Crystal Structure and Substrate Specificity Modification of Acetyl Xylan Esterase from Aspergillus luchuensis

ABSTRACT

Acetyl xylan esterase (AXE) catalyzes the hydrolysis of the acetyl bonds present in plant cell wall polysaccharides. Here, we determined the crystal structure of AXE from Aspergillus luchuensis (AlAXEA), providing the three-dimensional structure of an enzyme in the Esterase_phb family. AlAXEA shares its core α/β-hydrolase fold structure with esterases in other families, but it has an extended central β-sheet at both its ends and an extra loop. Structural comparison with a ferulic acid esterase (FAE) from Aspergillus niger indicated that AlAXEA has a conserved catalytic machinery: a catalytic triad (Ser119, His259, and Asp202) and an oxyanion hole (Cys40 and Ser120). Near the catalytic triad of AlAXEA, two aromatic residues (Tyr39 and Trp160) form small pockets at both sides. Homology models of fungal FAEs in the same Esterase_phb family have wide pockets at the corresponding sites because they have residues with smaller side chains (Pro, Ser, and Gly). Mutants with site-directed mutations at Tyr39 showed a substrate specificity similar to that of the wild-type enzyme, whereas those with mutations at Trp160 acquired an expanded substrate specificity. Interestingly, the Trp160 mutants acquired weak but significant type B-like FAE activity. Moreover, the engineered enzymes exhibited ferulic acid-releasing activity from wheat arabinoxylan.

IMPORTANCE Hemicelluloses in the plant cell wall are often decorated by acetyl and ferulic acid groups. Therefore, complete and efficient degradation of plant polysaccharides requires the enzymes for cleaving the side chains of the polymer. Since the Esterase_phb family contains a wide array of fungal FAEs and AXEs from fungi and bacteria, our study will provide a structural basis for the molecular mechanism of these industrially relevant enzymes in biopolymer degradation. The structure of the Esterase_phb family also provides information for bacterial polyhydroxyalkanoate depolymerases that are involved in biodegradation of thermoplastic polymers.

INTRODUCTION

Acetyl xylan esterase or acetylxylan esterase (AXE; EC 3.1.1.72) hydrolyzes ester linkages of acetic acid from acetylated hemicelluloses in the plant cell wall (1–3). Various types of xylans and other hemicelluloses are often acetylated (4, 5), and AXEs are involved in the microbial degradation system of the plant polysaccharides along with other hemicellulases. For example, the complete degradation of glucuronoarabinoxylan requires both enzymes for cleaving the main chain and side chains of the polymer (6), such as endo-β-1,4-xylanases (EC 3.2.1.8), β-xylosidases (EC 3.2.1.37), AXEs, feruloyl esterases (FAEs; EC 3.1.1.73) (7), α-l-arabinofuranosidases (EC 3.2.1.55), and α-glucuronidases (EC 3.2.1.139). Most of the carboxylesterases adopt the α/β-hydrolase fold as a versatile scaffold and have a Ser-His-Asp(Glu) triad or a Ser-His dyad in their active sites for the catalytic mechanism using Ser as the nucleophile (8). The ESTHER database is dedicated to the α/β-hydrolase fold enzymes and classifies more than 30,000 proteins into 87 families (9). In ESTHER, AXEs are classified into six families (Abhydrolase_7, Acetyl-esterase_deacetylase, Acetylxylan_esterase, Antigen85c, Esterase_phb, and Polyesterase-lipase-cutinase). CAZy (Carbohydrate-Active enZYmes) is also a reliable database dedicated to enzymes and binding modules acting on glycosidic bonds or ester linkages of carbohydrates (10). In CAZy, AXEs belong to the carbohydrate esterase (CE) families 1 to 7 and 16 (5, 11), and the CE1 to CE7 family enzymes adopt the α/β-hydrolase fold.

We previously reported gene cloning and characterization of AXEs from Aspergillus luchuensis (AlAXEA) (12, 13) and Aspergillus oryzae (AoAXE) (14), which belong to the Esterase_phb (ESTHER) and CE1 (CAZy) families. Notably, A. luchuensis was formerly designated A. awamori (15). The CE1 family in CAZy classifies a wider array of enzymes than those in ESTHER because CE1 contains both the Esterase_phb and the Antigen85c family enzymes, which share a very low or barely detectable sequence homology (identity, <20%). The three-dimensional structures of esterases belonging to Antigen85c in CE1 (mostly from bacteria) have been extensively studied (16–18), whereas the crystal structure of Esterase_phb family members is not yet available. The Esterase_phb family consists mainly of fungal AXEs (12, 14, 19, 20), fungal FAEs (21–27), bacterial FAEs (28), and bacterial polyhydroxyalkanoate (PHA) depolymerases (29, 30), and they form separate clades in the molecular phylogenetic tree (Fig. 1). In the recent classification system of fungal FAEs, the Esterase_phb enzymes are classified into the SF6 family, which constitutes the largest number of proteins among the 13 SF families (31). In this study, we report the crystal structure of AlAXEA, providing a three-dimensional view of the Esterase_phb family of enzymes. A structural comparison with fungal FAEs and a mutational analysis located a possible binding pocket for the acetyl group of the substrate and enabled endowing FAE activity to AlAXEA by widening the pocket.

FIG 1.

Phylogenetic tree of characterized enzymes in the Esterase_phb family. Labels are IDs of the ESTHER database except for AlAXEA, AoAXE, PfFAEB, NcFAE1, CQ31-FAE (FAE from Chaetomium sp. CQ31) (26), and ClFaeB2 (FaeB2 from Chrysosporium lucknowense C1) (25). Note that the source organism of pireq-faeb (Piromyces equi) is a fungus, but the protein is similar to modular esterases from bacteria (Pseudomonas species). GenBank/UniProt accession numbers: AlAXEA, Q92194; aspnc-axe1, XP_001395572; penpu-AXEI, Q8NJP6; AoAXE, XP_001826329; PfFAEB, Q9HE18; talce-faeB, AB937136; NcFAE1, Q9HGR3; CQ31-FAE, AFU88756; ClFaeB2, XP_003667253; pireq-faeb, Q9Y871; psele-pha1, P52090; alcfa-PHAZ, P94146; psele-Q9L7K1, Q9L7K1; psele-PHAZ4, Q51870; celju-b3pei5, B3PEI5; psele-PHAZ5, Q51871; marsp-PHAZ, Q8GRC2; psesp-PHBDP1, AAG00611; psele-PHAZ3, P72207; psele-PHAZ2, P72206. Bar, 5% sequence divergence.

RESULTS

Crystal structure.

The recombinant protein of AlAXEA was produced by the methylotrophic yeast Pichia pastoris. The molecular masses of the purified native AlAXEA protein (275-amino-acid [aa] mature region without the 29-aa signal sequence) as deduced from the amino acid sequence and estimated by SDS-PAGE and calibrated gel filtration chromatography were 29.5, 30, and 65 kDa, respectively, suggesting that this protein is dimeric in solution. Since the AlAXEA protein showed high solubility, the diffraction quality single crystals were obtained only when we set up the crystallization drop using protein solutions at concentrations higher than 66 mg/ml. In the amino acid sequence of the mature protein, Asn161 and Asn239 were predicted to be potential N-glycosylation sites. Treatment by α-mannosidase in the purification steps did not change the band size on SDS-PAGE, nor did it improve the diffraction quality of the crystals (data not shown). Therefore, we used α-mannosidase-untreated protein samples for the crystallography. The crystal structure of AlAXEA was solved by the single-wavelength anomalous dispersion method using a selenomethionine derivative, and a native structure was determined at 1.90-Å resolution (Table 1). Of note, molecular replacement trials using BALBES and MrBUMP programs, which comprehensively search the Protein Data Bank for templates, did not solve the structure.

TABLE 1.

Data collection and refinement statistics

Category and characteristic (unit)	SeMet AlAXEA	Native AlAXEA
Data collection
PDB entry		5X6S
Wavelength (Å)	0.9786	1.0000
Space group	R3 (H3)	R3 (H3)
Unit cell (Å)	a = b = 175.1 c = 51.1	a = b = 174.2; c = 50.7
Resolution (Å)a	50.00–2.20 (2.28–2.20)	87.08–1.90 (1.97–1.90)
Total no. of reflections	344,924	144,771
No. of unique reflections	59,332 (5,990)b	44,285 (4,308)
Completeness (%)a	100.0 (100.0)	98.1 (95.5)
Redundancya	5.8 (5.7)	3.3 (3.0)
Mean I/σ (I)a	31.8 (4.8)	16.4 (2.5)
Rmerge (%)a	5.8 (32.2)	7.1 (35.9)
Refinement
Resolution (Å)		87.08–1.90
No. of reflections		42,045
R factor/Rfree (%)		15.1/18.5
No. of atoms		4,754
RMSD from ideal values
Bond lengths (Å)		0.016
Bond angles (°)		1.52
Average B factor (Å2)
Protein (chain A/B)		21.5/22.1
GlcNAc (chain A/B)		35.8/29.8
MES (chain A/B)		21.1/20.4
Na+		29.8
Water		34.0
Ramachandran plot (%)
Favored		97.8
Allowed		2.2
Outlier		0.0

^aValues for highest-resolution shell are given in parentheses.

^bBijvoet pairs are counted separately.

The crystal contains two molecules in the asymmetric unit, and the final model contains residues Gly2 to Ala275 of both chains (amino acids are numbered for the mature protein). The dimer structure of AlAXEA is shown in Fig. 2A. The two molecules in the asymmetric unit are virtually the same, and the root mean square deviation (RMSD) for the Cα atoms between chains A and B is 0.18 Å. We will describe chain A unless otherwise noted. An N-linked glycosylation site with two GlcNAc residues was observed at Asn161 (Fig. 2C), and there are two disulfide bonds (Cys40-Cys75 and Cys149-Cys164). According to molecular interface analysis using the PDBePISA server (32), the dimer interface shown in Fig. 2A is predicted to be the most likely interface in solution among the crystallographic contacts. The interface area is 620 Ų and includes 6 hydrogen bonds and 4 salt bridges, and the estimated ΔⁱG value (solvation free energy gain upon formation of the interface) is −5.4 kcal/mol. The monomer structure of AlAXEA is shown in Fig. 2B. AlAXEA consists of a single domain adopting a typical α/β-hydrolase fold, in which a 10-stranded β-sheet is sandwiched by 8 α-helices and one 3₁₀ helix (Fig. 2D). As an additional structural element, there is a prominent loop connecting β6 and α5 (β6-α5 loop). The catalytic triad comprises Ser119, Asp202, and His259, and these residues are connected by two hydrogen bonds, as is commonly observed in serine hydrolases (33). The catalytic triad and four Cys residues forming the two disulfide bonds are completely conserved in fungal AXEs and FAEs in the Esterase_phb family (see Fig. S1 in the supplemental material).

FIG 2.

Crystal structure of AlAXEA. (A) Dimer structure. Chain A polypeptide is shown in rainbow colors, and chain B is shown in gray. (B) Monomer structure. The N and C termini are indicated. In panels A and B, N-glycans (cyan), the catalytic triad (magenta), and disulfide bonds (sulfur atoms in yellow and marked with red circles) are shown as sticks. To display the disulfide bond between Cys149 and Cys164, helix α5 is shown as transparent. (C) GlcNAc residues of the N-glycan at Asn161 of chain A with the mF_o-DF_c omit map (4.0σ). The β-Man residue linked to the C-4 atom of the distal GlcNAc residue was not clearly visible. (D) Topological diagram of secondary structure elements. The conserved core fold and β6-α5 loop are boxed with red and green dashed lines, respectively.

Structural homologs.

A database search using the Dali server revealed that AlAXEA displays structural similarities to those of esterases belonging to Abhydrolase_5, LYsophospholipase_carboxylesterase, AlphaBeta_hydrolase, and Antigen85c families (Z scores, >18.6; sequence identities, <22%) (Table 2). As shown in Fig. S2 in the supplemental material, these enzymes share a central part of the α/β-hydrolase fold, which contains a β-sheet with 7 strands flanking 9 helices (boxed in a red dotted line in Fig. 2D). In other words, the three flanking β-strands (β1, β8, and β9) and the β6-α5 loop are uniquely present in AlAXEA. AlAXEA also displays structural similarities to other enzymes having the α/β-hydrolase fold. The structurally known CE1 enzymes in CAZy, which all belong to the Antigen85c family in ESTHER, show relatively lower structural similarity to AlAXEA (Z scores, <17.8). AmCE1 from Anaeromyces mucronatus (34) showed the highest structural similarity among these enzymes, but it exhibits only 12% amino acid sequence identity to AlAXEA (Table 2).

TABLE 2.

Result of Dali structural similarity search using the AlAXEA structure (chain A) as a template

Protein name	ESTHER rank 1 family	PDB ID (chain)	Z score	RMSD (Å)	LALIa	%IDb
Esterase LC-Est1C from metagenome	Abhydrolase_5	3WYD (B)	20.1	2.1	175	22
Lysophospholipase-like 1 from Homo sapiens	LYsophospholipase_carboxylesterase	5KRE (A)	19.1	2.5	192	15
EstA from Thermotoga maritima	Abhydrolase_5	3DOH (A)	19.0	2.5	192	19
Acyl protein thioesterase from Homo sapiens	LYsophospholipase_carboxylesterase	1FJ2 (A)	19.0	2.4	193	16
PVA hydrolase from Sphingopyxis sp. 113P3	AlphaBeta_hydrolase	3WLA (A)	18.8	2.6	208	17
Est12 from metagenome	Antigen85c	4RGY (B)	18.7	3.3	205	17
AmCE1 from Anaeromyces mucronatus (CE1 top hit)	Antigen85c	5CXX (C)	17.7	3.1	202	12

^aNumber of aligned residues.

^bSequence identity.

Comparison with type A FAE.

Attempts at cocrystallization and soaking experiments with the products, substrates, and analogs (e.g., acetic acid, xylose, and xylooligosaccharides) were undertaken, but no electron densities were found in the active site. Therefore, the substrate-binding pocket of AlAXEA was estimated by comparison with the structure of FaeA (or FAE-III) from Aspergillus niger (AnFaeA), which is one of the best-characterized FAEs belonging to the Lipase_3 (ESTHER) and the SF7 (fungal FAE classification system) families (not listed in CAZy) (35). The substrate specificities of FAEs have been classified into four types (types A to D) based on the activities against four model substrates, methyl p-coumarate (MpCA), methyl caffeate (MCA), methyl ferulate (MFA), and methyl sinapate (MSA), as well as the releasing ability of diferulic acid (see Fig. S3 in the supplemental material) (36). In this functional classification, AnFaeA is a type A FAE (active on MFA, MSA, MpCA, and diferulic acid). Figure 3 shows the superposition of AlAXEA with AnFaeA complexed with ferulic acid (PDB ID 1UWC) (37). Although the overall structural similarity between these enzymes is very low (Dali score, <10), the catalytic triad (Ser133, His247, and Asp194 in AnFaeA) and residues forming the oxyanion hole (Leu134 and Thr68 in AnFaeA) can be superimposed. A local region around the catalytic serine and central β-strands of the α/β-hydrolase fold was also structurally aligned. In AnFaeA, the ferulic acid molecule forms hydrogen bonds with the main-chain nitrogen atoms of Leu134 and Thr68 and the side chain hydroxyl of Tyr80, and its phenolic group is surrounded by Asp77, Ile196, and Leu199. The catalytic elements of AlAXEA (the catalytic triad and oxyanion hole formed by Ser120 and Cys40) have locations similar to those of AnFaeA. The putative binding pocket of AlAXEA is narrowed by the side chains of Trp160 and Phe150. These observations indicate that the small pocket formed by Trp160 may bind the acetyl group. Tyr39 provides an aromatic platform at a position similar to that of Tyr25 in AnFaeA, and it possibly binds a xylose moiety of arabinoxylan.

FIG 3.

Stereo view of a comparison between AlAXEA (green) and AnFaeA (pale blue, PDB ID 1UWC). The catalytic residues, substrate-binding pocket residues, and ferulic acid (FA) in AnFaeA (yellow) are shown as sticks. The catalytic triad residues of AlAXEA and residues of AnFaeA are indicated by red and blue labels, respectively. Hydrogen bonds between the FA molecule and AnFaeA are shown as yellow dashed lines. The two structures were superimposed by fitting the Cα atoms of the catalytic Ser, His, and residues forming the oxyanion hole.

Homology modeling and substrate binding pocket.

Fungal FAEs in the Esterase_phb family show moderate sequence homology to AlAXEA (identity, ∼40%) with a few insertions/deletions (Fig. S1). Therefore, we were able to confidently build homology models of FAEs from Neurospora crassa (NcFAE1; global model quality estimation [GMQE], 0.76) (23) and Penicillium funiclosum (PfFAEB; GMQE = 0.68) (22), using the Swiss-Model server (38) with the crystal structure of AlAXEA as a template. Figure 4 shows a comparison of the molecular surface around the catalytic site of AlAXEA with those of the homology-modeled FAE structures. AlAXEA has a small pocket (surface-accessible volume, 7.8 ų) near the catalytic Ser119 and Trp160 (red circle in Fig. 4A), whereas the FAEs have a large pocket at the corresponding position. This is because Trp160 in AlAXEA is replaced with Pro and Ser in NcFAE1 (Fig. 4B) and PfFAEB (Fig. 4C), respectively (Fig. S1). The conserved disulfide bond (Cys40-Cys75 in AlAXEA) is located near the binding pocket. On the other side of the catalytic Ser, there is also a wide and shallow pocket in FAEs by replacement of Tyr39 in AlAXEA with Gly or Pro in FAEs.

FIG 4.

Molecular surfaces of AlAXEA (A, crystal structure), NcFAE1 (B, homology model), and PfFAEB (C, homology model). The catalytic triad, conserved disulfide bond, and aromatic residues forming the pockets are colored magenta, green, and yellow, respectively. Aromatic residues near the pockets of FAEs are colored gray. The putative binding pockets for the acetyl or ferulate groups are indicated by red circles. Surface-accessible area and volume calculated by the CASTp server (57) are indicated.

Mutational analysis.

To examine the roles of Tyr39 and Trp160 in the substrate specificity of AlAXEA, we constructed single and double mutants with site-directed mutations at these positions by replacing the residues with Ala or those in the FAEs in Esterase_phb (Gly, Pro, or Ser). Table 3 shows the activities of the wild-type and mutant enzymes toward α-naphthyl esters with different chain lengths (C₂ to C₁₀). The wild-type enzyme showed a strong preference for α-naphthyl acetate (C₂), as previously observed (13). All mutants showed a significant decrease in the basal activity for the C₂ substrate, the effect being more intense in the Trp160 mutants (<1.8 U/mg) than in the Tyr39 mutants (<5.1 U/mg). The wild-type enzyme exhibited less than one-half of the activity for α-naphthyl propionate (C₃) compared with the C₂ substrate and no detectable activity for the larger substrates α-naphthyl butyrate (C₄), octanoate (C₈), and decanoate (C₁₀). In contrast, FAEs typically show higher activity with the C₄ substrate than the smaller ones, as exemplified in the case of FAE A from A. luchuensis (AlFAEA) (39). The Tyr39 single mutants showed substrate specificity patterns similar to those of the wild-type AlAXEA, whereas the Trp160 mutants exhibited an FAE-like specificity, indicating that the pocket near Trp160 dictates the substrate specificity of AlAXEA. The activities of W160P, W160S, and W160A toward the C₃ substrate were slightly higher than those toward the C₂ substrate, and W160S and W160A mutants showed higher activity toward the C₄ substrate than did the W160P mutant. The double mutants (Y39G/W160P and Y39P/W160S mutants) were constructed to mimic the active sites of NcFAE1 and PfFAEB, as described above. However, the double mutants did not exhibit a higher activity to the C₄ substrate than did the single mutants, W160S and W160A mutants. All enzymes tested in this study showed no or barely detectable activities toward the substrates larger than C₈ substrates.

TABLE 3.

Activities toward 0.5 mM α-naphthyl esters^a

Enzyme	Sp act (U/mg)
	C2 (acetate)	C3 (propionate)	C4 (butyrate)	C8 (octanoate)
Wild type	21.9 ± 1.7	9.3 ± 0.3	NDb	ND
Y39G mutant	2.7 ± 0.2	0.87 ± 0.06	ND	0.03 ± 0.01
Y39P mutant	5.1 ± 0.2	1.5 ± 0.02	ND	ND
Y39A mutant	5.1 ± 0.3	1.6 ± 0.3	ND	ND
W160P mutant	1.1 ± 0.4	1.7 ± 0.2	0.31 ± 0.09	ND
W160S mutant	1.0 ± 0.1	1.5 ± 0.1	0.85 ± 0.09	0.05 ± 0.04
W160A mutant	1.1 ± 0.1	1.6 ± 0.1	0.84 ± 0.17	0.07 ± 0.06
Y39G/W160P mutant	1.8 ± 0.1	0.88 ± 0.06	0.59 ± 0.09	ND
Y39P/W160S mutant	0.49 ± 0.02	0.59 ± 0.06	0.04 ± 0.01	0.02 ± 0.01
AlFAEAc	1.3	1.4	3.1	0.070

^aActivities were measured in 85 mM potassium phosphate buffer (pH 7.0) at 37°C. Activity for α-naphthyl decanoate (C₁₀) was not detected for all enzymes.

^bND, not detected.

^cData from Koseki et al. (39).

The fungal FAEs in the Esterase_phb family, including NcFAE1 and PfFAEB, generally have the type B specificity (22–25), being active on MpCA, MCA, and MFA but not on MSA (Fig. S3). Table 4 shows the hydrolytic activities toward methyl hydroxycinnamates. Although the wild-type AlAXEA and Tyr39 single mutants did not hydrolyze the hydroxycinnamic acid substrates, mutations at Trp160 endowed the enzyme with weak but significant activity. The activities of the double mutants toward MFA were the highest among them (0.020 and 0.015 U/mg), but they were much lower than those of the canonical FAEs (>8 U/mg). We were not able to confidently determine the substrate specificity type of FAE of these mutants, but the relatively lower activities toward MSA indicate that they all show type-B-like specificity. The kinetic parameters of these mutants toward MFA were measured (Table 5). The K_m and k_cat values of the mutants were largely higher and lower, respectively, than those of the canonical FAEs. Finally, we measured ferulic acid-releasing activities from insoluble arabinoxylan (Table 6). Trp160 mutants showed activity, whereas other enzymes did not. The addition of xylanase significantly increased the ferulic acid-releasing activity from the polymer (>6-fold), indicating that the engineered FAE activity from AlAXEA also exhibits synergy with the main-chain-cleaving enzyme (xylanase) as observed for recombinant AoAXE (14). The Y39P/W160S mutant, which mimicked PfFAEB, exhibited higher activity than the single mutants, but the activity did not reach the level of a canonical FAE, AlFAEA.

TABLE 4.

Activities toward methyl esters of 1 mM methyl hydroxycinnamates^a

Enzyme	Sp act (U/mg)
	MpCA	MCA	MFA	MSA
Wild-type	ND	ND	ND	ND
Y39G mutant	ND	ND	ND	ND
Y39P mutant	ND	ND	ND	ND
Y39A mutant	ND	ND	ND	ND
W160P mutant	6.9 × 10−4 ± 0.5 × 10−4	1.8 × 10−4 ± 0.1 × 10−4	1.7 × 10−3± 0.1 × 10−3	1.2 × 10−4 ± 0.1 × 10−4
W160S mutant	1.1 × 10−3 ± 0.1 × 10−3	1.6 × 10−3 ± 0.03 × 10−3	5.7 × 10−3 ± 0.2 × 10−3	3.7 × 10−4 ± 0.2 × 10−4
W160A mutant	1.8 × 10−3 ± 0.2 × 10−3	5.5 × 10−4 ± 0.1 × 10−4	3.9 × 10−3 ± 0.2 × 10−3	1.7 × 10−4± 0.1 × 10−4
Y39G/W160P mutant	4.7 × 10−3 ± 0.4 × 10−3	1.4 × 10−2 ± 0.1 × 10−2	2.0 × 10−2 ± 0.1 × 10−2	2.3 × 10−3 ± 0.4 × 10−3
Y39P/W160S mutant	3.3 × 10−3 ± 0.6 × 10−3	8.7 × 10−3± 0.2 × 10−3	1.5 × 10−2 ± 0.04 × 10−2	1.5 × 10−3 ± 0.1 × 10−3
NcFAE1b	20.9	8.2	8.97	ND
PfFAEBc	36.1	31.7	22.9	ND

^aActivities were measured in 95 mM potassium phosphate buffer (pH 7.0) at 37°C. ND, not detected.

^bData from Crepin et al. (23).

^cData from Kroon et al. (22).

TABLE 5.

Kinetic parameters toward MFA^d

Enzyme	kcat (s−1)	Km (mM)	kcat/Km (s−1 mM−1)
W160P mutanta		>10	4.2 × 10−4 ± 0.3 × 10−4
W160S mutanta		>10	1.4 × 10−3 ± 0.5 × 10−3
W160A mutant	8.4 × 10−3 ± 1.1 × 10−3	8.3 ± 0.2	1.0 × 10−3 ± 0.4 × 10−3
Y39G/W160P mutant	3.2 × 10−2 ± 0.5 × 10−2	8.0 ± 2.0	4.0 × 10−3 ± 0.2 × 10−3
Y39P/W160S mutant	3.1 × 10−2 ± 0.3 × 10−2	6.6 ± 1.2	4.8 × 10−3 ± 0.1 × 10−3
NcFAE1b	5.2	0.25	21
PfFAEBc	16	0.047	340

^aThe K_m and k_cat values could not be confidently determined due to the solubility limits of the substrate (10 mM).

^bData from Crepin et al. (23).

^cData from Kroon et al. (22).

^dActivities were measured in 95 mM potassium phosphate buffer (pH 7.0) at 37°C.

TABLE 6.

Ferulic acid-releasing activities toward 1% feruloylated insoluble wheat arabinoxylan^c

Enzyme	Sp act (mU/mg)a
	After 24 h	After addition of xylanase (1 h)b
Wild type	ND	ND
Y39G mutant	ND	ND
Y39P mutant	ND	ND
Y39A mutant	ND	ND
W160P mutant	0.019 ± 0.001	0.20 ± 0.01
W160S mutant	0.055 ± 0.01	0.94 ± 0.46
W160A mutant	0.029 ± 0.01	0.44 ± 0.01
Y39G/W160P mutant	0.010 ± 0.007	0.060 ± 0.02
Y39P/W160S mutant	0.12 ± 0.02	1.5 ± 0.2
AlFAEA	7.5 ± 0.57	319 ± 21

^aCalculated from released ferulic acid after indicated time of incubation. ND, not detected.

^bXylanase from Thermomyces lanuginosus (10 μg/ml) was added.

^cActivities were measured in 50 mM potassium phosphate buffer (pH 7.0) at 37°C.

DISCUSSION

Homology modeling of PHA depolymerase.

In the PHA depolymerase engineering database (DED), enzymes belonging to the Esterase_phb family are classified as extracellular dPHA_SCL type 1 (30), which is the largest family, with 12 characterized members. Although crystal structures of some smaller PHA depolymerase families, e.g., extracellular dPHA_SCL type 2 (DED) or Esterase_phb_PHAZ (ESTHER) (40) and extracellular nPHA_SCL (DED) or PHAZ7_phb_depolymerase (ESTHER) (41), have been reported, the absence of a representative structure has hampered molecular understanding of the largest PHA depolymerase family. The crystal structure of AlAXEA was used as a template (sequence identity, 37.4%) to model the catalytic domain of a representative member of the extracellular dPHA_SCL type 1 family, PhaZ1 from Pseudomonas lemoignei (psele-pha1 in Fig. 1) (42) with relatively high quality (GMQE = 0.44; see Fig. S4A in the supplemental material). The modeled PhaZ1 structure has a wide cleft (Fig. S4B) formed by the catalytic triad (Ser154, His290, and Asp232), a conserved disulfide bond (Cys74 and Cys111), hydrophobic residues (Trp114, Leu153, Phe158, and Met195), and hydrophilic residues (Gln76 and Thr234). PhaZ1 and PhaZ4 can hydrolyze both poly(3-hydroxybutyrate) and poly(3-hydroxyvalerate), while other PHA depolymerases from P. lemoignei are specific for the former substrate (43), which has shorter side chains than the latter. Further studies will clarify the structure-function relationship of a wider array of PHA depolymerases.

Conclusion.

In this study, we determined the crystal structure of AlAXEA as the three-dimensional structure of the Esterase_phb family. The AlAXEA structure showed structural similarity to several esterase families, but structurally known CE1 enzymes in Antigen85c were less similar. The structural determination enabled us to build reliable homology models of fungal FAEs in the Esterase_phb family and to locate possible substrate binding sites. Structural comparison with type A FAE (AnFaeA) and the mutational analysis indicated that the small pocket of AlAXEA formed by Trp160 is a binding site for the acetyl group of the arabinoxylan substrate. Trp160 mutants exhibited a weak but significant FAE activity. Our protein engineering study successfully endowed FAE activity onto an AXE. Because Esterase_phb (SF6) accounts for a major family in fungal FAEs (31), the structural basis for the molecular mechanism of AXEs and FAEs in this family will contribute to biotechnological applications for enzymatic biomass degradation and conversion. Moreover, the Esterase_phb family contains a number of PHA depolymerases (42–47), and the present structure will also facilitate biotechnological applications of these enzymes for biodegradation of thermoplastic polymers such as polyhydroxybutyrate.

MATERIALS AND METHODS

Protein expression and purification.

The vector pPIC-αAXEA, which contains the α-factor secretion signal sequence and the mature protein region of AlAXEA from A. luchuensis IFO 4033/RIB 2601/NBRC 111188 (13), was used for protein expression. P. pastoris KM71H (Thermo Fisher Scientific, Waltham, MA) was transformed with the vector, and transformants were selected as described previously (48). Zeocin (100 μg/ml) was added to every culture. Selected colonies were grown by shaking overnight at 30°C in 10 ml YPG medium (1% yeast extract, 2% peptone, and 1% glycerol). The culture was inoculated in 200 ml YPG medium and subsequently incubated at 30°C overnight. The cells were harvested by centrifugation (1,500 × g for 5 min at 4°C), resuspended in 400 ml of YPM medium (1% yeast extract, 2% peptone, and 1% methanol), and incubated for another 72 h at 26°C. Methanol was added at a 1% concentration every 24 h during the period of induction. Three days after induction, the culture was spun down (30 min, 1,500 × g), and the supernatant was concentrated by ammonium sulfate precipitation (80% saturation). After centrifugation (10,000 × g for 30 min at 4°C), the precipitate was dissolved in 50 ml of 20 mM sodium phosphate (pH 7.0) and 1 M ammonium sulfate. The solution was incubated with 5% (wt/vol) bentonite (Wako Pure Chemical Industries, Osaka, Japan), and the bentonite was removed by centrifugation (10,000 × g for 30 min at 4°C). The solution was loaded onto a phenyl-Toyopearl (Tosoh Bioscience, Tokyo, Japan) column, which was equilibrated with 20 mM sodium phosphate (pH 7.0) and 1 M ammonium sulfate. After washing with the equilibration mixture, a linear gradient to 0 M ammonium sulfate was introduced. The fraction containing AlAXEA was desalted and concentrated against a 20 mM Tris-HCl (pH 7.5) buffer using Vivaspin 20 (Sartorius, Göttingen Germany). A portion of the protein sample was treated by 1 U α-mannosidase (New England BioLabs, Ipswich, MA) per 1 ml protein sample at 37°C for 12 h. The solutions were loaded on DEAE-Toyopearl (Tosoh Bioscience) columns equilibrated with 20 mM Tris-HCl (pH 7.5). After washing with the equilibration solution, a linear gradient to 1 M NaCl was introduced. The fraction containing AlAXEA was desalted against a 20 mM Tris-HCl (pH 7.5) buffer and concentrated to 1 ml using Vivaspin 20 (Sartorius). The sample was further purified using a gel filtration column (HiLoad 16/60 Superdex 200; GE Healthcare, Buckinghamshire, England) in 10 mM HEPES-KOH (pH 7.3). The relative molecular mass of the AlAXEA protein was determined with the gel filtration column using molecular standards for thyroglobulin (669 kDa), apoferritin (443 kDa), alcohol dehydrogenase (200 kDa), albumin (66 kDa), and carbonic anhydrase (29 kDa). Selenomethionine (SeMet)-labeled protein was expressed in buffered minimal methanol medium, consisting of 100 mM potassium phosphate (pH 6.0), 1.34% yeast nitrogen base with ammonium sulfate without amino acids (Wako Pure Chemical Industries), (4 × 10⁻⁵)% biotin, and 1% methanol, containing 0.1 mg · ml⁻¹ l-SeMet, 0.09 mg · ml⁻¹ l-Ile, 0.09 mg · ml⁻¹ l-Lys, and 0.6 mg · ml⁻¹ l-Thr using P. pastoris as described previously (49). The culture supernatant was concentrated by ultrafiltration using Stirred Cells Series 8000 and a Biomax membrane with a molecular weight cutoff (MWCO) of 5,000 (EMD Millipore, Darmstadt, Germany) instead of ammonium sulfate precipitation. The following protein purification procedures were the same as those for the native protein. The protein concentration was determined using the bicinchoninic acid (BCA) protein assay kit (Thermo Fisher Scientific) with bovine serum albumin as a standard. From 2-liter cultures, 42 mg and 19 mg of native and selenomethionine-labeled proteins, respectively, were purified.

Crystallography.

The crystals of native and SeMet-labeled AlAXEA were obtained at 25°C using the hanging-drop vapor diffusion method, by mixing 1.0 μl of protein solution containing 100 mg/ml and 10 mM HEPES-KOH (pH 7.3) with an equal volume of reservoir solution containing 21 to 22.5% polyethylene glycol (PEG) 8000, 0.1 M 2-(N-morpholino)ethanesulfonic acid buffer (MES)–NaOH (pH 5.0), 0.2 M calcium acetate, and 5% 2-methyl-2,4-pentanediol (MPD). Diffraction data were collected at beamline BL6A at the Photon Factory of the High Energy Accelerator Research Organization (KEK, Tsukuba, Japan). Crystals were cryoprotected in the reservoir solutions, whose MPD concentrations were increased to 30% and flash-cooled in a nitrogen gas stream at 100 K. Diffraction images were processed using HKL2000 (50). The initial phases were calculated using Solve/Resolve (51). Automated model building was performed using ARP/wARP (52). Manual model rebuilding and refinement were achieved using Coot (53) and REFMAC5 (54). The statistics for data collection and refinement are provided in Table 1. Molecular graphic images were prepared using PyMOL (Schrödinger, LLC, Portland, OR).

Mutagenesis and activity measurements.

Mutants were constructed using a QuikChange site-directed mutagenesis kit and Escherichia coli strain XL10-Gold (Stratagene, La Jolla, CA, USA) using the following primers and their complementary strands: 5′-TGGTTGCAATCCACGGTTGCACCGGCACC-3′ (Y39G), 5′-TGGTTGCAATCCACCCATGCACCGGCACC-3′ (Y39P), 5′-TGGTTGCAATCCACGCATGCACCGGCACC-3′ (Y39A), 5′-ACCAAGTAGACGGACCAAATTCCACCTG-3 (W160P), 5′-ACCAAGTAGACGGATCAAATTCCACCTG-3 (W160S), and 5′-ACCAAGTAGACGGAGCAAATTCCACCTG-3 (W160A) (mutated sites are underlined). The entire open reading frame (ORF) sequence was checked to verify that no base changes other than those designed had occurred. The mutant enzymes were expressed according to the same procedure as that used for the wild-type enzyme and were purified using a phenyl-Toyopearl column. Enzymatic activity measurement using α-naphthyl esters was performed basically as described previously (55). Twenty microliters of 5 mM α-naphthyl ester dissolved in methanol was added to 170 μl of 100 mM potassium phosphate buffer (pH 7.0). Following this, 10 μl of enzyme solution (0.17 to 0.85 μM) was added. After incubation at 37°C for 15 min, the reaction was arrested by adding 200 μl of a 10% SDS solution containing 0.1% Fast Gernet GBC salt. The release of α-naphthol was measured colorimetrically (absorbance at 560 nm). A standard curve was prepared using α-naphthol. FAE activities were determined as described previously (55). The reaction mixture (190 μl), consisting of 1 mM MpCA, MCA, MFA, or MSA (Apin Chemicals, Oxfordshire, UK) and 100 mM potassium phosphate buffer (pH 7.0), was added to 10 μl of enzyme solution (10 μM). After incubation at 37°C for 12 h, the reaction was arrested by freezing the mixture at −20°C. Aliquots of the reaction mixture (10 μl) were applied to a reversed-phase column (Pegasil ODS, 4.6-mm diameter by 250 mm; Senshu Kagaku, Tokyo, Japan) for high-pressure liquid chromatography (HPLC) analysis using UV detection (320 nm) with the mobile phase of acetonitrile (65%) and 50 mM acetate buffer (pH 4.0) (35%). Saturation curves for MFA were measured under the same conditions as those described above, but the substrate concentration was changed to 0.5, 1.0, 2.5, 4.0, 5.0, 6.0, 7.5, and 10 mM. Kinetic constants (K_m and k_cat) and their fitting errors were calculated according to nonlinear regression of the Michaelis-Menten equation to the saturation curve using Kaleidagraph (Synergy, Reading, PA, USA). Ferulic acid released from arabinoxylan was measured as follows. The reaction mixture (1 ml), consisting of 1% feruloylated insoluble arabinoxylan (Megazyme) in 50 mM potassium phosphate buffer (pH 7.0), was added to 10 μl of enzyme solution (170 to 680 μM). After incubation at 37°C for 1 h and 24 h, the reaction was terminated by incubating the mixture in boiling water for 5 min. Aliquots of the reaction mixture (10 μl) were applied to a reversed-phase column (TSKgel ODS-120T; Tosoh) for HPLC analysis using UV detection (320 nm) with the mobile phase of acetonitrile (65%) and 50 mM acetate buffer (pH 4.0) (35%). As a positive-control enzyme solution, 10 μl of FAE from A. luchuensis (23 μM) (56) was used. To investigate the synergy with xylanase, the reaction was performed with addition of 10 μl of xylanase from Thermomyces lanuginosus (2.5 units/ml; Sigma-Aldrich, St. Louis, MO). One unit of enzyme activity was defined as 1 μmol of reaction product (α-naphthol or hydroxycinnamic acid) released per minute.

Accession number(s).

The structure factors and coordinates have been deposited in the Protein Data Bank under accession number 5X6S.