Abstract

Plants are unique eukaryotes that can produce putrescine (PUT), a basic diamine, from arginine via a three-step pathway. This process starts with arginine decarboxylase that converts arginine to agmatine. Then, the consecutive action of two hydrolytic enzymes, agmatine iminohydrolase (AIH) and N-carbamoylputrescine amidohydrolase, ultimately produces PUT. An alternative route of PUT biosynthesis requires ornithine decarboxylase that catalyzes direct putrescine biosynthesis. However, some plant species lack this enzyme and rely only on agmatine pathway. The scope of this manuscript concerns the structural characterization of AIH from the model legume plant, Medicago truncatula. MtAIH is a homodimer built of two subunits with a characteristic propeller fold, where five αββαβ repeated units are arranged around the fivefold pseudosymmetry axis. Dimeric assembly of this plant AIH, formed by interactions of conserved structural elements from one repeat, is drastically different from that observed in dimeric bacterial AIHs. Additionally, the structural snapshot of MtAIH in complex with 6-aminohexanamide, the reaction product analog, presents the conformation of the enzyme during catalysis. Our structural results show that MtAIH undergoes significant structural rearrangements of the long loop, which closes a tunnel-shaped active site over the course of the catalytic event. This conformational change is also observed in AIH from Arabidopsis thaliana, indicating the importance of the closed conformation of the gate-keeping loop for the catalysis of plant AIHs.

Highlights

  • Biosynthesis of putrescine (PUT) starts from arginine (ARG) and follows one of the two pathways which comprise agmatine (AGM) or ornithine (ORN) biotransformation (Michael, 2017)

  • AAT and reverse: TTATCCACTTCCAATGTTACTAAATGGCTG GTTGTTGCTGAGTGAT and the cDNA from leaves of M. truncatula as a template were used in a polymerase chain reaction (PCR) prior to obtaining MtAIH open reading frame (MTR_4g112810) with encoded protein starting from codon number 11

  • agmatine iminohydrolase (AIH) are assigned by the Structural Classification of Proteins (SCOPe) (Fox et al, 2014) to the porphyromonas-type peptidylarginine deiminase family that is a part of the Superfamily of penteins, characterized by a propeller-like arrangement of five αββαβ units which form a narrow channel in the core (Hartzoulakis et al, 2007)

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Summary

Introduction

Biosynthesis of putrescine (PUT) starts from arginine (ARG) and follows one of the two pathways which comprise agmatine (AGM) or ornithine (ORN) biotransformation (Michael, 2017). ARG is decarboxylated to AGM by arginine decarboxylase (ADC). Plants are unique eukaryotes to biosynthesize PUT via the AGM biotransformation, which makes ADC, AIH, and CPA potential targets for herbicide design (Böger and Sandmann, 1989). AGM-to-PUT conversion is catalyzed by agmatine ureohydrolase (agmatinase) (Satishchandran and Boyle, 1986) and most of the cyanobacteria use this enzyme instead of AIH and CPA (Fuell et al, 2010). The second manner of PUT biosynthesis, the ORN route, is dominant for most eukaryotes, including animals and fungi, and involves ORN decarboxylation catalyzed by ornithine decarboxylase (ODC) (Janowitz et al, 2003). Other plants, which have preserved ODC, may obtain PUT either from AGM or ORN

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