RESUMO
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.
Assuntos
Alquil e Aril Transferases/metabolismo , Proteínas Arqueais/metabolismo , Ácido Azetidinocarboxílico/análogos & derivados , Methanobacteriaceae/enzimologia , Alquil e Aril Transferases/química , Alquil e Aril Transferases/genética , Sequência de Aminoácidos , Proteínas Arqueais/química , Proteínas Arqueais/genética , Ácido Azetidinocarboxílico/química , Ácido Azetidinocarboxílico/metabolismo , Sítios de Ligação/genética , Catálise , Cristalografia por Raios X , Ciclização , Ligação de Hidrogênio , Espectrometria de Massas , Methanobacteriaceae/genética , Modelos Químicos , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Molecular , Mutação , Dobramento de Proteína , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , S-Adenosilmetionina/química , S-Adenosilmetionina/metabolismo , Homologia de Sequência de Aminoácidos , Especificidade por SubstratoRESUMO
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 vapour-diffusion method using polyethylene glycol 3350 as precipitant. A complete native X-ray data set was collected to 1.7 A resolution at a synchrotron source.
Assuntos
Alquil e Aril Transferases/química , Proteínas Arqueais/química , Methanobacteriaceae/química , Proteínas de Plantas/química , Alquil e Aril Transferases/genética , Alquil e Aril Transferases/isolamento & purificação , Proteínas Arqueais/genética , Proteínas Arqueais/isolamento & purificação , Cromatografia em Gel , Cristalização , Cristalografia por Raios X , Primers do DNA , Methanobacteriaceae/genética , Proteínas de Plantas/genética , Reação em Cadeia da PolimeraseRESUMO
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.