His22 of TLXI plays a critical role in the inhibition of glycoside hydrolase family 11 xylanases

Abstract

Recently, a novel wheat thaumatin-like protein, TLXI, which inhibits microbial glycoside hydrolase family (GH) 11 xylanases has been identified. It is the first xylanase inhibitor that exerts its inhibition in a non-competitive way. In the present study we gained insight into the interaction between TLXI and xylanases via combined molecular modeling and mutagenic approaches. More specifically, site-specific mutation of His22, situated on a loop which distinguishes TLXI from other, non-inhibiting, thaumatin-like proteins, and subsequent expression of the mutant in Pichia pastoris resulted in a protein lacking inhibition capacity. The mutant protein was unable to form a complex with GH11 xylanases. Based on these findings, the interaction of TLXI with GH11 xylanases is discussed.

Keywords::

• Thaumatin-like protein
• xylanase inhibitor
• protein–protein interaction
• site-directed mutagenesis
• Pichia pastoris

Acknowledgements

The authors gratefully acknowledge financial support from the “Fonds voor Wetenschappelijk Onderzoek-Vlaanderen” (F.W.O.-Vlaanderen, Belgium) (post-doctoral fellowship K. Gebruers, A. Rabijns and S. Van Campenhout), the “Instituut voor de aanmoediging van Innovatie door Wetenschap en Technologie in Vlaanderen” (I.W.T., Brussels, Belgium) (Scholarship to S. Rombouts, E. Fierens and A. Voet, Xylafun GBOU project funding) and the Research Fund K.U. Leuven (post-doctoral fellowship to J. Beaugrand and S. Van Campenhout). We thank E. Lorent for the helping hand with CD.