10.1021/bi100036f.s001
Stephan Reitinger
Stephan
Reitinger
Ying Yu
Ying
Yu
Jacqueline Wicki
Jacqueline
Wicki
Martin Ludwiczek
Martin
Ludwiczek
Igor D’Angelo
Igor
D’Angelo
Simon Baturin
Simon
Baturin
Mark Okon
Mark
Okon
Natalie C. J. Strynadka
Natalie
C. J. Strynadka
Stefan Lutz
Stefan
Lutz
Stephen G. Withers
Stephen G.
Withers
Lawrence P. McIntosh
Lawrence P.
McIntosh
Circular Permutation of <i>Bacillus circulans</i> Xylanase: A Kinetic and Structural Study
American Chemical Society
2010
Structural StudyThe 20 kDa Bacillus circulans Bcx
glycoside hydrolysis
permutant
NMR spectroscopy
circularized Bcx gene
Such approaches
residue
PCR methods
salt bridge contact
permutation
termini
circularly permuted
loop regions
Initial experiments
xylanase activity
Circular Permutation
DNase cleavage
circularly permuted xylanases
Bacillus circulans Xylanase
2010-03-23 00:00:00
Journal contribution
https://acs.figshare.com/articles/journal_contribution/Circular_Permutation_of_i_Bacillus_circulans_i_Xylanase_A_Kinetic_and_Structural_Study/2782441
The 20 kDa <i>Bacillus circulans</i> Bcx is a well-studied endoxylanase with a β-jellyroll fold that places its N- and C-termini in salt bridge contact. Initial experiments verified that Bcx could be circularly permuted by PCR methods to introduce new termini in loop regions while linking its native termini directly or via one or two glycines. Subsequently, a library of circular permutants, generated by random DNase cleavage of the circularized <i>Bcx</i> gene, was screened for xylanase activity on xylan in Congo Red-stained agar. Analysis of 35 unique active circular permutants revealed that, while many of the new termini were introduced in external loops as anticipated, a surprising number were also located within β-strands. Furthermore, several permutations placed key catalytic residues at or near the new termini with minimal deleterious effects on activity and, in one case, a 4-fold increase. The structure of one permutant was determined by X-ray crystallography, whereas three others were probed by NMR spectroscopy. These studies revealed that the overall conformation of Bcx changed very little in response to circular permutation, with effects largely being limited to increased local mobility near the new and the linked old termini and to a decrease in global stability against thermal denaturation. This library of circularly permuted xylanases provides an excellent set of new start points for directed evolution of this commercially important enzyme, as well as valuable constructs for intein-mediated replacement of key catalytic residues with unnatural analogues. Such approaches should permit new insights into the mechanism of enzymatic glycoside hydrolysis.