Crystal structures of the oxygenase component of an aromatic monooxygenase in ligand-bound forms
Tamao Hisano, S.-H. Kim, Kazuki Takeda, W. Iwasaki, Akio Ebihara, Kunio Miki · Acta Crystallographica Section A Foundations of Crystallography · 2008
Poster SessionsArabinanase hydrolyzes the α-1,5-L-arabinofuranoside linkage of arabinan distributed in hemicelluloses, which comprise a large fraction of plant cell walls.AbnS1 from P.chrysogenum 31B and ABN-TS from Bacillus thermodenitrificans TS-3, which hydrolyze arabinan through an endo mechanism, show optimal activity at 333 and 343 K, respectively.The X-ray crystallographic analysis has revealed that the thermostable ABN-TS has a unique motif consisting of a five-bladed β-propeller fold.Since AbnS1 have 32% homology with ABN-TS, X-ray analysis of the mesophilic AbnS1 should provide information towards clarify the structural features that cause the difference in the optimum temperature.The recombinant AbnS1 was overexpressed in E. coli as a C-terminal His-Tagged protein (AbnS1-His).The first purification step was a Ni-affinitycolumn.Further purification steps were anion-exchange and gel filtration columns.The tag was not removed for the subsequent crystallization experiments, because AbnS1-His showed the same catalytic activity and optimum temperature as the native AbnS1.Crystals were obtained using PEG4000 as a precipitant.Data collection and structure analysis are under way.