Crystal structure determination and inhibition studies of a novel xylanase and α-amylase inhibitor protein (XAIP) from Scadoxus multiflorus.

A novel plant protein isolated from the underground bulbs of Scadoxus multiflorus, xylanase and α-amylase inhibitor protein (XAIP), inhibits two structurally and functionally unrelated enzymes: xylanase and α-amylase. The mature protein contains 272 amino acid residues which show sequence identities of 48% to the plant chitinase hevamine and 36% to xylanase inhibitor protein-I, a double-headed inhibitor of GH10 and GH11 xylanases. However, unlike hevamine, it is enzymatically inactive and, unlike xylanase inhibitor protein-I, it inhibits two functionally different classes of enzyme. The crystal structure of XAIP has been determined at 2.0 A resolution and refined to Rcryst and Rfree factors of 15.2% and 18.6%, respectively. The polypeptide chain of XAIP adopts a modified triosephosphate isomerase barrel fold with eight β-strands in the inner circle and nine α-helices forming the outer ring. The structure contains three cis peptide bonds: Gly33–Phe34, Tyr159–Pro160 and Trp253–Asp254. Although hevamine has a long accessible carbohydrate-binding channel, in XAIP this channel is almost completely filled with the side-chains of residues Phe13, Pro77, Lys78 and Trp253. Solution studies indicate that XAIP inhibits GH11 family xylanases and GH13 family α-amylases through two independent binding sites located on opposite surfaces of the protein. Comparison of the structure of XAIP with that of xylanase inhibitor protein-I, and docking studies, suggest that loops α3–β4 and α4–β5 may be involved in the binding of GH11 xylanase, and that helix α7 and loop β6–α6 are suitable for the interaction with α-amylase.