Structure of the catalytic core of the family F xylanase from Pseudomonas fluorescens and identification of the xylopentaose-binding sites.

Abstract Background: Sequence alignment suggests that xylanases evolved from two ancestral proteins and therefore can be grouped into two families, designated F and G. Family F enzymes show no sequence similarity with any known structure and their architecture is unknown. Studies of an inactive enzyme–substrate complex will help to elucidate the structural basis of binding and catalysis in the family F xylanases. Results We have therefore determined the crystal structure of the catalytic domain of a family F enzyme, Pseudomonas fluorescens subsp. cellulosa xylanase A, at 2.5 A resolution and a crystallographic R-factor of 0.20. The structure was solved using an engineered cata lytic core in which the nucleophilic glutamate was replaced by a cysteine. As expected, this yielded both high-quality mercurial derivatives and an inactive enzym e which enabled the preparation of the inactive enzyme–substrate complex in the crystal. We show that family F xylanases are eight-fold α / β -barrels (TIM barrels) with two active-site glutamates, one of which is the nucleophile and the other the acid-base. Xylopentaose binds to five subsites A–E with the cleaved bond between subsites D and E. Ca 2+ binding, remote from the active-site glutamates, stabilizes the structure and may be involved in the binding of extended substrates. Conclusion The architecture of P. fluorescens subsp. cellulosa has been determined crystallographically to be a commonly occurring enzyme fold, the eight-fold α / β -barrel. Xylopentaose binds across the carboxy-terminal end of the α / β -barrel in an active-site cleft which contains the two catalytic glutamates.