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.

Biochemical and structural characterization of TLXI, the Triticum aestivum L. thaumatin-like xylanase inhibitor

“…The circular dichroism (CD) spectrum of TLXI (Figure 3) confirmed the absence of a-helices in the TLXI structure, much as noted earlier [23] for its recombinant counterpart as the characteristic dual minimum at 208 and 222 nm for a-helices was not seen. The spectrum shows a single minimum between 210 and 215 nm and an increase at 200 nm, typical for antiparallel b-sheets.…”

“…The 3D structural model of TLXI [23] shows that it consists of mainly of b-strands and that the a-helices, present in the thaumatin structure [26], are absent in TLXI (Figure 1). The alignment of the amino acid sequence of TLXI with that of thaumatin shows a deletion of 50 amino acids (Figure 2), corresponding to domain II of thaumatin which contains the ahelices.…”

“…This model was used on the one hand to support biochemical properties of TLXI (this report) and to determine key residues in the interaction of TLXI with xylanases [23]. Mutational studies of selected surface exposed residues revealed His22 of TLXI to play a critical role in the inhibition of a GH11 xylanase from Trichoderma longibrachiatum (XTL1) [23].…”

Crystal structure of the noncompetitive xylanase inhibitor TLXI, member of the small thaumatin‐like protein family

“…The inactive H22A TLXI mutant from T. aestivum was expressed and purified as described previously 15. The purified TLXI mutant was concentrated to 3.3 mg/mL in 25 m M sodium acetate (pH 5.0).…”

“…Mutagenesis studies of TLXI have pointed to the histidine at position 22 as being important for inhibition of xylanases since mutation of this residue to alanine abolished the inhibition activity of TLXI 15…”