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.
Highlights
Polyamines (PAs) are small, aliphatic, polycationic compounds that bear at least two amino groups
MtCPA crystallizes in P21212 space group, with two octamers as the asymmetric unit
The octameric quaternary structure in the crystal lattice of MtCPA is in agreement with the assembly reported, based on native gel electrophoresis, for the very close ortholog from A. thaliana (85% identity, 92% similarity) (Piotrowski et al, 2003)
Summary
Polyamines (PAs) are small, aliphatic, polycationic compounds that bear at least two amino groups. In order to present the active sites in a clear manner, we divide the MtCPA cavities to four parts, each buried deeper in the protein core: (i) the entrance preceding the genuine active site; (ii) the tail amine-binding region, which binds the non-reacting amine group of PA; (iii) the non-polar section, interacting with the ligand aliphatic fragment; and (iv) the catalytic triad, which performs the reaction.
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