Glycoside Hydrolase Family 39 β-Xylosidases Exhibit β-1,2-Xylosidase Activity for Transformation of Notoginsenosides: A New EC Subsubclass

Article abstract of DOI:10.1021/acs.jafc.9b00027

β-1,2-Xylosidase activity has not been recorded as an EC subsubclass. In this study, phylogenetic analysis and multiple sequence alignments revealed that characterized β-xylosidases of glycoside hydrolase family (GH) 39 were classified into the same subgroup with conserved amino acid residue positions participating in substrate recognition. Protein-ligand docking revealed that seven of these positions were probably essential to bind xylose-glucose, which is linked by a β-1,2-glycosidic bond. Amino acid residues in five of the seven positions are invariant, while those in two of the seven positions are variable with low frequency. Both the wild-type β-xylosidase rJB13GH39 and its mutants with mutation at the two positions exhibited β-1,2-xylosidase activity, as they hydrolyzed o-nitrophenyl-β-d-xylopyranoside and transformed notoginsenosides R₁ and R₂ to ginsenosides Rg₁ and Rh₁, respectively. The results suggest that all of these characterized GH 39 β-xylosidases probably show β-1,2-xylosidase activity, which should be assigned an EC number with these β-xylosidases as representatives.

Full text of DOI:10.1021/acs.jafc.9b00027

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Biotechnology and Biological Transformations
Glycoside hydrolase family 39 #-xylosidases exhibit #-1,2-xylosidase
activity for transformation of notoginsenosides: a new EC subsubclass
Rui Zhang, Na Li, Shujing Xu, Xiaowei Han, Chunyan Li, xin wei,
Yu Liu, Tao Tu, Xianghua Tang, Junpei Zhou, and Zunxi Huang
J. Agric. Food Chem., Just Accepted Manuscript • Publication Date (Web): 05 Mar 2019
Downloaded from http://pubs.acs.org on March 5, 2019
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Glycoside hydrolase family 39 β-xylosidases exhibit β-1,2-xylosidase activity for
transformation of notoginsenosides: a new EC subsubclass
Rui Zhanga, b, †, Na Lia, b, †, Shujing Xu , Xiaowei Han , Chunyan Li , Xin Wei ,
a, b
a, b
a, b
c
Yu Liu , Tao Tu , Xianghua Tang , Junpei Zhoua, b, *, Zunxi Huanga, b, *
aEngineering Research Center of Sustainable Development and Utilization of Biomass
Energy, Ministry of Education, Yunnan Normal University, Kunming, 650500,
People’s Republic of China
a, b
d
a, b
bCollege of Life Sciences, Yunnan Normal University, Kunming, 650500, People’s
Republic of China
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cState Key Laboratory of Phytochemistry and Plant Resources in West China,
Kunming Institute of Botany, Chinese Academy of Sciences, Kunming, 650201,
People’s Republic of China
dKey Laboratory for Feed Biotechnology of the Ministry of Agriculture, Feed
Research Institute, Chinese Academy of Agricultural Sciences, Beijing, 100081,
People’s Republic of China
†R. Z. and N. L. contributed equally to this work
Running
title:
GH
39
β-1,2-xylosidases
*
Correspondence: Junpei Zhou, College of Life Sciences, Yunnan Normal
University, No. 768 Juxian Street, Chenggong, Kunming, Yunnan, 650500, People’s
Republic of China. Tel.: +86 871 65920830; Fax: +86 871 65920952; e-mail:
junpeizhou@ynnu.edu.cn; junpeizhou@126.com; ORCID: 0000-0001-6122-9851
Zunxi Huang, e-mail: huangzunxi@163.com; ORCID: 0000-0002-7998-3673
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ABSTRACT: β-1,2-Xylosidase activity has not been recorded as an EC subsubclass.
In this study, phylogenetic analysis and multiple sequence alignments revealed that
characterized β-xylosidases of glycoside hydrolase family (GH) 39 were classified
into the same subgroup with conserved amino acid residue positions participating in
substrate recognition. Protein-ligand docking revealed that seven of these positions
were probably essential to bind xylose-glucose, which is linked by a β-1,2-glycosidic
bond. Amino acid residues in five of the seven positions are invariant, while those in
two of the seven positions are variable with low frequency. Both the wild-type
β-xylosidase rJB13GH39 and its mutants with mutation at the two positions exhibited
β-1,2-xylosidase activity as they hydrolyzed o-nitrophenyl-β-D-xylopyranoside and
transformed notoginsenosides R and R to ginsenosides Rg and Rh , respectively.
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The results suggest that all these characterized GH 39 β-xylosidases probably show
β-1,2-xylosidase activity, which should be assigned an EC number with these
β-xylosidases as representatives.
KEYWORDS: β-xylosidase; β-1,2-glycosidic bond; enzyme commission;
notoginsenosides;
glycoside
hydrolase
family
39
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INTRODUCTION
Xylans, abundant in many agricultural biomasses, algae, and industrial wastes, are
polymerized mainly by xylose with β-1,4-glycosidic or β-1,3-glycosidic linkages.1–3
The role of β-1,4-xylosidases and β-1,3-xylosidases is to produce xylose from xylans
after degradation by endo-xylanases. Engineered Saccharomyces cerevisiae can
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utilize xylose to produce ethanol, xylitol, lactic acid, and other chemicals. Therefore,
β-xylosidases are of great importance in the food, beverage, bioenergy, feed, and
pharmaceutical industries.5
In addition to xylans, some saponins contain xylosyl moieties at their terminals.
Panax notoginseng, well known as “Sanqi” in Chinese, is recorded in the
Compendium of Materia Medica (Bencao Gangmu) and traditionally used for
hemostasis. In China, the plant is primarily cultivated in Yunnan and Guangxi
provinces. Both notoginsenosides R and R contain a xylosyl group linked by a
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2
β-1,2-glycosidic bond with a glucosyl group (Figure 1).
Removal of the xylosyl group from notoginsenosides R and R yields
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ginsenosides Rg and Rh , respectively, which possess anticancer, antioxidant, and
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1
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anti-inflammatory activities. Recently, Shin et al. reported that the β-xylosidase
(TtGH39) from Thermoanaerobacterium thermosaccharolyticum was the first
β-xylosidase exhibiting β-1,4-xylosidase activity as well as the ability to remove the
7
xylosyl group from notoginsenosides R and R . Li et al. also reported that the
1
2
β-xylosidase (Xln-DT) from Dictyoglomus thermophilum could efficiently degrade
xylobiose and remove the xylosyl group from notoginsenoside R . Both TtGH39 and
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Xln-DT are members of the glycoside hydrolase family (GH) 39. Shin et al. and Li et
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al. focused on the bioconversion of saponins, but they did not stress the key point that
the two β-xylosidases had the ability to hydrolyze β-1,2-glycosidic bonds.
To date, β-1,2-xylosidase or notoginsenoside xylohydrolase activity has not been
assigned an EC number (http://www.enzyme-database.org). Most β-xylosidases are
classified as EC 3.2.1.37 as they catalyze the hydrolysis of β-1,4-linked xylosyl
moieties from xylo-oligosaccharides. β-1,3-Xylosidases are classified as EC 3.2.1.72,
but seldom reported.2,3 To determine β-1,2-xylosidase activity, both notoginsenosides
and o-nitrophenyl-β-D-xylopyranoside are suitable substrates. For example, EC
3.2.1.191–195 show five types of ginsenoside glucohydrolases using various
ginsenosides as substrates.
This study aimed to determine whether other GH 39 β-xylosidases, like TtGH39
and Xln-DT, had the ability to hydrolyze β-1,2-glycosidic bonds for transformation of
notoginsenosides and further proposed that GH 39 β-xylosidases could be
representatives of the new EC subsubclass, β-1,2-xylosidases or notoginsenoside
xylohydrolases.
MATERIALS AND METHODS
Sequence, Strains, and Materials. Previously, we obtained a GH 39 β-xylosidase
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(JB13GH39) from Sphingomonas sp. JB13. The sequence of JB13GH39 can be
retrieved from the GenBank database (accession No. MG838204). Escherichia coli
BL21 (DE3) was purchased from TransGen (Beijing, China). p-Nitrophenol (pNP)
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and p-nitrophenyl-β-D-xylopyranoside (pNPXyl) were purchased from Sigma-Aldrich
(St. Louis, MO, USA). Notoginsenosides R and R , ginsenosides Rg and Rh , and
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1
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o-nitrophenyl-β-D-xylopyranoside (oNPXyl) were purchased from YuanYe
Bio-Technology (Shanghai, China). Mut Express® II Fast Mutagenesis Kit V2 was
purchased from Vazyme Biotech (Nanjing, China).
Enzyme Activity toward oNPXyl. Recombinant JB13GH39 (rJB13GH39) was
expressed in E. coli BL21 (DE3) cells using the pEASY-E2 vector and purified by
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+
Ni -NTA affinity chromatography. The details of enzyme preparation were described
in our previous study.8
o-Nitrophenol is not available to us because of its danger. Therefore, the pNP
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method was used as described previously to evaluate the activity of rJB13GH39
toward oNPXyl. One unit of β-1,2-xylosidase activity was defined as the amount of
enzyme that releases 1 µmol of pNP per minute at pH 4.5 and 50 °C.
Enzymatic Transformation of Notoginsenosides R and R . The 400 µL (pH
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2
4.5) reaction included 10 μg of rJB13GH39 and 4 mM substrate. The mixture was
incubated in a water bath at 30 °C for 24 h. The products were qualitatively detected
by electrospray ionization mass spectrometry (ESI-MS) before quantitative analysis.
ESI-MS was carried out in positive mode on a quadrupole time-of-flight
high-resolution mass spectrometer (6540 series, Agilent Technologies, Santa Clara,
CA, USA) with the following parameters: fragmentor, 135 V; capillary, 3500 V;
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skimmer, 65 V; gas (nitrogen) temperature, 350 °C; drying gas, 8 L/min; and
nebulizing gas, 30 psi. For quantitative analysis, the products from notoginsenosides
R1 and R2 were prepared as previously described6 and analyzed by
ultra-high-performance liquid chromatography (UHPLC) at 203 nm with a Hypersil
ODS column (4.0 mm × 250 mm, 5 µm; Agilent Technologies). The column was
eluted at room temperature with 20:80 (v/v) acetonitrile/water at 1.0 mL/min for R1
and Rg and with 20:80 to 55:45 (v/v) acetonitrile/water for 60 min at 1.5 mL/min for
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R and Rh .
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Phylogenetic Analysis. Phylogenetic analysis of GH 39 members was performed
by MEGA using the built-in Jones–Taylor–Thornton model for distance matrices
calculation and neighbor-joining algorithm for phylogenetic tree construction with
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1000 bootstrap replications. A total of 14 identified GH 39 β-xylosidases were
selected for phylogenetic analysis: TtGH39 and Xln-DT showing both
β-1,4-xylosidase and β-1,2-xylosidase activities for transformation of
notoginsenosides;6,7 XynB_thesa (accession no. AAA27369 or 1UHV) from
Thermoanaerobacterium saccharolyticum showing both β-1,4-xylosidase and
β-1,2-xylosidase activities using pNPXyl and oNPXyl as substrates;10,11 XynA
(AAB87373) and XynB (AAA23063) from Caldicellulosiruptor saccharolyticus as
well as BxyH (AEE47384) from Cellulomonas fimi showing β-1,2-xylosidase activity
using oNPXyl as substrate;12,13 XynB1 (ABI49941 or 2BS9) from Geobacillus
stearothermophilus
having
solved
crystal
structure
with
molecule
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2,5-dinitrophenyl-β-xyloside as ligand;14 JB13GH39, SlpA (AFK86459) from T.
saccharolyticum,15 XylBH39 (BAB04787) from Bacillus halodurans,16 WSUCF1
(EPR27580) from Geobacillus sp.,17 Bxl39A (CAD48308) from Clostridium
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stercorarium, Coxyl A (ADQ03734) from Caldicellulosiruptor owensensis, and
CcXynB2 (AAK24328 or 4EKJ) from Caulobacter crescentus20,21 showing
β-1,4-xylosidase activity.
Sequence Analysis. To illuminate patterns of amino acid conservation, the
multiple sequence alignment of the 14 identified GH 39 β-xylosidases were uploaded
2
2
to the Weblogo server to generate sequence logos.
Protein Structure Modeling. The protein homology model of JB13GH39 was
built using SwissModel (http://swissmodel.expasy.org/), followed by quality
estimation with GMQE, QMEAN Z-score, and a local quality plot.
Protein-ligand Docking. The ligand, designated as xylose-glucose, was a
compound linked by a β-1,2-glycosidic bond. Xylose-glucose was retrieved from the
PubChem database with the accessing number CID 3083150. Docking sites of
JB13GH39 were predicted using the Dock Ligands (CDOCKER) protocol by
Discovery Studio v2.5 software (Accelrys, San Diego, CA, USA).
Site-directed Mutagenesis. Site-directed mutagenesis was used to substitute
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Tyr257 and Tyr311 of JB13GH39 with Thr and Pro, designated as mutants
Y257(Ps266)T and Y311(Ps322)P, respectively. The mutation was introduced using
Mut Express® II Fast Mutagenesis Kit V2 according to the manufacturer’s
instructions.
Two
primer
sets
were
designed:
and
ACAGCaccGGCGTCGATGGCGGCTTTCTCGAC
ATCGACGCCggtGCTGTGCGTGGTGACGAAAT for mutant Y257(Ps266)T and
ATGGAGCACCAGCccaACGCCGCGCGATGCCGTG and
TtggGCTGGTGCTCCATTCGGTGAAATAGAGT for mutant Y311(Ps322)P.
Mutated nucleic acids were confirmed by DNA sequencing (Tsingke, Beijing, China).
Expression and purification of the mutated enzymes were performed as those of
rJB13GH39.
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Activity of Mutants. The pNP method was used as described previously to
determine the activity of the mutants toward pNPXyl. One unit of β-1,4-xylosidase
activity was defined as the amount of enzyme that releases 1 µmol of pNP per minute
at pH 4.5 and 50 °C. β-1,2-Xylosidase activity of the mutants toward oNPXyl and
notoginsenosides was detected by pNP method and UHPLC, respectively, as that of
rJB13GH39.
RESULTS AND DISCUSSION
β-1,2-Xylosidase Activity of rJB13GH39. rJB13GH39 was successfully
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expressed and purified as previously described. The specific activity of rJB13GH39
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toward oNPXyl was 31.7 ± 1.2 U/mg. The ESI-MS spectra indicated that
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notoginsenosides R and R were converted by the activity of purified rJB13GH39 to
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ginsenosides Rg and Rh , respectively (Figure S1). Xylose was also observed in
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those products (Figure S1). As shown in the UHPLC chromatograms, 100.0 % of
notoginsenoside R and 90.3 % of notoginsenoside R were individually hydrolyzed
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(Figures 2A, 3A).
β-Xylosidases have been found in 11 families, including GH 1, 3, 5, 30, 39, 43, 51,
52, 54, 116, and 120.5,23 Why are β-xylosidases so diverse? What are the differences
between these β-xylosidases? One explanation may be that β-xylosidases from each
glycoside hydrolase family have specific activity toward certain xylose-containing
substances. Previous studies have shown that the xylosyl moiety of notoginsenosides
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,7
R and R can be removed by GH 39 β-xylosidases TtGH39 and Xln-DT. This study
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confirmed that GH 39 β-xylosidase rJB13GH39 had the ability to degrade
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notoginsenosides R and R . Furthermore, Li et al. reported that three GH 3
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β-xylosidases, Tth XyB3, Tpe Xln3, and XlnD, as well as one GH 120 β-xylosidase,
Tth Xyl, were unable to degrade notoginsenoside R . Additionally, we found that the
1
GH 43 β-xylosidase HJ14GH43 did not function as a β-1,2-xylosidase (data not
shown). Therefore, the functional signature of GH 39 β-xylosidases may be their
ability to cleave the β-xylose-(1→2)-β-glucose bond. However, the function is not
exclusive to GH 39 β-xylosidases, as the GH 1 β-glucosidase from Pyrococcus
furiosus has β-xylosidase activity and can also convert notoginsenosides R and R to
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ginsenosides Rg and Rh , respectively.
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Transformation of notoginsenosides R and R by GH 39 β-xylosidases seems to
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be highly efficient. Notoginsenoside R is converted into ginsenoside Rg by GH 39
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β-xylosidase Xln-DT after 30 min with a corresponding molar conversion yield of 100
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% in total. GH 39 β-xylosidase TtGH39 completely converts notoginsenosides R
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and R to ginsenosides Rg and Rh after 4 and 18 h, respectively. Greater than 90 %
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of notoginsenosides R and R were hydrolyzed when the reactions occurred at 30 °C
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for 24 h using rJB13GH39.
Sequence and Phylogenetic Analyses. Previously, GH 39 enzymes were
regarded as α-L-iduronidases or β-xylosidases. According to the previous
phylogenetic analysis performed by Ali-Ahmad et al.,25 the GH 39 family has four
subgroups. In this study, GH 39 enzymes were also classified into 4 subgroups
(Figure 4). The results suggest that GH 39 enzymes should show at least 4 types of
activity. As shown in Figure 4, subgroup I includes two α-L-iduronidases from Homo
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sapiens and Mus musculus. NF2152, in subgroup III from the rumen anaerobic
fungus Neocallimastix frontalis, was reported to release β-1,2-arabinobiose from
sugar beet arabinan and both β-1,2-arabinobiose and α-1,2-galactoarabinose from rye
arabinoxylan.28 The GH 39 member in subgroup IV, PslG from Pseudomonas
aeruginosa found in chronic leg ulcers, was found to disrupt biofilm matrix
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exopolysaccharide in bacteria. GH39wh2 from the human gut bacteria Bacteroides
cellulosilyticus was classified in subgroup IV and predicted to harbor
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endo-glycosidase activity.25 TtGH39, Xln-DT, and JB13GH39, as well as other
characterized β-xylosidases, were classified into subgroup II.
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On the basis of the multiple sequence alignment of the 14 identified GH 39
β-xylosidases in subgroup II, conserved amino acid residue positions are shown in
Figure 5. According to the alignment and the three GH 39 β-xylosidases
XynB_thesa,10,11 XynB1, and CcXynB2
14
20,21
that have crystal structure data, Glu in
position (Ps) 317 corresponds to the catalytic nucleophile, Glu in Ps195 corresponds
to the general acid/base residue, and His in Ps88, Phe in Ps150, Asn in Ps194, Phe in
Ps201, His in Ps264, Tyr in Ps266, Tyr in Ps322, Trp in Ps356, Phe in Ps362, Glu in
Ps364, and Phe in Ps376 correspond to substrate recognition.
Protein-ligand Docking. To uncover which amino acid residue positions are
essential for β-1,2-xylosidase activity, protein-ligand docking was performed.
The homology model of JB13GH39 was built previously with CcXynB2 as a
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template. The quality of the model was high, taking the following values into
consideration: 66.5 % sequence identity, 0.78 GMQE score, -1.49 QMEAN Z-score,
and fewer than 4 residues (0.8 % of the total residues) showing a score below 0.6 in
the local quality plot.8
The xylose-glucose ligand successfully docked with the catalytic pocket of
JB13GH39 with 91 poses (Figure 6). Each pose was analyzed to show the hydrogen
bond interactions of amino acid residues with the ligand (Figure 7). The results
revealed that His88, Glu189, Tyr257, Glu306, Tyr311, Typ344, and Glu352 of
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JB13GH39 bound xylose-glucose with high frequency (Figure 7). These results
revealed that the seven conserved amino acid residue positions were probably
essential for β-1,2-xylosidase activity.
As shown in Figure 5, the positions of His88 (Ps88), Glu189 (Ps195), Glu306
(Ps317), Trp344 (Ps356), and Glu352 (Ps364) are not substituted with other amino
acid residues, while the positions of Tyr257 (Ps266) and Tyr311 (Ps322) can be
substituted with Thr and Pro with low frequency, respectively. As such, it is important
to figure out whether β-1,2-xylosidase activity is lost when the positions of Tyr257
(Ps266) and Tyr311 (Ps322) are substituted with Thr and Pro, respectively.
Expression and Purification of Mutants. The JB13GH39 residues Tyr257 and
Tyr311 were successfully substituted with Thr and Pro, respectively, and confirmed
by DNA sequencing (Figure S2). Under the same conditions as those of the
expression and purification of rJB13GH39, mutants Y257(Ps266)T and
Y311(Ps322)P were expressed in cells and purified to an electrophoretically pure state
(Figure S3).
Activity of Mutants. Both Y257(Ps266)T and Y311(Ps322)P showed
β-1,4-xylosidase activity, with specific activities of 0.106 ± 0.002 and 28.641 ±
1.380 U/mg toward pNPXyl, respectively. The specific activities of Y257(Ps266)T
and Y311(Ps322)P were reduced by 99.7 % and 24.2 %, respectively, compared with
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that of rJB13GH39 (37.8 ± 0.9 U/mg). The decrease in the activity of Y257(Ps266)T
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may be attributed to the function of Y257 (Ps266): the tyrosine enables the catalytic
nucleophile to attack the anomeric center correctly.10
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Both Y257(Ps266)T and Y311(Ps322)P showed β-1,2-xylosidase activity. The
specific activities of Y257(Ps266)T and Y311(Ps322)P toward oNPXyl were 0.073 ±
0.030 and 7.619 ± 0.429 U/mg, respectively. More than 90 % of notoginsenosides R1
and R were converted by purified Y311(Ps322)P to ginsenosides Rg and Rh ,
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1
1
respectively (Figures 2B, 3B). The rates at which Y311(Ps322)P hydrolyzes
notoginsenosides R and R are almost the same as those of rJB13GH39. However,
1
2
just as there was a substantial reduction in β-1,4-xylosidase activity, a substantial
reduction in Y257(Ps266)T β-1,2-xylosidase activity was observed: 57.0 % of
notoginsenoside R and 7.5 % of notoginsenoside R were individually hydrolyzed by
1
2
purified Y257(Ps266)T (Figures 2C, 3C).
Phylogenetic analysis revealed that characterized β-1,4-xylosidases and
β-1,2-xylosidases of GH 39 were classified into the same subgroup. Multiple
sequence alignments revealed that these GH 39 β-xylosidases have conserved amino
acid residue positions that participate in substrate recognition. Among these conserved
amino acid residue positions, seven were probably essential to xylose-glucose binding
revealed by protein-ligand docking. Amino acid residues in five of the seven positions
are invariant, while those in two of the seven positions are variable with low
frequency. Mutation at the two positions maintained β-1,4-xylosidase and
β-1,2-xylosidase activity at the expense of decreased activity. These results suggest
that all GH 39 β-xylosidases (GH 39 subgroup II) probably show β-1,2-xylosidase
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activity for transformation of notoginsenosides. As such, we proposed that GH 39
β-xylosidases could be representatives of a new EC subsubclass.
In conclusion, based on molecular analysis, conserved amino acid residue
positions of characterized GH 39 β-xylosidases involved in β-1,2-xylosidase activity
were revealed. Activity determination showed that both wild-type rJB13GH39 and its
mutants exhibited β-1,2-xylosidase activity. These results confirm the cleavage of
terminal β-xylose-(1→2)-β-glucose bond by these GH 39 β-xylosidases for
transformation of notoginsenosides, and such cleavage activity should be assigned an
EC number. These characterized GH 39 β-xylosidases could be representatives of the
new EC subsubclass.
Funding
This work was supported by the National Key Research and Development Program of
China (Grant No. 2017YFB0308401), the National Natural Science Foundation of
China (Grant No. 31660445), the Yunling Scholars (Grant No. 2015 56), the Yunling
Industry Leading Talents (Grant No. 2014 1782), the Reserve Talents Project for
Young and Middle-Aged Academic and Technical Leaders of Yunnan Province
(Grant No. 2015HB033), the Ten Thousand Talents Program of Yunnan Province for
Excellent Young Talents (YNHRSS 2018 73), and the National Innovation and
Entrepreneurship Training Program for College Students of China (Grant Nos.
201710681016, 201810681017).
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Notes
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The authors declare that they have no conflict of interest.
Supporting Information
The Supporting Information is available free of charge on the ACS Publications
website at DOI: .Figure S1 shows the ESI-MS spectra of the enzymatic
transformation of notoginsenosides R and R using rJB13GH39. Figure S2 shows
1
2
the DNA sequencing results for mutants Y257(Ps266)T and Y311(Ps322)P using the
universal sequencing primers T7 and T7t. Figure S3 shows SDS-PAGE analysis of
rJB13GH39 and its mutants Y257(Ps266)T and Y311(Ps322)P.
ACKNOWLEDGMENTS
We thank Prof. Florence Vincent and Ahmad Ali-Ahmad from Aix Marseille
University, Marseille, France, for help in building the phylogenetic tree of GH 39.
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FIGURE CAPTIONS
Figure 1. The diagram of enzymatic transformation of notoginsenosides R and R to
1
2
ginsenosides Rg and Rh , respectively.
1
1
Figure 2. UHPLC analysis for the conversion of notoginsenosides R to ginsenosides
1
Rg using rJB13GH39 (A), Y257(Ps266)T (B), and Y311(Ps322)P (C).
1
Figure 3. UHPLC analysis for the conversion of notoginsenosides R to ginsenosides
2
Rh using rJB13GH39 (A), Y257(Ps266)T (B), and Y311(Ps322)P (C).
1
Figure 4. The phylogenetic tree constructed on the basis of GH 39 enzymes.
The enzyme name, accession number, and source are given. Bootstrap values (n =
1
000 replicates) are reported as percentages. The scale bar represents the number of
changes per amino acid position.
Figure 5. Sequence logos generated on the basis of the multiple sequence alignment
of the 14 identified GH 39 β-xylosidases in subgroup II shown in Figure 4.
Arrows show the putative catalytic and substrate recognition residues. Asterisks show
the conserved docking positions shown in Figure 7.
Figure 6. Xylose-glucose ligand docking with the catalytic pocket of JB13GH39 with
9
1 poses.
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Hydrogen bonds are represented by green-dashed lines.
Figure 7. Heat plot showing the hydrogen bond interactions of amino acid residues
with the 91 poses of the xylose-glucose ligand.
Arrows
show
the
conserved
docking
positions.
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R1 Notoginsenoside R2
GH 39 β-1,2-xylosidase
Rg1 Ginsenoside Rh1
Figure 1.
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A
Notoginsenoside R1
Notoginsenoside R1
hydrolyzed by rJB13GH39
Ginsenoside Rg1
B
Notoginsenoside R1
Notoginsenoside R1
hydrolyzed by Y257(Ps266)T
Ginsenoside Rg1
C
Notoginsenoside R1
Notoginsenoside R1
hydrolyzed by Y311(Ps322)P
Ginsenoside Rg1
Figure
2.
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A
Notoginsenoside R2
Notoginsenoside R2
hydrolyzed by rJB13GH39
Ginsenoside Rh1
B
Notoginsenoside R2
Notoginsenoside R2
hydrolyzed by Y257(Ps266)T
Ginsenoside Rh1
C
Notoginsenoside R2
Notoginsenoside R2
hydrolyzed by Y311(Ps322)P
Ginsenoside Rh1
Figure
3.
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9
9
NF2523, ASF57709, Neocallimastix frontalis
NF2152, ASF57707 or 5U22, Neocallimastix frontalis
Hypothetical β-xylosidase, SHJ25262, Fibrobacter sp.
Hypothetical β-xylosidase, SDM81094, Streptomyces guanduensis
Hypothetical β-xylosidase, SIR88575, Microbispora rosea
XynB_thesa, AAA27369 or 1UHV, Thermoanaerobacterium saccharolyticum
SlpA, AFK86459, Thermoanaerobacterium saccharolyticum
TtGH39, ADL68513, Thermoanaerobacterium thermosaccharolyticum
Xln-DT, ARO76324, Dictyoglomus thermophilum
1
00
0.2
III
1
00
Containing CBM
Families
1
00
9
9
1
00
9
8
5
3
9
1
00
XynA, AAB87373, Caldicellulosiruptor saccharolyticus
XylBH39, BAB04787, Bacillus halodurans
9
9
XynB1, ABI49941 or 2BS9, Geobacillus stearothermophilus
0
II
1
00 WSUCF1, EPR27580, Geobacillus sp.
Bxl39A, CAD48308, Clostridium stercorarium
BxyH, AEE47384, Cellulomonas fimi
β-Xylosidases
1
00
8
4
Coxyl A, ADQ03734, Caldicellulosiruptor owensensis
XynB, AAA23063, Caldicellulosiruptor saccharolyticus
1
00
1
00
JB13GH39, MG838204, Sphingomonas sp.
CcXynB2, AAK24328 or 4EKJ, Caulobacter crescentus
hIDUA, P35475 or 3W81, Homo sapiens
1
00
8
9
I
1
00
IDUA, AAC42044, Mus musculus
α-L-Iduronidases
PslG, AAG05625 or 4ZN2, Pseudomonas aeruginosa
IV
GH39wh2, WP_029426617 or 5JVK, Bacteroides cellulosilyticus Endo-glycosidases
Figure 4.
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Figure 5.
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91 poses of xylose-glucose
Tyr311
Glu352
Glu189
Tyr257
Trp344
His88
Glu306
Figure 6.
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Hydrogen bonds
0
1
2
3
Pose
His88
Glu189
Tyr257
Glu306 Tyr311
Typ344 Glu352
Residue
Figure 7.
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