Functional Global Flexibility Demonstrated for the Multi-Domain Extracellular GH43 Arabinanase from Geobacillus stearothermophilus

Yuval Shoham ¹ Shifra Lansky ² Rachel Salama ¹ Xevi Biarnes ³ Omer Shwartshtein ² Dina Schneidman ⁴ Antoni Planas ³ Gil Shoham ²

¹Biotechnology and Food Engineering, Technion-Israel Institute of Technology, Israel
²Institute of Chemistry, The Hebrew University of Jerusalem, Israel
³Institut Químic De Sarrià, Universitat Ramon Llull, Spain
⁴The Alexander Silberman Institute of Life Sciences, The Hebrew University of Jerusalem, Israel

Arabinanases are glycosidases that hydrolyze alpha-(1,5)-arabinofuranosidic linkages found in the backbone of the pectic polysaccharide arabinan. Geobacillus stearothermophilus T-6 is a thermophilic Gram-positive soil bacterium that possesses an extensive hemicellulolytic system [1,2]. To utilize arabinan, the bacterium secretes an extracellular endo-arabinanase (AbnA) which hydrolyzes the high molecular weight arabinan polymer to produce short substituted arabino-oligosaccharides. These products enter the cell via specific ABC sugar transporters and their final degradation is accomplished by the action of intracellular enzymes. The 3D structure of AbnA has recently been determined by X-ray crystallography, representing the largest structure reported so far in the GH43 family, and revealing a unique “pincer-shaped” architecture composed of four domains. One of these domains represents a new carbohydrate-binding module (CBM) family, to which an arabinopentaose substrate was found bound. Three different crystallographic conformational states have been determined for AbnA, differentiated by approximately 13 Å movement in location of the third and fourth domains. Complementary analysis conducted by small angle X-ray scattering (SAXS), molecular dynamics (MD), metadynamics, and dynamic light scattering (DLS) suggest that these conformation changes are significantly larger in solution, involving movements of up to 100 Å. Mutagenesis and kinetics experiments indicate that such global flexibility is also functional, suggesting a "harpoon" catalytic mechanism for arabinan degradation by AbnA.

[1] Shulami, S. et al., (2011) The L-arabinan utilization system of Geobacillus stearothermophilus. J. Bacteriol. 193:2838-2850.

[2] Alhassid, A. et al., (2009) Crystal structure of an inverting GH43 1,5-α-L-arabinanase from Geobacillus stearothermophilus complexed with its substrate. Biochem. J. 422:73-82.