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.