Enzymology: Crystal structure and characterization of the glycoside hydrolase family 62 α -L-arabinofuranosidase from Streptomyces coelicolor Tomoko Maehara, Zui Fujimoto, Hitomi Ichinose, Mari Michikawa, Koichi Harazono and Satoshi Kaneko J. Biol. Chem. published online January 30, 2014

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Crystal structure and characterization of the glycoside hydrolase family 62 α-Larabinofuranosidase from Streptomyces coelicolor Tomoko Maehara‡1, Zui Fujimoto§1, Hitomi Ichinose‡2 Mari Michikawa‡, Koichi Harazono¶, and Satoshi Kaneko‡3 From the ‡Food Biotechnology Division, National Food Research Institute, 2-1-12 Kannondai, Tsukuba, Ibaraki 305-8642, Japan §Biomolecular Research Unit, National Institute of Agrobiological Sciences, 2-1-2 Kannondai, Tsukuba 305-8602, Japan, and ¶Enzyme Division, Bio Chemicals Department, Nagase ChemteX Corporation, 1-52 Osadanocho, Fukuchiyama, Kyoto, 620-0853,Japan 1

T.M. and Z.F equally contributed to this work. Current address: Applied Bacteriology Division, National Food Research Institute, 2-1-12 Kannondai, Tsukuba, Ibaraki 305-8642, Japan

2

Running title: α-L-Arabinofuranosidase from Streptomyces coelicolor 3

Keywords: α-L-arabinofuranosidase; crystal structure; substrate specificity; glycoside hydrolase family 62; Streptomyces coelicolor

arabinofuranosidases have been

Background: Glycoside hydrolase family 62 α-L-arabinofuranosidases specifically release L-arabinose from arabinoxylan. Result: The crystal structure of glycoside hydrolase family 62 α-L-arabinofuranosidase from Streptomyces coelicolor was determined. Conclusion: L-Arabinose and xylohexaose complexed structures revealed the mechanism of substrate specificity of this enzyme. Significance: Efficient catalysis by glycoside hydrolase family 62 α-Larabinofuranosidase requires its binding to terminal xylose sugars at the substratebinding cleft.

determined, although the structures, catalytic mechanisms, and substrate specificities of GH62 enzymes remain unclear. To evaluate the substrate specificity of a GH62 enzyme, we determined the crystal structure of α-Larabinofuranosidase, which comprises a carbohydrate-binding module family 13 domain at its N-terminus and a catalytic domain at its C-terminus, from Streptomyces coelicolor. The catalytic domain was a five-bladed β-propeller

ABSTRACT α-L-Arabinofuranosidase, which

comprising five radially oriented anti-

belongs to the glycoside hydrolase family

parallel β-sheets. Sugar complex

62 (GH62), hydrolyzes arabinoxylan but

structures with L-arabinose, xylotriose,

not arabinan or arabinogalactan. The

and xylohexaose revealed five subsites in

crystal structures of several α-L-

the catalytic cleft and an L-arabinose-

1

Copyright 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

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Address correspondence to: Satoshi Kaneko, National Food Research Institute, 2-1-12 Kannondai, Tsukuba, Ibaraki 305-8642, Japan; Phone. +81-29-838-8063; Fax+81-29-838-7996; E-mail: [email protected]

binding pocket at the bottom of the cleft.

their substrate specificities toward

The entire structure of this GH62 family

structurally defined substrates (5-16).

enzyme was very similar to that of

However, the relationships between the

glycoside hydrolase 43 family enzymes,

amino acid sequences of these enzymes and

and the catalytically important acidic

their substrate specificities remain to be

residues found in family 43 enzymes were

resolved. Glycoside hydrolases (GHs) are

conserved in GH62. Mutagenesis studies

currently classified into 132 families based

catalytic residues, and Trp270, Tyr461, and

on their amino acid sequence similarities,

Asn462 were involved in the substrate-

implying that there are both structural and

binding site for discriminating the

mechanistic relationships (17, 18). α-L-

substrate structures. In particular,

Arabinofuranosidases are divided into 5 GH

hydrogen bonding between Asn462 and

families: GH3, 43, 51, 54, and 62. The

xylose at the non-reducing end subsite +2

crystal structures of GH 43, 51, and 54

was important for the higher activity of

family enzymes have been resolved;

substituted arabinofuranosyl residues

however, those of GH3 and GH62 enzymes

than that for terminal arabinofuranoses.

are not resolved. Thus, the substrate specificity and catalytic mechanism of these enzyme families are poorly understood. It is

Hemicellulose comprises approximately 20% of lignocellulose and is a

known that GH62 enzymes belong to the

key component for effective utilization of

clan GH-F together with GH43 enzymes and

plant biomass (1-3). Although the content of

are thought to operate an inversion

L-arabinose (Ara)

mechanism, as demonstrated in GH43

in the plant cell walls is

low, Ara residues are frequently found in

enzymes. To date, the crystal structures of 24

plant cell walls as arabinoxylan, arabinan,

GH43 enzymes have been resolved (16, 19-

arabinogalactan, and others. Because the

28), and the inversion catalytic mechanism

structures of L-arabinose-containing

was proposed for some enzymes based on

polysaccharides are highly variable and

their crystal structures and mutagenesis

complex, various α-L-arabinofuranosidases

studies (16, 20, 22, 27, 29). Here, we present the three-

(EC 3.2.1.55) with varying substrate specificities are necessary to hydrolyze these

dimensional structure of α-L-

polysaccharides (4). We previously purified

arabinofuranosidase from Streptomyces

few α-L-arabinofuranosidases and elucidated

coelicolor (ScAraf62A) and the results of

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revealed that Asp202 and Glu361 were

mutagenesis studies based on the structures

its mutants was secreted into the culture

of the enzymes–Ara and –

medium and purified by affinity

xylooligosaccharides complexes. These

chromatography using lactosyl–Sepharose as

results clearly demonstrate the substrate

previously described (32). The purity of each

specificity mechanism of GH62 α-L-

protein was confirmed by SDS-PAGE.

arabinofuranosidase and should facilitate Substrates—p-Nitrophenyl-α-L-

further studies on GH62 enzymes.

arabinofuranoside (PNP-α-L-Araf), larch wood arabinogalactan, oat spelts xylan, and

EXPERIMENTAL PROCEDURES

birchwood xylan were purchased from

Generation—The primers used in this study

Sigma Chemical Company (St. Louis, MO,

are listed in Table 1. ScAraf62A was

USA). Debranched arabinan, xylotriose (X3),

expressed as a mature protein using the S.

xylohexaose (X6), and wheat arabinoxylan

lividans expression system (30). The gene

(Low Viscosity; 2cSt.) were obtained from

encoding the putative α-L-

Megazyme International (Wicklow, Ireland).

arabinofuranosidase from S. coelicolor A3(2)

Oligosaccharides, including O-α-L-

(SCO5932; GenBank accession number

arabinofuranosyl-(1→3)-O-β-D-

CAA16189) (31) was amplified from S.

xylopyranosyl-(1→4)-D-xylopyranose

coelicolor genomic DNA by PCR and cloned

(A1X2), and O-β-D-xylopyranosyl-(1→4)-[O-

into a Streptomyces expression vector (30).

α-L-arabinofuranosyl-(1→3)]-O-β-D-

Sco5932 (ScAraf62A) has a signal sequence

xylopyranosyl-(1→4)-D-xylopyranose

at its N-terminus, and the mature protein can

(A1X3), were prepared using previously

be secreted by a cell. The strategy to

described methods (33). Gum arabic was

construct ScAraf62A mutants was as

obtained from Nacalai Tesque (Kyoto, Japan).

follows: we used site-directed mutagenesis to

Nihon Shokuhin Kako (Fuji, Japan) provided

generate amino acid substitutions in

corn hull arabinoxylan. Sugar beet arabinan

ScAraf62A. Site-directed mutagenesis was

was prepared using previously described

performed by PCR with KOD-plus-neo

methods (34).

polymerase (Toyobo, Osaka, Japan) using the primer pairs shown in Table 1. Each

Enzyme Assays—The catalytic activities of

constructed plasmid was transformed into S.

ScAraf62A and its mutants were assayed

lividans 1326, which was used as the host

using 100 nM–1 μM of enzyme incubated

strain. Recombinant ScAraf62A or each of

with 0.5–5 mM PNP-α-L-Araf in McIlvaine

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Expression of ScAraf62A and Mutant

buffer, pH 5.5, in a total volume of 0.5 ml at

membrane (Merck-Millipore). The protein

30°C for up to 30 min. The amount of PNP

was crystallized by the sitting-drop vapor-

released was determined at 400 nm with an

diffusion method at 293 K using a precipitant

extinction coefficient of 2165 M−1cm−1. The

solution comprising 20% (w/v) PEG3350

effects of pH and temperature on enzyme

and 0.2 M tripotassium citrate, pH 8.3. Rod-

activity were investigated as previously

shaped crystals with dimensions of

described (13). Each assay was performed in

maximum 0.5 × 0.2 × 0.1 mm were obtained

triplicate.

using 50 μl of reservoir solution with a drop consisting of 1.2- μl protein solution and 1.0μl reservoir solution.

Substrate Specificity for Polysaccharides—

ScAraf62A toward polysaccharides, we

Data Acquisition and Structure

selected wheat arabinoxylan, corn hull

Determination—Diffraction experiments for

arabinoxylan, oat spelts xylan, birchwood

native and Hg-derivative crystals were

xylan, arabinan, debranced arabinan, larch

performed at the beamline NE3A of the

arabinogalactan, and gum arabic as

Photon Factory (PF), High Energy

substrates. ScAraf62A was mixed with 0.5%

Accelerator Research Organization, Tsukuba,

(w/v) substrate in a buffer (pH 6) containing

Japan. Crystals were mounted on a quartz

0.1% (w/v) bovine serum albumin (BSA)

glass capillary (diameter, 0.3 mm) and then

and incubated at 45°C for up to 20 min. The

flash-cooled under a nitrogen stream at 95 K.

reaction was stopped by heating at 100°C for

Diffraction data were collected at a

20 min. Hydrolytic activity was determined

wavelength of 1.0031 Å with a Quantum 270

on the basis of the amounts of reducing

CCD detector (Area Detector Systems Corp.,

sugars as determined by the Somogyi–

Poway, CA, USA). An Hg-derivative crystal

Nelson method (35).

was prepared by adding 0.2- μl 10 mM ethyl mercuric phosphate to the crystal drop and

ScAraf62A Crystallization—The purified

incubation for 10 min. For structural

protein solution was dialyzed against 2 mM

analyses of the enzyme in complex with Ara,

Tris-HCl buffer, pH 7.0, containing 20 mM

X3, and X6, ScAraf62A crystals were soaked

NaCl, concentrated to 8.3 mg ml−1 (A2801 mm

in a drop that contained 5% (w/v)

= 2.0 U) by ultrafiltration using a YM-30

carbohydrates in the precipitant solution at

membrane (Merck-Millipore, Billerica, MA,

10 min before the diffraction experiments.

USA), and filtered through a 0.1-µm

Diffraction data for the sugar complexes

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To evaluate the substrate specificity of

were acquired at the PF beamline NW12A

RESULTS

(36) or BL-5A. Diffraction data were

ScAraf62A Expression and Purification—

collected with a Quantum 210 CCD detector

The DNA sequence of the S. coelicolor

(ADSC). All data were integrated and scaled

Sco5932 gene was 1,428-bp long, and the

using the programs DENZO and

gene putatively encoded a 475-amino acid

SCALEPACK in the HKL2000 program

protein comprising N-terminal carbohydrate-

suite (37).

binding module family 13 (CBM13) and Cterminal GH62 domains. The deduced amino

The crystal structure was determined

acid sequence was compared with sequences

dispersion method using an Hg-derivative

in the protein database using a BLAST

crystal. Three Hg atom positions were

search (National Center for Biotechnology

determined, and the initial phases were

Information:

calculated using the program

http://www.ncbi.nlm.nih.gov/BLAST/).

SOLVE/RESOLVE (38, 39). The initial

The ScAraf62A (Sco5932) sequence

model building was conducted by the auto-

resembled that of AbfB, an α-L-

modeling program ARP/wARP (40)

arabinofuranosidase from S. lividans (46),

incorporated in the CCP4 program suite (41).

and the amino acid similarity was 97% (data

Manual model building and molecular

not shown). To express ScAraf62A, the DNA

refinement were performed using COOT

fragment encoding the full-length protein

(42) and REFMAC5 (43, 44).

was cloned. Recombinant ScAraf62A was

To analyze sugar complex structures,

successfully expressed in the secreted form

structural determinations were performed

in S. lividans and purified as a single band

using the ligand-free structure as the starting

with an apparent molecular size of 44 kDa on

model, and the bound sugars were observed

SDS-PAGE (data not shown).

in the difference electron density map. Data acquisition and refinement statistics are

ScAraf62A Characterization and Substrate

shown in Table 3. The model stereochemistry

Specificities—The effects of pH and

was determined with the program

temperature on ScAraf62A activity and

RAMPAGE (45). Structural drawings were

stability were determined using PNP-α-L-

prepared using the program PyMol (DeLano

Araf as the substrate (Fig. 1). Maximal

Scientific LLC, Palo Alto, CA, USA).

enzyme activity was detected at pH 5.5 at 35°C. ScAraf62A was stable between pH 7.0 and 9.0 at 35°C for 1 h. It was also stable up

5

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by the single-wavelength anomalous

to 30°C at pH 5.5 for 1 h. The optimum

(51). Interestingly, ScAraf62A barely reacted

temperature for ScAraf62A was lower than

with arabinan and possibly reacted with

that for AbfB α-L-arabinofuranosidase from

arabinoxylan, although both arabinoxylan

S. lividans (55°C) (46). However, these could

and arabinan possess α-1,3-linked L-

not be simply compared because the

arabinofuranose. To evaluate the mechanisms

experimental conditions, such as the

of substrate specificity, we analyzed the

substrates used, were different.

crystal structure of ScAraf62A.

Next, we examined the activities of Overall Structure of ScAraf62A—The crystal

polysaccharides. ScAraf62A released Ara

structure of ScAraf62A was determined by

from wheat arabinoxylan, corn hull

the single-wavelength anomalous dispersion

arabinoxylan, and oat spelts xylan but not

method using the Hg-derivative crystal data.

from arabinan or arabinogalactan (data not

Native and Ara, X3, and X6 complex

shown). The substrate specificity of

structures were successively determined. The

ScAraf62A was similar to that of other GH62

structure refinement statistics are

enzymes from S. lividans, Penicillium

summarized in Table 3. Recombinant

chrysogenum, and Pseudomonas cellulosa

ScAraf62A comprised a single polypeptide

(46-49).

chain of 438 amino acids, Ala38–Arg475, in which the N-terminal residues Met1–Ala37

The kinetic parameters of ScAraf62A for PNP-α-L-Araf and wheat

constituted the signal peptide. The 136 N-

arabinoxylan were determined (Table 2). The

terminal residues of Ala38–Asp173 comprising

kcat/Km value for wheat arabinoxylan was

the linker and CBM13 domain were not

approximately 4-fold higher than that for

identified because of a lack in the electron

PNP-α-L-Araf, suggesting that ScAraf62A

density. Therefore, only the structure of the

specifically hydrolyzed arabinoxylans.

C-terminal GH62 catalytic domain was

It has been reported that arabinoxylan

determined. The final model included one

comprises a β-1,4-linked-xylan backbone

ScAraf62A catalytic domain in the

that is partially substituted with 2- or 3-, or

asymmetric unit, accompanied by one cation

2,3-linked L-arabinofuranoses (50). In

located at the center of the domain that was

contrast, arabinan comprises an α-1,5-linked

set as a calcium ion in this model, one

L-arabinofuranose backbone that

chloride ion, and one citrate molecule.

is

substituted with α-1,3-L-arabinofuranose

ScAraf62A is a monomer protein in the

and/or α-1,2-L-arabinofuranose side chains

native state. One disulfide bond (Cys176–

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ScAraf62A for arabinose-containing

Cys444) and one cis-peptide (Phe340–Pro341)

was observed in a β-anomeric configuration.

were in the protein structure. The ligand-free

This was docked in the pocket with four

structure contained one Tris molecule, the

hydroxyl oxygen atoms oriented toward the

Ara complex structure contained one Ara

bottom of the pocket, which were recognized

molecule, and the X3 complex structure

by hydrogen bonds with the protein residues.

contained one X3 molecule. The X6 complex

The O1 atom of bound L-arabinofuranose

structure contained one X6 molecule, of

(Ara-O1) was within the hydrogen bond

which five xylose moieties were observed in

distances with Asp202-Oδ1, Lys201-Nζ, Gln451-

the electron density map.

Nε2, and Tyr461-Oη atoms. The Ara-O2 atom hydrogen bonded to Asp309-Oδ2 and His427-

The ScAraf62A catalytic domain

Nδ1 atoms. One water molecule that was

five radially oriented anti-parallel β-sheets,

coordinated to Glu361-Oε2, Glu379-Oε1, and

designated as blades I–V in Fig. 2; each

Ser426-Oγ atoms hydrated the Ara-O1 and

blade essentially comprised four β-strands.

Ara-O2 atoms. The Ara-O3 atom hydrogen

The domain structure was primarily a β-

bonded to Asp309-Oδ1 and Gln269-Nε2 atoms.

structure with six 310-helices and no α-

The Ara-O5 atom hydrogen bonded to

helices. A fold such as this was first reported

Asp202-Oδ2 and Lys201-Nζ atoms. The Ara-

for tachylectin (52) and is observed so far in

O4 atom, a component of the furanosyl ring,

three glycoside hydrolase families: GH32,

was within the hydrogen bond distances with

GH43, and GH68 (19, 53, 54). Overall, the

the Lys201-Nζ atom. Overall, the bound Ara

structures of sugar complexes were nearly

molecule in the catalytic pocket was

identical to the ligand-free structure with

recognized by 11 direct hydrogen bonds and

root-mean-square differences of 0.12–0.15 Å,

one water-mediated interaction. The C5 atom

implying little effect of ligand binding on the

was sequestered in the hydrophobic hollow

overall structure.

that comprised Tyr224, Trp270, and Val251. The flat surface of Ara was held from the top by

Crystal Structure of ScAraf62A in Complex

the side-chain of Ile308 by hydrophobic

with L-arabinofuranose—L-Arabinofuranose

interaction.

was found in the catalytic pocket located at the central depression of the five blades (Figs.

Crystal Structure of ScAraf62A in Complex

2 and 3). Bound L-arabinofuranose assumed

with Xylooligosaccharides—To elucidate the

a structure of an envelope 2E conformation,

substrate-binding mechanism of this enzyme,

and its O1 atom in the C-1 hydroxyl group

we used X3 and X6 for enzyme–substrate

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was a five-bladed β-propeller comprising

+2NR was hydrophobically situated with

xylooligosaccharides were observed in the

residues Tyr224, Trp270, Thr305, and Ile308. One

cleft at the surface of the catalytic domain

hydrogen bond was observed between the

lying above the catalytic pocket (Fig. 2A).

Asn462-Oδ1 atom and the O3 atom of the

Five xylose moieties were observed in the X6

xylose at subsite +2NR. The xylose moiety at

complex structure. The orientation of X6 was

subsite +3NR had a stacking interaction with

resolved on the basis of the B-factors of the

the indole group of Trp270, and the xylose

C5 and O5 atoms of these xylose moieties,

moiety at subsite +4NR was embedded in the

and the non-reducing and reducing ends of

peptide bond plane between Trp270 and Gly271.

these sugars were the lower and upper

However, direct interactions were not

moieties, respectively, as shown in Fig 4B.

observed with the protein atoms besides

The second xylose moiety from the reducing

these stacking interactions. The average B-

end was located at the entrance of the

factors of the six cyclic atoms of the xylose

catalytic pocket, the position of which was

rings were 63.3, 50.9, 38.2, 28.0, and 38.8 Å2

designated as subsite +1. One xylose moiety

for xylose moieties at subsites from +4NR to

was present at the reducing end, and three

+2R. The electron density for xylose at

moieties were present at the non-reducing

subsite +4NR was slightly ambiguous. In the

end through β-1,4-glycoside bonds. These

X3 complex structure, three xylose moieties

positions were designated as subsites +3NR,

were observed and located at nearly the same

+2NR, +1NR, +1, and +2R (Fig. 4A and 4D).

positions as those at subsites +2NR, +1, and

One surface of the xylose at subsite +1 was

+2R in the xylotriose complex structure.

embedded in the aromatic ring of Tyr461,

Protein side-chains around the sugar-

which formed a stacking interaction, and

binding sites were mostly conserved among

another side faced the side-chains of Asp326

these structures, although an apparent

and Phe360. The O2 and O3 atoms faced

structural difference was observed for Ile308

toward the bottom of the cleft. The O2 atom

(Fig. 4C). In the ligand-free form, the major

hydrogen bonded to Glu361-Oε2 and Arg386-

conformation of the Ile308 side-chain was

Nη2 atoms, and the O3 atom bonded to the

observed with a χ-1 value of 51° (green stick

Glu361-Oε1 atom. Xylose at subsite +2R was

model in Fig. 4C), but it was approximately

half-sandwiched by residues Phe360, Met381,

between −161° and −163° in the Ara or

and Tyr461, but the other half was exposed to

xylooligosaccharide complex structures. The

solvent regions, and polar contact was not

entrance to the catalytic pocket was wider in

observed between the protein atoms. Subsite

the former structure, and therefore, the

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complex analyses. Bound

closed conformation in the latter structure

formed a stacking interaction with xylose

appeared to be induced by the binding of

located at subsite +1, Y461A, Y461F, and

sugar ligands either in the catalytic pocket or

Y461W mutants were tested. Although all

the catalytic cleft.

these mutants completely or significantly lost their activity for PNP-α-L-Araf, Y461A and Y461F showed an apparent activity for

evaluate the substrate specificity mechanism,

wheat arabinoxylan (kcat/Km of 0.2–0.8 for

some amino acids in ScAraf62A that were

wild-type). The Y461W mutant lost its

possibly involved in enzyme catalysis and

activity for PNP-α-L-Araf and wheat

substrate binding were mutated, and these

arabinoxylan, probably because the side

mutant enzymes were characterized (Table 2). Three possible catalytic residues,

chain of the amino acid was too large and

Asp, Asp, and Glu, which are conserved in

binding cleft in ScAraf62A.

destroyed the structure of the substrate-

GH43 enzymes, were also conserved in 202

309

In contrast, Asn462 mutations did not

361

ScAraf62A (Asp , Asp , Glu ; Fig. 3). It was predicted that Asp

202

and Glu

affect the activity for PNP-α-L-Araf but

361

apparently affected the activity for

behave as general base and acid catalytic

arabinoxylan. For the hydrogen bonding of

residues, respectively (29). No enzyme

the Oδ1 atom of Asn462 with xylose-O3 at

202

activities for the mutant Asp

and Glu

361

subsite +2NR, an N462Q mutant was

enzymes, such as D202N and E361Q, were

constructed to investigate the effect of this

detected when enzyme activities were tested

interaction on enzyme activity. The kcat/Km of

using PNP-α-L-Araf.

N462Q for PNP-α-L-Araf was 0.71 mM−1·min−1 (1.2 relative to that of wild-

Regarding substrate binding, the indole group of Trp270 had a stacking

type), whereas it was 1.8 mg−1·ml·min−1 (0.7

interaction with xylose at +3NR and formed

relative to that of wild-type) for arabinoxylan.

a part of the pocket wall at subsite −1. When

More critical differences were observed

Trp

270

was replaced by Ala, Tyr, or Phe, the

when arabinoxylooligosaccharides such as

Km for PNP-α-L-Araf significantly increased

A1X2 and A1X3 were used as substrates for

(5–9-fold), and the kcat for the substrate

N462Q (Table 4). This mutant had the same

slightly decreased (0.6–0.3-fold), whereas

level of catalytic efficiency for A1X2 with

these values for wheat arabionoxylan were

wild-type ScAraf62A, whereas the catalytic

not significantly affected (3–5-fold for Km

efficiency of the mutant for A1X3 was about

and 0.9–1.7-fold for kcat). Because Tyr

461

one-third of that for the wild-type enzyme,

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ScAraf62A Mutagenesis Studies—To

suggesting that the hydrogen bonding

the bound Ara molecule, and the bound water

between Asn462 and xylose at subsite +2NR

molecule, as described below. In general, two acidic amino acids,

had an important role in locating arabinoxylan at the exact position in the

either aspartate or glutamate, are employed

substrate-binding cleft of ScAraf62A.

by most inverting GHs to catalyze hydrolysis. One carboxylate protonates the scissile glycosidic oxygen atom and the other

Catalytic Mechanisms and Substrate

coordinates one water molecule, which acts

Specificities of GH62 enzymes—The detailed

as the nucleophile (55). The ScAraf62A–Ara

reaction mechanisms of GH62 enzymes have

complex structure showed that the C1

not yet been determined. Here we present the

hydroxyl group of the bound L-

structure of a GH62 enzyme. L-

arabinofuranose molecule assumed a β-

Arabinofuranose bound to ScAraf62A aided

anomeric conformation and that the distance

in surmising the catalytic mechanism of this

between the Glu361-Oε1 and the Ara-C1

GH62 enzyme. In the ScAraf62A–Ara complex

atoms was 3.8 Å. However, the distance

structure, the bound Ara molecule was found

would be within the hydrogen-bonding

in the catalytic pocket situated in the

distance, assuming that the Ara-O1 atom was

catalytic domain, as it is often observed with

in the α-anomeric conformation. Furthermore,

exo-acting enzymes. This pocket provided

Glu361-Oε1 atom hydrogen bonded to the

between the Ara-O1 and Glu361-Oε1 atoms

space for only one Ara moiety and a cleft

Xyl-O3 atom of the bound X6 at subsite +1

specific for a xylan backbone was present

in the ScAraf62A–X6 complex structure. L-

outside the pocket. The docked Ara molecule

Arabinofuranoside often substitutes the 3′-

assumed the 2E conformation, and its O1

hydroxyl group of the xylan backbone; thus,

atom in the C1 hydroxyl group was in a βanomeric

configuration,

although

the atom at the O3 position could be

the

considered as the scissile bond position of

amounts of Ara α- and β-anomers are almost

the substrate. Therefore, Glu361 appeared to

same in the solution. Because the scissile bond was an α-glycosidic bond, the bound

play the proton donor role for this enzyme.

Ara

However, one catalytic water molecule was

molecule

having

the

β-anomeric

conformation appeared to represent the

necessary to invert the α-anomeric

product state of the inverted catalytic process.

conformation to the β-configuration by the

This inverting mechanism is deduced from

catalytic process. In the ligand-free or

the relative positions of the catalytic residues,

ScAraf62A–X6 complex structure, one

10

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DISCUSSION

bound water molecule was observed in

backbone, although the 2-hydroxyl group is

proximity to the C-1 atom of the bound Ara

occasionally substituted. In the ScAraf62A–

molecule in the direction of the β-anomeric

X6 complex structure, the O2 atom of the

side. This water molecule was closely

bound xylose moiety at subsite +1 faced

supported by Asp202-Oδ1, Lys201-Nζ, Gln451-

toward the catalytic cleft. However, the

Nε2, and Tyr461-Oη atoms, and its position

distance to the Ara-C1 atom in the

was close to the Ara-O1 atom of the

ScAraf62A–Ara complex structure was

ScAraf62A–Ara complex structure.

rather long (4.9 Å) compared with that from

Therefore, it appears that the nucleophile

the O3 atom (3.1 Å). Therefore, this enzyme

water caused the inversion of the hydrolyzed

did not appear to catalyze the α-1,2-

and Asp202

glycosidic bond in this situation. However, it seems plausible that the xylan backbone

appeared to be the catalytic general base.

could be reversely docked in the substrate

Besides the catalytic residues, acidic residues in the catalytic site often play

cleft so that the atom at the O2 position

important roles during catalysis, and their

moves to the O3 position in the ScAraf62A–

mutations cause drastic decreases in the

X6 complex structure, owing to the high

hydrolytic activity of several GH enzymes.

structural symmetry of D-xylopyranoside and

In ScAraf62A, Asp309 was located at the

a β-1,4-xylan backbone. In fact, in our

bottom of the catalytic pocket, bound to the

previous study, we showed that

Ara molecule by its O2 and O3 atoms, and

xylooligosaccharides can reversibly bind in

appeared to be important for transition-state

the xylan-binding pocket of CBM13 linked

stabilization. Asp309 also appeared to

to β-1,4-xylanase using decorated

modulate the pKa of Glu361 and to keep it in

xylooligosaccharides (56). Moreover,

the correct orientation relative to the

cleavage of arabinofuranosyl side-chains

substrate, as reported for Asp285 in

linked to O2 and O3 of single-substituted

Geobacillus stearothermophilus xylosidase

xylose residues in arabinoxylan by GH62

(22). Three acidic residues in GH62 enzymes

arabinofuranosidase was previously reported

are considered to be catalytically important

(49). The possibility that arabinan can

residues, and our present structural study highlights the roles of these residues in

bind to the substrate-binding cleft of

catalysis.

ScAraf62A was analyzed by molecular

L-Arabinofuranoside

modeling. As shown in Fig. 6, arabinan is

often

unable to fit into the cleft because of

substitutes the 3-hydroxyl group of the xylan

11

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L-arabinofuranose conformation,

for Ara binding. Therefore, this mutation

having an elongated structure with a typical

would drastically affect small substrates that

3-fold screw helix, an arabinan chain is

use only subsites +1 and −1 than

extended with a turn to nest Ara residues in a

polysaccharides in which only one subsite is

different orientation with xylan. When the

lost among five subsites. Similarly, the

orientations of O-2 and O-3 of Ara located at

affinity of subsite +1 should have been lost

subsite +1 were superimposed on the xylose

in the Tyr461 mutant, which would result in

of subsite +1 in the X3 binding model of

the reduction of activity against small

ScAraf62A, the α-1,5-linked arabinan

substrates but not against polysaccharides.

backbone did not fit into the substrate-

Asn462 mutations break the hydrogen bond

binding cleft. Thus, ScAraf62A could

between Asn462 and xylose at subsite +2NR

discriminate sugar structures at the cleft,

and affect the activity only for

thereby enabling the hydrolysis of only

polysaccharides. Furthermore, a reduction in

arabinoxylan.

the kcat/Km value for arabinoxylan was

As described above, the substrate-

observed with the N426Q mutant, despite

binding cleft of ScAraf62A appeared to play

that the kcat/Km value for PNP-α-L-Araf was

an important role in the substrate specificity

unchanged. The importance of this hydrogen

of this enzyme. Our mutagenesis studies

bond was also confirmed using a different

were in agreement with the structural

approach from the substrate side.

insights into the substrate recognition

Wild-type ScAraf62A cleaved A1X3

mechanism of ScAraf62A. The three selected

at a 5.4-fold faster rate than A1X2 (Table 4).

amino acids Trp270, Tyr461, and Asn462 are

Although xylose residues docked into

thought to be important amino acids for

subsites +2R, +1, and +2NR when the

substrate binding. Trp270 and Tyr461 interact

enzyme hydrolyzed A1X3, only +2R and +1

in a stacking manner with a xylose ring at

were used for A1X2 hydrolysis. This

subsites +3NR and +1, respectively. A

difference in catalytic efficiency is caused by

significant reduction in the activity for PNP-

the affinity for subsite +2NR, which

α-L-Araf and a lesser effect on the activity

primarily comprises hydrogen bonding

for arabinoxylan was observed when Trp270

between Asn462 and xylose. A more dramatic

and Tyr461 were mutated. For the Trp270

reduction in the catalytic efficiency of

mutants, a mutation may have caused a loss

N426Q was observed for A1X3 than that for

of the stacking interaction with the xylose at

A1X2.

+3NR and disruption of the pocket structure

12

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structural issues. In contrast to a xylan chain

Comparisons with GH51, 54

kawachi showed that GH54 comprises a

Arabinofuranosidases—Beldman et al. (57)

catalytic domain with a β-sandwich fold and

divided arabinofuranosidases into 3 types:

an Ara-binding domain belonging to CBM42

type-A (cannot degrade polymers), type-B

(59). CBM42 binds to arabinosyl side chains

(can degrade polymers), and type AXH

in arabinan and arabinoxylans, and this

(specifically degrades arabinoxylan). Type-A

property explains why GH54 enzymes can

and type-B correspond to GH51 and GH54,

possibly degrade polysaccharides, although

respectively, and type AXH activity has been

the enzymes in this family possess similar

observed with GH43 and GH62 family

pocket topology for substrate binding (60).

enzymes.

An Ara soaking study elucidated a small catalytic pocket in the catalytic domain,

arabinofuranosidase AbfA from G.

although the location of subsite +1 could not

stearothermophilus was the first structure

be confirmed. A loose interaction at subsite

resolved for an arabinofuranosidase. AbfA is

+1 may enable the hydrolysis of both α-1,2-

a homohexamer and each AbfA monomeric

and α-1,3-arabinofuranosyl linkages

subunit is organized into a (β/α)8-TIM barrel

observed in the arabinose side-chains of

catalytic domain at the N-terminus and a 12-

arabinoxylans.

stranded β-sandwich with a jelly-roll

Thus, the substrate recognition

topology at the C-terminus (58). GH51

mechanism of GH62 is distinguishable from

enzymes have a substrate-binding pocket that

that of GH51 and GH54 enzymes by having

is suitable for binding to a single

the substrate-binding cleft that can fit the

arabinofuranose residue. The pocket

xylan backbone along with the

topology is not amenable for discriminating

arabinofuranose-binding pocket.

the structures of aglycons; therefore, this enzyme has broad substrate specificity for

Comparisons with GH43

arabinose-containing substrates. However,

Arabinofuranosidases—GH62 enzymes

there have been no detailed studies related to

belong to the clan GH-F together with GH43

its structure and substrate specificity. In

enzymes and fold into a five-bladed β-

contrast, GH54 arabinofuranosidase can

propeller (19). As described above, a

degrade polysaccharides without

glutamate and two aspartates were conserved

discriminating between xylan and arabinan.

between GH62 and GH43 as the catalytic

The crystal structure of the

amino acids. In addition to three conserved

arabinofuranosidase from Aspergillus

acidic amino acids, Ala252 and Ser217 in

13

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The structure of the GH51 α-L-

ScAraf62A were relatively conserved

catalytic domains of ScAraf62A and these

between GH43 and GH62. These amino

GH43 AXHs were relatively low at

acids formed the substrate-binding pocket at

approximately 16%–20%, the arrangements

subsite −1. It is interesting to note that a

of their β-sheets constructing the five-bladed

tryptophan residue, which is located before

β-propellers were well conserved (Fig. 5A).

the above mentioned alanine residue, is

In the catalytic pockets, three catalytically

highly conserved in GH43 enzymes and

important acidic residues were also well

provides the wall of subsite –1, was replaced

conserved among these enzymes (Fig. 5B).

by Val251 in ScAraf62A.

Catalytic proton donors were conserved and located at similar positions. Complex

To date, three arabinofuranosidases

structures with xylooligosaccharides

α-1,3-L-arabinofuranohydrolase from

revealed that the orientations of the bound

Bacillus subtilis (XHm2,3s); double-

xylooligosaccharides were similar among

substituted xylan α-1,3-L-

these enzymes. However, the amino acid

arabinofuranohydrolase from Humicola

residues constructing the xylan-binding clefts

insolens (AXHd3); and exo-1,5-

on the catalytic domains were different (Fig.

arabinofuranosidase from S. avermitilis (14,

5C).

25, 29). Exo-α-1,5-L-arabinofuranosidase

These differences result in different

specifically cleaves the α-1,5-linked

topologies of their substrate-binding clefts.

arabinofuranosyl linkage but is not involved

At least three xylose residues can bind to the

in any xylan-degrading enzyme system.

cleft of AXHm2,3, similar to ScAraf62A,

However, AXHm2,3 and AXHd3 target

whereas AXHd3 has a shallow arabinose-

arabinoxylan as the substrate, similar to

binding pocket adjacent to the deep active

GH62 enzymes. AXHm2,3 is an

site pocket, and the orientation of the xylan

arabinoxylan-specific arabinofuranosidase

backbone with the O2-linked

that cleaves an Ara linkage of O2- or O3-

arabinofuranose is a critical specificity

single substitutions in xylan. In contrast,

determinant for AXHd3 through extensive

AXHd3 hydrolyzes only an O3-linked L-

interactions with this enzyme. The ten-loop

arabinosyl linkage of O2- and O3-doubly

regions from the five-bladed β-propellers

substituted xylans (25).

have various structures, even among GH43 enzymes, and these enzymes exhibit original

We compared the structures of AXHm2,3, AXHd3, and ScAraf62A (Fig. 5).

binding properties for their substrates, which

Although the sequence identities of the

were previously discussed (16). These loops

14

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types have been reported: An arabinoxylan

in GH62 appear to possess additional

arabinooligosaccharides were soaked into a

structural variations and have a unique

ScArf62A crystal, we did not observe a clear

recognition property for arabinoxylan.

electron density in the cleft. This discrimination of substrates was also apparent from the data of our mutagenesis

manuscript, the first structures for GH62

study. As shown for the N462 mutant (Table

enzymes were reported (61). In this study,

4), xylan recognition at subsite +2NR is

although the N-terminal CBM13 was not

important for substrate specificity in this

observed because of a lack of electron

enzyme, and this enzyme exhibits high

density, we determined the crystal structure

hydrolysis activity toward arabinoxylan but

of the ScAraf62A catalytic domain. L-

lesser activity toward arabinan. These results

Arabinofuranose is bound in the catalytic

clearly explain the substrate discrimination

pocket, whereas xylooligosaccharide is

mechanisms of GH62 enzymes that

bound in the cleft lying over the catalytic

specifically hydrolyze arabinoxylans but not

pocket. When α-1,5-linked L-

arabinans.

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52. Beisel, H. G., Kawabata, S., Iwanaga, S., Huber, R., and Bode, W. (1999) Tachylectin-2: crystal structure of a specific GlcNAc/GalNAc-binding lectin involved in the innate immunity host defense of the Japanese horseshoe crab Tachypleus tridentatus. EMBO J. 18, 2313-2322

53. Meng G and Fütterer K. (2003) Structural framework of fructosyl transfer in Bacillus subtilis levansucrase. Nat Struct Biol. 10, 935-941

54. Alberto, F., Bignon, C., Sulzenbacher, G., Henrissat, B., and Czjzek, M. (2004) The threedimensional structure of invertase (β-fructosidase) from Thermotoga maritima reveals a bimodular arrangement and an evolutionary relationship between retaining and inverting glycosidases. J Biol Chem. 279, 18903-18910

55. Chiba, S. (2012) A historical perspective for the catalytic reaction mechanism of glycosidase; so 56. Fujimoto, Z., Kaneko, S., Kuno, A., Kobayashi, H., Kusakabe, I., and Mizuno, H. (2004) Crystal structures of decorated xylooligosaccharides bound to a family 10 xylanase from Streptomyces olivaceoviridis E-86. J Biol Chem. 279, 9606-9614

57. Beldman, G., Schols, H. A., Pitson, S. M., Searle-van Leeuwen, M. J. F., and Voragen, A. G. J. (1997) Arabinans and arabinan degrading enzymes. Adv. Macromol. Carbohydr. Res. 1, 1–64

58. Hövel, K., Shallom, D., Niefind, K., Belakhov, V., Shoham, G., Baasov, T., Shoham, Y., and Schomburg, D. (2003) Crystal structure and snapshots along the reaction pathway of a family 51 α-L-arabinofuranosidase. EMBO J. 22, 4922-4932

59. Miyanaga, A., Koseki, T., Matsuzawa, H., Wakagi, T., Shoun, H., and Fushinobu, S. (2004) Crystal structure of a family 54 α-L-arabinofuranosidase reveals a novel carbohydrate-binding module that can bind arabinose. J. Biol. Chem. 279, 44907-44914

60. Miyanaga, A., Koseki, T., Miwa, Y., Mese, Y., Nakamura, S., Kuno, A., Hirabayashi, J., Matsuzawa, H., Wakagi, T., Shoun, H., and Fushinobu, S. (2006) The family 42 carbohydratebinding module of family 54 α-L-arabinofuranosidase specifically binds the arabinofuranose side chain of hemicellulose. Biochem J. 399, 503-511

61. Siguier, B., Haon, M., Nahoum, V., Marcellin, M., Burlet-Schiltz, O., Coutinho, PM., Henrissat, B., Mourey, L., O., Donohue, M. J., Berrin, J. G., Tranier, S., Dumon, C. (2014) First structural insights into α-L-arabinofuranosidases from the two GH62 glycoside hydrolase subfamilies. J. Biol. Chem.

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as to bring about breakthrough in confusing situation. Biosci Biotechnol Biochem. 76, 215-231

Acknowledgements—This work was supported by JSPS KAKENHI Grant Number 25450140. For the use of synchrotron radiation, we thank the beamline researchers at PF and SPring-8. We thank Ms. Mariko Honda from NFRI for assistance with DNA cloning and sample preparation for crystallization. FOOTNOTES Abbreviations: BSA, bovine serum albumin; CAZy, Carbohydrate-Active enZymes; GHs, glycoside hydrolases; GH62, glycoside hydrolase family 62; PNP, p-nitrophenol; PNP-α-L-Araf, p-nitrophenylα-L-arabinofuranoside; ScAraf62A, Streptomyces coelicolor α-L-arabinofuranosidase, X3, xylotriose; X6, xylohexaose; A1X2, O-α-L-arabino-furanosyl-(1→3)-O-β-D-xylopyranosyl-(1→4)-Dxylopyranose; A1X3, O-β-D-xylopyranosyl-(1→4)-[O-α-L-arabinofuranosyl-(1→3)]-O-β-Dxylopyranosyl-(1→4)-D-xylopyranose. FIGURE LEGENDS

FIGURE 2. Structure of α-L-arabinofuranosidase from Streptomyces coelicolor (ScAraf62A) catalytic domain. A, Stereoview of the ribbon model of the ScAraf62A–L-arabinofuranose complex structure. The bound sugar, catalytically important residues, and disulfide bridge are shown as stick models, and the bound sodium ion is shown as a purple sphere. B, Topological diagram of ScAraf62A. 310-Helices and β-strands are shown as shaded cylinders and filled arrows, respectively. FIGURE 3. Stereo view of the α-L-arabinofuranosidase from Streptomyces coelicolor (ScAraf62A)– L-arabinose (Ara) complex catalytic pocket. Yellow stick model, bound Ara; pale red, three catalytically important residues; broken lines, estimated hydrogen bonds; magenta, 2Fo–Fc electron density map of bound Ara (contour level, 1σ). Sugar carbon atoms are numbered. FIGURE 4. Bound ligand structures of α-L-arabinofuranosidase from Streptomyces coelicolor (ScAraf62A). A, Surface model of the ScAraf62A–xylohexaose (X6) complex catalytic cleft. Gray stick model, bound X6; pale red, gray stick model, bound Tris molecule; broken lines, estimated hydrogen bonds; magenta, 2Fo–Fc electron density map of bound X6 (contour level, 1 σ). B, Superimposed model of L-arabinose (Ara) of ScAraf62A–Ara complex structure (yellow stick model) on the ScAraf62A–X6 complex structure around the catalytic center. C, Superimposed model of the ligand-free structure (light green stick model) on the ScAraf62A–Ara complex structure (yellow– orange–pale red) around the catalytic center. D, Schematic representation of the binding model of arabinoxylan to ScAraf62A. Hexagon, xylose; pentagon, Ara. FIGURE 5. Superimposition of α-L-arabinofuranosidase from Streptomyces coelicolor (ScAraf62A) and two glycoside hydrolase (GH)43 AXH catalytic domain structures. A, Overall ribbon model. ScAraf62A–xylahexaose (X6) complex, orange and pink; Bacillus subtilis arabinoxylan α-L-1,3arabinofuranohydrolase complexed with xylotetraose (BsAXH-m2,3; PDB code: 3C7G) (25), cyan and blue; Humicola insolens double substituted xylan α-L-1,3-arabinofuranosidase complexed with 32-α-L-arabinofuranosyl-xylotriose (AXHd3; PDB code: 3ZXK) (28), pale green and green. B, Closeup view of the three catalytically important residues superimposed on the bound L-arabinose (Ara) molecule in the ScAraf62A–Ara complex. C, Close-up view of the catalytic cleft with bound xylooligosaccharides. FIGURE 6. The active site cleft of α-L-arabinofuranosidase from Streptomyces coelicolor

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FIGURE 1. Enzymatic properties of wild-type α-L-arabinofuranosidase from Streptomyces coelicolor (ScAraf62A). A, Optimum pH; B, optimum temperature; C, stability for pH; D, stability for temperature. Symbols: black triangle, Glycin-HCl buffer; black circle, McIlvaine buffer; black square, Atkins-Pantin buffer.

(ScAraf62A). Yellow, xylotriose; pink, arabinoheptaose.

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TABLE 1. Primers used in this study *The underlined sequences represent mutation sites. Primer Nucleotide sequence* Araf62A

F: 5′-CATATGCACAGAGGAAGTCTCAGCCGCGGG-3′ R: 5′-AAGCTTTCAGCGCCGCAGGGTGAGGACTCC-3′

D165N

F: 5′-GTCGCGCTGAAGAACTTCACCACGGTGACGCAC-3′ R: 5′-CGTGGTGAAGTTCTTCAGCGCGACCCACCCG-3′

D165E

F: 5′-GTCGCGCTGAAGGAGTTCACCACGGTGACGCAC-3′ R: 5′-CGTGGTGAACTCCTTCAGCGCGACCCACCCG-3′

W233A

F: 5′-CTGGCGTACCAGGCGGGCTCGTGGCCGTTCATCTA-3′ R: 5′-CCACGAGCCCGCCTGGTACGCCAGCACCCA-3′

W233Y

F: 5′-CTGGCGTACCAGTATGGCTCGTGGCCGTTCATCTA-3′ R: 5′-CCACGAGCCATACTGGTACGCCAGCACCCA-3′ F: 5′-CTGGCGTACCAGTTTGGCTCGTGGCCGTTCATCTA-3′ R: 5′-CCACGAGCCAAACTGGTACGCCAGCACCCA-3′

E324Q

F: 5′-GCCAACCTGTTCCAGGGCGTACAGGTCTACAAGG-3′ R: 5′-ACCTGTACGCCCTGGAACAGGTTGGCCTTCGT-3′

Y424A

F: 5′-GCGGGCGGGGACGCCAACTCGCTGCCGTGGC-3′ R: 5′-CGGCAGCGAGTTGGCGTCCCCGCCCGCGTTCG-3′

Y424W

F: 5′-GCGGGCGGGGACTGGAACTCGCTGCCGTGGC-3′ R: 5′-CGGCAGCGAGTTCCAGTCCCCGCCCGCGTTCG-3′

Y424F

F: 5′-GCGGGCGGGGACTTCAACTCGCTGCCGTGGC-3′ R: 5′-CGGCAGCGAGTTGAAGTCCCCGCCCGCGTTCG-3′

N425Q

F: 5′-GGGGACTACCAATCGCTGCCGTGGCGGCCGGGA-3′ R: 5′-CGGCAGCGATTGGTAGTCCCCGCCCGCGTTCGGG-3′

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W233F

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TABLE 2. Activity of wild type and mutants of α-L-arabinofuranosidase from Streptomyces coelicolor (ScAraf62A) against p-nitrophenyl-α-Larabinofuranoside (PNP-α-L-Araf) and wheat arabinoxylan N.D., no activity detected; N.A., not analyzed

The kinetic parameters kcat and Km were determined by Lineweaver–Burk plots from three independent experiments and with five substrate concentrations.

Substrates

Wheat arabinoxylan

PNP-α-L-arabinofuranoside Km

kcat

Km

kcat

(mM)

(min−1)

(mg·ml−1)

(min−1)

Wild type

1.9 ± 0.2

1.2 ± 0.1

7.3 ± 0.0

18 ± 2

D202N

N.D.

N.D.

N.A.

N.A.

E361Q

N.D.

N.D.

N.A.

N.A.

W270A

18 ± 1

0.71 ± 0.13

0.038

0.06

27 ± 9

W270Y

11 ± 2

0.69 ± 0.08

0.068

0.1

W270F

9.4 ± 0.8

0.36 ± 0.03

0.039

0.06

Y461A

N.D.

N.D.

Y461W

1.5E+15 ± 0.0

7.2E+11 ± 0.0

0.47E−3

Y461F

2.9 ± 0.2

0.0038 ±

Enzyme

kcat/Km

Relative to WT

(mM−1·min−1)

0.61

1

kcat/Km

Relative to WT

(mg−1·ml·min−1)

2.5

1

16 ± 2

0.65

0.3

26 ± 3

16 ± 3

1.2

0.5

36 ± 8

25 ± 6

0.72

0.3

15 ± 4

30 ± 9

2.0

0.8

0.8E−3

N.D.

N.D.

0.0013

0.002

7.0 ± 0.6

3.0 ± 1.05

0.43

0.2

0.71

1.2

28 ± 13

51 ± 23

1.8

0.7

0.000 N462Q

0.88 ± 0.00

0.62 ± 0.0

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TABLE 3. Data acquisition and structure refinement statistics of α-L-arabinofuranosidase from Streptomyces coelicolor (ScAraf62A) Values in parentheses refer to the highest resolution shell. Data PDB code Data collection Unit-cell parameters (Å) Beam Line

Native 3WMY P41212 a = b = 97.2, c = 102.8 PF-AR NE3A

Hg derivative (Peak) 3WMZ P41212 a = b = 97.3, c = 103.5 PF-AR NE3A

Ara complex 3WN0 P41212 a = b = 98.2, c = 103.7 PF BL-5A

X3 complex 3WN1 P41212 a = b = 97.7, c = 104.0 PF BL-5A

X6 complex 3WN2 P41212 a = b = 97.4, c = 103.2 PF-AR NW12A

Wavelength (Å)

1.0031

1.0031

1.0000

1.0000

1.0000

Resolution (Å)

100.0–1.40 (1.43–1.40)

100.0–1.90 (1.97–1.90)

100.0–1.90 (1.97–1.90)

100.0–2.00 (2.07–2.00)

100.0–2.1 (2.18–2.10)

Rsym

0.061 (0.441)

0.077 (0.414)

0.106 (0.433)

0.155 (0.451)

0.117 (0.512)

Completeness (%)

95.9 (99.8)

99.9 (100.0)

99.9 (100.0)

99.9 (100.0)

100.0 (100.0)

Multiplicity

5.3 (5.0)

28.5 (27.3)

12.0 (11.3)

17.3 (17.3)

Average I/σ (I)

23.1 (4.6)

42.9 (13.8)

20.4 (7.2)

42.9 (13.8)

16.1 (8.5)

Unique reflections

92 684 (6 323)

39 646 (3 898)

40 681 (3 984)

34 678 (3 400)

29 718 (2 910)

Observed reflections

488 849

1 131 521

487 017

599 796

635 625

Resolution

22.1–1.4 (1.44–1.40)

32.7–1.9 (1.95–1.90)

29.8–1.9 (1.95–1.90)

31.1–2.0 (2.05–2.00)

34.5–2.1 (2.16–2.10)

R-factor

0.205 (0.378)

0.182 (0.200)

0.175 (0.243)

0.179 (0.198)

0.167 (0.191)

Rfree-factor

0.218 (0.399)

0.198 (0.250)

0.184 (0.253)

0.204 (0.237)

0.191 (0.227)

Bond lengths (Å)

0.005

0.006

0.006

0.007

0.007

Bond angles (°)

1.064

1.072

1.122

1.510

1.138

No. of water molecules

380

185

375

308

256

Average B-value

17.4

27.4

17.0

19.2

22.3

21.2 (21.4)

Structure refinement

r.m.s. deviations from ideal value

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favored region (%)

96.0

96.3

96.3

96.7

allowed region (%)

3.3

3.0

3.0

2.7

outlier region (%)

0.7

0.7

0.7

0.7

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Ramachandran plot

96.3 3.0 0.7

TABLE 4. Activity of wild-type and mutants of α-L-arabinofuranosidase from Streptomyces coelicolor (ScAraf62A) against the arabino-oligosaccharides A1X2 and A1X3 Substrates A1X2

A1X3 kcat/Km (mM−1·min−1)

Enzyme Wild-type

33 ± 9

178 ± 7

N462Q

24 ± 4

66 ± 14

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Fig. 1.

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Fig. 2.

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3.

30

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Fig.

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Fig. 4.

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Fig. 5.

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Fig. 6

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