Structure of the catalytic core of the family F xylanase from Pseudomonas fluorescens and identification of the xylopentaose-binding sites.
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.
Harris
G W
GW
Protein Engineering Department, Institute of Food Research, Reading Laboratory, UK.
Jenkins
J A
JA
Connerton
I
I
Cummings
N
N
Lo Leggio
L
L
Scott
M
M
Hazlewood
G P
GP
Laurie
J I
JI
Gilbert
H J
HJ
Pickersgill
R W
RW
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
D017931:DNA Primers; D004269:DNA, Bacterial; D014995:Xylosidases; D043364:Endo-1,4-beta Xylanases