Expression, purification, crystallization and preliminary X-ray analysis of an archaeal protein homologous to plant nicotianamine synthase

Abstract

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.

Crystallographic snapshots of iterative substrate translocations during nicotianamine synthesis in archaea

“…1A). This is consistent with the redox-dependent dimerization previously revealed by size exclusion chromatography (25). Many disulfide bonds were identified and found to contribute significantly to the protein thermostability in some archaea (26).…”

“…MtNAS was cloned, expressed, and purified as described by Dreyfus et al (25). MtNAS mutants were constructed using the QuikChange II XL Site-Directed Mutagenesis kit from Stratagene following the manufacturer's manual.…”

The ancient roots of nicotianamine: diversity, role, regulation and evolution of nicotianamine-like metallophores

“…Indeed, for still unknown reason, in-vitro study of a NAS enzyme from eukaryotic origin is challenging and, in this context, a distantly related enzyme from an archaea, which shares less than 20% sequence identity with a plant enzyme was investigated. [32][33][34] Solving the three-dimensional structure of such an enzyme from M. thermotautrophicus (MtNAS) revealed an original fold composed of an N-terminal helix bundle combined with a Rosmann fold. Unexpectedly, it was shown that the protein co-purified and cocrystallized with its product.…”

Detrimental effect of the 6 His C-terminal tag on YedY enzymatic activity and influence of the TAT signal sequence on YedY synthesis

“…His-tag fusion simplifies protein purification, but it may also impair protein expression [7] or be detrimental to either the protein’s function or crystal structure [8]. It is thus advisable to examine expression and activity, either between C- and N-terminal fusions or after tag removal by enzymatic cleavage.…”

“…To date, all E. coli YedY biochemical studies have been performed using a purified protein labeled with a 6 histidine-tag at its C-terminus [ 4 - 6 ]. His-tag fusion simplifies protein purification, but it may also impair protein expression [ 7 ] or be detrimental to either the protein’s function or crystal structure [ 8 ]. It is thus advisable to examine expression and activity, either between C- and N-terminal fusions or after tag removal by enzymatic cleavage.…”