Structural basis of the substrate specificity of the FPOD/FAOD family revealed by fructosyl peptide oxidase from Eupenicillium terrenum

Abstract

The FAOD/FPOD family of proteins has the potential to be useful for the longterm detection of blood glucose levels in diabetes patients. A bottleneck for this application is to find or engineer a FAOD/FPOD family enzyme that is specifically active towards α-fructosyl peptides but is inactive towards other types of glycated peptides. Here, the crystal structure of fructosyl peptide oxidase from Eupenicillium terrenum (EtFPOX) is reported at 1.9 Å resolution. In contrast to the previously reported structure of amadoriase II, EtFPOX has an open substrate entrance to accommodate the large peptide substrate. The functions of residues critical for substrate selection are discussed based on structure comparison and sequence alignment. This study reveals the first structural details of group I FPODs that prefer α-fructosyl substrates and could provide significant useful information for uncovering the mechanism of substrate specificity of FAOD/FPODs and guidance towards future enzyme engineering for diagnostic purposes.

Keywords: Eupenicillium terrenum; FAOD/FPOD family; fructosyl peptide oxidase.

Figure 1

Structure-guided sequence alignment of FAOD/FPODs. The secondary-structure elements in the structures of EtFPOX (above) and amadoriase II (below) are indicated. Residues are coloured according to their conservation. The sequences are classified into three groups according to their substrate specificity (Ferri et al., 2009; Lin & Zheng, 2010 ▶). Sequence alignment was performed and presented using ClustalX (Larkin et al., 2007 ▶) and ESPript3 (Robert & Gouet, 2014 ▶). The sequences used for alignment are EtFPOX from E. terrenum ATCC 18547 (BAD00185; Hirokawa, Gomi & Kajayima, 2003 ▶), PnFPOX from Phaeosphaeria nodorum SN15 (XP_001798711; Kim, Ferri et al., 2010A), FPOX-C from Coniochaeta sp. NISL 9330 (BAD00186; Hirokawa, Gomi & Kajayima, 2003 ▶), FAOD-P from Penicillum janthinelum AKU 3413 (CAA70219; Yoshida et al., 1995, 1996 ▶), FAOD-U from Ulocladium sp. JS-103 (BAE93140; Fujiwara et al., 2006 ▶), N1-1 FAOD from Pichia sp. N1-1 (AAP83789; Ferri et al., 2004 ▶), FAOD-Ao2 from A. oryzae (BAE58870; Akazawa et al., 2004 ▶), amadoriase II from A. fumigatus (AAC49711; Collard et al., 2008; Takahashi et al., 1997a ,b ▶), FAOD-F from Fusarium oxysporum NBRC 9972 (Fujiwara et al., 2007 ▶), amadoriase I from A. fumigatus (AAB88209; Takahashi et al., 1997a ,b ▶) and FAOD-Ao1 from A. oryzae (BAD54824; Akazawa et al., 2004 ▶).

Figure 2

Stereoview of structure comparison of EtFPOX (green) and amadoriase II (magenta). The fragments Gln91–Gly157 in EtFPOX and Asp93–Ala155 in amadoriase II (loop A1 and loop A2), the conformations of which differ in these two enzymes, are shown in cartoon mode. The other peptide chains, which superimpose well, are shown as lines. FAD and FSA molecules are shown as sticks.

Figure 3

Electrostatic surface diagrams of (a) EtFPOX, (b) the FSA-bound form of amadoriase II and (c) free amadoriase II viewed from the top of the substrate entrance. An FSA molecule shown in stick representation is placed into the active site of EtFPOX and free amadoriase II in the same position as in the FSA-bound structure of amadoriase II, in which the active pocket is completely covered by loop A1, loop A2 and helix 3. (d) The same as in (b) but the surface is shown with transparency to show FSA; loop A1, loop A2 and helix 3 are coloured green, magenta and yellow, respectively.

Figure 4

Comparison of the active-site residues of (a) EtFPOX, in which an FSA molecule is present as in amadoriase II, and (b) amadoriase II bound to FSA. Residues involved in substrate binding and critical for activity are labelled. FSA and FAD are shown as stick models coloured cyan and grey, respectively.