Crystal structure of a β-fructofuranosidase with high transfructosylation activity from Aspergillus kawachii

Figures & data

Table 1. Data collection and refinement statistics.

	Unliganded	Glycerol complex	Fru complex
Data collection
Beamline	PF-AR NW12A	PF-AR NW12A	PF-AR NW12A
Wavelength (Å)	1.0	1.0	1.0
Space group	P3121	P3121	P3121
Cell dimensions
a = b (Å)	108.8	108.8	108.4
c (Å)	153.2	152.0	152.9
Resolution range (Å)	50.0–2.30 (2.34–2.30)^a	47.1–2.10 (2.14–2.10)^a	50.0–2.10 (2.14–2.10)^a
Measured reflections	423,545	677,785	336,897
Unique reflections	47,061	61,305	61,268
Redundancy	9.0 (8.4)^a	11.1 (11.1)^a	5.5 (5.3)^a
Completeness (%)	99.9 (99.8)^a	99.9 (100)^a	99.8 (99.9)^a
<I/σ(I)>	24.5 (5.8)^a	53.6 (7.9)^a	36.1 (4.8)^a
Rmerge	0.093 (0.365)^a	0.076 (0.480)^a	0.076 (0.469)^a
Refinement
Rwork	0.189	0.171	0.179
Rfree	0.219	0.202	0.210
Root mean square deviation
Bond lengths (Å)	0.010	0.011	0.008
Bond angles (°)	1.396	1.426	1.307
Ramachandran plot (RAMPAGE)
Favored (%)	95.3	96.7	95.8
Allowed (%)	4.5	3.1	4.0
Outliers (%)	0.2	0.2	0.2
Number of atoms
Protein	4,656	4,664	4,664
Glycerol	–	36	–
Fru (1)–(4)	–	–	47
Acetate ion	–	8	–
Sodium ion	1	1	1
Water	186	342	300
Average B (Å^2)
Protein	36.8	33.8	39.1
Glycerol	–	45.3	–
Fru (1)–(2)	–	–	50.3
Fru (3)–(4)	–	–	76.7
Acetate ion	–	53.0	–
Sodium ion	27.1	25.2	27.3
Water	28.0	34.8	38.6
PDB ID	5XH9	5XH8	5XHA

^a The values for the highest-resolution shells are listed in parentheses.

Note: “–” Not applicable.

Figure 1. Enzymatic properties of AkFFase.

Notes: (A, B) TLC analysis of the reactions for sucrose (A) and fructose (B). Symbols: S, sucrose; F, fructose; K, 1-kestose; M, maltooligosaccharide markers; G1, glucose; G2, maltose; G3, maltotriose; G4, maltotetraose. Numbers indicate reaction times (min). Arrow represents the accumulation of 1-kestose. (C) Gel filtration chromatogram of AkFFase. (D) Determination of the molecular mass of AkFFase by gel filtration. The size markers (indicated by white circles) are: 1, blue dextran 2,000 (2,000 kDa); 2, thyroglobulin (669 kDa); 3, ferritin (440 kDa); 4, catalase (230 kDa); 5, bovine serum albumin (68 kDa); 6, ovalbumin (43 kDa). AkFFase is indicated by a black square.

Figure 2. Overall structure of AkFFase.

Notes: (A) A ribbon model of AkFFase-glycerol. Component NS (blue), a β-propeller domain (gray), an α-helical linker (orange), a C-terminal β-sandwich domain (magenta), a sodium ion (purple), and six glycerol molecules (cyan) are indicated. (B) Stereo view of component NS (blue) and the β-propeller domain of AkFFase. Five blades (I–V) are shown in cyan, green, yellow, orange, and red, respectively. Extra regions of blade V are indicated by red arrows. (C, D) Comparison of the domain composition of AkFFase (C) and MsFFase (D; PDB ID, 3WPU). The models are represented as Cα backbones. β-Propeller domains and other portions are shown in gray and magenta, respectively.

Table 2. Summary of structural similarity search using the DALI server.

Protein	PDB id	CAZy	Z-score	rmsd^a(Å)	Aligned residues	Sequence identity (%)
β-propeller domain
AjFFase	3LIG	GH32	44.0	1.1	412	67
Cellvibrio japonicus α-1,2-arabinofuranosidase	3QED	GH43	23.2	3.3	264	11
MsFFase	3WPY	GH68	20.2	2.5	278	15
Listeria innocua β-1,2-mannobiose phosphorylase	5B0R	GH130	19.5	3.4	260	10
Bacteroides plebeius anhydro-α-L-galactosidase	4AK6	GH117	18.8	3.9	255	15
Coprinopsis cinerea α-L-arabinofuranosidase	5B6S	GH62	18.6	3.6	251	13
C-terminal β-sandwich domain
AjFFase	3LIG	GH32^b	29.1	0.7	174	68
Aspergillus ficuum endo-inulinase	3SC7	GH32^b	18.9	2.5	150	15
Human thrombospondin-2 lectin-like protein	2RHP	–	14.1	2.7	141	9
Phaseolus vulgaris arcelin-1 lectin-like protein	1AVB	–	13.1	2.7	145	14
Lotus tetragonolobus fucose-specific lectin	2EIG	–	13.0	3.1	144	11
Arachis hypogaea galactose-binding lectin	1V6L	–	12.8	3.1	144	15

^a rmsd, root mean square deviation.

^b C-terminal β-sandwich domain is not the catalytic domain, therefore the CAZy classification of the catalytic domain is shown.

Note: “–” Not applicable.

Figure 3. Comparison of the Cα backbones of AkFFase and related GH32 enzymes.

Notes: (A) Stereo view of the superimposition of AkFFase (red), AjFFase (green; PDB ID, 3LF7), and XdFFase (cyan; PDB ID, 5ANN). (B) Stereo view of the superimposition of AkFFase (red) and Thermotoga maritima FFase (blue; PDB ID, 1UYP). (C) The predicted dimer structure of AkFFase composed of Mol-A and Mol-B. (D) Comparison of the dimer structures of AkFFase, XdFFase, and SoFFase. The Mol-A parts of AkFFase, XdFFase, and SoFFase are superimposed and shown in gray. The Mol-B parts of AkFFase (red), XdFFase (cyan), and SoFFase (orange) are shown.

Figure 4. Structure of the AkFFase-Fru complex.

Notes: (A) The tunnel at the bottom of the catalytic cleft of AkFFase. The route of the tunnel (cyan) was calculated using Caver. Blue balls indicate water molecules; magenta indicates the model of Fru-β-(2 → 1)-Fru bound at the catalytic cleft. (B) Stereo view of the positions of four Fru-binding sites (1)–(4) in AkFFase. The Fru molecules are shown in red. (C) The |F_o| − |F_c| omit maps for the four Fru molecules (1)–(4) at the 2.5 σ contoured level.

Figure 5. Schematic drawing of the amino acid residues interacting with Fru −1 and Fru +1.

Notes: Black circle, carbon atom; gray circle, nitrogen atom; red circle, oxygen atom; cyan circle, water molecule; green dashed line, hydrogen bond; red crown, hydrophobic interaction.

Table 3. Amino acid residues in the catalytic cleft of AkFFase and the corresponding residues in other clan GH-J enzymes.

Enzyme	Abbreviation	GH	PDB	Identity (%)	Subsite −1
A. kawachii FFase	AkFFase	32	5XH9	–	L82	F121	D122	R193	W370
A. japonicus FFase	AjFFase	32	3LF7	68	L78	F118	D119	R190	W398
X. dendrorhous FFase	XdFFase	32	5ANN	35	Q97	F145	D146	R220	W393
S. occidentalis FFase	SoFFase	32	3KF3	21	Q68	F110	S111	R178	W310
T. maritima FFase		32	1UYP	25	Q33	F74	S75	R137	W256
A. awamori exo-inulinase		32	1Y9G	21	Q57	F102	S203	R188	W331
Cicory fructan 1-exohydrolase		32	2ADD	18	Q38	W82	S83	R146	W292
M. saccharophilum FFase	MsFFase	68	3WPU	16	L135	W199	S200	R281	F470
B. subtilis levansucrase		68	1OYG	12	L109	W136	S164	R246	Y429
L. johnsonii inulosucrase		68	2YFT	15	M296	W339	S340	R424	Y619
Consensus					L/Q/M	F/W	D/S	R	W/F/Y

Enzyme	Catalytic			Subsite +1						Other key residue
A. kawachii FFase	D64	D194	E271	Y342	H310	I146	H147	W148	E296	Q61	G63	–	–
A. japonicus FFase	D60	D191	E292	Y369	H332	I143	H144	W145	E318	Q57	G59	–	–
X. dendrorhous FFase	D80	D221	E303	Y376	H343	L170	G171	A172	E334	W77	N79	W105	W314
S. occidentalis FFase	D50	D179	E230	Y293	N254	Q176	–	N227	Q228	W47	N49	W76	W335
T. maritima FFase	D17	D138	E190	Y240	N211	E101	–	E188	T208	W14	N16	W41	–
A. niger exo-inulinase	D41	D189	E241	Y313	N265	–	–	–	V239	W38	N40	W65	W260
Cicory fructan 1-exohydrolase	D22	D147	E201	Y274	G235	D143	–	–	–	W19	N21	F46	–
M. saccharophilum FFase	D112	D282	E374	Y457	H392	Y232	–	T395	Q372	R146	W111	H147	–
B. subtilis levansucrase	D86	D247	E342	Y411	R360	–	K363	K363	E340	R433	W85	–	–
L. johnsonii inulosucrase	D272	D425	E524	Y600	R542	–	N420	R544	E522	E269	W271	R623	–
Consensus	D	D	E	Y

Notes: Residues that are identical in AkFFase are underlined. Bars indicate no corresponding residues identified.

Figure 6. Structure of the catalytic cleft of AkFFase.

Notes: (A, B) Surface models of the catalytic clefts of SoFFase (A) and AkFFase (B). Fru −1 is shown in magenta. Some key amino acid residues are indicated. (C) Stereo view of the catalytic residues (red) and the IHW sequence (green) of AkFFase. Models of nystose (magenta) and lactosucrose (cyan) are placed on the structures.