Crystal structure of a natural circularly-permuted jellyroll protein: 1,3-1,4-b-D-glucanase from Fibrobacter succinogenes

Li-Chu Tsai^1,2, Lie-Fen Shyur³, Shu-Hua Lee³, Su-Shiang Lin³ and Hanna S. Yuan^1*

¹Institute of Molecular Biology, Academia Sinica, Taipei, Taiwan, ROC.

²Department of Molecular Science and Engineering, National Taipei University of Technology, Taipei, Taiwan, ROC.

³Institute of BioAgricultural Sciences, Academia Sinica, Taipei, Taiwan, ROC.

Abstract

The 1,3-1,4-b-D-glucanase from Fibrobacter succinogenes (Fsb-glucanase) is classified as one of the family 16 glycosyl hydrolases.  It hydrolyzes the glycosidic bond in the mixed-linked glucans containing b-1,3- and b-1,4-glycosidic linkages.  We constructed a truncated form of recombinant Fsb-glucanase containing the catalytic domain from amino acid residues 1 to 258, which exhibited a higher thermal stability and enzymatic activity than the full-length enzyme.  The crystal structure of the truncated Fsb-glucanase was solved at a resolution of 1.7 Å by the multiple wavelength anomalous dispersion (MAD) method using the anomalous signals from the seleno-methionine-labeled protein.  The overall topology of the truncated Fsb-glucanase consists mainly of two eight-stranded anti-parallel b-sheets arranged in a jellyroll b-sandwich, similar to the fold of many glycosyl hydrolases and carbohydrate-binding modules.  Sequence comparison with other bacterial glucanases showed that Fsb-glucanase is the only naturally-occurring circularly-permuted b-glucanase with reversed sequences.  Structural comparison shows that the engineered circular-permuted Bacillus enzymes are more similar to their parent enzymes with which they share ~70 % sequence identity, than to the naturally-occurring Fsb-glucanase of similar topology with 30 % identity.  This result suggests that protein structure relies more on sequence identity than topology.  The high-resolution structure of Fsb-glucanase provides a structural rationale for the different activities obtained from a series of mutant glucanases and a basis for the development of engineered enzymes with increased activity and structural stability.

http://www.idealibrary.com on J. Mol. Biol. 330, 607-620 (2003)