Glycosyl Cations versus Allylic Cations in Spontaneous and Enzymatic Hydrolysis

Article abstract of DOI:10.1021/jacs.7b05628

Enzymatic prenyl and glycosyl transfer are seemingly unrelated reactions that yield molecules and protein modifications with disparate biological functions. However, both reactions employ diphosphate-activated donors and each proceed via cationic species: allylic cations and oxocarbenium ions, respectively. In this study, we explore the relationship between these processes by preparing valienyl ethers to serve as glycoside mimics that are capable of allylic rather than oxocarbenium cation stabilization. Rate constants for spontaneous hydrolysis of aryl glycosides and their analogous valienyl ethers were found to be almost identical, as were the corresponding activation enthalpies and entropies. This close similarity extended to the associated secondary kinetic isotope effects (KIEs), indicating very similar transition state stabilities and structures. Screening a library of over 100 β-glucosidases identified a number of enzymes that catalyze hydrolysis of these valienyl ethers with k_cat values up to 20 s^-1. Detailed analysis of one such enzyme showed that ether hydrolysis occurs via the analogous mechanisms found for glycosides, and through a very similar transition state. This suggests that the generally lower rates of enzymatic cleavage of the cyclitol ethers reflects evolutionary specialization of these enzymes toward glycosides rather than inherent reactivity differences.

Full text of DOI:10.1021/jacs.7b05628

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Communication
Glycosyl Cations versus Allylic Cations in
Spontaneous and Enzymatic Hydrolysis
Phillip M. Danby, and Stephen G. Withers
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.7b05628 • Publication Date (Web): 24 Jul 2017
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Glycosyl Cations versus Allylic Cations in Spontaneous and
Enzymatic Hydrolysis
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Phillip M. Danby and Stephen G. Withers*
Department of Chemistry, University of British Columbia, Vancouver, British Columbia 2036 Main Mall, Canada
Supporting Information Placeholder
phosphate synthase (OtsA), which is the enzyme responsible for
the assembly of trehalose from UDPꢀglucose and glucoseꢀ6ꢀ
phosphate. The mechanistic analogy between these two enzymes
is clear and it is likely that VldE employs an allylic cationꢀlike
transition state in place of the oxocarbenium ionꢀlike transition
state found in traditional GT mechanisms (Figure 1a and c).6 In a
broader sense we saw valienolꢀbased substrates as an opportunity
to directly compare the stabilities of allylic and oxocarbenium
ionꢀlike transition states within an otherwise identical molecular
construct.
ABSTRACT: Enzymatic prenyl and glycosyl transfer are seemꢀ
ingly unrelated reactions that yield molecules and protein modifiꢀ
cations with disparate biological functions. However, both reacꢀ
tions employ diphosphateꢀactivated donors and each proceed via
cationic species: allylic cations and oxocarbenium ions, respecꢀ
tively. In this study, we explore the relationship between these
processes by preparing valienyl ethers to serve as glycoside mimꢀ
ics that are capable of allylic rather than oxocarbenium cation
stabilization. Rate constants for spontaneous hydrolysis of aryl
glycosides and their analogous valienyl ethers were found to be
almost identical, as were the corresponding activation enthalpies
and entropies. This close similarity extended to the associated
secondary kinetic isotope effects (KIEs), indicating very similar
transition state stabilities and structures. Screening a library of
over 100 βꢀglucosidases identified a number of enzymes that cataꢀ
lyze hydrolysis of these valienyl ethers with kcat values up to 20
secꢀ1. Detailed analysis of one such enzyme showed that ether
hydrolysis occurs via the analogous mechanisms found for glycoꢀ
sides, and through a very similar transition state. This suggests
that the generally lower rates of enzymatic cleavage of the cyclitol
ethers reflects evolutionary specialization of these enzymes toꢀ
wards glycosides rather than inherent reactivity differences.
Enzymatic transfer reactions enable organisms to assemble
complex molecules by modular or incremental coupling of key,
activated donor substrates. Two examples are the prenyl transferꢀ
ases1 responsible for transfer of isoprene molecules and the glycoꢀ
syl transferases (GTs) that assemble oligosaccharides and append
glycosyl moieties to proteins.2 While these two enzyme subclasses
act on distinct substrates, they accomplish transfer through analoꢀ
gous mechanisms. Not only do both enzymes employ pyrophosꢀ
phateꢀcontaining donor substrates but both reactions also proceed
through resonanceꢀstabilized cationic species: oxocarbenium ions
during glycosyl transfer (Figure 1a) or allylic cations during isoꢀ
Figure 1. a. Trehaloseꢀ6ꢀphosphate synthase (OtsA) transfers a
glucosyl moiety from UDPꢀGlc to glucoseꢀ6ꢀphosphate (HOR1)
via oxocarbenium ionꢀlike transition states. b. Geranyl pyrophosꢀ
phate synthase (GPPS) transfers a prenyl moiety from dimethylalꢀ
lyl pyrophosphate to isoprenyl pyrophosphate (IPP) via an allylic
cation intermediate. c. VldE from Streptomyces hygroscopicus sp.
catalyzes transfer of valienol from a GDP (guanosine diphosꢀ
phate) valienol to validamineꢀ7ꢀphosphate (R3) via a proposed
allylic cationꢀlike transition state.
4
prene transfer (Figure 1b).3, Despite the clear similarities beꢀ
tween these systems, direct comparison of reactivities and ion
stabilities has been complicated by the core structural differences
between glycosides and isoprenoids, such as cyclic vs acyclic and
substituent effects.
Given these analogies, and since glycoside hydrolases (GHs)
likewise effect hydrolysis via oxocarbeniumꢀionꢀlike transition
states, we investigated whether GHs could carry out hydrolysis of
unsaturated cyclitol ethers, and if so, how such reactions compare
with those of glycosides. This report describes both the nonꢀ
enzymatic (spontaneous) solvolysis and enzymatic hydrolysis of
valienyl ether substrates, alongside an analysis of their mechaꢀ
nisms.
The similarity of these mechanisms was recently highlighted by
the discovery that a key step in the biosynthesis of the trehalase
inhibitor validamycin A is carried out by an enzyme (VldE),
which transfers a 5,6ꢀunsaturated Cꢀ7 cyclitol (valienol) moiety
from a GDP (Guanosine DiPhosphate) donor onto the amino
group of validamine phosphate.5 Structural analysis of VldE reꢀ
vealed a close similarity to the glycosyltransferase trehaloseꢀ6ꢀ
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Figure 2: a. Product stereochemistry determined by HꢀNMR, following reaction of Abg with TFMUꢀValienol. Green trace is the
αꢀ
anomer, red is the βꢀanomer and in black the reaction mixture. The region shown corresponds to the chemical shift of the alkenyl proton.
Also present is a peak corresponding to unreacted TFMUꢀVal. b. Intact mass spectrometry of wild type Abg (left) and Abg incubated with
DNPꢀValienol for 20 min (centre) or 14 h (right). Observed MW increase of 158 corresponds to covalent ValienylꢀEnzyme intermediate.
Synthesis of valienol from δꢀDꢀgluconolactone was achieved
following published protocols.7 Installation of chromogenic leavꢀ
ing groups at the Cꢀ1 position (see Figure 1c for numbering conꢀ
vention) was completed using Mitsunobu chemistry8 to couple
trifluoromethylumbelliferyl alcohol (to give TFMUꢀValienol,
TFMUꢀVal) or by nucleophilic aromatic substitution with fluoroꢀ
2,4ꢀdinitrobenzene to produce the dinitrophenyl ether (DNPꢀ
Table 1. Rate Constants and Activation Parameters for Het-
erolysis of DNP Valienol and DNP Glucoside
Substrate:
DNPꢀVal
ꢀ
4.91x10ꢀ6
108 (1.5)
112 (1.5)
13.1 (0.3)
DNPꢀGlc
5.58 x 10ꢀ6
7.41x10ꢀ6
106 (1.3)
111 (1.3)
14.4 (0.3)
*Lit. k (37oC) /sꢀ1
Exp. k (37oC) /sꢀ1
ꢁGǂ /kJ·molꢀ1
ꢁH(37oC)/kJ·molꢀ1
ꢁS /J·molꢀ1·Kꢀ1
Valienol, DNPꢀVal). We focused on substrates that mimic
glycosides as a large body of reliable data is available on both
βꢀ
their nonꢀenzymatic and enzymatic hydrolysis.9, 10
We first established the rate constants for nonꢀenzymatic hyꢀ
drolysis to determine relative intrinsic reactivities by comparison
with data on the spontaneous hydrolysis of 2,4ꢀdinitrophenyl
*Literature values from Namchuk and Withers.9 Error in parentheꢀ
ses propagated from linear regression of Eyring plots (Figure S4).
11ꢀ13
(DNP)
β
ꢀglucosides.9,
Use of a 2,4ꢀdinitrophenyl leaving
group simplified interpretation of kinetic data on glucoside hyꢀ
drolysis, for which rates were shown to be pHꢀindependent beꢀ
tween 2 and 8.5.13 Thus, within this range, rate constants reflect
the simple heterolysis of the CꢀO bond, without any involvement
of acidꢀ/baseꢀcatalyzed processes. We measured rate constants for
The nonꢀenzymatic solvolysis of aryl glycosides is known to
proceed via an oxocarbenium ionꢀlike transition state, as supportꢀ
ed by the alphaꢀdeuterium KIE of 1.09 ± .02 on spontaneous hyꢀ
20
drolysis of DNPꢀGlc.11 Likewise, Bennet and Sinnott reported
°
nonꢀenzymatic hydrolysis of DNPꢀVal at 70 C at a series of pH
the same value (kH/kD = 1.089 ± 0.006) for acidꢀcatalyzed cleavꢀ
age of methyl βꢀDꢀglucopyranoside concluding that the reaction
values and showed them to be independent of pH between pH 4
and 7.5 (Fig S2). All further studies of uncatalyzed hydrolysis
were performed at pH 6.5 under the same conditions used for
DNPꢀGlc hydrolysis.13
proceeds via a SN1ꢀlike (DN*AN) process with a shortꢀlived oxꢀ
carbenium ion intermediate. To enable a direct comparison of
transition state structures we synthesized the analogous 1ꢀ2Hꢀ
DNPꢀVal by stereospecific reduction of the protected valienone
using sodium borodeuteride under Luche reduction conditions,
followed by coupling with fluorodinitrobenzene and deprotection
as described above. Integration of the corresponding 1HꢀNMR
confirmed >96 % deuteration. The KIE measured in this case was
(kH/kD = 1.11 ± 0.01), fully consistent with the anticipated allylic
cationꢀlike transition state with sp2ꢀhybridization at C1.
Heterolysis of the DNPꢀValienol ether bond was followed by
monitoring dinitrophenol release at a range of temperatures, over
at least 3 halfꢀlives. Rate constants were determined by fitting
these progress curves to a first order equation. The resultant
Eyring plots (Figure S4) were of good quality (r2 = 0.9995) and
yielded enthalpies and entropies of activation as well as allowing
interpolation of rate constants at any temperature (Table 1).9, 14
Rate constants for solvolysis of the analogous DNPꢀglucoside
were also reꢀdetermined, under the same conditions, and are in
agreement with previously published data (Table 1).9, 11, 15, 16
The stereochemical outcome of the solvolysis of DNPꢀVal was
evaluated by 1HꢀNMR analysis of the product mixture, revealing a
3:1 ratio of the αꢀ and βꢀ ‘anomers’ (using carbohydrate terminolꢀ
Remarkably, the rate constants for solvolysis of the two comꢀ
pounds are extremely similar, as are the activation enthalpies and
entropies. In previous work Cocker and Sinnott showed that terꢀ
tiary carbocations and oxocarbenium ions had broadly similar
stabilities.12 This result was reinforced by nearly identical lifeꢀ
times observed for glycosyl oxocarbenium cations17, 18 and tꢀbutyl
cations.19 To our knowledge allyl cations or hydroxylꢀsubstituted
systems have not been explored.
ogy). Also present, as approximately 50% of the total, were the
products of water attack at the tertiary C5 position of the allylic
cation (Figure S5). Such an outcome is not, of course, possible for
pyranosyl systems, but should not confound the kinetic analysis
since the rateꢀlimiting step, heterolysis, precedes the productꢀ
determining step, as was confirmed by the KIE. These are indeed
kinetically controlled products since incubation of pure βꢀvalienol
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product at 70 °C for 3 hours under the same conditions revealed no
mutant missing the catalytic nucleophile (mutation: E358G) had
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interconversion to products of different stereochemistry at C1 nor
positional isomerization (Figure S6). Alongside the KIE measꢀ
urements these results are consistent with a reaction mechanism
that is largely dissociative (SN1ꢀlike). Assuming similar ground
state energies these conclusions suggest that the thermodynamic
stabilities of the transition states preceding the allylic and oxoꢀ
carbenium ions are also nearly identical.
no valienyl ether cleavage activity, but that cleavage could be
rescued by addition of an exogenous nucleophile (1 M azide),
(Figure S11, Table S4), exactly as had been shown previously for
glucoside cleavage.25, 26 Direct detection of the intermediate and
its turnover were achieved by mass spectrometry (Figure 2b)
showing both free Abg and its covalent valienyl–enzyme intermeꢀ
diate. Further incubation for 14 hours resulted in complete turnoꢀ
ver as seen by the return of free enzyme.
Having explored the nonꢀenzymatic hydrolysis, we turned our
attention towards enzymeꢀcatalyzed hydrolysis of these cyclitol
The successful observation of the valienylꢀenzyme intermediate
strongly suggests that the subsequent ‘deglycosylation’ step of
this process, which releases valienol, is rate limiting, as also do
the comparably low Km values for the two substrates, likely due to
accumulation of the covalent intermediate. Confirmation of this
was sought through nucleophilic competition experiments parallel
to those performed on DNPꢀGlc hydrolysis in which addition of
DTT increased the steady state rate.15 Accordingly, addition of
DTT also increased the turnover of TFMUꢀVal (Fig. S13): control
experiments confirmed that this does not arise from background
solvolysis. The reagents therefore function as transient inactivaꢀ
tors. Finally, we confirmed that the enzymatic transition state also
has substantial allylic cation character by measuring a secondary
KIE, under Vmax conditions, for 1ꢀ2HꢀDNPꢀValienol hydrolysis
of kH/kD = 1.15 ± 0.03. This is again very similar to that of kH/kD
= 1.10 ± 0.02 measured for Abgꢀcatalyzed DNPꢀGlc hydrolysis.15
Taken together these results suggest the overall reaction is double
displacement with the most rateꢀlimiting transition state having
considerable allylicꢀcationꢀlike character. It must be noted that the
hydrolysis of the enzymeꢀsubstrate complex may be SN2ꢀlike
(ANDN) (with significant dissociative character) or SN1ꢀlike
(DN*AN or DN + AN); both are consistent with the KIEs.
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ethers. Substrates were tested initially with the βꢀglucosidase from
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Agrobacterium sp. (Abg), a very well characterized member of
CAZY family GH1 (http://www.cazy.org/).15, 16, 21, 22
Kinetic studies with fluorogenic TFMUꢀVal, revealed that Abg
is indeed able to cleave the pseudoꢀglycosidic bond, yielding the
kinetic parameters shown in Table 2 alongside those for the analꢀ
ogous TFMUꢀglucoside substrate. As can be seen, the kcat value is
approximately 103ꢀfold lower than that for the corresponding
glucoside Given their similar Km values this difference extends to
the specificity constants (kcat/Km) as well. Since the inherent reacꢀ
tivities of the two substrates are the same, this substantial variaꢀ
tion presumably reflects differences in recognition of the two
substrates and their transition states as a consequence of the evoꢀ
lutionary optimization of Abg towards glucoside hydrolysis.
Table 2. Kinetic Parameters for Hydrolysis of TFMU-Valienol
by Abg at 25oC
kcat/Km
Substrate:
kcat /minꢀ1
Km /ꢂM
/minꢀ1·ꢂMꢀ1
0.004 ± 0.001
13.1 ± 2.1
In order to assess whether these enzymatic studies were generꢀ
ally representative we screened a library of over 100 expressed
GH1 glycosidases, made available from the Joint Genome Instiꢀ
tute, for their ability to hydrolyze TFMUꢀVal.27 Seven of these
enzymes exhibited activities that are >2 standard deviations above
the average activity across the library. Since at least 23 members
of the library cleave only 6ꢀphosphoꢀsugars and are thus unlikely
to work in our assay this represents an initial hit rate of close to
5%. These hits were purified, concentrations determined by active
site titration, and kinetic parameters determined (Table 3).
TFMUꢀVal
TFMUꢀGlc
1.44 ± 0.13
2682 ± 170
356 ± 80
204 ± 30
Given this considerable difference in rates it was necessary to
establish whether the enzyme employs the same mechanisms for
the two substrates. We thus first established that catalysis was, in
fact, occurring at the normal active site by showing that preꢀ
incubation of Abg with the active siteꢀdirected mechanismꢀbased
inactivator cyclophellitol abolishes hydrolase activity (Figure
S9).23, 24 Secondly, we confirmed the ‘retaining’ stereochemical
outcome of Abgꢀcatalyzed valienyl ether cleavage by NMR analꢀ
ysis against prepared standards (Figure 2a). Only the equatorial β
product is observed, confirming that the enzyme completes vaꢀ
lienyl ether cleavage by a retaining mechanism (Figure 3), in conꢀ
trast to the product distribution observed for heterolysis.
Table 3. Kinetic Parameters for Cleavage of TFMU-Val by
GH1 enzymes.
kcat (Glc)/
kcat(Val)
ID*
kcat /minꢀ1
Km /µM
A.flav
Bac.
1.81
0.26
0.95
0.63
0.58
2.67
0.89
1.44
112
27
481
6007
141
C.sacc
Exi.
82
271
41
1810
1004
24
P.chrys
Ssβ
117
710
355
Figure 3. Proposed general mechanism of enzymeꢀcatalyzed hyꢀ
V.vul
Abg
1316
1863
drolysis of
βꢀvalienyl ethers. Substituent hydroxyls as well as
transition states or intermediates are omitted for clarity.
* See Table S8 for description of species, parameters and errors.
We next confirmed that the same active site residues were inꢀ
volved in hydrolysis of valienol substrates by demonstrating that a
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Turnover numbers (Table 3) fall roughly within one order of
ACKNOWLEDGMENT
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magnitude but more interesting are the relative rates between the
two substrates. Unsurprisingly all enzymes were better able to
process the TFMUꢀglycoside substrate over the valienol derivaꢀ
tive. Nevertheless, several enzymes appear to be less highly speꢀ
cific for the glucoside, exhibiting a greater tolerance for valienyl
Financial support by NSERC, the Natural Sciences and Engineerꢀ
ing Research Council of Canada is acknowledged. Dr. Feng Liu’s
assistance is gratefully appreciated. We thank Jason Rogalski for
intact protein MS analyses, Dr. Miriam Kӧtzler and Emily Kwan
for expression/purification of Abg and Abg2F6 and Dr. Hongꢀ
Ming Chen for synthesis of DNPꢀGlc and Cyclophellitol as well
as the Joint Genome Institute for the GH1 library.
ether cleavage. We selected the thermostable βꢀglucosidase from
Sulfolobus solfataricus (SsβꢀGlc) for further study since it has
undergone substantial characterization.28,29 We were interested in
seeing what turnover numbers could be achieved at higher temꢀ
peratures. The kinetic parameters we determined are found in
Table 4. As anticipated the kcat value increases 20ꢀfold from 25 oC
to 70 oC, arriving at a very respectable value of almost 20 secꢀ1.
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Table 4. Kinetic Parameters for Hydrolysis of DNP-Val and
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kcat /sꢀ1
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/sꢀ1·ꢂMꢀ1
0.001
DNPꢀVal (25o)
DNPꢀVal (70oC)
0.92 ± 0.04
18.9 ± 1.2
938 ±95
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44.7 ± 0.4
645 ± 36
373 ±8
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Namchuk, M. N. Substituted aryl glycosides as probes of the
In conclusion, relative to ground states, the transition states
leading to the allylic and oxocarbenium ions are nearly identical,
when isolated within similar molecules. Allylic isoprenyl cations
are invoked as formal intermediates for prenyl transfer whereas
oxocarbenium ions feature as transition states in glycosyl transfer.
This difference is not due to allylic cations being inherently more
stable than their oxocarbenium cousins. Rather, we suggest the
differences lie in the electronegative hydroxyl substituents on
glycosides, which inductively destabilize the positive charge, in
conjunction with constraints on planar geometry imposed by the
ring. We have also shown the first examples, to our knowledge, of
valienyl ether hydrolysis by glycoside hydrolases. Indeed, several
such enzymes were identified from screening efforts, demonstratꢀ
ing that this capability is not limited to a single enzyme. Further,
enzymes with sufficient activity to be of practical utility in effectꢀ
ing valienol transfer were identified. Detailed characterization of
Cocker, D.; Sinnott, M. L., J. Chem. Soc., Chem. Commun.
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Huang, X. C.; Surry, C.; Hiebert, T.; Bennet, A. J J. Am. Chem.
Toteva, M. M.; Richard, J. P., J. Am. Chem. Soc. 1996, 118,
Bennet, A. J.; Sinnott, M. L., J. Am. Chem. Soc. 1986, 108,
one model enzyme, the
βꢀglucosidase from Agrobacterium sp.
implies that the mechanism of hydrolysis is the same as that
shown for glycoside cleavage, cementing notions of the mechanisꢀ
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AUTHOR INFORMATION
Corresponding Author
*withers@chem.ubc.ca
Notes
The authors declare no competing financial interests.
Pisani, F. M.; Rella, R.; Raia, C. A.; Rozzo, C.; Nucci, R.;
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