Characterization and Structural Analysis of a Novel exo-Type Enzyme

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Characterization and structural analysis of a novel exo-type enzyme acting on #-1,2-glucooligosaccharides from Parabacteroides distasonis Hisaka Shimizu, Masahiro Nakajima, Akimasa Miyanaga, Yuta Takahashi, Nobukiyo Tanaka, Kaito Kobayashi, Naohisa Sugimoto, Hiroyuki Nakai, and Hayao Taguchi Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.8b00385 • Publication Date (Web): 15 May 2018 Downloaded from http://pubs.acs.org on May 16, 2018

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Biochemistry

Characterization and structural analysis of a novel exo-type enzyme acting on β-1,2-glucooligosaccharides from Parabacteroides distasonis

Hisaka Shimizu†, Masahiro Nakajima*,†, Akimasa Miyanaga‡, Yuta Takahashi§, Nobukiyo Tanaka†, Kaito Kobayashi†, Naohisa Sugimoto§, Hiroyuki Nakai§, and Hayao Taguchi†



Department of Applied Biological Science, Faculty of Science and Technology, Tokyo

University of Science, 2641 Yamazaki, Noda, Chiba 278-8510, Japan ‡

Department of Chemistry, Tokyo Institute of Technology, 2-12-1, O-okayama, Meguro-ku,

Tokyo 152-8551, Japan §

Faculty of Agriculture, Niigata University, Niigata 950-2181, Japan

ABSTRACT β-1,2-Glucan is a polysaccharide produced mainly by some Gram-negative bacteria as a symbiosis and infectious factor. We recently identified endo-β-1,2-glucanase from Chitinophaga pinensis (CpSGL) as an enzyme comprising a new family. Here, we report the characteristics and crystal structure of a CpSGL homolog from Parabacteroides distasonis, an intestinal bacterium (BDI_3064 protein), which exhibits distinctive properties of known β-1,2-glucan-degrading

enzymes.

BDI_3064

hydrolyzed

linear

β-1,2-glucan

and

β-1,2-glucooligosaccharides with degrees of polymerization (DP) of 4 or more to produce sophorose specifically, but did not hydrolyze cyclic β-1,2-glucan. This result indicates that BDI_3064 is a new exo-type enzyme. BDI_3064 also produced sophorose from β-1,2-glucooligosacharide analogs that have a modified reducing end, indicating that BDI_3064 acts on its substrates from the non-reducing end. The crystal structure showed that BDI_3064 possesses additional N-terminal domains 1 and 2, unlike CpSGL. Superimposition of BDI_3064 and CpSGL complexed with ligands showed that R93 in the domain 1 overlapped subsite –3 in CpSGL. Docking analysis involving a β-1,2-glucooligosacharide with DP4 showed that R93 completely blocks the non-reducing end of the docked β-1,2-glucooligosacharide. This indicates that BDI_3064 employs a distinct mechanism of recognition at the non-reducing end of substrates to act as an exo-type enzyme. Thus, we propose 2-β-D-glucooligosaccharide sophorohydrolase (non-reducing end) as a systematic name for BDI_3064.

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Introduction β-1,2-Glucan is a homopolymer composed of glucose. β-1,2-Glucan mainly exists as a cyclic form in some Gram-negative bacteria, such as Agrobacterium, Rhizobium, Shinorhizobium, and Brucella1–5. Cyclic β-1,2-glucan is known as a commensal factor in Rhizobium and as an infectious factor in Brucella abortus as to their hosts6,7. Production of this glucan in the periplasm or the extracellular space for osmotic regulation has also been reported 8–10. The chain lengths of cyclic β-1,2-glucans are usually around the degrees of polymerization (DP) of 20, while it has been reported that Rhizobium meliloti produces longer cyclic β-1,2-glucans (DP of around 40)11. Cyclic β-1,2-glucan synthases are known as membrane proteins and its reaction mechanism are well-characterized12–16. Linear β-1,2-glucan (unless otherwise noted, β-1,2-glucan represents the linear form) is found in Acetobacter and Xanthomonas17,18. Production of β-1,2-glucan with β-1,6-glucosyl branches in Escherichia coli and Pseudomonas syringae has been reported as well8,10,19. In addition, sophorosides such as kaempherol 3-O-sophoroside and stevioside are found in some plants20,21. In spite of such distribution and important physiological roles of β-1,2-glucan, small abundance of β-1,2-glucan has limited investigation of β-1,2-glucan degrading enzymes. We first identified a glycoside phosphorylase acting on β-1,2-glucan from Listeria innocua as a β-1,2-glucan degrading enzyme. This enzyme was named 1,2-β-oligoglucan phosphorylase (SOGP) and was given a new EC number (EC3.2.1.333)22. SOGP enables us to prepare β-1,2-glucan in large-scale23. Then, two kinds of glycoside hydrolase family (GH) 3 β-glucosidases acting on β-1,2-gluco (sophoro)-oligosaccharides (Sopns, n represents DP) were identified24–27. However, they all are enzymes acting on non-reducing end β-glucoside. Recently, a bacterial β-1,2-glucanase (SGL) from Chitinophaga pinensis (CpSGL) was identified28. CpSGL is specific for β-1,2-glucosidic linkage and prefers β-1,2-glucan over Sop5 as substrates. The enzyme follows anomer-inverting reaction mechanism. The complex structure of CpSGL with Sop3 and Glc provides structural evidence for binding of Sopns. CpSGL was classified into a new GH family, GH144, because CpSGL shows no significant sequence similarity with known GHs. Though SGL homologs are widely distributed in Gram-negative bacteria and are divided into more than 10 subgroups phylogenetically, biochemical functions of GH144 proteins other than CpSGL have not been reported. Therefore, investigation of GH144 homologs is important for finding other β-1,2-glucan-related enzymes. Parabacteroides distasonis, one of the major intestinal bacteria29, has two SGL homologs (BDI_3064 protein and BDI_3066 protein). BDI_3064 is expected to have different

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Biochemistry

functions from CpSGL, because it possesses an additional N-terminal region of unknown function. In this study, we identified BDI_3064 as an exo-type enzyme acting on β-1,2-glucooligosaccharides and revealed the structural features related to the exolytic property of the enzyme.

Materials and methods Materials β-1,2-Glucan (average DP of 77) was prepared as described by Nakajima et al.22. Cyclic β-1,2-glucan (DP 17 to 24) was kindly supplied by Prof. Hisamatsu. Sop2–6 was prepared using SOGP from L. innocua and CpSGL22,28. The reducing-ends of Sopns were reduced by adding 20 µl of 1 M NaBH4 to 10% (w/v) Sopn solutions. After incubation at room temperature over 5 min, 15 µl of 3 M acetate was added to stop the reaction. Then, 1 ml of 2-propanol was added to the samples. After incubation of the samples on ice for 5 min, pellets were collected by centrifugation at 15,000 rpm for 4 min and then dissolved with a small amount of water. Precipitation of 2-propanol was repeated to remove salts and the pellet was dried in air. Sopn modified at its reducing end is denoted as rSopn. Laminarin, carboxymethyl (CM)-cellulose, and hydroxyethyl-cellulose were purchased from Sigma-Aldrich (MO, USA). Pachyman, CM-pachyman, CM-curdlan, lichenan, tamarind xyloglucan, glucomannan, arabinogalactan, arabinan, polygalacturonate, laminarioligosaccharides, cellooligosaccharides, and gentiobiose were purchased from Megazyme (Wicklow, Ireland).

Preparation of recombinant BDI_3064 A signal peptide of BDI_3064 protein was searched for with SignalP version 4.030. The signal peptide was excluded when the BDI_3064 gene was cloned. Genomic DNA of P. distasonis ATCC 8503 was purchased from Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures (Braunschweig, Germany). PCR was performed using the primer

pair

of

5’-CAGCTTGCATATGGTGGAGAAACCTTAC-3’

5’-GCACTCGAGTTTTATGGATTGAATCTTTTG-3’

(Reverse)

(Forward)

(restriction

sites

and are

underlined). The amplified gene was inserted into pET30a (Novagen) to fuse a His6-tag at the C-terminus. The resulting plasmid was transformed into E. coli Rosetta2 (DE3). The was cultured at 37°C in LB medium containing 30 µg/ml of kanamycin until OD600 reached 0.8. After addition of 0.1 mM isopropyl-1-thio-β-D-galactopyranoside (final concentration), the

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transformant was incubated at 20°C overnight. The cells were collected by centrifugation at 3,900 × g for 10 min at 4°C. The cells suspended in 50 mM MOPS-NaOH (pH 7.0) containing 300 mM NaCl (buffer A) were then sonicated by Branson sonifier 450. The supernatant was collected by centrifugation at 27000 × g for 15 min at 4°C. The sample was loaded onto a HisTrap FF crude column (GE Healthcare, Buckinghamshire, UK) equilibrated with buffer A. Then, the column was washed with buffer A containing 10 mM imidazole until the unbound compounds were almost completely removed. The target protein was eluted with a linear gradient of 10–300 mM imidazole in buffer A. The sample was buffered with 50 mM MOPS buffer (pH 7.0) using Amicon Ultra 30,000 molecular weight cut-off (Merck Millipore, MA, USA). Protein concentrations were calculated using the absorbance at 280 nm and extinction coefficient (187,855 M−1 ·cm−1)31 calculated from the theoretical molecular mass of the protein (83614 Da). For crystallization, the sample was loaded onto a UnoQ column equilibrated with 50 mM MOPS (pH 7.0). After the column had been washed with the same buffer, the sample was eluted with a linear gradient of 0–300 mM NaCl in 50 mM MOPS. The sample was purified using Superdex 200 (Hiload 16/60, GE Healthcare) equilibrated with 20 mM MES buffer (pH 6.5) containing 150 mM NaCl. Ovalbumin (44 kDa), conalbumin (75 kDa), aldolase (158 kDa), ferritin (440 kDa), and thyroglobulin (669 kDa) (GE Healthcare) were used as molecular weight markers. The sample was concentrated to 10 mg/ml and buffered with 5 mM MOPS buffer (pH 7.0) using Amicon Ultra 30,000 molecular weight cut-off (Merck Millipore). The purity of the enzyme was check by a sodium dodecyl sulfate–polyacrylamide gel electrophoresis. For preparation of R93A mutant and domain 1–2-truncated mutant, PrimeSTAR Mutagenesis Basal Kit (TakaraBio, Osaka, Japan) was used in accordance with a manufacturer’s

instruction.

Primer

pairs

used

were

5’-

ATTGCCAGCGACCGGGCAATATTGTATC

-3’

(Forward)

and

5’-

CCGGTCGCTGGCAATGACTTCTTTCGTG

-3’

(Reverse),

and

5’-

and

5’-

ATATGGTGACGGACGAGCAATTGCTG

-3’

(Forward)

CGTCCGTCACCATATGTATATCTCC -3’ (Reverse), respectively.

Assay The substrate specificity toward various polysaccharides was examined by the PAHBAH method using p-hydroxybenzhydrazide32,33. Each polysaccharide (0.0125% lichenan, 0.0125% laminarin,

0.006%

arabinogalactan,

0.1%

pachyman,

0.05%

CM-curdlan,

0.025%

CM-pachyman, 0.1% glucomannan, 0.2% tamarind xyloglucan, 0.1% polygalacturonate, 0.1%

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Biochemistry

CM-cellulose, 0.1% hydroxyethyl-cellulose, or 0.2% arabinan) in 50 mM MES buffer (pH 6.5) was incubated with 0.1 mg/ml (1.2 µM) of BDI_3064 at 30°C for 24 h. The samples were heated at 100°C for 5 min to stop the reaction. After the samples had been mixed with 4 volumes of a PAHBAH solution and heated at 100°C for 5 min, the absorbance at 410 nm was measured. Glucose was used as a standard. The substrate specificity toward various oligosaccharides and β-1,2-glucans was examined by TLC. The substrate solution (0.2%) in 50 mM MES-NaOH (pH 6.5) was incubated with BDI_3064 at 30°C. After the reaction had been stopped by the same heat treatment, the reaction products were analyzed by TLC as described below. The activity toward β-1,2-glucan (average DP of 77), Sop4 and Sop5 was measured using the GOPOD method using the GOPOD solution (Megazyme). As standard conditions, the reaction was performed at 30°C in 20 mM MES buffer (pH 6.5). After the reaction had been stopped by heat treatment at 100°C for 5 min, an equal volume of β-glucosidase from almonds (Oriental Yeast, Tokyo, Japan) in 200 mM sodium acetate buffer (pH 5.0) was added to the samples to degrade only released Sop2 at 50°C for 2 h. Then, the samples mixed with six volumes of the GOPOD solution were incubated at 45°C for 20 min. The absorbance at 510 nm was measured to determine the glucose concentrations in the samples. Glucose was used as a standard.

Temperature and pH profiles The enzymatic reaction was performed for 15 min in 20 mM buffer containing 3 mM Sop4 and 3.25 µg/ml of BDI_3064. The released Sop2 was quantified using the GOPOD method as described above. The optimum temperature and pH were determined by ones changing the temperature and buffer from the standard conditions, respectively. When temperature and pH stability were determined, 32.5 µg/ml (0.39 µM) of the enzyme solutions were incubated at various temperatures in 20 mM MES buffer (pH 6.5) and at 30°C in various buffers, respectively, for 1 h. The enzymatic reaction was performed for 15 min in 20 mM MES buffer (pH 6.5) containing 3 mM Sop4 and one-tenth volume of the incubated BDI_3064 solution. These experiments were performed triplicate.

TLC analysis The reaction solutions (1 µl) and markers (1 µl) were spotted onto TLC Silica gel60 F254 plates (Merck Millipore). Marker solutions containing 0.2% of each compound are used. The samples were developed with a solution (acetonitrile : water = 3:1, v/v). Then, the plates were heated

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with an oven until sugars were detected clearly. Only for analysis of degradation patterns, the samples were developed twice using a solution (acetonitrile : acetate : 2-propanol : water = 17:4:4:3, v/v).

Kinetic analysis The enzymatic reaction and quantification of Sop2 was performed under the standard conditions as described above. The concentrations of BDI_3064, Sop4, Sop5, and β-1,2-glucan used for the kinetic analysis were 3.3 µg/ml, 0.25–4.0 mM, 0.3–10 mM, and 0.05–0.5 mM, respectively. The kinetic parameters were calculated by non-linear regression of the experimental data to the Michaelis-Menten equation using KaleidaGraph version 3.51. Standard deviations of kcat/Km values are calculated using a modified Michaelis-Menten equation as follows; ‫ݒ‬/[‫ܧ‬଴ ] =

݇ୡୟ୲ [S] × ‫ܭ‬୫ [S] 1 + 1 ‫ܭ‬୫

, where v is the initial reaction velocity of Sop2 release, [E0] the enzyme concentration, and [S] a concentration of a substrate.

Crystallography The screening of crystallization conditions was performed by the sitting-drop vapor diffusion method using Wizard Classic 1&2 (Emerald Biosystems, WA, USA), PEG/Ion Screen (Hampton Research, CA, USA), and Crystal Screen HR2-110 (Hampton Research). The reservoir solution (1 µl) and an equal volume of 10 mg/ml BDI_3064 in 5 mM MOPS buffer (pH 7.0) were mixed and incubated at 25°C on CrystalQuick plates, 96-well, PS, round wells, and U-bottom (Greiner Bio-One, Kremsmünster Austria), with the corresponding reservoir solutions (70 µl). Crystals were grown with a reservoir solution (200 mM calcium acetate, 100 mM imidazole/ hydrochloric acid, pH 8.0, and 20% (w/v) PEG 1000). Before X-ray data collection, crystals were transferred to the reservoir solution supplemented with 25% glycerol as a cryoprotectant. Each crystal was cooled and then kept at 100 K in a nitrogen-gas stream during data collection. A set of X-ray diffraction data for the crystal was collected using a CCD detector (ADSC Q210) with a beamline NW12A at Photon Factory (Tsukuba Japan). The diffraction data set was processed using XDS34,35. The initial phase was determined by MOLREP36 using the structure of BACCAC_03554 (PDB ID: 4QT9, 36% amino acid sequence identity with BDI_3064) as a search model. Automated model building was performed with

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Biochemistry

Buccaneer37. Automated and manual refinement of the BDI_3064 structure were performed using Refmac538 and Coot39, respectively. The figures were prepared using MacPyMOL (DeLanoScientific; http://www.pymol.org).

Docking analysis The docking study was carried out using the AutoDock 4.2 program40. The Sop4 molecule was generated using Avogadro software41. Chain B of the crystal structure of BDI_3064 was used for the docking study. Although most of the water molecules and all glycerol molecules in the BDI_3064 structure were removed for the docking study, two water molecules that interact with D408 were retained. These two water molecules occupy almost the same positions as the water molecules involved in the interaction with the Sop3 molecule at subsite +1 in the CpSGL crystal structure28. Retaining these two water molecules was essential for Sop4 docking into the subsites from +2 to –2. Using AutoDockTools, polar hydrogen atoms were added to amino acid residues, and Gasteiger charges were assigned to all atoms of the protein. The 24 torsion angles of Sop4 were rotatable but the sugar rings were fixed in the 4C1 conformation. All of the protein residues were kept rigid. Grid maps were prepared with 40 × 40 × 40 points covering the substrate binding pocket with a point spacing of 0.375 Å. A total of 256 docking runs were performed with the Lamarckian genetic algorism, and the resulting binding modes were ranked into clusters based on their binding energies. The first-ranked cluster was reasonably large (47 of 256 docking runs) and showed a significantly lower binding energy (–5.80 kcal/mol) compared with the second-ranked cluster (–4.11 kcal/mol).

Results and Discussion Sequence profile BDI_3064 consists of a C-terminal homologous region with CpSGL and an additional N-terminal region. The C-terminal region (residues 300–721) shows 47% amino acid sequence identity

with

CpSGL

according

to

NCBI

BLAST

search

(https://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE=Proteins). The N-terminal region is predicted to consist of galactose-binding lectin (residues 15–197, CATH superfamily 2.60.120.430) and immunoglobulins (residues 201–314, CATH superfamily 2.60.40.10) based on InterPro analysis (https://www.ebi.ac.uk/interpro/).

According

to

the

LipoP

1.0

server

(http://www.cbs.dtu.dk/services/LipoP/)42, BDI_3064 possesses a type-I signal peptide, suggesting periplasmic localization of the enzyme.

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Substrate specificity Since CpSGL is an enzyme acting on polymers, we first examined the substrate specificity of BDI_3064 using 13 kinds of polysaccharides. As a result, BDI_3064 showed hydrolytic activity only toward β-1,2-glucan among polysaccharides examined, indicating that the enzyme is highly specific to β-1,2-glucan. However, its specific activity on β-1,2-glucan (average DP25) was much lower (0.1 U/mg) than that of CpSGL (26 U/mg)28. Therefore, the chain length specificity of the enzyme was examined. BDI_3064 hydrolyzed Sop4 to release Sop2, and hydrolyzed Sop5 to release Sop2 and Sop3 (Figure 1). The reaction velocities with Sop4 and Sop5 were similar. Consumption rate of Sop6 was approximately 5 times slower than those of Sop4 and Sop5. Sop2 was detected as the main product in the reaction with Sop6. The amount of detected Sop4 was significantly smaller than that of Sop2 (Figure 1). This is probably because Sop4 released from Sop6 on the first hydrolysis is a more preferable substrate than Sop6. Sop3 was not hydrolyzed at all. The enzyme did not act on laminari-oligosaccharide (-biose, -triose, and -tetraose), cello-oligosaccharide (-biose, -triose, and -tetraose), or gentiobiose (Figure 1). These results indicate that the enzyme is specific to Sopns with a DP of 4 or more.

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Biochemistry

Sop3

Sop4

Sop5

Sop6

1 2 3 4 5 6 7

M 0 1 6 24 M 0

5 10

Reaction time (h)

(min)

60

M0

5 10

60

M0

5 10

(min)

Laminaribiose

Laminaritriose Laminaritriose

M 0 1 6 24

M 0 1 6 24

60

(min)

1 2 3 4

Reaction time (h) Cellobiose

M 0 1 6 24 (h)

(h) Cellotriose

Cellotetraose

Gentiobiose 1

1 2

2

3 4

M 0 1 6 24 Reaction time (h)

M 0 1 6 24

M 0 1 6 24

M 0 1 6 24

(h)

(h)

(h)

Figure 1. Activity of BDI_3064 toward glucooligosaccharides. Markers are indicated by M. The linkage positions of glucooligosaccharides used as markers correspond to those of the oligosaccharides shown above the TLC plates. Markers contain 0.2%

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of each compound. DPs of the markers are shown at left of the plates. Reaction times are shown below the TLC plates. Units of time are shown in parentheses. The reaction times for Sop4–6 were 0, 2.5, 5, 7.5, 10, 15, 30, and 60 min. The concentrations of the enzyme were 6.5 µg/ml (0.078 µM) for Sop4 and Sop5, 32.5 µg/ml (0.39 µM) for Sop6, and 0.325 mg/ml (3.9 µM) for the other substrates. All concentrations of substrates used for the reactions were 0.2%. Detail reaction conditions are shown in Materials and Methods section.

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General properties BDI_3064 migrated at approximately 80 kDa on an SDS-PAGE gel (Figure S1). The enzyme was eluted at the position of 110 kDa on size-exclusion chromatography, which is relatively larger than that on the SDS-PAGE. However, considering a surface contact area in the crystal structure of BDI_3064 (described below), the enzyme is a monomer. The enzyme showed the highest activity at pH 6.0 and was stable at pH 6.0–10.0. The optimum temperature of the enzyme was 40°C and it showed more than 90% residual activity up to 30°C (Figure 2).

(A)

(B) 100

100

Relative activity (%)

Relative activity (%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

80 60 40 20 0

2

4

6 pH pH

8

10

80 60 40 20 0 0

10

20

30

40

50

60

Temperature (℃)

Figure 2. pH (A) and temperature (B) profiles. Error bars represent standard error. (A) Closed symbols and lines indicate the pH optimum, and open symbols and dashed lines the pH stability. Citrate (circles), MES-NaOH (triangles), MOPS-NaOH (squares), HEPPSO-NaOH (inverted triangles), Bicine-NaOH (rhomboids), and glycine-NaOH (arrowheads) were used for creation of the profiles. Hydrolytic activity in MES buffer (pH 6.5) is defined as 100%. (B) The temperature optimum is shown by closed circles and lines, and the temperature stability by open circles and dashed lines. The highest hydrolytic activities for optimum and stability are defined as 100%.

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Action pattern In order to determine whether BDI_3064 is exo-type or endo-type, hydrolytic activity toward linear and cyclic β-1,2-glucan was examined. The enzyme hydrolyzed linear β-1,2-glucan to produce only Sop2, while it did not hydrolyze cyclic β-1,2-glucan (Figure 3). This result indicates that the enzyme cleaves β-1,2-glucan from either end by Sop2 unit. Thus, BDI_3064 is an exo-type enzyme. In order to determine the direction of hydrolysis by the enzyme, the reaction of BDI_3064 was analyzed with Sopn analogs (rSopns), each of which was prepared from the corresponding Sopn by reducing the aldehyde group of the reducing end Glc moiety. As a result, BDI_3064 degraded rSop6 into Sop2 and rSop4, and also degraded rSop5 into Sop2 and rSop3 without a significant decrease in reaction velocity (Figures 1 and 3). These results suggest that the enzyme releases Sop2 from the non-reducing end of Sopns. Among Prokaryotes, only endo-type enzymes (SGL from C. arvensicola and CpSGL) have been reported28,43. SGLs from Eukaryotes such as Acremonium sp.15 was reported to produce Sop2 as a main product but cleave cyclic β-1,2-glucan44, indicating that they are endo-type enzymes like the bacterial SGLs. Therefore, the action pattern of BDI_3064 is apparently different from those of the other reported SGLs.

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Biochemistry

Figure 3. Action pattern of BDI_3064 as to cyclic and linear β-1,2-glucans, rSop5, and rSop6. The concentrations of BDI_3064 in the reaction solutions were 325 µg/ml (3.9 µM) for cyclic and linear β-1,2-glucan, 6.5 µg/ml (0.078 µM) for rSop5, and 32.5 µg/ml (0.39 µM) for rSop6. Markers are indicated by M. DPs of markers are shown as numbers and letters “r” indicates modification of markers at the reducing end. Markers contain 0.2% of each compound. The reaction times were 0, 2.5, 5, 7.5, 10, 15, 30, and 60 min from the second lane from the left. In the case of cyclic β-1,2-glucan, the reaction times were 0, 1, 6, and 24 h. Detail reaction conditions are shown in Materials and Methods section. Cyclic β-1,2-glucan was hydrolyzed with CpSGL in 50 mM MES-NaOH (pH 6.5) containing 0.325 mg/ml (6.3 µM) of CpSGL for 0.5 h at 30°C. All concentrations of substrates used for the reactions were 0.2%.

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Kinetic properties In order to understand the chain length preference of BDI_3064, kinetic analysis of the enzyme as to Sop4, Sop5, and β-1,2-glucan was performed. The enzyme showed similar Km values for these substrates (Table 1 and Figure S2). The kcat value for Sop4 is twice and 100 times higher than those for Sop5 and β-1,2-glucan, respectively. The differences in kcat/Km values among the substrates depend on these kcat values. This finding suggests that the most preferable substrate is Sop4 and the hydrolytic activity decreases in accordance with the increase in DP.

Table 1. Kinetic parameters of BDI_3064. Substrate

kcat (s–1)

Km (mM)

kcat/Km (s–1 mM–1)

Sop4

54 ± 3

1.8 ± 0.2

28 ± 2

29 ± 2

1.6 ± 0.3

18 ± 2

0.48 ± 0.09

1.0 ± 0.3

0.47 ± 0.03

(13 ± 3) b

(0.037 ± 0.003) b

Sop5 β-1,2-Glucan

a

(average DP77) a

These values are calculated using the data with only lower substrate concentrations than the

Km value. b

Parentheses indicate that units for Km and kcat/Km are mg/ml and s–1 mg–1 ml, respectively.

Overall structure The crystal structure of apo BDI_3064 was determined at 2.1 Å resolution (Table 2). There are two molecules in an asymmetric unit. The rmsd of the two molecules is 0.1 Å, indicating that the structures of the two molecules in the asymmetric unit are almost identical. The buried surface area between the two molecules is approximately 900 Å2 (only 3.6% of whole surface area of a monomer)

according

to

analysis

of

macromolecular

interface

by

PDBePISA

(http://www.ebi.ac.uk/pdbe/pisa/pistart.html)45, suggesting that a biological assembly of BDI_3064 is a monomer. The structure of BDI_3064 comprises three domains; domain 1 (residues 19–198), domain 2 (residues 211–299), and domain 3 (residues 300–721) (Figure 4).

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Biochemistry

Domains 1 and 2 are connected by a loop (199–210). Then, structurally similar proteins to the domains were searched by Dali server46. Domain 1 shows structural similarity to the family 30 carbohydrate binding module (CBM30) in the endoglucanase from Clostridium thermocellum (PDB ID: 2C24) (Z score, 17.7; rmsd, 2.5 Å; sequence identity, 19%) (Figures 4B and S3A). A calcium ion presumably derived from the crystallization buffer is bound to the domain 1 with 7-coordination geometry (Figure S4). Domain 2 shows structural similarity to a fibronectin III domain, which is the C-terminal domain of a hedgehog protein from Drosophila melanogaster (PDB ID: 2IBB) 47 (Z score, 13.1; rmsd, 1.8 Å; sequence identity, 18%) (Figures 4C and S3B). Domain 3 shows structural similarity to the (α/α)6 barrel of CpSGL (PDB ID: 5GZK) (Z score, 60.5; rmsd, 1.2 Å; sequence identity, 49%) (Figures 4D and 5), and its homologs from Bacteroides species (PDB ID: 4GL3) (Z score, 62.2; rmsd, 1.0 Å; sequence identity, 50%).

Table 2. Data collection and refinement statistics of BDI_3064. Data collection statistics Refinement statistics Beamline PF AR-NW12A R/Rfree (%) No. of non-hydrogen Wavelength (Å) 1.0000 atoms 46.73-2.10 Protein Resolution (Å) (2.14-2.10) Ca2+ Space group C2 Glycerol a = 130.08 Å, Solvent b = 123.34 Å, Average B-factors (Å2) Unit cell dimensions c = 101.38 Å Protein β = 107.88° Ca2+ Observed reflections 334365 (16964) Glycerol Unique reflections 86780 (4410) Solvent Completeness (%) 97.9 (94.0) r.m.s.d from ideality I/σ(I) 14.1 (2.2) Bond length (Å2) Redundancy 3.9 (3.8) Bond angle (°) Rmerge 0.068 (0.570) Chiral volume CC1/2 (0.739) Ramachandran plot Favored region (%) Allowed region (%) Outliers (%) Values in parentheses are for the outermost shell. PDB ID of the structure of BDI_3064 is 5Z06.

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17.8/21.9 11333 2 36 550

33.7 30.8 45.1 33.4 0.011 1.478 0.084 96.3 3.6 0.1

Biochemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

(A)

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Domain 1 (magenta)

N

Y-axis

Domain 3 (cyan)

Loop (grey)

90° C Domain 2 (orange)

(B)

W68 D82(BDI)

(C)

(D)

n

c C C

c C

n c W27 W78 Q41(BDI)

N

N

n N

R93 (BDI)

Figure 4. Overall structure of BDI_3064. (A) Domain constitution of the BDI_3064 monomer. The colors of domains and a linker in BDI_3064 are shown as the figure. The positions of the N and C-termini are indicated. (B–D) Comparison of domains 1, 2, and 3 with CBM30 from C. thermocellum (PDB ID: 2C24) (B), N-terminal domain of a hedgehog protein from D. melanogaster (PDB ID: 2IBB) (C), and CpSGL (PDB ID: 5GZK) (D), respectively. (B) CBM30 is shown in yellow. W27, W68 and W78 important for binding to xylan in the CBM30 are shown as sticks. Q41 and D82 in BDI_3064 are residues corresponding to W27 and W68, respectively, and are shown as green sticks. R93 in BDI_3064 is also shown as a stick. (C) The N- and C-terminal domains of the hedgehog protein are shown in light brown and light blue, respectively. (D) CpSGL and an inserted helix (residues 511–526) in BDI_3064 are shown in grey and blue, respectively. In the other figures, the same color usage as for BDI_3064 and CpSGL is applied.

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Biochemistry

β20

α1 BDI_3064 BDI_3064 BDI_3066 CpSGL CpSGL

β21

298 44 1 23 1

P.M.TDEQLLDMVQEANFRYYWEGAEPNSGLARENIP......G.RNDMIATGASGFGIM LDFQSDDEFLTYIQKAHFNYMWEGAEPTSGLACERIHTDGVYPENDADIVTTGGSGFGIA A.L.SADSIFNIVEEQTFQYFWDGAEPVSGMARERYHVDGNYPENDMNVVTSGGSGFGVM TTT TT TT TT α1 β1 β2 α2 α3

β22

BDI_3064 BDI_3064 BDI_3066 CpSGL CpSGL

β23

TT 349 104 61 81 59

TT TT

AIVAGIERGFITREEGVQRFLKITSFLEKADKFHGAVSHFIDGTTGKTVAFFGPKDNGGD GLLVGIERGFITREEGVARFHQIADYLAKADRFHGVWPHWMHGPTGKVKP.FGQKDNGGD ALLVGIERGYISREQGLERLMKIVSFLEKADRFHGAWPHWLYGETGKVKP.FGQKDNGGD TT TT α3 β3 β4 α4

α5

η5

β24

BDI_3064 BDI_3064 BDI_3066 CpSGL CpSGL

TT 409 120 163 118 140

LVETSFLFQGLLTARQYFNQENDKEKQIRKSIDNLWKNVEWSWYKQFKDSPYLYWHWSPD LVESAFLMQGLLCVRQYFKDGNASEKALAAKIDQLWREMEWTWYLNG..QDVLYWHWSPN LVETSFMIQGLLCVRQYFANGNEQEKALAARIDQLWKAVEFSWYRNG..KNVLYWHWSPN α4

α5

η1

α6 469 221 178 198 176

QAWVINHKLIGWNETMITYMLAIMGPKYGISPEMYYSGWASQEEYAQEYRADWGRVEDGK YAWEMNFPLEGYNECLITYILAASSPTYPIPASAYHNGWARKG................. YKWQMNFPVTGYNECLIMYILAAASPTHGIPAEVYHEGWAKSG................. TTT α6 α7 η2 β25 β26 TT

529 264 221 241 219

η6

β27 TT 585 324 281 301 279

α11 645 382 339 361 339

η8

α9

β28

η9

β29

α10

TT

QRYCIENQGGYVGYGEDCWGLTASDFAWNYQAQEPMPHRDNGTMAPTGALASFPYTPDAS YRYCAENPKGFKGYSDACWGLTASYSVKGYNAHMPS..NDHGVITPTAALSSYPYTPEES REWCIQNPKHYKGYGPDSWGLTASYSVKGYAAHAPGENNDLGVISPTAALSSMPYTPEYS TT TT TT β8 β9 α9 α12 β30 β31 TT

BDI_3064 BDI_3064 BDI_3066 CpSGL CpSGL

η7

T..T

MYTNGNTYYGENLKV.GV.S..NGGPLFFIHYSYLGLDPHKFTDKYTNYFENNQKMAKIN GIKSDAVAYDLPLILKHNYAEEYGGPLFWAHYSYIGLCPVGLSDRYANYWDLNRNHVLID AIKDSINAYGHTLKLSHNFAKEYGGPLFWSHYSYLGLDPHGLKDRYADYWENNLNHVLIN TT TT β6 β7 η3 η4 α8

BDI_3064 BDI_3064 BDI_3066 CpSGL CpSGL

α8

TTT

BDI_3064 BDI_3064 BDI_3066 CpSGL CpSGL

β5

α7

BDI_3064 BDI_3064 BDI_3066 CpSGL CpSGL

α2

TTT

β32

β33

β34

α13

α14

MKALRNYYRNHGSFLWGEYGFRDAFNLTVNWVSPLFMGLNQAPVTVMIENYRTNLLWNLF SKALKHFYFNLGDSIWGKYGFYDAFSEGENWYPRRYLAIDQCTIAPMIENYRTGLLWRLF KQAMVHWYNDMRTKIFGKYGFYDAFSETENWYPQQYLAIDQGPIVVMMENYRSGLLWKLF TT α10 β10 β11 β12 α11 α12 α15

BDI_3064 BDI_3064 BDI_3066 CpSGL CpSGL

. 705 442 399 421 399

MSHPDVQKGIQKI.QS...I MSCPEIQDGLKKLGF..... MSCPEVQAGLKKLDFQSPYL TT α13

Figure 5. Multiple alignment of the catalytic domains of BDI_3064, BDI_3066 and CpSGL. Structure-based pairwise alignment of BDI_3064, BDI_3066 and CpSGL (PDB ID, 5GZK) was performed using MATRAS (http://strcomp.protein.osaka-u.ac.jp/matras/matras_pair.html)58,59. A model structure of BDI_3066 used for the alignment was constructed using CpSGL (PDB ID, 5GZK).

The

figure

was

prepared

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using

ESPript

3.0

Biochemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

(http://espript.ibcp.fr/ESPript/cgi-bin/ESPript.cgi)60. Residues mentioned in this paper are boxed with blue dashed lines. Among these residues, residues conserved in the three enzymes and residues which are not conserved in BDI_3066 and CpSGL are indicated as blue and magenta arrowheads, respectively.

Catalytic pocket In the catalytic pocket of BDI_3064, three glycerol molecules were observed (Figure 6A). Glycerol molecule 1 undergoes a stacking interaction with F400 and Y614, and forms hydrogen bonds with R93. Glycerol molecule 2 forms hydrogen bonds with E411. Glycerol molecule 3 forms hydrogen bonds with N481, E482, and Y517. To elucidate the precise binding modes of the substrates of BDI_3064, we attempted to obtain complex structures of BDI_3064 with substrates. However, these attempts were unsuccessful despite tremendous effort. Therefore, automated docking analysis was carried out. A Sop4 molecule was docked well into the catalytic pocket of BDI_3064, as shown in Figure 6BC. The Glc moiety at the reducing end of the Sop4 molecule well superimposes on glycerol molecule 3. The Glc moiety at the non-reducing end of the Sop4 molecule forms hydrogen bonds with R93 as in the case of glycerol molecule 1. This fact suggests that glycerol molecules 1 and 3 bind to BDI_3064 by mimicking Glc moieties. The second Glc moiety from the non-reducing end of the Sop4 molecule is also located near glycerol molecule 2, though deviated around 2 Å. R93 and the non-reducing end Glc moiety in the Sop4 molecule should be noted. R93 blocks the cleft at the non-reducing end side. The 2-OH group in the non-reducing end Glc moiety apparently faces the wall of the binding site (Figure 6D). This observation strongly suggests that there is no subsite beyond the 2-OH group. Because the enzyme is completely an exo-type enzyme releasing Sop2, the Sop4 molecule is considered to be docked at subsites –2 to +2. The Glc moieties in the Sop4 molecule except at subsite –1 seem to be recognized by BDI_3064 residues through several interactions (Figure 6BC). The Glc moiety at subsite –2 undergoes a stacking interaction with F400 and Y614, and forms three hydrogen bonds with R93 (Figure 6B). The Glc moiety at subsite +1 undergoes a stacking interaction with W463, and

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Biochemistry

forms four hydrogen bonds including water-mediated ones with the enzyme (Figure 6C). In addition, an oxygen atom in the pyranose ring of the Glc moiety forms intramolecular hydrogen bonds. The Glc moiety at subsite +2 forms six hydrogen bonds with N481, E482, and Y517 as in the case of glycerol molecule 3. Contrarily, the Glc moiety at subsite –1 undergoes only two inter- and intra-molecular hydrogen bond interactions. This is probably because all of the sugar rings of the model molecule were fixed in the 4C1 conformation for the docking analysis despite that general β-glucanases adopt twisted conformations at subsite –148,49. This is consistent with the difference in the positions between glycerol molecule 2 and the Glc moiety at subsite –1.

Figure 6. Catalytic pockets of apo (A) and Sop4-docked (B-D) BDI_3064. (A) Residues derived from domains 1 and 3 in BDI_3064 are shown in red and blue letters, respectively. Glycerol molecules are shown as light pink sticks. The Fo−Fc electron density maps of the glycerol molecules are presented as a grey mesh (contoured at 3.0 σ). Hydrogen bonds are shown as blue dashed lines. (B, C) A docked Sop4 molecule is shown as a thick light green stick. Subsite numbers are shown in bold letters. Parenthesis indicates that the position of a subsite is likely deviated. Inter- and intra-molecular hydrogen bonds, and hydrogen bond

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interactions mediated by water molecules are shown as blue, grey, and green dashed lines, respectively. Water molecules are shown as orange spheres. (C) Subsites –1 and –2 are omitted. (D) BDI_3064 is shown as a grey semi-transparent surface model. Only the region in the surface model related with domain 1 is shown in red. The residues except R93 and Y614 are omitted.

Comparison of the catalytic pockets In CpSGL, it has been reported that Sop3 and Glc bind to subsites +1 to +3 and –3, respectively28. In the catalytic pocket of BDI_3064, there are five acidic residues (E329, D404, D408, E411, and E482), which are conserved in CpSGL (Figure 5). This observation implies that both enzymes are presumed to follow the same reaction mechanism, though CpSGL seems not to follow a canonical reaction mechanism of glycoside hydrolases. Mutational analysis of the corresponding acidic residues in CpSGL did not narrow down candidates for catalytic residues28. Therefore, we focused on comparison of the substrate binding modes of BDI_3064 and CpSGL. The Sop2 moiety of the docked Sop4 molecule at subsites +1 and +2 in BDI_3064 well superimposes on that of the Sop3 molecule at the same subsites in CpSGL (Figure 7A). This suggests that the binding mode of Sop2 at subsites +1 and +2 is retained in BDI_3064. Actually, many of the residues involved in substrate binding around subsites +1 and +2 are conserved in both enzymes. Though N258 and W269 forming hydrogen bonds in CpSGL are not conserved in BDI_3064, Y517 provides two hydrogen bonds in BDI_3064 instead. D404 loses a direct hydrogen bond, while H475 provides a hydrogen bond unlike CpSGL. The position of subsite +3 in BDI_3064 is predicted to be the same as CpSGL. There is sufficient space to accommodate a Glc moiety at the presumed subsite +3 in BDI_3064. This is consistent with sufficient hydrolytic activity toward Sop5. However, the 1-OH group of the Glc moiety of the reducing end of the Sop3 molecule in CpSGL faces an inserted helix (α8, residues 511–526, Figure 5) in BDI_3064, though the space beyond the reducing end is not completely blocked (Figures 4D and 7A). This observation implies that longer substrates have to be twisted for binding to the enzyme, being consistent with the low activity toward Sop6 and β-1,2-glucan.

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Biochemistry

The Glc moiety at subsite –2 in BDI_3064 is clearly deviated from the Glc molecule in CpSGL (Figure 7B). This is caused by R93 in domain 1 apparently, because R93 overlaps the Glc molecule in CpSGL. There are several structural differences between the two enzymes around R93 as shown in Figure 7BC. The loop containing R93 in domain 1 overlaps a loop (residues 57–66) in CpSGL. The corresponding loop (residues 331–334) in domain 3 of BDI_3064 is short, making a space for the loop in domain 1 (Figures 5 and 7C). The position of the side chain of R93 is stabilized by salt bridges (or hydrogen bonds) with E329 and D609. N95 and N330 also form hydrogen bonds with the main chain of R93. While E54 and R55 in CpSGL interact with the Glc molecule (Figure 7BC), the corresponding E329 and N330 in BDI_3064, respectively, lose the role in the interaction with the substrate. Y325 stabilizing the position of R55 in CpSGL is replaced with D609 in BDI_3064. In addition, BDI_3064 has no residue at the position corresponding to N95 in CpSGL (Figure 7C). R93 also changes the substrate binding mode indirectly. R93 likely pushes Y614 away from the corresponding position of Y330 in CpSGL. This deviation can be associated with a difference in substrate positioning, since these residues stack on the substrates. Such deviation is consistent with the fact that the side-chain orientation of F400 stacking on the Glc moiety in BDI_3064 is different from that of F151 in CpSGL. It should also be noted that the orientation of the Glc moiety at subsite –2 is apparently different from that of the Glc molecule in CpSGL. While the 2-OH group in the Glc moiety at subsite –2 in BDI_3064 faces the wall of its catalytic pocket, the 2-OH group in the Glc molecule at subsite –3 in CpSGL faces the solvent28 (Figures 6C and 7B). Overall, these observations well reflect the fact that BDI_3064 is an exo-type enzyme while CpSGL is an endo-type one. In the cases of GH74 oligoxyloglucan reducing end-specific cellobiohydrolase from Geotrichum sp. M128 (OXG-RCBH), GH16 β-transglycosylase from Paecilomyces thermophila (PtBgt16A), and GH26 mannobiohydrolase from Cellvibrio japonicus, loops allow the enzymes to be exolytic50–52. However, these loops are derived from their catalytic domains, while the loop blocking the non-reducing end in BDI_3064 protrudes from the N-terminal additional domain.

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(A) D135 D404 (F131)

H193 H464

Page 22 of 36

none Y517

F204 H475

+3

W192 W463

N258 none

+1

D139 D408

+2 N210 N481 none W521

Sop3 Sop2 moiety H119 H387 E142 E411

E211 E482

CpSGL BDI_3064

W269 F553

(B) F131 F400 R55 N330

(–1)

E54 E329

Glc (Cp)

Sop4 (–3) (BDI) –2

none R93 Y325 D609 Y330 Y614

(C)

E54 E329 none N95

(–3)

R55 E329

Glc (Cp) none R93

Loop (BDI) Y325 (331-334) D609 Loop (Cp) (57-66) Loop (BDI) (domain 1)

Y330 Y614

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Biochemistry

Figure 7. Comparison of catalytic pockets of Sop4-docked BDI_3064 and CpSGL-Sop3 -Glc complex. Catalytic pockets around Sop3 (A) and Glc (B) in CpSGL. Domain 3 of BDI_3064 and CpSGL are superimposed. Ligands in CpSGL are shown as thin yellow sticks. A docked Sop4 molecule in BDI_3064 is shown as a thick light green stick as used in Fig. 6. Subsite numbers in CpSGL are shown as bold black letters. Hydrogen bonds related with substrate binding in BDI_3064 and CpSGL are shown as thick blue and thin green dashed lines, respectively. Residue numbers and substrates derived from BDI_3064 and CpSGL are shown in bold light blue letters and black letters, respectively. Residues in domain 1 of BDI_3064 are shown in red letters. (A) Water molecules needed in BDI_3064 for docking analysis and water molecules in CpSGL involved in substrate recognition are shown as orange and red spheres, respectively. The side chain of F131 (in a parenthesis) in CpSGL and the non-reducing end Sop2 moiety in the docked Sop4 molecule are omitted for visibility. (B) (–3) represents the presumed subsite number of a Glc molecule in CpSGL. Semi-transparent red circles represent the 2-OH groups in the Glc molecule and the Glc moiety in the Sop4 molecule. The reducing end Sop2 moiety in the docked Sop4 molecule is omitted. (C) Catalytic pocket around R93 in BDI_3064. Domain 3 in BDI_3064 is shown as surface representation. Loops in BDI_3064 (residues 331–334) and CpSGL (residues 57–66) are shown in cyan and black cartoons, respectively. Hydrogen bonds in both BDI_3064 and CpSGL are shown in thick dashed lines.

Role of N-terminal region Structural analysis revealed that the N-terminal region of BDI_3064 consists of a CBM30-like domain and an FNIII domain. The CBM30-like domain is localized near the non-reducing end of a substrate and is connected with the catalytic domain by the FNIII domain. In the CBM30 of the Clostridium endoglucanase, three tryptophan residues (W27, W68, and W78) located along β-sheets form a hydrophobic environment and are responsible for binding of xyloglucan53. However, these residues are not conserved in BDI_3064 and the corresponding positions are located beyond the blocked non-reducing end (Figure 4C). This observation implies that the CBM30-like domain loses the original ability to bind polysaccharides. Instead

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of the three Trp residues, a loop protruding into the catalytic pocket is considered to participate in substrate recognition according to docking analysis. R93 in the loop plays a critical role in product specificity due to blockage at the non-reducing end (Figures 6C and 7B). This residue is conserved in many homologs, though some homologs have an arginine residue at a different position or no arginine residue in the loop. To evaluate the function of R93, we constructed a R93A mutant and a truncated protein that lacks domains 1 and 2. However, both proteins were expressed as insoluble proteins. Interactions between R93 and domain 3 might be important for folding and/or stability of the enzyme.

Prediction of physiological role of BDI_3064 The distribution of SGL homologs is limited to some Bacteroidales species among genome-sequenced intestinal bacteria. Since P. distasonis is a member of the Bacteroidales, the bacterium is expected to play an important role in β-1,2-glucan metabolism in the large intestine54. Therefore, we predicted the physiological role of BDI_3064. In the Polysaccharide-Utilization Loci DataBase (http://www.cazy.org/PULDB/)55, a region from BDI_3060 to BDI_3071 is annotated as a PUL (Figure 8A). Judging from the array of genes, the region from BDI_3062 to BDI_3069 is likely an actual PUL. Another SGL homolog (BDI_3066) in the P. distasonis genome is also located in this gene cluster. BDI_3066 has no N-terminal domain unlike BDI_3064, and resembles CpSGL (63% sequence identity) rather than BDI_3064 (47% sequence identity). The inserted helix (α8) in BDI_3064 is absent in BDI_3066 as well as CpSGL (Figure 6). All of the residues related to substrate binding are conserved between BDI_3066 and CpSGL. These facts imply that BDI_3066 shares the same substrate specificity as CpSGL. In fact, the functional analysis showed that BDI_3066 is an endo-type SGL releasing Sopns (data not shown). Two putative GH3 β-glucosidases (BDI_3067 and BDI_3068) in the gene cluster are close homologs of Lin1840 from L. innocua, which predominantly prefers Sop2 as a substrate24, and BT_3567 from Bacteroides thetaiotaomicron, which acts on Sopns with a wide range of DPs25. R572 in Lin1840 and R622 in BT_3567 were shown to be quite important residues for recognition of Sop2 among gluco-disaccharides24,25. This arginine residue is conserved in BDI_3067 and BDI_3068 (Figure S5). An asparagine residue that was shown to be important for accommodating longer Sopns in BT_356725 is conserved in BDI_3068. In contrast, the asparagine residue is replaced by glycine in BDI_3067 as well as Lin1840 (Figure S5). These facts imply that Sop2 and longer Sopns are preferable

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Biochemistry

substrates for BDI_3067 and BDI_3068, respectively. As shown by LipoP1.0 analysis, BDI_3067 possesses a type-I signal peptide, and BDI_3066 and BDI_3068 possess a type-II signal one (Figure 8B). These facts suggest that BDI_3066 and BDI_3068 preferring longer Sopns are located extracellularly and BDI_3064 and BDI_3067 preferring shorter Sopns are located in the periplasm. BDI_3062 and BDI_3063 are annotated as SusC and SusD, respectively, implying that these proteins are transporters responsible for the uptake of extracellular hydrolysates of β-1,2-glucan55. In the gene cluster, there are two GH43 proteins (subfamily 28)56. Prediction of their functions is difficult, since no homolog in the subfamily has been characterized. Nevertheless, a complete β-1,2-glucan degradation pathway can be speculated without the GH43 proteins, as shown in Figure 8C. In this predicted pathway, BDI_3066 degrades β-1,2-glucan to Sopns. Then, BDI_3064 supplies Sop2 to BDI_3067 preferring Sop2 as a substrate. Overall, BDI_3064 may accelerate β-1,2-glucan degradation.

(A)

3060

3061

GH16

(B)

Protein

3062

3063

SusC

SusD

Annotation

3064

3065

3067

3068

GH144 GH43_28 GH144 GH3 +CBM32

GH3

Localization

3066

(C)

3069 3070 3071

GH43_28

β-1,2-Glucan BDI_3066 (GH144)

BDI_3060 GH16

Sopns

BDI_3061 hypothetical protein BDI_3063 SusD (outer membrane protein) Extracellular

Extracellular

BDI_3064 This study

Periplasm

BDI_3065 GH43_28 + CBM32 module

Cytosol

BDI_3066 GH144

Extracellular

BDI_3067 GH3

Periplasm

BDI_3068 GH3

Extracellular

BDI_3069 GH43_28

Periplasm

BDI_3070 hypothetical protein BDI_3071 hypothetical protein

Glc BDI_3068 (GH3)

BDI_3062 SusC (outer membrane protein) Periplasm

BDI_3062, 3063 (SusC, SusD)

Sopns BDI_3067 BDI_3064 (GH3) (GH144) Sop2 Periplasm

Glc

Cytosol

Figure 8. A putative PUL around the BDI_3064 gene. (A) The gene constitution of a putative PUL around BDI_3064 gene. Annotations for the gene products are based on Polysaccharide-Utilization Loci DataBase and are shown below the genes. Numbers above the gene represents locus numbers. (B) Localization of the gene products in the PUL. Localization of the proteins is based on LipoP. (C) A predicted metabolic pathway for β-1,2-glucan.

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Conclusion In this study, we identified BDI_3064 as an enzyme releasing Sop2 from Sopns with a DP 4 or more. The enzyme acted on the non-reducing end exolytically unlike the other known SGLs. Structural analysis well explained the exolytic reaction mechanism of BDI_3064. Though β-glucosidases from L. innocua and B. thetaiotaomicron, and SOGP are exo-type enzymes acting on Sopns and β-1,2-glucan, their reaction products are glucose and α-glucose 1-phosphate, respectively24,25,57. Therefore, BDI_3064 is a novel enzyme catalyzing a unique hydrolytic reaction on β-1,2-glucan, and should be given a new EC number. Considering the remarkable preference of the enzyme for Sopns over β-1,2-glucan, we propose 2-β-D-glucooligosaccharide sophorohydrolase (non-reducing end) as a systematic name for the enzyme. Sophorohydrolase (non-reducing end) and sophorosylhydrolase (non-reducing end) are possible as well. The finding of this novel enzyme is an important achievement for understanding the molecular evolution of SGLs. It is also beneficial for large scale preparation of Sop2 and exploration of the metabolic pathway for β-1,2-glucan. This study will lead to further study of β-1,2-glucan-related enzymes, their physiological roles, and utilization of Sopns and β-1,2-glucan.

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Biochemistry

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website at XXX. Figures S1-S5 (PDF)

Accession codes The atomic coordinates and structure factors have been deposited in the Protein Data Bank (PDB ID, 5Z06).

AUTHOR INFORMATION Corresponding author *E-mail: [email protected]

ORCID Masahiro Nakajima: 0000-0002-3182-0859

Author contributions M.N., H.N., and H.T. designed the experiments; H.S., M.N., A.M., Y.T., N.T., K.K., and N.S. performed the experiments; H.S., M.N., A.M., H.N., and H.T. analyzed the data, and M.N. wrote the manuscript with help of the other authors.

Funding This work was supported in part by an academic research grant from the Mishima Kaiun Memorial Foundation (2016).

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENTS We thank the staff of the Photon Factory for the X-ray data collection (2016G010 and 2016G619). We also thank Dr. M. Hisamatsu and Dr. N. Isono for providing the cyclic β-1,2-glucan, and Dr. M. Kitaoka for helping with the substrate preparation.

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ABBREVIATIONS SOGP,

1,2-β-oligoglucan

β-1,2-glucooligosaccharides;

phosphorylase; LiBGL,

DP,

degree

β-glucosidase

from

of

polymerization;

Listeria

inocua;

Sopns, BtBGL,

β-glucosidase from Bacteroides thethaiotaomicron; GH, glycoside hydrolase; SGL, β-1,2-glucanase;

CpSGL,

β-1,2-glucanase

from

Chitinophaga

carbohydrate-binding module.

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pinensis;

CBM,

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TOC graphic For Table of Contents Use Only

Characterization and structural analysis of a novel exo-type enzyme acting on β-1,2-glucooligosaccharides from Parabacteroides distasonis

Hisaka Shimizu†, Masahiro Nakajima*,†, Akimasa Miyanaga‡, Yuta Takahashi§, Nobukiyo Tanaka†, Kaito Kobayashi†, Naohisa Sugimoto§, Hiroyuki Nakai§, and Hayao Taguchi†



Department of Applied Biological Science, Faculty of Science and Technology, Tokyo

University of Science, 2641 Yamazaki, Noda, Chiba 278-8510, Japan ‡

Department of Chemistry, Tokyo Institute of Technology, 2-12-1, O-okayama, Meguro-ku,

Tokyo 152-8551, Japan §

Faculty of Agriculture, Niigata University, Niigata 950-2181, Japan

Corresponding author *E-mail: [email protected]

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