Novel Dextranase Catalyzing Cycloisomaltooligosaccharide Formation and Identification of Catalytic Amino Acids and Their Functions Using Chemical Rescue Approach

Results :

GH 66 enzymes are composed of 5 regions from the N-to the C-terminus (7,8) as follows: N-terminal variable region (N-VR), conserved region (CR), CITase-specific region (CIT-SR), C-terminal conserved region (C-CR), and the C-terminal variable region (C-VR).All dextranases reported so far are devoid of CIT-SR.
CIT-SR, which forms carbohydrate-binding module (CBM) 35, contributed to the CI-formation of CITase (7).More recently, we resolved the three-dimensional (3D) structure of Dex from Streptococcus mutans (SmDex), which is truncated at N-VR and C-VR (9,10).Our X-ray studies demonstrated that CR forms a catalytic (/) 8 barrel as has been observed in the GH 13, 27, and 31 proteins, indicating that they probably share a common evolutionary origin (11).Our studies also predicted that 3 acidic amino acids at CR of PsDex (Asp189, Asp340, and Glu412; PsDex, Dex from Paenibacillus sp.) are candidates for catalytic residues (9,10,12), although the functions of those candidates have yet to be elucidated.
Chemical rescue (ChR) is a reaction that is used to recover the activity of mutant enzyme by an exogenously added organic or inorganic compound that functions instead of the mutated residue.For example, the catalytic residue-mutated glycosylase displays its activity on a fluoride substrate by the addition of an anion (e.g., N 3 -or formate), which remains at the position of the altered residue.The glycosylase, mutated at its so-called nucleophile, catalyzes the formation of a product (glycosyl azide) that has an anomeric configuration opposite that of the substrate.The ChR of glycosylases, mutagenized at the acid/base catalyst, forms a product (glycosyl azide) with the same anomeric configuration as that observed in the intact enzyme-catalyzed hydrolytic reaction (13,14).Therefore, the use of ChR is among the most convenient approaches to identifying catalytic residues functions.
The amino-acid sequence of PsDex harbors CBM 35 at its CIT-SR, permitting the classification of the GH 66 enzymes into 3 types: (i) pure Dex; (ii) Dex with low cyclization activity; and (iii) CITase with low high cyclization activity and low hydrolytic activity.As mentioned before, the 3D structure analysis of SmDex (9, 10) implicated 3 acidic PsDex residues as candidates for catalytic residues (Asp189, Asp340, and Glu412).However, the function of those 3 residues remains unclear.To address this, we applied the ChR approach to their functional analysis.This study, for the first time, identifies the function of the catalytic residues of GH 66 enzymes.

EXPERIMENTAL PROCEDURES
Bacterial strains and plasmids.Paenibacillus sp. was isolated from soil on the Hokkaido University campus (Sapporo, Japan).This bacterium was aerobically incubated in 5 ml of pre-culture medium containing 0.1% dextran T2000 (Amersham Biosciences, Uppsala, Sweden), 1% Bacto peptone (Becton Dickinson and Company, Sparks, MD, USA), 0.01% yeast extract (Becton Dickinson and Company), and 0.05% NaCl (pH 7.0) at 30°C.For the production of PsDex, the pre-cultured Paenibacillus sp. was added to a fermenter containing 1,000 ml of medium [3 g of Na 2 HPO 4 , 1.5 g of KH 2 PO 4 , 0.25 g of NaCl, 0.5 g of NH 4 Cl, 0.5 mg of thiamine HCl, 0.5 mM MgSO 4 , 0.05 mM of CaCl 2 , 100 mM MES-NaOH buffer (pH 7.0), and 0.5% dextran T2000 per liter] and cultivated aerobically 3 times at 30°C for 20 h.Escherichia coli DH5 and E. coli BL21 (DE3)-CodonPlus-RIL (Stratagene, La Jolla, CA, USA) were used for the construction of expression plasmids and for the production of recombinant PsDex and its C-terminal-truncated PsDex (Ala39-Ser1304; PsDex-CT), respectively.Plasmids of pBluescript II SK (+) (Stratagene) and pET-23d (Novagen, Darmstadt, Germany) were used for the subcloning of DNA fragments amplified by polymerase chain reaction (PCR).E. coli was grown in LB medium [10 g of Bactotryptone (Becton Dickinson and Company) 5 g of yeast extract (Becton, Dickinson), and 5 g of NaCl in 1 l of H 2 O, pH 7.0] containing ampicillin (50 g/ml).Purification of native PsDex.Paenibacillus sp. cells were collected by centrifugation (8,000 x g for 10 min at 4°C), resuspended in 500 ml of 20 mM potassium phosphate buffer (pH 6.5; buffer-A), homogenized by an ice-chilled French press (Ohtake, Tokyo, Japan; 1,350 kg/cm 2 , 3 times), and centrifuged (14,000 x g for 20 min at 4°C) to discard the insoluble components.Supernatants were pooled as crude extract containing 183 U of PsDex with 0.0178 U/mg.Solid ammonium sulfate was slowly added to the crude extract up to 30% saturation, and the turbid solution was maintained at 4°C overnight.The resulting precipitant was collected by centrifugation (12,000 x g for 20 min at 4°C), dissolved in 600 ml of buffer-A, dialyzed against buffer-A, and centrifuged (8,000 x g for 10 min at 4°C) to remove insoluble materials.The supernatant (865 ml, 178 U, 0.0548 U/mg) received a 0.02% final concentration of sodium azide.The solution was applied to a column of DEAE-TOYOPEARL 650M (Tosoh, Tokyo, Japan; 3.1 × 66 cm) equilibrated with buffer-A, followed by elution with a 0-1 M sodium chloride of linear gradient.The active fractions (65.2 U, 0.688 U/mg) were dialyzed against buffer-A containing 1.0 M ammonium sulfate (buffer-B), loaded onto a column of Butyl-TOYOPEARL 650M (Tosoh; 2.2 × 55 cm) equilibrated with buffer-B, and eluted with a linear gradient of 1.0-0 M ammonium sulfate.Active fractions (40.0 U, 8.51 U/mg) were dialyzed against buffer-A containing 0.05 M sodium chloride (buffer-C), concentrated to 3.5 ml, and added to a gel-filtration column using Sepharose 6B (Amersham Biosciences; 2 × 110 cm) equilibrated with buffer-C.The active fractions (17.9 U, 14.3 U/mg) were dialyzed against buffer-A.All purification steps were performed at 4°C.Protein concentration of the crude extract and ammonium sulfate separation was measured using the Bradford method (15) with bovine serum albumin as a standard.
A computer-based sequence analysis to find homologous regions was performed using the BLAST network service (http://www.isb-sib.ch.) (20) with the Swiss-Prot/TrEMBL database.Prediction of a protein signal peptide was done using the Signal P server (http://www.cbs.dtu.dk/services/SignalP/)(21).

Construction of expression vectors of PsDex and
PsDex-CT.After C761 was replaced by G to remove an inner NcoI site without amino acid substitution, NcoI and NotI sites were introduced to the 5'-and 3'-termini of the ORF, respectively, and the ORF was then introduced to the NcoI-NotI site of pET23d to produce a PsDex protein that lacked the original signal sequence (Met1-Ala38).PCR was performed using psdexRs (5'-TACCATGGCCAATCAGGAAGAGAAGC-133 -3', where the NcoI site is underlined; sense primer) and psdexRa (5'-773-TGATAGGCCATGCCCGCTGAG CCG-750 -3', where the bold-faced letter corresponds to C761-replacement by G, antisense primer).The resultant DNA fragment was used as a primer for megaprimer PCR (22) with psdexRCA2 (5'-AAGCGGCCGCTTCGATCAGATCCAACAA -5071-3', where the NotI site is underlined, antisense primer).The PCR product and pET-23d were digested by NcoI and NotI (Takara, Kyoto, Japan), followed by connection with a DNA Ligation Kit ver. 2 (Takara) to construct an expression vector to produce PsDex (Ala39-Ala1696, an original Glu1696 replaced by Ala) with His6-tag at its C-terminus.
Eleven kinds of mutations at Asp189, Asp340, Glu412, Asp1254, and Cys1124 of PsDex-CT, having a His6-tag at the C-terminal, was performed by megaprimer PCR (22) using the appropriate primers.The amplified DNA fragment was digested by SacI and NotI, followed by introduction to the corresponding restriction sites of PsDex-CT gene-carrying pET-23d.

Production and purification of PsDex and PsDex-CT.
PsDex, PsDex-CT, and the mutants of PsDex-CT at Asp189, Asp340, Glu412, Asp1254, and Cys1124 were produced in E. coli transformants carrying the respective expression plasmid.Each transformant was cultured in 1,000 ml of LB medium containing 50 g/ml ampicillin at 37°C until the absorbance at 600 nm reached approximately 0.5.Protein production was induced with 0.2 mM isopropyl -thiogalactoside, followed by further incubation with vigorous shaking at 18°C for 20 h.After E. coli cells were disrupted by sonication using a Sonifier 250 (Branson, Danbury, CT, USA), PsDex and PsDex-CT were purified to homogeneity by Ni-chelating chromatography using Chelating Sepharose Fast Flow (Amersham Biosciences).Active fractions were dialyzed against buffer-A and concentrated using a CentriPrep YM-50 apparatus (Millipore, Bedford, MA, USA).The concentration of purified protein was measured using 12.2 (recombinant PsDex) and 5.54 (PsDex-CT and its 11 mutants) of A 1% at 280 nm determined by the aforementioned analysis of their amino acid compositions.Enzyme reaction.The effects of pH on dextranolytic activity were examined by incubating each enzyme (26.0 nM PsDex and 30.0 nM PsDex-CT) with 0.4% dextran T2000 at 35°C in 16 mM modified Britton-Robinson buffer (pH 2.60-11.4;pH of mixture of acetate, phosphate and glycine was adjusted by NaOH; buffer-D).For pH-stability, PsDex (190 nM) and PsDex-CT (203 nM) were maintained at 4°C for 18 h in 36 mM buffer-D, followed by assay of residual enzyme activity under the aforementioned conditions.For thermal stability, PsDex (25.0 nM) and PsDex-CT (42.2 nM) were maintained at 25-60°C and 20 mM sodium acetate buffer (pH 5.5; Na-AB) for 15 min, and residual activity was then measured under the assay conditions.
The remaining dextran and long oligosaccharides were discarded by the addition of ethanol (2 volumes) to 25 ml of the reaction mixture, forming a precipitate at 4°C for 1 h.The supernatant was concentrated to 2 ml by a vacuum evaporator, reacted with buckwheat -glucosidase (7.6 U/ml) and Aspergillus niger -glucosidase (12 U/ml) at 37°C and 20 mM Na-AB for 15 h to digest short linear oligosaccharides, passed through a small column of Amberlite MB-4 (Organo, Tokyo, Japan), and applied to the Sep-PackC18 cartridge (Waters; Milford, MA, USA) followed by a first elution with milli-Q water (5 ml) for washing the remaining short linear oligosaccharides and by second elution with 20% ethanol (5 ml) to recover the CIs.The ethanol fraction was concentrated to 0.4 ml and analyzed by HPLC using a model D-2000 refractive index detector (Hitachi, Tokyo, Japan) using a Shodex RS Pak DC-613 column (6 × 150 mm; Showa Denko, Tokyo, Japan), followed by elution at 60°C with 61% acetonitrile.Molecular masses of the CIs were determined using model JMS-SX102A electrospray ionization-mass spectrometry (JEOL, Tokyo, Japan).13 C-NMR spectra were recorded using a Bruker AMX-500 Spectrometer at 125 MHz with an external standard of trimethylsilyl propionate.
The ChR reaction was performed by the reaction of the mutated PsDex-CT at Asp189, Asp340, or Glu412 (see Table 2 for mutants used) with 6 mM IG4F and salt (0.2-2.4 M NaN 3 , sodium formate or NaNO 3 ; Table 2) at 35°C and 200 mM Na-AB.Fluoride ions liberated from IG4F were measured by estimating the formation of a lanthanum complex (25).The ChR product was analyzed using thin-layer chromatography (TLC) with a silica-gel plate (60F 254 ; Merck, Darmstadt, Germany) and a solvent of nitromethane/ 1-propanol/water (4/10/3, v/v; 2-time development), followed by visualization at 110°C for 5 min after dipping of the TLC plate in 5% sulfuric acid in methanol containing 0.03% -naphthol.Purification of 2 ChR-products formed by D340A and E412Q was done using TLC.After development, each product was recovered from the TLC plate.Their structures were analyzed on a model JMS-SX102A fast atom bombardment (FAB)-MS and a model AMX-500 1 H-NMR at 500 MHz.

Purification and characterization of PsDex.
A dextran-degrading bacterium was isolated from soil.DSMZ (Braunschweig, Germany) identified this strain as Paenibacillus sp. on the basis of the 16S rRNA sequence and cellular fatty acid composition analyses.
1cm Dextranolytic activity at 24 h after cultivation reached 100 U per ml of medium, 3% and 97% of which were found in the supernatant and cell-homogenized fraction, respectively.The cell suspension, which reacted directly with 0.4% dextran T2000 at 35°C and 50 mM sodium acetate buffer (pH 5.5; Na-AB), displayed almost the same activity as that observed with the cell-homogenized fraction, implying that PsDex resided at the cell surface.Microscopic observation confirmed that the cells were not disrupted during incubation with dextran.
PsDex was isolated from the cell-disrupted fraction by 4 purification procedures; the final step entailed gel-filtration and purified a 200 kDa-protein with a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (Fig. 1A; lane 1).This protein band was positive for periodic acid-Schiff staining (PAS) (Fig. 1A; lane 2).Both monosaccharide composition and sugar contents analyses confirmed that PsDex was a glycoprotein that had 5.1% D-galactose without glucosamine, galactosamine, or N-acetyl sugars thereof.PsDex was the most active at pH 5.5 and was stable at pH 5.0-9.0 and up to 40°C.

Expression and characterization of recombinant PsDex and PsDex-CT. C-VR of PsDex contains a
Pro/Ser-rich region (PSRR, Pro1305-Pro1329), followed by 3 sets of putative surface-layer homology domains (SLHDs; Tyr1518-Ala1685; refer to the Discussion section for details).
Therefore, we constructed a PSRR/SLHD-truncated PsDex (PsDex-CT carrying Ala39-Ser1304) to elucidate the influence of those regions on catalytic function.Full-size PsDex or PsDex-CT was produced in Escherichia coli cells, and enzyme activities in the medium were increased to be 162 U/1,000 ml and 433 U/1,000 ml, which were larger than that of native PsDex (100 U/1,000 ml) by 1.6-times and 4.3-times, respectively.Each enzyme was purified as a single band protein (Figs.1A and 1B) with a specific activity of 14.0 U/mg for PsDex and 7.20 U/mg for PsDex-CT.The N-terminal sequence started at Ala37 in each protein, and electrospray ionization-mass spectrometry (ESI-MS) and SDS-PAGE analyses determined the molecular sizes of 186 kDa for PsDex and 143 kDa for PsDex-CT (Figs. 1B and 1C), indicating the absence of proteolytic digestion during production, which occurred in the production of SmDex (8).PsDex and PsDex-CT showed the same pH (stable at pH 5.2-10; optimum at pH 5.5) and temperature (stable at < 37°C) properties, together with the formation of mainly IG-4 from dextran T2000.All these properties were identical to those of native PsDex, allowing us to use those expressed enzymes for further experiments.Investigation of CI-production.Since the primary structure of PsDex included CIT-SR, which is only found in CITase, we investigated the PsDex-CT-associated CI production from dextran T10 (Fig. 2C).Structures of 7 purified CIs were analyzed using ESI-MS and NMR.The mass signals of [M + Na] + were 1157.29, 1319.57, 1481.67, 1644.11, 1806.28, 1968.21, 2130.62, and 2292.91, the estimated masses of which were multiples of 162.14, corresponding to a mass of 1 glucosyl unit, indicating that each carbohydrate was a non-reducing sugar with cyclic form. 13C-NMR (Fig. 2F) also supported the PsDex-CT-catalyzed CI formation of CI-7 to CI-14 (3,6).Prolonged incubation with the enzyme resulted in CI degradation (Fig. 2D), since PsDex-CT had endo-wise dextranolytic activity, which could cleave the CIs with almost equal efficiency.Native and recombinant PsDexs without truncation also catalyzed the same CI production, which was followed by degradation.
ChR was applied to Asp189, Asp340, and Glu412 mutants (Fig. 3).Sodium azide or sodium formate enhanced the fluoride ion-releasing velocity from 6.0 mM -isomaltotetraosyl fluoride (IG4F) by 1.5-times to 13-times (Table 2).Structures of products formed in the presence of the azide ion (Figs.3A, 3B,  and 4C) were analyzed by 3 approaches, namely, the reducing power test, FAB-MS, and 1 H-NMR.D340G and E412Q synthesized the non-reducing sugars that had the same molecular mass (691 Da), while they exhibited different J 12 values of 8.5 Hz and 2.5 Hz in 1 H-NMR, respectively, indicating that D340G formed -isomaltotetraosyl azide and E412Q formed -isomaltotetraosyl azide.On the other hand, D189A with 0.4 M NaN 3 or 0.4 M sodium formate produced IG-4 or/and several IG-n from 6.0 mM IG4F, 20 mM IG-5 and 0.40% dextran T2000 (Figs. 3D and 3E), suggesting that a ChR of D189A catalyzed the hydrolytic reaction.The same products were also observed in the presence of 0.4 M NaNO 3 (lanes 11, 16, and 21 of Figs.3D and 3E).Interestingly, the formation of IG-4 from IG4F and dextran T2000 appeared by D189A without any salt (lane 8 of Fig. 3D; lane 18 of Fig. 3E), and those IG-4 productions were markedly enhanced in the presence of salts (lanes 9-11 of Fig. 3D; lanes 19-21 of Fig. 3E).
D189A displayed hydrolysis at its ChR, suggesting that transglycosylation might also occur.D189A was reacted with 30 mM IG4F at a high concentration in the presence of 0.4 M NaN 3 , and we found the product had a large molecular size, since this sugar stayed at its original position on the thin-layer chromatography (TLC) plate.The addition of ethanol to the reaction mixture resulted in the formation of turbid material (Fig. 3F).This turbid material was identified as a dextran due to the production of IG-n by SmDex-treatment.
Its size was analyzed by estimating DP using reducing power and total sugar, indicating an average DP of about 100.This size can be categorized as a small dextran.D189C/C1124Y activity restoration by KI-treatment.Asp189 of PsDex-CT was substituted by a Cys residue, followed by KI oxidation.Prior to this mutation, an original and sole Cys1124 of PsDex-CT was replaced with a Tyr residue to construct C1124Y, and the further mutation at Asp189 was then introduced to form D189C/C1124Y. C1124Y maintained the same dextranolytic activity (7.22 U/mg) as observed in PsDex-CT (7.20 U/mg), while D189C/C1124Y decreased its specific activity (0.0768 U/mg) by 1/94-times.An SH-group of D189C/C1124Y was probably converted to a sulfinate moiety by KI-treatment (26).
No free Cys residue was confirmed using Ellman's titration method (33).As shown in Fig. 2E, dextranolytic activity increased and reached its greatest value at 250 mM KI (0.522 U/mg; 6.8-fold higher than the original activity of D189C/C1124Y), followed by reduction by incubation with KI exceeding at more than 250 mM.

Relationship between structure and function of PsDex.
In C-VR of PsDex, are 3 tandem 55 residue-repeated SLHDs (Tyr1518-Thr1560, Phe1578-Ala1620, and Tyr1642-Ala1685), that share great similarity to the N-terminal 3-repeat SLHDs (Phe34-Ala197) of the Bacillus anthracis surface array protein (Fig. 4A).This array protein remains at the cell surface-layer of Gram-positive bacteria by SLHD-associated non-covalent binding to a secondary cell wall carbohydrate in the bacterial cell wall (34).SLHDs of PsDex seem to be active, since 97% dextranolytic activity of the native enzyme during cultivation was found on the cell surface of Paenibacillus sp. of Gram-positive bacteria.A possible linker of PSRR connects SLHR to C-CR.
Both recombinant PsDex and PsDex-CT exhibited properties identical to those of the native enzyme, indicating that the C-terminal PSRR and 3 SLHRs of PsDex do not affect any catalytic functions.
Interestingly, PsDex is a glycoprotein with only galactose units.Many N-glycosylated and/or O-glycosylated proteins have been discovered among approximately 25 kinds of bacterial genera (39).To our knowledge, PsDex is the first glycoprotein in the genus Paenibacillus that contains only galactose.The O-glycosylation at the Tyr residue has been reported on an S-layer protein from Paenibacillus alvei, which has a polysaccharide composed of Glc, Gal, ManNAc, and Rha (40).The galactose residue in PsDex is not considered to be involved in dextranolytic activity, since the enzyme activity is available in the E. coli-expressing wild-type PsDex.This host lacks the glycosylation system.Catalytic residues and their functions.This study determined, for the first time, the catalytic residues of GH 66 enzyme and their functions.The 3 findings of (i) point mutation approaches at Asp189, Asp340, and Glu412 (Table 2), (ii) ChR reaction using mutated enzymes (Table 2, Fig. 3), and (iii) our 3D structure analysis (9,10) are evidence that Asp340 and Glu412 are catalytic residues.Reaction products derived from ChR (Figs. 3A, 3B, and 3C) clearly revealed that Asp340 is a so-called catalytic nucleophile and Glu412 is an acid/base catalyst by virtue of the formation of -isomaltotetraosyl azide and -isomaltotetraosyl azide, respectively.Asp189 exhibited the interesting ChR phenomena involving catalysis of the hydrolytic reaction (Figs.3D and 3E), meaning that Asp189 mutants display the ordinal ChR reaction simply by occupying the Asp189 position with an external anion of N 3 -, formate, or NO 3 -.We thought that an Asp189-mutated enzyme (D189A) would catalyze the transglycosylation on IG4F, since D189A becomes a simple hydrolase in the presence of an anion.Surprisingly, a dextran-type polysaccharide was formed (Fig. 3F), indicating that D189A catalyzed a "sequential ChR reaction"; this, to our knowledge, is a novel discovery in the endo-lytic hydrolase reaction reacting on -glucan.The reaction mechanism of sequential ChR still cannot be explained clearly, but the produced short IG-n was used for further D189A-mediated ChR.A negative charge is essential for the position of Asp189 since D189C/C1124Y enhanced its dextranolytic activity by KI-treatment (Fig. 2E), which oxidizes the SH-group of C1124 to form a possible anionic sulfinate (26).

Table 1 . Kinetic parameters for IG-n and dextran T2000 by PsDex and products with their levels.
* +++, ++, and + indicate strong, moderate, and weak production levels, respectively, tested by TLC.