Creation of an α-mannosynthase from a broad glycosidase scaffold

Article abstract of DOI:10.1002/anie.201201081

α-Mannosides made easy: Mutation of a family-GH31 α-glucosidase that displays plasticity to alterations at the 2-OH position of donor substrates created an efficient α-mannoside-synthesizing biocatalyst. A simple fluoride donor reagent was used for the synthesis of a range of mono-α-mannosylated conjugates using the α-mannosynthase displaying low (unwanted) oligomerization activity. Copyright

Full text of DOI:10.1002/anie.201201081

Angewandte
Chemie
DOI: 10.1002/anie.201201081
Biocatalysts
Creation of an a-Mannosynthase from a Broad Glycosidase Scaffold**
Keisuke Yamamoto and Benjamin G. Davis*
Dedicated to Professor Kunio Ogasawara
Glycosynthases provide a rare and practical source of
catalytic synthetic utility for oligosaccharide[1–3] and other
glycoside[4–6] synthesis. These biocatalysts, nucleophile-less
mutants of retaining glycosidases,[2,7] are typically exquisitely
stereoselective (inverting) enzymes and some can also attach
monosaccharides with good regioselectivity.[7–10] Many exam-
ples now exist,[2] but in most cases b-glycosides are synthe-
sized; a-glycosynthases are rare[11–14] and have not been
applied widely to practical synthetic use, and some important
linkages in biology, such as a-mannosides, have been inacces-
sible.[2]
In synthesis glycosynthases advantageously obviate some
of[15] the typical need in oligosaccharide chemistry for
hydroxy protection.[16] However, when glycosynthases are
derived from exo-glycosidases (Figure 1a) their mode of
action often leads to repetitive condensation of these
unprotected substrates, sometimes resulting uncontrollably
in the creation of a mixture of varying oligomers as
products[7,8,17–20] (Figure 1b). This limitation may, at first
sight, seem an inherent consequence of ꢀreversingꢁ the natural
Figure 1. exo-Glycosidase trims oligosaccharides sequentially from
exo-glycosidase mechanism (Figure 1a), since an enzyme that
evolved to bind the repetitive sugar structure may then
readily accommodate product containing this sugar type at its
nonreducing terminus. However, we describe herein a strategy
for discrete, controlled glycoside synthesis in which a glyco-
synthase is created that can bind one sugar type as a donor
substrate in the À1 (or D) subsite but then bind it less
favorably as an acceptor when displayed in any ensuing
product (Figure 1d). This strategy has allowed the discovery
of a novel a-mannosynthase that does not uncontrollably
catalyze oligomerization.
nonreducing termini (a), and its glycosynthase can catalyze the reverse
reaction, which may result in the formation of unwanted oligomers
(b). When the enzyme has plasticity at its D site (c,d), the selection of
an appropriate donor could, in principle, prevent oligomerization (d).
A site and D site denote acceptor binding site(s) (+1 (,2,3…)) and
donor binding site (À1), respectively. Green circle=preferred glycan
substrate residues; yellow motif=donor substrate that binds to the
donor site but less favorably to the acceptor site; red triangle indicates
the site of hydolysis between the sites À1 and +1.
identifying a nonpreferred substrate for a glycosidase with
plasticity in its D site (Figure 1c,d). Validation for this
approach came from the contrasting behavior of the Agro-
bacterium sp. b synthase[7] towards Gal and Glc substrates.
Although a priori prediction of enzyme sugar specificity and
plasticity is difficult, Nishio et al. have reported an interesting
feature of the glycoside hydrolase family 31 (GH31) in the
carbohydrate-active enzyme (CAZy)[21] classification: these
hydrolases can display their activity toward both a-glucosides
and 2-deoxy derivatives.[22] This activity suggested that the 2-
OH group of a-d-glucose might be less important for
substrate recognition and implied that the epimer of a-d-
glucose at the 2-OH position, a-d-mannose, might also be
accommodated at the D site of these enzymes. If this were
true, and d-mannose is also not favorable as an acceptor, our
design concept (Figure 1d) might be realized.
Our design necessitated the combination of a glycosidase
and a monosaccharide with good mutual affinity at the donor
binding site (D site) but poor affinity at the acceptor-binding
site (A site). This goal could, in principle, be achieved by
[*] K. Yamamoto, Prof. B. G. Davis
Department of Chemistry
University of Oxford
Chemistry Research Laboratory
Mansfield Road, Oxford OX1 3TA (UK)
E-mail: ben.davis@chem.ox.ac.uk
[**] We thank our sources of financial support: VTU (http://www.vtu.-
com/) and B.G.D. is also a recipient of a Royal Society Wolfson
Research Merit Award. We thank Mitul Patel for preparation of
2,3,4,6-tetra-O-acetyl b-mannosyl fluoride, Dr. Tim Claridge for
assistance with NMR spectroscopy analysis, and Dr. Thomas
Purkarthofer, Dr. Roland Weis, and Dr. Iskandar Dib at VTU for
useful discussions.
To test this hypothesis, we selected and expressed GH31
open reading frames (orfs) and discovered previously
unknown[23] a-mannosidase activity for the product of GH31
gene malA[24,25] from Sulfolobus solfataricus. The enzymeꢁs
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201201081.
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preferred substrate was glucose (para-nitrophenyl a-d-gluco-
pyranoside (pNP-a-d-Glc); KM = (1.7 Æ 0.2) mm, Table S1 in
the Supporting Information) yet its affinity for pNP-a-d-Man
was comparable (KM = (3.8 Æ 0.3) mm, Table S1 in the Sup-
porting Information) as we desired, albeit with lower
turnover.[26–29] After this identification of a promising candi-
date glycosidase scaffold with breadth, a nucleophile-less
mutant D320G was prepared (using site-directed mutagene-
sis, see the Supporting Information), which expressed well
(10–14 mgLÀ1 from E. coli). Putative a-glucosynthase activity
and a-mannosynthase activity were tested using b-d-glucosyl
fluoride (1) and b-d-mannosyl fluoride (2) as donors,
respectively. b-Fluorides were chosen since, as for all syn-
thases derived from retaining (double inversion step) GHs,
inverting (single step) synthase activity would be logically
anticipated[2,7] from removal of the nucleophile D320. Both
donors 1 and 2 were readily prepared (see the Supporting
Information), are stable as solids at 48 C, and even in aqueous
solution have half lives longer than 11 h;[30] their sufficient
stability has been previously noted.[31]
para-Nitrophenyl b-d-glucoside (3a) was used as an
acceptor to allow ready reaction monitoring by UV spectros-
copy at 300 nm (alternative methods based on quantitative
MS, high-performance anion exchange chromatography with
pulsed amperometric detection (HPAEC-PAD), evaporation
light-scattering detection (ELSD), and NMR spectroscopy
were less precise), and we were delighted to see effective
glycosylation of this acceptor. Under near-identical condi-
tions with stoichiometric amounts of donor and acceptor
(20 mm each with 0.5 mgmLÀ1 enzyme in 100 mm sodium
phosphate buffer, pH 6.0), similar conversion levels were
observed for a-gluco- and a-manno-synthase activities
(Figure 2). However, oligomerization was observed in the
glucosynthase reaction using donor 1. In striking contrast, no
oligomerized product was observed in the mannosynthase
reaction using donor 2 (Figure 2) at similar conversion. This
initial result therefore critically demonstrated the validity of
our design to develop an enzyme for discrete glycoside
synthesis (Figure 1d). The kinetics of this mannosylation were
determined (Table S2 in the Supporting Information) and
indicated that this designed, unnatural biocatalytic reaction
displays parameters that are comparable[32] or superior to
several other synthases[5,18,20,33] (kcat(3a) = (5.3 Æ 0.1) minÀ1,
KM(3a) = (9.1 Æ 0.7) mm at [2]0 = 20 mm; kcat(2) = (3.8 Æ
0.1) minÀ1, KM(2) = (2.2 Æ 0.1) mm at [3a]0 = 20 mm). Notably,
those values determined from hydrolytic kinetic analyses (see
above) of the scaffold GH31 were indicative of useful (indeed
superior) synthetic parameters in the resulting synthase
(KM(synthesis) = (2.2 Æ 0.1) mm for donor 2 at [3a]0 = 20 mm
confer KM(hydrolysis) = (3.8 Æ 0.3) mm for pNP-a-d-Man).
The values of kcat = [(3.8–5.3) Æ 0.1] minÀ1 for mannosylation
compare well with those for spontaneous hydrolysis of donor
2 (kapp ca. (0.8–1.1) ꢂ 10À3 minÀ1).[30] Glucosylation kinetics
were also determined (see the Supporting Information); these
showed kinetic profiles (Figure S7 in the Supporting Infor-
mation) that could not be interpreted by discrete reaction. In
contrast, mannosynthase kinetics (Figures S5 and S6 in the
Supporting Information) are consistent with a model (see the
Supporting Information) of minimal oligomerization as
Figure 2. Chromatograms of the gluco- or mannosynthase reactions
(both shown at the same ca. 30% conversion; 20 mm of both donor
and acceptor with 0.5 mgmLÀ1 enzyme MalA D320G in 100 mm
sodium phosphate buffer, pH 6.0). In the glucosynthase reaction using
donor 1 (top), oligomerization was observed (highlighted) even at this
partial level of conversion, while none was observed in the mannosyn-
thase reaction using donor 2 (bottom). The hemispheres in the
structures of the products indicate that mixtures of regioisomers were
formed.
a result of weak affinity of terminal mannosides to the
acceptor site. Similar relative turnovers also showed that
reduced oligomerization for mannosylation was not simply
due to lower activity.
After having established this promising utility, we inves-
tigated next the potential substrate scope of the a-manno-
synthase-catalyzed reaction using 2 as a donor. A represen-
tative range of pNP glycosides was screened as acceptors and
revealed sufficient tolerance within the A site to allow the
processing (a-mannosylation) of a variety of biologically
important sugars (Glc, Man, Xyl, l-Fuc, l-Rha, and glucur-
onic acid (GlcA)) whilst others (Gal, galacturonic acid
(GalA), N-acetyl-glucosamine (GlcNAc)) were not efficient
acceptor substrates (Figure S4 in the Supporting Informa-
tion).
After optimization for synthetic use, the mannosynthase
allowed successful preparation (Scheme 1) of a variety of
discretely a-mannosylated products in overall yields of up to
77% with effectively absolute stereoselectivity at carbon
atom C-1 (de > 98% a). In most cases good regioselectivities
(> 90%) were also observed, typically for a(1,3)-linked
product. Moreover, consistent with the intended design,
typical oligomerization ratios were low (OF < 20% for
several of these products) despite the use of an excess of
donor. Notably the mannosynthase catalyst is highly robust
and could be readily recovered after separation from small
molecules using ultrafiltration and dialysis and was readily
recyclable for other reactions without any significant deteri-
oration in its activity (Figure S10 in the Supporting Informa-
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Scheme 1. a-Mannosynthase-catalyzed reactions (scale: from ca. 2.5 up to ca. 20 mg product). [a] Yields after purification or isolation. These
yields are not based on recovered starting material, and starting material was typically also recovered in the acceptor mass balance.
[b] Determined by HPLC or Man(1-H) 1H NMR integration. [c] Oligomerization factor (OF) defined as% of oligomannosylated products in total
mannosylated products. [d] Isolated as peracetate. Reaction conditions: acceptor (3a–i; 20 mm, 1 equiv), donor (2) 2 equivꢀ3, sodium phosphate
buffer (1 mL, 300 mm, pH 7), room temperature, synthase (2.0 mgmLÀ1); DMF (5–15%, v/v) used as cosolvent as needed to aid solubility.
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tion). Moreover, the catalyst could be used in the presence of
up to 15% DMF (v/v), as an organic cosolvent for starting
materials that were sparingly soluble in water, again with little
effect upon overall efficiency (Figure S11 in the Supporting
Information) over several cycles.
necessitates an average of up to seven additional manipu-
lation steps for each corresponding formation of a glycosidic
bond in total oligosaccharide synthesis;[48] this reduces
average yields by more than 50% even if individual yields
for these steps are more than 90%. Therefore, the use of
methods, such as those explored here, that minimize or avoid
the use of protecting groups are valuable and give rise to
alternative and competitive overall synthetic strategies. For
comparative balance, it should also be noted that, if regiose-
lectivity can be achieved, the a-mannoside linkage is more
readily synthesized than several others using chemical syn-
thesis.
Together these syntheses allowed the ready preparation of
a number of naturally occurring a-mannoside motifs: Man-
a(1,4)-Glc[34] (4a-I), Man-a(1,3)-Glc[35] (4b), Man-a(1,3)-l-
Rha[36] (4e), Man-a(1,3)-l-Fuc[37] (4 f), and Man-a(1,3)-Cel[38]
(4h-II) found in bacterial polysaccharides. These motifs also
included the dimannosides Man-a(1,2)-Man (4c-II, 4i-I) and
Man-a(1,3)-Man (4c-I, 4i-II) found in N-glycans. In this way
a strategy designed to select against uncontrolled oligoman-
noside formation can nonetheless be used for discrete
dimannosides (and even higher) through suitable control of
substrate concentrations. Notably, the preparation of 4c-I also
allowed its use as a starting material in a subsequent discrete
mannosylation reaction, which afforded easy access to the
core trisaccharide (4i-I) of the D1-arm of high-mannose N-
linked protein glycans. Although the yield was lower than for
some of the other mannosynthase-catalyzed reactions, use of
minimally protected building blocks (and avoidance of
associated protection/deprotection steps) makes this a com-
petitive overall route. This product is a key ligand motif in the
binding of neutralizing human antibodies (e.g., 2G12) to HIV-
1[39] and, as such has been used by us[40] and others as
a building block in anti-HIV vaccine design.[41]
Finally, the utility of the a-mannosynthase was tested in
highly crowded substrates: the target synthesis of a-manno-
sylated inositols. Mannosylated myo-inositols are components
of the cell wall glycolipids found in Mycobacterium tuber-
oculosis and, as such, have been the targets of traditional
glycoside syntheses.[42,43] Reaction of more hindered acceptor
myo-inositol (5) was more sluggish than those for some of the
other acceptors. Nonetheless, reaction provided a 41% yield
of mono-a-mannosylated inositols from which Man-a(1,5)-
myo-inositol (6b)[44,45] and, intriguingly, Man-a(1,1)-myo-
inositol (6a)[46] were isolated (Scheme 2) in approximately
3:2 ratio. The latter was formed from a rare example of
simultaneous glycosylation and desymmetrization of such
a complex meso polyol acceptor.[47]
In nature glycosidic bonds are made, without protection,
by specialized glycosyltranferases that are typically selective
for one bond type. That is, they sometimes lack the plasticity/
breadth needed for pragmatic synthetic application. The
specialized glycosyltranferases also usually employ rare and
difficult to access sugar phosphate donors (nucleotides e.g.,
mannose guanosine diphosphate (ManGDP), or lipids) as the
source of glycosylation. This requirement, as well as lack of
enzyme availability (to our knowledge no a-mannosyltrans-
ferases are commercially available), has prevented their
widespread use in synthetic chemistry. One of the most
common biologically relevant glycoside motifs is the a-
mannoside bond. Therefore, it is notable and striking that this
bond is rarely made through biocatalysis[49–53] in current
synthetic routes. Herein we have presented a catalyst that will
now efficiently and selectively form a-mannosides in a broad
manner using a simple mannosyl fluoride reagent. Recently,
other strategies for engineering enzymes (e.g. glycoligases[54]
)
have allowed efficient xylosylation. The logic that we have
shown here, of conversion (through rational engineering) of
a glycosidase that preferentially cleaves a linkage of one type
to a glycosynthase that catalyzes the formation of another,
may be a broadly useful strategy that we are exploring in
other systems.
Experimental Section
In conclusion, we have created a rationally designed a-
mannosynthase using an a-glucosidase as a scaffold for
discrete mono-mannoside synthesis. This is the first example
of a glycosynthase for this biologically important linkage and
a rare example of a glycosynthase that constructs a-anomers.
It has been highlighted that the use of protecting groups
Representative procedure for a-mannosynthase reaction: b-d-Man-F
(2; 200 mL of 200 mm) in sodium phosphate buffer (400 mm, pH 7.0)
and pNP-sugar (3; 500 mL of 40 mm) in sodium phosphate buffer
(400 mm, pH 7.0) were mixed and then synthase solution in sodium
phosphate buffer (100 mm, pH 6.0), sodium phosphate buffer
(400 mm, pH 7.0), and water were added so that the final concen-
trations of the enzyme and the buffer became 2.0 mgmLÀ1 and
300 mm, respectively (the final volume was 1 mL). When the acceptor
substrate was sparingly soluble in the aqueous buffer, it was added as
a solution in DMF, giving the same substrate concentration. The
reaction solution was incubated at room temperature and, if needed,
additional aliquots of 2 were added (200 mL of 200 mm b-d-Man-F (2)
in 300 mm sodium phosphate buffer, pH 7.0). After completion, the
reaction solution was loaded onto an ultrafiltration device (Vivaspin
6, MWCO 10000, Sartorius, pretreated with 0.1% Triton X-100) to
separate the protein fraction and the small-molecule fraction. The
recovered protein fraction was dialyzed against sodium phosphate
buffer (100 mm, pH 6.0) for recycled use. The small-molecule fraction
was purified by HPLC (Synergi 4u Fusion-RP 80A, 100 ꢂ 21.20 mm
(Phenomenex) then Luna 5u NH2, 250 ꢂ 21.20 mm, 100 A (Phenom-
Scheme 2. Synthesis of mannoinositols. Reagents and conditions:
a) a-mannosynthase, sodium phosphate buffer (pH 7, 300 mm), 2;
b) Ac2O, pyridine (1:1 v/v).
4
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enex)) and lyophilized prior to analysis or, when necessary, addition-
ally peracetylated and analyzed as their peracetates.
[26] It should be noted that it is the first/glycosylation step and not
subsequent steps that are suggested to be rate-limiting in GH31
enzymes (see following references [27], [28], and [29]). The KM
hydrolysis values were therefore envisaged to be a good measure
of relative affinities that will be of relevance to subsequent
engineered enzymes.
Received: February 8, 2012
Revised: May 5, 2012
Published online: && &&, &&&&
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Keywords: biocatalysis · glycosylation · glycosynthases ·
mannosylation · synthetic methods
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Angew. Chem. Int. Ed. 2012, 51, 1 – 6
ꢀ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.angewandte.org
5
These are not the final page numbers!

.
Angewandte
Communications
Communications
Biocatalysts
a-Mannosides made easy: Mutation of
a family-GH31 a-glucosidase that dis-
plays plasticity to alterations at the 2-OH
position of donor substrates created an
efficient a-mannoside-synthesizing bio-
catalyst. A simple fluoride donor reagent
was used for the synthesis of a range of
mono-a-mannosylated conjugates using
the a-mannosynthase displaying low
(unwanted) oligomerization activity.
K. Yamamoto,
B. G. Davis*
&&&& — &&&&
Creation of an a-Mannosynthase from
a Broad Glycosidase Scaffold
6
www.angewandte.org
ꢀ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2012, 51, 1 – 6
These are not the final page numbers!