title: 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 catalytic core in which the nucleophilic glutamate was replaced by a cysteine. As expected, this yielded both high-quality mercurial derivatives and an inactive enzyme which enabled the preparation of the inactive enzyme-substrate complex in the crystal. We show that family F xylanases are eight-fold alpha/beta-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. Ca2+ binding, remote from the active-site glutamates, stabilizes the structure and may be involved in the binding of extended substrates.
  
  
  CONCLUSIONS
  The architecture of P. fluorescens subsp. cellulosa has been determined crystallographically to be a commonly occurring enzyme fold, the eight-fold alpha/beta-barrel. Xylopentaose binds across the carboxy-terminal end of the alpha/beta-barrel in an active-site cleft which contains the two catalytic glutamates.
authors.affiliation: Protein Engineering Department, Institute of Food Research, Reading Laboratory, UK.
authors.forename: G W
authors.initials: GW
authors.lastname: Harris
authors.forename: J A
authors.initials: JA
authors.lastname: Jenkins
authors.forename: I
authors.initials: I
authors.lastname: Connerton
authors.forename: N
authors.initials: N
authors.lastname: Cummings
authors.forename: L
authors.initials: L
authors.lastname: Lo Leggio
authors.forename: M
authors.initials: M
authors.lastname: Scott
authors.forename: G P
authors.initials: GP
authors.lastname: Hazlewood
authors.forename: J I
authors.initials: JI
authors.lastname: Laurie
authors.forename: H J
authors.initials: HJ
authors.lastname: Gilbert
authors.forename: R W
authors.initials: RW
authors.lastname: Pickersgill
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D017931:DNA Primers / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics; D004563:Electrochemistry; D043364:Endo-1,4-beta Xylanases; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D011551:Pseudomonas fluorescens / Q000201:enzymology* / Q000235:genetics; D017386:Sequence Homology, Amino Acid; D014995:Xylosidases / Q000737:chemistry* / Q000235:genetics
chemical_list: D017931:DNA Primers; D004269:DNA, Bacterial; D014995:Xylosidases; D043364:Endo-1,4-beta Xylanases