A Mechanism-Based Approach to Screening Metagenomic Libraries for Discovery of Unconventional Glycosidases

Article abstract of DOI:10.1002/anie.201806792

Functional metagenomics has opened new opportunities for enzyme discovery. To exploit the full potential of this new tool, the design of selective screens is essential, especially when searching for rare enzymes. To identify novel glycosidases that employ cleavage strategies other than the conventional Koshland mechanisms, a suitable screen was needed. Focusing on the unsaturated glucuronidases (UGLs), it was found that use of simple aryl glycoside substrates did not allow sufficient discrimination against β-glucuronidases, which are widespread in bacteria. While conventional glycosidases cannot generally hydrolyze thioglycosides efficiently, UGLs follow a distinct mechanism that allows them to do so. Thus, fluorogenic thioglycoside substrates featuring thiol-based self-immolative linkers were synthesized and assessed as selective substrates. The generality of the approach was validated with another family of unconventional glycosidases, the GH4 enzymes. Finally, the utility of these substrates was tested by screening a small metagenomic library.

Full text of DOI:10.1002/anie.201806792

A Journal of the Gesellschaft Deutscher Chemiker
Angewandte
Chemie
International Edition
www.angewandte.org
Accepted Article
Title: A mechanism based approach to screening metagenomic
libraries for discovery of unconventional glycosidases
Authors: Stephen Withers, Seyed Nasseri, Leo Betschart, Daria
Opaleva, and Peter Rahfeld
This manuscript has been accepted after peer review and appears as an
Accepted Article online prior to editing, proofing, and formal publication
of the final Version of Record (VoR). This work is currently citable by
using the Digital Object Identifier (DOI) given below. The VoR will be
published online in Early View as soon as possible and may be different
to this Accepted Article as a result of editing. Readers should obtain
the VoR from the journal website shown below when it is published
to ensure accuracy of information. The authors are responsible for the
content of this Accepted Article.
To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.201806792
Angew. Chem. 10.1002/ange.201806792
Link to VoR: http://dx.doi.org/10.1002/anie.201806792
http://dx.doi.org/10.1002/ange.201806792

10.1002/anie.201806792
Angewandte Chemie International Edition
COMMUNICATION
A mechanism based approach to screening metagenomic
libraries for discovery of unconventional glycosidases
Seyed Amirhossein Nasseri, Leo Betschart, Daria Opaleva, Peter Rahfeld and Stephen G. Withers*[a]
Abstract: Functional metagenomics has opened up exciting new
opportunities for enzyme discovery. To take advantage of the full
potential of this new tool, the design of suitable, selective screens is
essential, especially when searching for rare enzymes. In order to
identify novel glycosidases that employ cleavage strategies other than
fluorophores such as 6-chloro-4-methylumbelliferone (ClMU) that
have low pKa values, are very useful tools in such screens.[5]
As part of our long standing interest in studying
unconventional glycosidases[6,7], namely those that do not follow
canonical Koshland mechanisms (Figure 1a), we decided to
the conventional Koshland mechanisms, we needed a suitable screen. screen metagenomic libraries with the end goal of discovering
Focusing initially on the unsaturated glucuronidases (UGLs) we
showed that use of simple aryl glycoside substrates did not allow
sufficient discrimination against β-glucuronidases, which are
widespread in bacteria. While conventional glycosidases are
generally not able to hydrolyze thioglycosides efficiently, UGLs follow
a distinct mechanism that allows them to do so. Thus, fluorogenic
thioglycoside substrates featuring thiol-based self-immolative linkers
were synthesized and successfully assessed as selective substrates.
The generality of the approach was validated by expanding the
concept to another family of unconventional glycosidases, the GH4
enzymes. Finally, the utility of these substrates was put to the test and
confirmed by screening a small metagenomic library.
new and unusual glycosidases to add to the CAZy GH families.[8]
Although the main hope is to find new classes of enzymes with
novel mechanisms, these screens will also identify further
members of established families of mechanistically unusual
glycosidases and hence provide a better understanding of the
different roles of these often-understudied enzymes.
The main issues facing any screen for these rare
glycosidases are as follows: Firstly, the expected hit rate will, by
definition, be extremely low, thus use of high-throughput
screening methods is essential. Secondly, and more importantly,
the use of conventional substrates for these screens will give rise
to many false positive hits, from conventional glycosidases. Not
only will this cause much unnecessary work, but it will also mask
the desired enzymatic activity. Consequently, novel substrates
that are selectively hydrolyzed by the target enzymes are required.
This study describes the development of a class of
substrates that would be cleaved only by unconventional
glycosidases. As a first model, we focused on developing
substrates for unsaturated glucuronidases (UGLs) of CAZy
glycoside hydrolase families GH88 and GH105.[9,10] We then
briefly describe the extension of this concept to the NAD+-
dependent glycosidases of GH4 and GH109.[11,12] Enzymes from
these two groups have in common the fact that their mechanism
for cleavage of the glycosidic bond does not involve the simple
protonation and substitution events of the Koshland mechanism.
The native substrates of UGLs, 4,5-unsaturated
glucuronides, are rare glycosides that are only formed in the
process of enzymatic degradation of uronic acid-containing
polysaccharides by polysaccharide lyases.[13] The mechanism of
UGLs from GH88 and GH105 families[14] (Figure 1b) involves
hydration of the C4-C5 double bond in the first stage. This is
initiated by addition of a proton onto C4 of the double bond to form
Metagenomics has proven to be an invaluable gate for entering
a previously unexplored territory, the world of uncultured bacteria.
While simple sequencing of environmental DNA provides
valuable insights, a high percentage of the identified genes have
no known functions (30-40%[1]). In functional metagenomics
however, environmental DNA is expressed heterologously and
the resulting proteins are screened for a desired activity. This
assaying step provides direct access to information on the
function of the expressed proteins, thereby helping to bridge the
gap between sequence and function. Since new enzyme families
are unlikely to be found through sequencing alone, such high-
throughput enzyme assays are needed. Indeed, the development
of novel and much more affordable ultrahigh-throughput
methods[2] has rendered functional metagenomics an ever more
attractive approach for the discovery of new enzymes especially
as, by definition, any enzyme so identified can be expressed in
the host organism used for screening.
So far, focus on discovery of enzymes from metagenomic
screens has primarily been upon industrially relevant enzymes[3]
and among the glycosidases in this category, cellulases,
pectinases and xylanases are the most sought after enzymes.[4]
These screens usually involve a chromogenic or more often a
fluorogenic glycoside substrate that will give rise to a strong signal
upon hydrolysis by the enzyme of interest. We have found that
methylumbelliferyl(MU)-glycoside substrates, particularly with
a
carbocation intermediate and then enzyme-assisted
nucleophilic attack of water onto the positively charged carbon.
The second part of the mechanism involves rearrangement of the
hemiketal followed by decomposition of the resulting hemiacetal
to afford the keto-aldehyde, which subsequently hydrates. Note
that this relatively simple mechanism does not explain all the
kinetic and crystallographic findings, and other variations on this
mechanism have been proposed, but despite extensive studies
not yet established[13,15]. However, it is clear that the initial
mechanistic step is the hydration of the C4-C5 double bond as
shown in Figure 1b[7,9,10]. Moreover, these studies suggest that
this is the rate-determining step of this mechanism.[14]
Interestingly, this makes UGLs relatively promiscuous enzymes
that are capable of accepting a range of substrates with different
[a]
S. A. Nasseri, Dr. L. Betschart, D. Opaleva, Dr. P. Rahfeld, Prof. S.
G. Withers
Department of Chemistry, University of British Columbia
Vancouver, British Columbia, V6T 1Z1 (Canada)
E-mail: withers@chem.ubc.ca
Supporting information for this article is given via a link at the end of
the document.
This article is protected by copyright. All rights reserved.

10.1002/anie.201806792
Angewandte Chemie International Edition
COMMUNICATION
Figure 1: a) Retaining Koshland mechanism of a GH2 β-glucuronidase, b) putative mechanism of UGLs c) structure of the substrates used in kinetic studies
unactivated thioglycosides corresponding to their natural
aglycones, since the aglycone is not directly involved in the rate
determining step of this mechanism.[15,16]
[18–22]
substrate.
However, UGLs follow a distinct mechanism in
which the glycosidic bond is cleaved via an elimination
mechanism. Accordingly, it had been previously shown that at
least one member of the GH88 family is able to hydrolyze
activated thioglycosides[7] and as shown in the present study they
are also able to hydrolyze alkyl thioglycosides. Likewise members
of the mechanistically unusual GH4 family have been shown to
cleave thio-linked disaccharides.[23]
Thus, our initial approach to differentiating the expected
UGL hits from the false positive hits resulting from β-
glucuronidases was to replace the O-glycosides typically used in
screening with the corresponding S-glycoside. To this end, we
synthesized the conventional screening substrates for UGL 1, 2
along with a similar thioglycoside substrate 3 (Figure 1c). In
parallel, we assembled the analogous substrates for a β-
glucuronidase 4-6, for a direct comparison. The Michaelis-Menten
kinetic parameters for hydrolysis of these substrates were
determined with one model UGL (the unsaturated glucuronidase
from Clostridium perfringens, UGC) and two commercially
available β-glucuronidases; I) BGE and II) β-glucuronidase from
bovine liver[24] (BGB), which is reported to hydrolyze UGL
substrates.[25]
UGLs represent an excellent and challenging test case for
design of screens for unconventional glycosidases. First of all,
they are relatively rare enzymes, with only 1449 entries in the
GH88 family and 2562 entries in the GH105 family (compared to
for example more than 12000 entries in the GH2 family, the family
that contains most of the known β-glucuronidases). Secondly, as
shown in the present study, the conventional MU-glycoside
screening substrates of UGLs are cleaved, albeit slowly, by β-
glucuronidases. Since β-glucuronidase activity occurs much more
frequently than UGL activity in metagenomic genes, screening for
UGL activity with MU-glycosides of unsaturated glucuronides can
lead to false positive hits that contain genes for β-glucuronidases
and not UGLs. Further, since our metagenomic libraries are
expressed in the E. coli strain EPI300, β-glucuronidase from E.
coli[17] (BGE) will always be present in the screening media, giving
rise to a high background activity (although this could be avoided
in future screens by using BGE deficient strains of E. coli).
While in the literature there are examples for hydrolysis of
aryl glycosides that contain phenols of low pKa by Koshland
glycosidases, none of these enzymes have been shown to cleave
Table 1: Summary of Michaelis-Menten kinetic parameters
Entry
Enzyme
UGC
Sub.
Km (µM)
280 ± 25
160 ± 21
1390 ± 181
kcat (s-1)
kcat/Km (s-1µM-1)
(3.0 ± 0.4) × 10-2
(6.7 ± 1.2) × 10-2
(5.7 ± 1.0) × 10-3
Entry
9
Enzyme
BGE
Sub.
Km (µM)
580 ± 90
n.d.
kcat (s-1)
kcat/Km (s-1µM-1)
(9.5 ± 2.1) × 10-6
n.d.
1
2
3
1
2
3
8.2 ± 0.3
10.8 ± 0.4
7.9 ± 0.4
6
1
2
0.0055 ± 0.0004
n.d.
10
BGB
11
1260 ± 123
0.0146 ± 0.0008
(1.2 ± 0.2) × 10-5
4
5
6
7
8
BGE
1
2
3
4
5
750 ± 130
6.7 ± 0.7
--
0.037 ± 0.002
0.048 ± 0.001
--
(4.9 ± 1.1) × 10-5
(7.2 ± 0.9) × 10-3
--
12
13
14
15
3
4
5
6
--
--
--
280 ± 24
109 ± 9
1.39 ± 0.05
1.36 ± 0.03
0.060 ± 0.006
(5.0 ± 0.6) × 10-3
(1.2 ± 0.1) × 10-2
(2.4 ± 0.6) × 10-5
140 ± 12
63 ± 4
13.6 ± 0.4
12.8 ± 0.2
(9.7 ± 1.1) × 10-2
(2.0 ± 0.2) × 10-1
2500 ± 380
-- stands for no or negligible activity, n.d.: not determined
This article is protected by copyright. All rights reserved.

10.1002/anie.201806792
Angewandte Chemie International Edition
COMMUNICATION
C-O, while releasing a stable fluorophore
that is easy to detect. Thus, we designed
and synthesized two substrates, 7 and 8,
with two distinct types of self-immolative
linkers (Figure 2).
The synthetic routes to these two
substrates are detailed in the SI. Note that
two different schemes for the synthesis of
substrates containing an S,O-acetal linker
are presented (Schemes S3 and S4), either
of which could be applied to the synthesis of
this linker for future applications.
The first candidate, 7, contains a
thioquinone methide-generating linker,
which will decompose as shown in Figure
2b. The generated thioquinone methide is
an unstable intermediate that either reacts
with surrounding nucleophiles or gets
reduced.[27] Quinone methides are well
known reactive intermediates that have
been central to a number of self-immolative
linker strategies, and other different
applications.[28]
Thioquinone
methides
however are far less studied, although they
have been used as self-immolative linkers
previously. [27,29]
Figure 2: a) Structures of the selective screening substrates; mechanism of disintegration of b) 7 c) 8
The kinetic parameters determined are given in Table 1. As
The linker implemented in substrate 8 features an S,O-
acetal that will decompose to thioformaldehyde and release ClMU
as shown in Figure 2c. Thioformaldehyde is an unstable
molecule[30] that has never been isolated and is reported to
spontaneously trimerize to 1,3,5-trithiane.[31] O,O-Acetal linkers
feature much less prominently in the literature than those
involving quinone methides. They have mainly been reported as
acid-labile linkers[32] for application as prodrugs[33,34] and as
protein cross linkers.[35,36] S,S-acetals have been reported as
stable linkers to attach glycosides to peptides[37] and have been
isolated as part of a group of β-thioglucosides from seeds of
Afrostyrax lepidophyllus, a genus of tree native to equatorial
Africa.[38] Finally, S,O-acetals have been reported as useful
intermediates in synthesis of azidomethyl and O,O-acetal
glycosides.[39] The structure of our substrate was inspired by a
molecule that was tested unsuccessfully as a substrate of
glycosaminidases[40] and more recently, a FRET-based substrate
for glycosidases.[41] However, the aforementioned studies feature
O,O-acetal linkers and the present work is the first report of a self-
immolative S,O-acetal linker to the best of our knowledge.
anticipated, the β-glucuronidases are indeed capable of
hydrolyzing unsaturated-glucuronides, although with activities 2
to
3 orders of magnitude lower than the corresponding
glucuronide (entries 4 & 7, 5 & 8 and 11 & 14). Notably, although
the β-glucuronidases are able to hydrolyze the thioglucuronide
substrate 6, the kcat/Km values are 104 and 102 fold lower than for
the corresponding O-glycoside 4, respectively, for BGE (entries 7
and 9) and BGB (entries 13 and 15). In contrast for UGC the
kcat/Km for 3 is only 5 times lower than that of 1 (entries 1 and 3).
Further, hydrolysis of 3 by the β-glucuronidases is negligible.
These results may suggest that 3 would be a good substrate
for screening metagenomic libraries for UGL activity. However,
the use of 3 faces other serious problems: First, the aglycone of
3, 4-methyl-7-thioumbelliferone (SMU), is not fluorescent, though
it has a relatively large extinction coefficient at 370 nm so that UV-
Vis could be used instead. However, UV-Vis-based detection is
inherently less sensitive and is therefore generally less desirable
than fluorescence. Secondly and more importantly, thiophenols
such as the aglycone of 3 are very prone to oxidation and the
disulfide formed is not differentially detectable by UV/Vis with any
sensitivity. The oxidation is relatively rapid; a half-life of 2 hours
was determined for the dimerization of SMU at pH 7 in open
aqueous solution at room temperature (see SI section 2.2.). Since
the standard protocol for screening metagenomic libraries
involves incubation of the substrates with cell lysates for several
hours[26], even a fluorescent derivative of SMU would not be
suitable for high throughput screening of these libraries.
The main limitation of using substrates with linkers is that
the enzymes must still be able to accommodate the substrates in
their active site. The S,O-acetal linker is appealing in the sense
that it is probably the smallest possible self-immolative linker. On
the other hand, if the enzyme requires an activated leaving group
for efficient cleavage, then the linker of 7 may be more suitable:
its reactivity can be further increased by modification of the aryl
group if necessary. Finally, the linker should be stable in the
To overcome these issues, we explored the incorporation of
thiol-based self-immolative linkers into our substrates. The thiol
linker connects the sugar moiety and fluorophore, and will release
the fluorophore upon enzymatic hydrolysis of the anomeric bond.
This should allow us to retain the specificity of cleavage of C-S vs
This article is protected by copyright. All rights reserved.

10.1002/anie.201806792
Angewandte Chemie International Edition
COMMUNICATION
Figure 3: a) Fluorescence signal from incubation of the strains of E. coli with 2, 7 and 8 b) Z-Score of fluorescence readings from screening a beaver fecal library
with compounds 2, 7 and 8. Data points are jittered for better visibility. c) the genes present in the hits from screening.
presence of other reagents present in the screen (e.g.
nucleophiles, reducing agents, etc. from the cell lysate).
Next, to demonstrate that the general structure of these
screening substrates can be applied to other unconventional
glycosidases, we synthesized compound 9 as a substrate for a
GH4 glycosidase, a family that cleaves via an NAD+-dependent
elimination mechanism.[11] 9 is a 6-phospho-β-glucoside featuring
the more sensitive of the two self-immolative linkers, the S,O-
acetal, along with a chromogenic probe, para-nitrophenol. As
seen for the GH88 enzymes, this substrate is selectively
hydrolyzed by the 6-phosphoglucosidases from GH4 and not by
those that follow Koshland mechanisms (See section 3.2 of SI).
Finally, a subset of our beaver fecal metagenomic fosmid
library[42] (one 96-well plate, but containing up to 2000 ORFs),
which we knew contained at least one gene encoding a GH88
enzyme, was screened for UGL activity. This plate was screened
using substrates 2, 7 and 8, and the resulting fluorescence
readings are reported in terms of Z-scores (see SI for details) in
Figures S3 and S4 and also summarized in Figure 3b. Screening
with the less selective O-glycoside 2 revealed two stand-out hits
(wells D9, F9) (Figure 3b and Figure S4a) while screening with
the more selective substrates 7 (Figure 3b and Figure S5a) and 8
(Figure 3b and FigureS5b) gave rise to only one hit (well F9). We
anticipated that the one hit with our selective substrate should
contain a UGL gene, while the other hit identified from screening
with 2 would be a false positive.
Armed with the new compounds and in order to see if they
will fulfill the above conditions to be useful as screening
substrates, we moved straight to testing them with crude cell
lysates according to standard screening procedures. E. coli
strains BL21(DE3) carrying, individually, the plasmids for
expressing UGC, BGE and ABG (Agrobacterium sp. β-
glucosidase as negative control) were grown overnight in auto-
induction LBE 5052 media along with the strain of E. coli that our
metagenomic libraries are expressed in, E. coli strain EPI300.
Each of these overnight cultures was then mixed with a lysis
buffer (phosphate buffer 50 mM pH = 7, containing 1% Triton X-
100) along with 50 µM of one of the three substrates bearing the
same fluorophore: 2, 7 and 8. After overnight incubation with cell
lysate, fluorescence was measured as summarized in Figure 3a.
As expected, 2 does not usefully distinguish between UGC
and BGE. Further, even the background β-glucuronidase activity
of the EPI300 strain shows up as a positive hit with this substrate
after overnight incubation. On the other hand, both of our selective
substrates, 7 and 8, show only UGC as a positive hit. Of note is
that the resulting fluorescence signal from incubation of 7 with the
cell lysate is lower than that of 8. Our observations suggest that
this is because a fraction of the free thiophenol that is formed after
hydrolysis of 7 gets oxidized and will not release the fluorophore,
leading to the lower fluorescence signal. This assumption was
further tested and affirmed, as detailed in the SI, section 3.1.
Analysis of the sequences of these hits identifies the open
reading frames (ORFs) and further BLAST analysis of each of
these ORFs identifies those that belong to GH families (Figure 3c).
Indeed, the fosmid within F9 does include a GH88 gene, which is
This article is protected by copyright. All rights reserved.

10.1002/anie.201806792
Angewandte Chemie International Edition
COMMUNICATION
Henrissat, Nucleic Acids Res. 2014, 42, 490–495.
S. Mori, S. Akao, H. Nankai, W. Hashimoto, B. Mikami, K. Murata,
Protein Expr. Purif. 2003, 29, 77–84.
T. Itoh, A. Ochiai, B. Mikami, W. Hashimoto, K. Murata, J. Mol. Biol.
2006, 360, 573–585.
V. L. Yip, S. G. Withers, Biocatal. Biotransformation 2006, 24, 167–
176.
Q. P. Liu, G. Sulzenbacher, H. Yuan, E. P. Bennett, G. Pietz, K.
Saunders, J. Spence, E. Nudelman, S. B. Levery, T. White, et al.,
Nat. Biotechnol. 2007, 25, 454–464.
S. A. K. Jongkees, S. G. Withers, Acc. Chem. Res. 2014, 47, 226–
235.
presumably responsible for the UGL activity. By contrast, D9
contains no known gene for expression of a UGL, but does
contain a GH2 gene – a family known to include -glucuronidases.
In order to confirm that the observed activities arise from these
two genes, they were expressed and their proteins purified.
Activity assays confirmed that the UGL activity indeed derives
from the anticipated GH88 gene in F9 (Figure S7a). Moreover, the
GH2 enzyme derived from D9 was found to be a β-glucuronidase
with a UGL side activity as predicted (Figure S7b).
In conclusion, we have designed two classes of substrate for
selective screening of metagenomic libraries for unconventional
glycosidases, based on the different mechanisms of these
enzymes. These substrates were then tested and the practicality
and generality of this approach was established by identifying a
new enzyme and confirming its activity. Large scale screening of
metagenomic libraries with a number of such substrates with
different sugar structures will be carried out in the future. This will
hopefully lead to the discovery of novel glycosidase families
featuring new mechanisms as well as further representatives of
known families, thereby helping to reduce the number of
unassigned genes and close the gap between predicted and
characterised enzyme activities.
[9]
[10]
[11]
[12]
[13]
[14]
[15]
[16]
[17]
T. Itoh, W. Hashimoto, B. Mikami, K. Murata, J. Biol. Chem. 2006,
281, 29807–29816.
S. A. K. Jongkees, H. Yoo, S. G. Withers, ChemBioChem 2014, 15,
124–134.
S. A. K. Jongkees, H. Yoo, S. G. Withers, J. Biol. Chem. 2014, 289,
11385–11395.
K. J. Wilson, S. G. Hughes, R. A. Jefferson, in GUS Protoc. Using
GUS Gene as Report. Gene Expr., Academic Press Inc., 1992, pp.
7–22.
A. G. Day, Purif. Prelim. characterisation β-glucosidase from
Alcaligenes faecalis (ATCC 21400) 1985, MSc Thesis.
M. S. Macauley, K. A. Stubbs, D. J. Vocadlo, J. Am. Chem. Soc.
2005, 127, 17202–17203.
A. A. Narine, J. N. Watson, A. J. Bennet, Biochemistry 2006, 45,
9319–9326.
H. Shen, L. D. Byers, Biochem. Biophys. Res. Commun. 2007, 362,
717–720.
C. A. De Leon, P. M. Levine, T. W. Craven, M. R. Pratt,
Biochemistry 2017, 56, 3507–3517.
V. L. Y. Yip, S. G. Withers, Angew. Chemie - Int. Ed. 2006, 45,
6179–6182.
M. Himeno, Y. Hashiguchi, K. Kato, J. Biochem. 1974, 76, 1243–
1252.
M. Azoulay, N. Ivastchenko, J.-C. Florent, Claude Monneret,
Bioorganic Med. Chem. Lett. 1997, 7, 1307–1310.
Z. Armstrong, P. Rahfeld, S. G. Withers, Discovery of New
Glycosidases From Metagenomic Libraries, Elsevier Inc., 2017.
A. Choi, S. C. Miller, Org. Biomol. Chem. 2017, 15, 1346–1349.
S. Rokita, in Quinone Methides, John Wiley & Sons, Inc., Hoboken,
NJ, USA, 2009, pp. i–xviii.
[18]
[19]
[20]
[21]
[22]
[23]
[24]
[25]
[26]
Acknowledgements
We would like to thank Dr. Zach Armstrong, Dr. Hongming Chen,
Emily Kwan, Julia Dallmann, Phillip Danby and Kyle Robinson for
discussions and assistance. This work was supported by the
Canadian Institutes for Health Research (CIHR), the Swiss
[27]
[28]
[29]
[30]
P. D. Senter, W. E. Pearce, R. S. Greenfield, J. Org. Chem. 1990,
55, 2975–2978.
National
Science
Foundation
(Early
Postdoc.Mobility
D. J. Clouthier, D. A. Ramsay, Annu. Rev. Phys. Chem. 1983, 34,
31–58.
M. R. DuBois, J. Am. Chem. Soc. 1983, 105, 3710–3711.
B. Liu, S. Thayumanavan, J. Am. Chem. Soc. 2017, 139, 2306–
2317.
E. R. Gillies, A. P. Goodwin, J. M. J. Fréchet, Bioconjug. Chem.
2004, 15, 1254–1263.
L. F. Tietze, M. Neumann, T. Möllers, R. Fischer, K. H.
Glüsenkamp, M. F. Rajewsk, E. Jähde, Cancer Res. 1989, 49,
4179–4184.
K. Srinivasachar, D. M. Neville, Biochemistry 1989, 28, 2501–2509.
D. M. Neville, K. Srinivasachar, R. Stone, J. Scharff, J. Biol. Chem.
1989, 264, 14653–14661.
X. Zhu, K. Pachamuthu, R. R. Schmidt, Org. Lett. 2004, 6, 1083–
1085.
A. N. Ngane, M. Lavault, D. Séraphin, A. Landreau, P. Richomme,
Carbohydr. Res. 2006, 341, 2799–2802.
D. Crich, F. Yang, Angew. Chemie Int. Ed. 2009, 48, 8896–8899.
A. Chibba, S. Dasgupta, N. Yakandawala, S. Madhyastha, M. Nitz,
J. Carbohydr. Chem. 2011, 30, 549–558.
P2EZP2_168757 to L.B.) and the Canada Council Killam
Foundation.
[31]
[32]
Keywords: enzyme discovery • functional metagenomics •
high-throughput screening • hydrolases • self-immolative linker
[33]
[34]
[1]
[2]
M. Y. Galperin, E. V. Koonin, Trends Biotechnol. 2010, 28, 398–
406.
P.-Y. Colin, B. Kintses, F. Gielen, C. M. Miton, G. Fischer, M. F.
Mohamed, M. Hyvönen, D. P. Morgavi, D. B. Janssen, F. Hollfelder,
Nat. Commun. 2015, 6, 10008.
[35]
[36]
[37]
[38]
[3]
[4]
P. Lorenz, J. Eck, Nat. Rev. Microbiol. 2005, 3, 510–516.
Y. Bashir, S. Pradeep Singh, B. Kumar Konwar, Chinese J. Biol.
2014, 2014, 1–7.
[5]
[6]
H.-M. Chen, Z. Armstrong, S. J. Hallam, S. G. Withers, Carbohydr.
Res. 2016, 421, 33–39.
V. L. Y. Yip, A. Varrot, G. J. Davies, S. S. Rajan, X. Yang, J.
Thompson, W. F. Anderson, S. G. Withers, J. Am. Chem. Soc.
2004, 126, 8354–8355.
[39]
[40]
[41]
[42]
S. Cecioni, D. J. Vocadlo, J. Am. Chem. Soc. 2017, 139, 8392–
8395.
Z. Armstrong, K. Mewis, F. Liu, M. Scofield, W. Durno, H. Chen, K.
Mehr, N. Hanson, K. Konwar, S. G. Withers, et al., ISME J. 2018, in
Press, DOI 10.1038/s41396-018-0215-9.
[7]
[8]
S. A. K. Jongkees, S. G. Withers, J. Am. Chem. Soc. 2011, 133,
19334–19337.
V. Lombard, H. Golaconda Ramulu, E. Drula, P. M. Coutinho, B.
This article is protected by copyright. All rights reserved.

10.1002/anie.201806792
Angewandte Chemie International Edition
COMMUNICATION
Entry for the Table of Contents
Layout 2:
COMMUNICATION
Seyed Amirhossein Nasseri,
Leo Betschart, Daria
Opaleva, Peter Rahfeld and
Stephen G. Withers*
Page No. – Page No.
Title
Design of novel selective substrates for high-throuput screening makes it possible to identify rare, unconventional glycosidases from
metagenomic DNA amongst a myriad of other similar enzymes. Using traditional susbtrates faces a high back ground activity from
these similar enzymes that can mask the desired hits and/or give rise to false positive hits.
This article is protected by copyright. All rights reserved.