|Ph.D Student||Rachel Salama|
|Subject||Biochemical and Structure-Function Studies of|
Geobacillus Stearothermophilus Enzymes
Involved in the Utilization of Arabinan
|Department||Department of Biotechnology and Food Engineering||Supervisor||Full Professor Shoham Yuval|
|Full Thesis text|
The degradation of the plant biomass is a pivotal step in the Earth’s carbon cycle and is carried out mostly by microorganisms. Plant-based biomass is considered a readily available renewable energy source that can be fermented into liquid biofuel for transportation without contributing net CO2 to the atmosphere. The plant cell-wall is a complex structure composed mainly of polysaccharides and their hydrolysis is catalyzed by the synergistic action of glycoside hydrolases (GHs), a widespread group of enzymes that hydrolyze the glycosidic bond between carbohydrates.
Geobacillus stearothermophilus is a Gram-positive, thermophilic, soil bacterium that possesses an extensive system for the utilization of plant cell-wall polysaccharides including xylan, galactan and arabinan. In this study, we determined the substrate specificities and structure-function relationships of three novel glycoside hydrolases from the L-arabinan utilization system: b-L-arabinopyranosidase, GsAbp27, b-L-arabinofuranosidase, GsAra127N, and the extracellular arabinanase, GsAbn43A.
GsAbp27 was shown to release b-L-arabinopyranose residues from p-nitrophenyl b-L-arabinopyranoside, sugar beet arabinan and arabinogalactan. The three-dimensional structure of wild-type GsAbp27 and its catalytic mutant GsAbp27-D197A with and without a bound L-arabinose product were determined by X-ray crystallography. The enzyme is tetrameric in solution and its monomer consists of two main domains: an N-terminal TIM-barrel domain and a C-terminal all-b domain. While most of GH27 enzymes are a-D-galactopyranosidases, GsAbp27 has a unique specificity towards b-L-arabinopyranoside, representing a distinct clade in GH27 family. An isoleucine residue (Ile67) located at a key position in the active site was shown to play a critical role in the substrate specificity of GsAbp27.
GsAra127N has a unique b-L-arabinofuranosidase activity and it belongs to the novel GH127 family. It showed activity towards p-nitrophenyl b-L-arabinofuranoside and towards pectic polysaccharides extracted from olive's cell. GsAra127N is a dimer in solution and its monomer is organized in three domains: catalytic (a/a)6 barrel domain and two b-sandwich domains. Metal content analysis revealed that the enzyme contains one Zn2 ion per protein molecule, most likely coordinated by glutamate and three cysteine residues. The structure of GsAra127N-E311A bound to Zn2 was solved and revealed that the Zn2 is located near the active site. A metal dependent retaining activity was hypothesized, by which the Zn2 reduces the pKa of a cysteine thiol side chain, thus allowing it to act as the nucleophile.
GsAbn43A is folded into four domains: Domain 1 accommodates the active site, Domain 2 acts as accessory to the catalytic domain, Domain 3 is a linker that functions as a hinge and Domain 4 was suggested to be a carbohydrate-binding module (CBM). X-ray crystallography and isothermal titration calorimetry (ITC) analysis demonstrated that Domain 4 binds arabino-oligosaccharides and thus, represents a new CBM family for arabinan. Removing Domain 4 did not reduce the activity of GsAbn43A towards soluble arabinan. The results suggest that Domain 4 acts as an arabinan-binding module as to allow the enzyme to degrade efficiently insoluble plant-cell arabinan. The significance of Domain 4 for the degradation of insoluble arabino-polysaccharides has yet to be determined.