Access to both anomers of rutinosyl azide using wild-type rutinosidase and its catalytic nucleophile mutant

Article abstract of DOI:10.1016/j.catcom.2020.106193

Rutinosidases hydrolyze β-rutinosylated flavonoids. As retaining glycosidases they also have a transglycosylation activity. Here we show that two newly identified wild-type rutinosidases, which are members of the glycoside hydrolase family 5–23, are capable of glycosylation of an inorganic azide with rutin as a glycosyl donor, yielding rutinosyl β-azide. On the other hand, rutinosyl α-azide was synthesized by the catalytic nucleophile mutant of the rutinosidase from Aspergillus niger, which also belongs to GH5–23. Thus, we were able to synthesize at a preparatory scale both anomers of rutinosyl azide from rutin using either wild-type or mutant rutinosidases of GH5–23.

Full text of DOI:10.1016/j.catcom.2020.106193

CatalysisCommunications149(2021)106193
Contents lists available at ScienceDirect
Catalysis Communications
journal homepage: www.elsevier.com/locate/catcom
Short communication
Access to both anomers of rutinosyl azide using wild-type rutinosidase and
its catalytic nucleophile mutant
Michael Kotik⁎, Katerina Brodsky, Petr Halada, Hana Javůrková, Helena Pelantová,
Dorota Konvalinková, Pavla Bojarová, Vladimír Křen
Institute of Microbiology of the Czech Academy of Sciences, Vídeňská 1083, CZ-142 20 Prague 4, Czech Republic
A R T I C L E I N F O
A B S T R A C T
Keywords:
Rutinosidases hydrolyze β-rutinosylated flavonoids. As retaining glycosidases they also have a transglycosylation
activity. Here we show that two newly identified wild-type rutinosidases, which are members of the glycoside
hydrolase family 5–23, are capable of glycosylation of an inorganic azide with rutin as a glycosyl donor, yielding
rutinosyl β-azide. On the other hand, rutinosyl α-azide was synthesized by the catalytic nucleophile mutant of
the rutinosidase from Aspergillus niger, which also belongs to GH5–23. Thus, we were able to synthesize at a
preparatory scale both anomers of rutinosyl azide from rutin using either wild-type or mutant rutinosidases of
GH5–23.
Transglycosylation
Rutinosyl azide
Glycosidase
Activity rescue
Rutin
Glycoside hydrolase family 5
1. Introduction
use in the versatile Cu(I)-catalyzed azide–alkyne 1,3-dipolar cycload-
dition reaction. This so-called “click-chemistry” approach enabled the
Rutinosidases (6-O-α-L-rhamnopyranosyl-β-D-glucopyranosidases;
EC 3.2.1.168) catalyze the hydrolysis of rutin and other structurally
related flavonoid glycosides by cleaving the glycosidic bond between
the rutinose moiety and the aromatic aglycone (Fig. 1A). The vast
majority of characterized rutinosidases are retaining glycosidases of
fungal origin. By virtue of their transglycosylation activity, novel ruti-
nosides with various aglycones can easily be prepared [1–3]. Im-
portantly, the synthesis of novel rutinose-containing glycosides is of a
high relevance to biomedical research [4,5].
synthesis of a large number of glycoconjugates [15,16]. Glycosyl azides
can also be used in various other synthetic applications [17,18]. In-
organic azide has been previously used in so-called activity-rescue re-
actions as an external nucleophile with glycosidase mutants that lack
either the catalytic nucleophile or the acid/base catalyst [19]. The ac-
tivity rescue concept has been employed for the enzymatic synthesis of
monoglycosyl azides such as β-L-fucopyranosyl, α-D-glucopyranosyl,
and β-D-glucopyranosyl azides [18,20,21].
There exist a few synthetic approaches for the preparation of spe-
cific glycosyl β-azides [22,23]. On the other hand, the preparation of
glycosyl α-azides is usually very challenging [22,24]. Especially, azido-
functionalized disaccharides are not readily accessible. Therefore, en-
zymatic synthesis of diglycosyl α-azides is a highly attractive alter-
native to the synthetic chemistry approach.
Rutinosylated flavonoids such as rutin or hesperidin occur in sub-
stantial quantities in various plants and their fruits [6,7]. Rutin is a
convenient glycosyl donor for glycosylations because the process of its
extraction and purification has been optimized due to its importance as
a nutritional supplement [8,9]. Thus, rutin has become an inexpensive
natural glycoside that is readily available in food-grade quality.
Numerous glycosylations of hydroxyl-containing compounds with
activated carbohydrate donors using retaining glycosidases have been
described [10–14]. In contrast to the purely chemical approach to
carbohydrate synthesis, which is usually a multi-step process, enzy-
matic glycosylations often exhibit higher overall yields and high to
absolute stereo- and regioselectivities. Azide-functionalized carbohy-
drates are useful building blocks in carbohydrate chemistry due to their
We report here the synthesis of both anomers of rutinosyl azide from
rutin using wild-type or mutant rutinosidases of GH5–23. Through their
azide-based transglycosylation activities, the newly identified rutino-
sidases McRut and PcRut catalyzed the formation of rutinosyl azide
with β-anomeric configuration (Fig. 1B). The synthesis of rutinosyl α-
azide was achieved using the catalytic nucleophile mutant of AnRut as a
catalyst (Fig. 1B). Both rutinosyl α-azide and rutinosyl β-azide were
synthesized at a preparatory scale and fully characterized.
Abbreviations: AnRut, rutinosidase from Aspergillus niger CCIM K2; McRut, rutinosidase from Mucor circinelloides CCF 2598; PcRut, rutinosidase from Penicillium
chrysogenum CCF 1269
⁎ Corresponding author.
E-mail address: kotik@biomed.cas.cz (M. Kotik).
https://doi.org/10.1016/j.catcom.2020.106193
Received 17 August 2020; Received in revised form 23 September 2020; Accepted 7 October 2020
Availableonline10October2020
1566-7367/©2020TheAuthors.PublishedbyElsevierB.V.ThisisanopenaccessarticleundertheCCBY-NC-NDlicense
(http://creativecommons.org/licenses/BY-NC-ND/4.0/).

M. Kotik, et al.
CatalysisCommunications149(2021)106193
Fig. 1. Reaction scheme for hydrolysis of rutin (quercetin 3-O-β-rutinoside) and transglycosylation of inorganic azide with rutin as a donor, catalyzed by McRut,
PcRut and AnRut or the catalytic nucleophile mutant E319A of AnRut. (A) The hydrolysis of rutin results in the formation of the disaccharide rutinose (6-O-α-L-
rhamnosyl-D-glucose) and quercetin. (B) Using inorganic azide as an acceptor and rutin as a glycosyl donor, rutinosyl β-azide (α-L-rhamnopyranosyl (1 → 6) β-D-
glucopyranosyl azide) is produced in the presence of wild-type AnRut, McRut or PcRut as a catalyst. In contrast, the catalytic nucleophile mutant E319A of AnRut
catalyzes the formation of rutinosyl α-azide (α-L-rhamnopyranosyl (1 → 6) α-D-glucopyranosyl azide) in the presence of inorganic azide as an acceptor and rutin as a
glycosyl donor. (C) Hesperidin (hesperetin 7-O-β-rutinoside) as an alternative rutinosyl donor in transglycosylations.
2. Experimental
(pH 4.0). McRut or PcRut was eluted using a linear gradient of NaCl
(0 → 1.0 M). The active fractions were pooled and subjected to ultra-
filtration using a cellulose membrane with a 10 kDa cut-off (Amicon;
Merck). The enzymes were further purified using size-exclusion chro-
matography (Superdex 200 10/300 GL, Merck; 25 mM Tris-HCl,
150 mM NaCl, pH 8.5) and chromatofocusing (Mono P 5/200 GL
column, Merck; 25 mM Tris-HCl, pH 8.5; elution with Polybuffer 96
(pH 6.0), followed by 1.0 M NaCl).
2.1. Materials
Rutin (quercetin 3-O-rutinoside) was purchased from Alchimica
s.r.o. (Czech Republic). Hesperidin (hesperetin 7-O-rutinoside), he-
speretin and quercetin were obtained from Sigma-Aldrich. Rutinose was
prepared from rutin using AnRut as a catalyst [25]. Sodium azide was
from Alfa Aesar (USA). Anthrone was obtained from PanReac Ap-
pliChem (USA).
2.4. In-gel digestion and MALDI-TOF mass spectrometry
2.2. Cultivations of fungal strains
The protein bands were excised from the acrylamide gels and pre-
pared for trypsin or AspN protease treatment as previously described
[1]. The MALDI mass spectra of the generated peptides were acquired
with an Ultraflex III instrument (Bruker Daltonics) as described [1].
The strains Mucor circinelloides CCF 2598 and Penicillium chryso-
genum CCF 1269 are deposited in the Czech Collection of Fungi (CCF),
Charles University in Prague. The strains were cultivated at 28 °C for
6–12 days using the previously described growth medium [1].
2.5. Cloning of McRut and PcRut-encoding genes
2.3. Purification of wild-type McRut and PcRut
Total RNA and chromosomal DNA were isolated from freeze-dried
fungal biomass according to the manufacturer's protocol using an RNA
PowerSoil total RNA isolation kit (Mo Bio Laboratories, USA). A
Herculase Hotstart DNA polymerase (Agilent Technologies, USA) was
used with the degenerate primers, which were designed based on the
peptide sequences (Table S1). RACE experiments (Fig. S1) were
The fungal culture was filtered through a cellulose filter, and the
filtrate was dialyzed against 10 mM acetate buffer (pH 4.0). The dia-
−
lyzed sample was loaded on
a
Fractogel EMD SO3
column
(1.5 × 10.0 cm; Merck) equilibrated with 10 mM acetate buffer
2

M. Kotik, et al.
CatalysisCommunications149(2021)106193
MN393234.1 Aspergillus niger K2
BAE61018.1 Aspergillus oryzae RIB40
AMD11613.1 Acremonium sp. DSM 24697
BAG70961.1 Penicillium multicolor TS-5
100
26
GH5-23
100
55
MN562485.1 Mucor circinelloides CCF 2598
XP 002559889.1 Penicillium chrysogenum CCF 1269
ABD03937.1 Vicia sativa
74
100
99
ABN70849.1 Dalbergia nigrescens
BAC78656.1 Camellia sinensis
100
GH1
98
BAD14925.1 Viburnum furcatum
XP 747510.1 Aspergillus fumigatus Af293
G2NFJ9.1 Streptomyces sp.
GH30-3
GH55
69
BAL86042.1 Actinoplanes missouriensis
BAF52916.1 Arthrobacter sp. NHB-10
MK941183.1 Acremonium sp. DSM 24697
BAE62006.1 Aspergillus oryzae RIB40
BAM08953.1 Oerskovia sp. Y1
100
85
GH3
100
71
0.10
Fig. 2. Phylogenetic relationships between rutinosidases (α-L-rhamnosyl-β-D-glucosidases) and other disaccharide-specific glycosidases. The accession numbers of
each entry are indicated. The clades are colored according to the CAZy classification of glycoside hydrolases: GH5–23 (fungal origin, red), GH3 (fungal origin, pink),
GH55 (bacterial origin, blue), GH30-3 (fungal origin, black), and GH1 (plant origin, green). The bar represents 0.1 amino acid substitutions per site. The entries of
AnRut, McRut and PcRut are marked as follows: ▲, ● and ■, respectively. Two β-1,3-glucanase sequences of GH55 were added (△). The following protein sequence
identities were determined after sequence alignment: AnRut–McRut (54.8%) and AnRut–PcRut (58.6%). Multiple sequence alignments were performed using
CLUSTALW [28]. The phylogenetic tree was constructed in the program MEGA7 using the Neighbor-Joining method [29]. The evolutionary distances were computed
using the p-distance method. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
performed using a 5′/3’ RACE kit of Roche and the RACE primers in
Table S2. The full-length McRut and PcRut-encoding genes were am-
plified from cDNA using a Q5 DNA polymerase (New England Biolabs,
USA) and the primer pairs B and C (Table S2), respectively. The se-
quence of the McRut-encoding gene has been deposited at the National
Center for Biotechnology Information (NCBI) under the accession
number MN562485. The PcRut-encoding nucleotide sequence was
found to be identical to the sequence of a hypothetical protein (acces-
sion number XP_002559889) from Penicillium rubens Wisconsin
54–1255. The McRut and PcRut-encoding genes without their predicted
signal sequences [26] were PCR-amplified using the primer pairs D and
E (Table S2). After treatment with XbaI and XhoI, the restricted PCR
products were inserted into the prepared pPICZα A vector downstream
of the α-factor signal sequence encoded by the vector.
chromatography (TLC) using silica gel 60 F254 plates (Merck) and the
anthrone reagent (7 mM) dissolved in ethanol/water/sulfuric acid
(5:1:5; v/v/v).
2.8. Preparative transglycosylation reactions
Rutinosyl β-azide (α-L-rhamnopyranosyl (1 → 6) β-D-glucopyranosyl
azide): A slurry with a total volume of 16 mL was formed by adding
sodium azide (66 mmol) and purified wild-type AnRut, McRut or PcRut
(2.0 mg) to rutin (2.0 g, 3.28 mmol) in 10 mM acetate buffer (pH 3.6).
The reaction mixtures were incubated at 600 rpm for 5–7 h at 36 °C.
The progress of the enzymatic conversions was monitored by TLC
analysis as stated above. The reaction mixtures were filtered through
filter paper, centrifuged (15 min at 4500 ×g), concentrated by eva-
poration at 45 °C, and loaded on a size-exclusion chromatography
column using a Biogel P2 Fine stationary phase (Bio-Rad, USA; ø:
2.5 cm, length: 95 cm; flow rate: 7.0 mL h−1) and water as a mobile
phase. Fractions containing the product were combined and freeze-
dried. The product was then further purified by silica gel chromato-
graphy (ethyl acetate/methanol/water, 7:3:1; v/v/v).
2.6. Heterologous expression and purification
The transformation of Pichia pastoris KM71H cells with the linear-
ized McRut and PcRut-encoding plasmids and the activity screening of
the transformants were performed as described [1,3]. During cultiva-
tion of the recombinant yeasts, AnRut, its E319A and E210A mutants
[27], McRut and PcRut were secreted to the medium. The enzymes were
purified by cation exchange chromatography using a Fractogel EMD
SO3− column (Fig. S2). Protein concentrations were determined using a
Qubit protein assay kit and a Qubit fluorometer (Life Technologies,
USA).
Rutinosyl α-azide (α-L-rhamnopyranosyl (1 → 6) α-D-glucopyranosyl
azide): The reaction mixture (8 mL) contained rutin (0.4 g, 0.66 mmol),
sodium azide (7.7 mmol) and the purified E319A mutant of AnRut
(25 mg) in 10 mM acetate buffer (pH 3.6). The slurry was incubated at
600 rpm and 30 °C for 48 h. After the enzymatic reaction, the reaction
mixture was processed as stated above for rutinosyl β-azide.
2.7. Activity determination and product detection
3. Results and discussion
Initial hydrolytic rates with rutin or hesperidin as substrate were
determined in McIlvaine buffer (pH 3.4) at 37 °C in the presence of 10%
(v/v) dimethyl sulfoxide. The incubation time was 10 min, after which
the enzyme was inactivated at 95 °C for 30 min. The amount of the
aglycone (quercetin or hesperetin) released in the reaction was de-
termined by HPLC using calibration curves for quercetin and hesperetin
(Fig. S3). The reaction products were also analyzed by thin-layer
3.1. Cloning and overexpressions
A previous screening resulted in the discovery of fungal strains with
rutinosidase activity, among them were M. circinelloides and P. chryso-
genum [1]. The sequence of the McRut-encoding gene was obtained in
three consecutive steps: (i) PCR with degenerate primers based on
peptide sequences of McRut, (ii) RACE experiments that accessed the 5′
3

M. Kotik, et al.
CatalysisCommunications149(2021)106193
60.0
50.0
40.0
30.0
20.0
10.0
0.0
and 3′ coding and non-coding regions, and (iii) PCR with specific pri-
mers derived from the RACE sequences using cDNA as a template. In
the case of PcRut, the entire coding region was amplified from cDNA
using specific primers derived from the non-coding regions of the gene
Pc13g14840 encoding XP_002559889.
In a phylogenetic analysis of experimentally verified diglycosidases,
McRut and PcRut clustered together with other fungal rutinosidases of
glycoside hydrolase family 5, subfamily 23, including AnRut (Fig. 2).
All rutinosidases, i.e., AnRut and its mutants E319A and E210A
[27], McRut and PcRut, were overexpressed in Pichia pastoris KM71H.
The enzymes were purified to apparent homogeneity (Fig. S4).
3.2. Biotransformations using wild-type AnRut and its mutants
Transglycosylations with retaining glycosidases are usually per-
formed with activated carbohydrate donors and hydroxyl-containing
acceptors. We discovered the azide-mediated transglycosylation ac-
tivity of wild-type AnRut while investigating activity-rescue reactions of
its catalytic nucleophile and acid/base catalyst mutants E319A and
E210A, respectively (Fig. S5). We were surprised to detect high con-
centrations of rutinosyl azide in the reaction mixtures with wild-type
AnRut compared with its mutant E319A (Fig. 3A). Azide-based trans-
glycosylation reactions are not limited to wild-type AnRut, since the
production of rutinosyl azide was also observed in reactions with McRut
and PcRut (Fig. 3B). Importantly, no formation of transglycosylation
products was observed in the absence of enzyme or with denatured
enzyme (Fig. S6). The detection of azide-based transglycosylation ac-
tivities with wild-type rutinosidases was unexpected because normally,
in wild-type glycosidases, the anomeric center of the glycosyl-enzyme
intermediate is supposed to be shielded from the attack of anionic nu-
cleophiles such as azide by the presence of charged glutamate residues
acting as the acid-base catalyst or the catalytic nucleophile [30]. As
predicted, virtually no hydrolytic activity was detected with the E319A
mutant due to the missing catalytic nucleophile, as evidenced by the
HPLC-based analysis of the reaction product (quercetin) (Fig. S7) and
the absence of rutinose spots on the TLC plate (Fig. 3A). Hesperidin
(Fig. 1C) can be used as an alternative glycosyl donor to rutin for the
synthesis of rutinosyl azide (Fig. 3). However, there is a lower specific
activity (comprising both hydrolysis and azide-based transglycosyla-
tion) for hesperidin as a substrate compared with rutin (Fig. 4).
0.0
0.2
0.4
0.6
0.8
1.0
Azide [M]
Fig. 4. Conversion of rutin (open symbols) or hesperidin (filled symbols) via
hydrolysis and transglycosylation catalyzed by wild-type McRut (○, ●) or
PcRut (□, ■) in the presence of various concentrations of inorganic azide. The
initial velocity measurements were performed in at least triplicate. The reaction
conditions used: McIlvaine buffer, pH 3.4; 37 °C; 625 ng mL−1 of wild-type
rutinosidase; 5.0 mM rutin or 2.0 mM hesperidin (i.e., saturating concentrations
with the exception of the McRut-hesperidin enzyme-substrate pair). See
Materials and methods for details on the HPLC-based method for the activity
determination. Only positive or negative error bars are shown for clarity rea-
sons.
Preparative transglycosylations with azide, wild-type AnRut, McRut or
PcRut and rutin as a donor afforded rutinosyl azide (480–565 mg) with
isolated yields of 40–47% relative to the donor (Fig. S8 and S9 for NMR
spectra). The configuration at the anomeric center for all three isolated
products was determined by NMR spectroscopy to be beta as inferred
from the H-1/H-2 coupling constant (JH1-H2 = 8.7 Hz; Table S3). In
addition, the presence of the azide aglycone was confirmed by the
heteronuclear correlation between the anomeric proton and nitrogen at
83 ppm, detected in a 1He15N HMBC NMR experiment. The structures
were fully congruous with their MS spectra (Fig. S10 and S11).
The preparative transglycosylation reaction with inorganic azide,
the E319A mutant of AnRut and rutin as a donor afforded rutinosyl α-
azide (100 mg; 44% isolated yield) with inverted anomeric configura-
tion compared with rutin. Here, alpha configuration of the CeN gly-
cosidic linkage was proved by the H-1/H-2 coupling constant (JH1-
H2 = 4.2 Hz; Table S4; Fig. S12–S13 for NMR spectra), as expected for
catalytic nucleophile mutants of retaining glycosidases [19]. The in-
tegrity of the structure was supported by its HR-MS spectrum (Fig. S14).
Hence, both α- and β-anomers of rutinosyl azide are accessible in
preparative amounts: wild-type AnRut, McRut and PcRut catalyze the
formation of the β-anomer, whilst the α-anomer is produced by the
catalytic nucleophile mutant of AnRut (Fig. 1B).
Despite their hydrolytic activities (see Table S5), wild-type AnRut,
McRut and PcRut with their pronounced azide-based transglycosylation
activities are highly attractive catalysts for the facile synthesis of ruti-
nosyl β-azide from inexpensive rutin. This is fundamentally different
from the retaining wild-type β-glucosidase from Agrobacterium faecalis,
where azide-based transglycosylations were not observed [30].
The lost assistance of the acid/base catalyst in the E210A mutant of
AnRut can be somewhat substituted by external nucleophiles, but only
to a much lower extent compared to the E319A mutant [27]. Indeed,
the rescued activity of E210A in the presence of azide is so low that it
cannot be used in any production of rutinosyl β-azide (Fig. S6A). This is
in contrast to the expected rescue activity of an acid/base catalyst
mutant with a glycoside such as p-nitrophenyl glucoside [19].
Fig. 3. TLC analysis of azide-based transglycosylation reactions catalyzed by
wild-type AnRut, its E319A nucleophile mutant, McRut and PcRut. (A) Wild-
type AnRut was used with rutin (lane 1) or hesperidin (lane 2) as a rutinosyl
donor; the same donors (rutin: lane 3; hesperidin: lane 4) were used with the
E319A mutant. (B) Products of azide-based transglycosylation reactions with
wild-type McRut and PcRut in the presence of rutin (lanes 1 and 2) or he-
speridin (lanes 3 and 4), respectively. The reaction products were visualized
70 min after the initiation of the reaction using the anthrone reagent. The
quercetin spots are colored yellow; spots of rutinose, rutinosyl azide and re-
sidual hesperidin are colored blue. Residual rutin is colored green. The spots of
rutin and hesperidin are boxed. Identical reaction conditions and catalyst
concentrations were used for all reaction mixtures (McIlvaine buffer, pH 3.4;
10 mM rutinosyl donor; 250 mM sodium azide; 1.0 mg mL−1 of enzyme). (For
interpretation of the references to colour in this figure legend, the reader is
referred to the web version of this article.)
4

M. Kotik, et al.
CatalysisCommunications149(2021)106193
3.3. Rutinosyl β-azide as a substrate
flavonoid degrading fungus Acremonium sp. DSM 24697 produces two diglycosi-
dases with different specificities, Appl. Microbiol. Biotechnol. 103 (2019)
9493–9504.
Transglycosylation products can be substrates, leading to product
hydrolysis, which lowers the yields. To test this hypothesis, we in-
cubated wild-type McRut with rutinosyl β-azide under reaction condi-
tions established for rutin hydrolysis. Interestingly, it turned out that
practically no hydrolysis of rutinosyl β-azide was detectable by TLC
analysis (Fig. S15). Based on this outcome, we examined a potential
inhibitory effect of rutinosyl β-azide on rutin hydrolysis. An analysis of
the initial rate data revealed that the hydrolytic activity of wild-type
McRut towards rutin was virtually unaffected by the presence of the
transglycosylation product (Fig. S16). The data indicate that rutinosyl
β-azide interacts only very weakly with McRut, which probably ex-
plains the determined high transglycosylation yield.
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azide, thereby forming a β-N-glycosidic bond between the aglycone and
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structurally characterize both β- and α-anomers of rutinosyl azide. Our
results suggest that azide-based transglycosylations may be a general
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Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influ-
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Appendix A. Supplementary data
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.catcom.2020.106193.
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