Nicotianamine (NA), a small molecule ubiquitous in plants, is an important divalent metal chelator and the main precursor of phytosiderophores. Nicotianamine synthase (NAS) is the enzyme catalyzing NA synthesis by the condensation of three aminopropyl moieties of S-adenosylmethionine (SAM) and the cyclization of one of them to form an azetidine ring. Here we report five crystal structures of an archaeal NAS from Methanothermobacter thermautotrophicus, either free or in complex with its product(s) and substrate(s). These structures reveal a two-domains fold arrangement of MtNAS, a small molecule related to NA (named here thermoNicotianamine or tNA), and an original mechanism of synthesis in a buried reaction chamber. This reaction chamber is open to the solvent through a small inlet, and a single active site allows the selective entrance of only one substrate at a time that is then processed and translocated stepwise. metal homeostasis ͉ reaction mechanism

The crystal structure of Ap-ThrRS-1 has been successfully determined at 2.3 Å resolution, as the first example. Ap-ThrRS-1 is a dimeric enzyme, the two identical subunits being associated to each other. Each subunit is composed of the two domains for the catalytic reaction and for the anticodon-binding, as expected. Their structures are similar to those of Ec-ThrRS. The amino acid residues essential for the catalysis and for the anticodon recognition are highly conserved at the positions and in the orientations. The essential editing domain of ThrRS is completely missing in Ap-ThrRS-1 as expected, suggesting the necessity of the second enzyme ThrRS-2 for editing. Since the N-terminal sequence of Ap-ThrRS-2 is similar to the sequence of the editing domain of Pa-ThrRS, Ap-ThrRS-2 is expected to catalyze de-aminoacylation of the misacylated serine moiety at the CCA terminus.

In plants, nicotianamine synthase (NAS) plays a key role in metal homeostasis as it catalyzes the formation of nicotianamine, an important iron and nickel chelator and a precursor of plant phytosiderophores. Here, the crystallization of a protein from Methanothermobacter thermoautotrophicus (MTH675; referred to here as MtNAS) that appears to be homologous to plant NAS is reported. Purification of this protein showed a monomer-dimer equilibrium that could be displaced by using a reducing agent such as DTT. Crystals belonging to space group P2 1 2 1 2 1 and containing dimers of MtNAS were grown by the vapourdiffusion method using polyethylene glycol 3350 as precipitant. A complete native X-ray data set was collected to 1.7 Å resolution at a synchrotron source.

We determined the three-dimensional structure of a complex between an archaeal nicotianamine synthase homologue and a chemically synthesised reaction intermediate. This structure suggests that the enzymes cavity allows both an ordered substrate binding and provides energetic coupling of the reaction intermediate formation and translocation.