon dioxide, absorbed by the crystallization solution from air. The process was however stopped at the geminal diol intermediate, stabilized by the protein oxyanion hole, observed in the structure of PDB ID: 5h8i.…”