Abstract
Siderophore-binding proteins (SIPs) perform a key role in iron acquisition in multiple organisms. In the genome of the marine bacterium Shewanella frigidimarina NCIMB 400, the gene tagged as SFRI_RS12295 encodes a protein from this family. Here, the cloning, expression, purification and crystallization of this protein are reported, together with its preliminary X-ray crystallographic analysis to 1.35 Å resolution. The SIP crystals belonged to the monoclinic space group P21, with unit-cell parameters a = 48.04, b = 78.31, c = 67.71 Å, α = 90, β=99.94, γ = 90°, and are predicted to contain two molecules per asymmetric unit. Structure determination by molecular replacement and the use of previously determined ∼2 Å resolution SIP structures with ∼30% sequence identity as templates are ongoing.
Highlights
The Great Oxygenation Event (GOE) was marked by a profound transition in ocean chemistry, where the previously readily available iron became a limiting nutrient worth battling for (Holland, 2006)
The polymerase chain reaction (PCR) product was ligated into the expression vector pETBlue-1 (Novagen) and transformed into competent E. coli Tuner (DE3) pLacI cells for expression
The best diffracting crystal grew in 22% PEG 3350, 0.22 M magnesium formate dehydrate, and data to 1.35 Aresolution were collected on beamline I04 at Diamond Light Source (DLS), UK
Summary
The Great Oxygenation Event (GOE) was marked by a profound transition in ocean chemistry, where the previously readily available iron became a limiting nutrient worth battling for (Holland, 2006). To circumvent low iron bioavailability, organisms have found diverse strategies for importing and utilizing iron, including direct extracellular reduction (Deneer et al, 1995), the acquisition of iron-bound or haem proteins using specific receptors (Wandersman & Delepelaire, 2004) and the synthesis and extracellular release of small molecules with high affinity for ferric iron named siderophores (Neilands, 1981) These iron siderophores are strong ferric chelators and can be used in various applications, including the cultivation of uncultured microorganisms, as ecofriendly substitutes for pesticides, as enhancers in the bioremediation of heavy metals, in iron-overload therapy, in cancer therapy and as Trojan horse antibiotics (Saha et al, 2016).
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