Decoupled Roles for the Atypical, Bifurcated Binding Pocket of the ybfF Hydrolase

Article abstract of DOI:10.1002/cbic.201300085

Serine hydrolases have diverse intracellular substrates, biological functions, and structural plasticity, and are thus important for biocatalyst design. Amongst serine hydrolases, the recently described ybfF enzyme family are promising novel biocatalysts with an unusual bifurcated substrate-binding cleft and the ability to recognize commercially relevant substrates. We characterized in detail the substrate selectivity of a novel ybfF enzyme from Vibrio cholerae (Vc-ybfF) by using a 21-member library of fluorogenic ester substrates. We assigned the roles of the two substrate-binding clefts in controlling the substrate selectivity and folded stability of Vc-ybfF by comprehensive substitution analysis. The overall substrate preference of Vc-ybfF was for short polar chains, but it retained significant activity with a range of cyclic and extended esters. This broad substrate specificity combined with the substitutional analysis demonstrates that the larger binding cleft controls the substrate specificity of Vc-ybfF. Key selectivity residues (Tyr116, Arg120, Tyr209) are also located at the larger binding pocket and control the substrate specificity profile. In the structure of ybfF the narrower binding cleft contains water molecules prepositioned for hydrolysis, but based on substitution this cleft showed only minimal contribution to catalysis. Instead, the residues surrounding the narrow binding cleft and at the entrance to the binding pocket contributed significantly to the folded stability of Vc-ybfF. The relative contributions of each cleft of the binding pocket to the catalytic activity and folded stability of Vc-ybfF provide a valuable map for designing future biocatalysts based on the ybfF scaffold.

Full text of DOI:10.1002/cbic.201300085

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DOI: 10.1002/cbic.201300085
Decoupled Roles for the Atypical, Bifurcated Binding
Pocket of the ybfF Hydrolase
Elizabeth E. Ellis,[a] Chinessa T. Adkins,[b] Natalie M. Galovska,[a] Luke D. Lavis,[b] and
R. Jeremy Johnson*[a]
Serine hydrolases have diverse intracellular substrates, biologi-
cal functions, and structural plasticity, and are thus important
for biocatalyst design. Amongst serine hydrolases, the recently
described ybfF enzyme family are promising novel biocatalysts
with an unusual bifurcated substrate-binding cleft and the abil-
ity to recognize commercially relevant substrates. We charac-
terized in detail the substrate selectivity of a novel ybfF
enzyme from Vibrio cholerae (Vc-ybfF) by using a 21-member li-
brary of fluorogenic ester substrates. We assigned the roles of
the two substrate-binding clefts in controlling the substrate se-
lectivity and folded stability of Vc-ybfF by comprehensive sub-
stitution analysis. The overall substrate preference of Vc-ybfF
was for short polar chains, but it retained significant activity
with a range of cyclic and extended esters. This broad sub-
strate specificity combined with the substitutional analysis
demonstrates that the larger binding cleft controls the sub-
strate specificity of Vc-ybfF. Key selectivity residues (Tyr116,
Arg120, Tyr209) are also located at the larger binding pocket
and control the substrate specificity profile. In the structure of
ybfF the narrower binding cleft contains water molecules pre-
positioned for hydrolysis, but based on substitution this cleft
showed only minimal contribution to catalysis. Instead, the res-
idues surrounding the narrow binding cleft and at the en-
trance to the binding pocket contributed significantly to the
folded stability of Vc-ybfF. The relative contributions of each
cleft of the binding pocket to the catalytic activity and folded
stability of Vc-ybfF provide a valuable map for designing
future biocatalysts based on the ybfF scaffold.
Introduction
Serine hydrolases (EC: 3.1) constitute a large class of ubiqui-
tous and highly conserved cellular enzymes with broad sub-
strate specificity and broad biological function.[1] The diverse
members of the serine hydrolase family are grouped together
based on their conserved protein fold (a/b hydrolase protein
fold) and conserved catalytic machinery (a catalytic triad of
serine, histidine, and an acidic residue).[2] This conserved cata-
lytic triad can, however, catalyze a range of chemical transfor-
mations with a diverse group of biological substrates, depend-
ing on the structural orientation of the active site and binding
pocket.[3] This plasticity in the reactivity of serine hydrolases
has been exploited by protein engineers to design novel and
commercially applicable reactivity on serine hydrolase scaf-
folds.[3a,b,4] Bioengineered serine hydrolases have found appli-
cations in variety of processes, including biofuel production,
pharmaceutical synthesis, and commercial resolution of chiral
esters.[3a,b,5] Expanded understanding of the serine hydrolase
structure and comprehensive databases of engineered hydro-
lases have greatly aided the development of novel serine hy-
drolase biocatalysts.[1b,3c,6] New structural and enzymatic fami-
lies of serine hydrolases, however, continue to be discovered,
thus presenting new scaffolds and reactivities for the contin-
ued development of serine hydrolase biocatalysts.[3c]
Amongst the serine hydrolase family, the serine hydrolase
ybfF from Escherichia coli (Ec-ybfF) was shown to be a promis-
ing target for biocatalyst development, as it catalyzes impor-
tant chemical transformations and contains an unusual bifur-
cated binding pocket that can accommodate a range of sub-
strates.[7] The ybfF protein is also highly conserved across bac-
teria, with homologues in a variety of pathogenic bacteria, in-
cluding Vibrio cholerae, but it has low sequence similarity
(<20%) with other serine hydrolases.[7a] The crystal structure of
Ec-ybfF confirmed that it is a serine hydrolases with the canon-
ical a/b hydrolase fold and a GxSxG catalytic motif, where the
central serine is the catalytic nucleophile required for the hy-
drolysis reaction.[7a]
The structure also revealed an unusual, buried substrate-
binding pocket that bifurcates into two distinct binding clefts
(Figure 1). A charged malonate molecule was bound tightly
into the broader cleft of the substrate-binding pocket, in close
proximity to the catalytic serine, thus leading to the classifica-
tion of ybfF as a malonyl-CoA thioesterase (Figure 1).[7a] The
narrower cleft of the substrate-binding pocket contained three
highly ordered water molecules, but was measured to contain
only space sufficient to accommodate an unbranched four-
atom chain.[7a] Additionally, the structure of Ec-ybfF showed
that the enzyme contains a separate helical cap domain atop
[a] E. E. Ellis, N. M. Galovska, Dr. R. J. Johnson
Department of Chemistry, Butler University
4600 Sunset Ave, Indianapolis, IN 46208-3443 (USA)
E-mail: rjjohns1@butler.edu
[b] Dr. C. T. Adkins, Dr. L. D. Lavis
Janelia Farm Research Campus, The Howard Hughes Medical Institute
19700 Helix Dr, Ashburn, VA 20147-2408 (USA)
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cbic.201300085.
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and stability of ybfF, thus suggesting the ability to decouple
the thermal stability and substrate selectivity of ybfF in future
biocatalyst designs.
Results
Serine hydrolase substrate library
The binding pocket and active site of ybfF is located at the in-
terface between the a/b hydrolase domain and the cap
domain (Figure 1). The entrance to the bifurcated binding
pocket contains the nucleophilic serine (Ser86), which is posi-
tioned to initiate the classic catalytic mechanism of serine hy-
drolases (Figure 1B and C). The larger binding cleft (Figure 1B
and C, left) contains a well-positioned malonate in the Ec-ybfF
structure, which is held in position by hydrogen bonds to
Arg120 and Tyr209.[7a] The larger cleft was proposed to accom-
modate the incoming carboxylic acid portion of the ester sub-
strate, with space to accommodate substrates of up to six car-
bons.[7a] The larger cleft was also modeled to fit IPG esters of
four and eight carbons with stereoselectivity for the R iso-
mer.[7b] In comparison, the narrower binding cleft (right side in
Figure 1B and C) has sufficient space to accommodate only
straight chains of approximately four atoms and contains three
well-positioned water molecules in the Ec-ybfF structure.[7a]
Along the exposed interface between the a/b hydrolase
domain and the cap domain, a hydrophobic cleft is present
that can accommodate longer chains, including palmitoyl-CoA
or the CoA moiety of malonyl-CoA. The distinct binding surfa-
ces and diversity of currently identified substrates for ybfF
complicate the assignment of the function of ybfF and compli-
cate manipulation of the ybfF scaffold in biocatalyst designs.
Given the current list of substrates hydrolyzed by ybfF, we
set out to clarify the substrate specificity of Vc-ybfF by using
a library of diverse fluorogenic esters (Scheme 1). Each of the
fluorogenic ester substrates contains an invariant fluorescein
core with various R-groups attached to the phenolic oxygens
as acyloxymethyl ethers. The acyloxymethyl ether attachment
provides lower background fluorescence and increased sensi-
tivity over traditional esterase substrates, thus permitting
rapid, precise measurement of full enzyme kinetics in micro-
plate format.[10] A library of 21 acyloxymethyl ether substrates
(Scheme 1) was synthesized by a previously published proce-
dure,[10a,b] and correct synthesis was confirmed by NMR and
LC-MS (see the Experimental Section).
Figure 1. Structure of ybfF and its binding pocket. A) General architecture of
E. coli ybfF (PDB ID: 3BF8) showing a/b hydrolase domain (light grey) and
helical cap domain (dark grey). B) Dual-cleft substrate binding pocket. The
exterior surface has been removed to show the nucleophilic serine (Ser86)
and the bifurcated binding pocket of ybfF: broader binding cleft (left) and
narrower binding cleft (right). C) Interior view of the binding pocket with
bound malonate (MAL, ball and stick) and water molecules (spheres). All fig-
ures were made with PyMOL.
the a/b hydrolase domain and likely serves to recognize coen-
zyme A.[7a] A similar structural architecture (hydrolase and cap
domains) was also observed in Rv0045c, a ybfF homologue
from Mycobacterium tuberculosis. In the structure of Rv0045c
(PDB ID: 3P2M) the majority of the cap domain was, however,
disordered, thus making comparisons of the substrate-binding
pocket architecture between the bacterial homologues diffi-
cult.[8] The unique nature of the ybfF binding pocket was also
modeled to facilitate the rapid, stereospecific hydrolysis by
ybfF of 1,2-O-isopropyideneglycerol (IPG) esters, a potential
pharmaceutical starting material in the production of b-block-
ers, prostaglandins, and leukotrienes.[7b] Focused substitutions
around the entrance to the binding cleft endowed Ec-ybfF
with the highest known enantioselectivity for R-IPG butyrate
and caprylate.[9]
Here, we characterized the role of the dual substrate-binding
pocket of a novel ybfF enzyme from V. cholera (Vc-ybfF) to de-
termine the enzymatic activity and substrate specificity of ybfF
proteins. Using a library of 21 fluorogenic ester substrates, we
measured the substrate specificity of Vc-ybfF, by varying chain
length, steric hindrance, polarity, and degree of saturation.
Against the optimal substrates, the dual substrate-binding sur-
face of Vc-ybfF was then mapped by using alanine-scanning
mutagenesis to assess the relative contributions of different
residues to the enzymatic activity and structural stability of
ybfF. Focused substitutions of key residues of the substrate-
binding pocket were then used to identify residues essential to
the catalytic activity of Vc-ybfF. The global picture of the sub-
strate-binding pocket of ybfF shows that residues key to cata-
lytic activity and thermal stability of ybfF are located at the in-
terface between the a/b hydrolase domain and helical cap
domain. Each of the dual lobes of the substrate-binding
pocket provides distinct contributions to the overall activity
The ester functionalities of the fluorogenic substrates were
chosen to mimic distinct biological classes of ester-based sub-
strates, including alkyl esters, polar esters (alkyl ether esters),
unsaturated esters, aryl esters (cycloalkyl esters), tertiary esters,
and fluoroesters (Scheme 1).[1a] The library also focused on
basic building blocks for each of the classes, by favoring
simple ester structures over more complex, branched struc-
tures. Certain classes in the library (alkyl esters and cycloalkyl
esters) were more highly populated than other classes, based
on the relative abundance of similar substrates amongst natu-
ral esters and their potential for clarifying the binding pocket
structure of ybfF. A limitation of the current library is the inabil-
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Scheme 1. Library of fluorogenic esterase substrates. Each substrate is composed of diacyloxymethyl ether fluorescein (center) and R groups. The differing R
groups are organized into classes based on chemical functionality. All substrates were synthesized as described previously[10b,c] (see the Experimental Section).
ity to classify serine hydrolases into the subcategories carboxyl
esterase and lipase, as solubility limitations restricted the maxi-
mum length of the alkyl chain to six carbons, although seven
atoms are possible after addition of ethers within the chain
(substrates 6–8). To confirm the specificity of Vc-ybfF with sub-
strates of longer carbon-chain length, a commercial series of p-
nitrophenyl substrates of varying lengths was also tested for
reactivity. The substrate-specificity profile of Vc-ybfF was then
defined by mapping its precise enzymatic activity toward the
entire 21-member fluorogenic library and the four p-nitrophen-
yl substrates.
active against a variety of ester-based substrates, with a broad
pH tolerance and classic Michaelis–Menten kinetics (Figure S2).
For each of the 21 fluorogenic substrates, the kcat, Km, and kcat/
Km values were determined by microplate analysis of the puri-
fied enzyme (Table S1). Enzymatic efficiency (kcat/Km) was used
for comparisons between the different substrates (Figure 2).
The global landscape of Vc-ybfF substrate specificity showed
high selectivity toward alkyl ether esters (substrates 6–8;
Figure 2). Vc-ybfF cleaved the alkyl ether esters with a catalytic
efficiency more than ten times higher than for the other fluo-
rogenic substrates (Table S1). Within the alkyl ether esters, Vc-
ybfF had the highest activity with 6, which contains four
atoms, consistent with the predicted size selectivity of ybfF
(four atom units; Figure 2C).[7a] The selectivity of Vc-ybfF for
polar esters matched the significant activity of Ec-ybfF toward
IPG esters.[7b]
Substrate specificity of ybfF enzymes
We characterized the activity of a homologue of Ec-ybfF to
expand the limited knowledge about this unusual enzyme
family. We chose to study ybfF from V. cholerae (Vc-ybfF) be-
cause of the global health epidemic of cholera and the se-
quence conservation (45% identity) between Vc-ybfF and Ec-
ybfF. Vc-ybfF was cloned into a bacterial expression vector, ex-
pressed in E. coli, and purified to homogeneity by Ni-metal af-
finity chromatography (Figure S1 in the Supporting Informa-
tion). The wild-type Vc-ybfF enzyme had an unfolding curve
(Tm =488C; Table 1) and size exclusion chromatogram indica-
tive of a well-folded enzyme (Figure S3). Vc-ybfF appeared to
be an extended dimer in solution; this is consistent with the
crystallographic state of Ec-ybfF (Figure S3). Vc-ybfF was highly
Although the activity was significantly lower with the re-
maining library members, distinct patterns of substrate selec-
tivity emerged when comparing the enzymatic efficiency
values within different subgroups (Figure 2). Within alkyl esters
(1–5), Vc-ybfF shows bell-shaped selectivity with maximal activ-
ity against C4 (3; Figure 2B), again confirming the preference
of ybfF for substrates of four atoms.[7a] This level of detail
about substrate selectivity was not available with the p-nitro-
phenyl substrates, but the global picture again confirms the
selectivity of ybfF as highest with four-carbon substrates (p-ni-
trophenyl butyrate; Figure 2E). Similar size preferences were
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showed significantly higher ac-
tivity than substrates with six-
membered rings (Figure 2D).
The selectivity for smaller cyclo-
alkyls was independent of aro-
maticity or flexibility in ring at-
tachment, although adding one
extra carbon between the ester
and phenyl ring (15 versus 16)
did increase the activity over
50-fold (Table S1).
Table 1. Biochemical characterization of Vc-ybfF and variants.
Substrate 3
Substrate 6
kcat/Km [mꢀ1 sꢀ1
ybfF variant
Km [mm][a]
kcat/Km [mꢀ1 sꢀ1
]
Km [mm]
]
Tm [8C][c]
WT
6.6ꢁ0.8
3.3ꢁ0.5
3.6ꢁ0.5
n.d.[b]
210ꢁ40
2.7ꢁ0.4
120ꢁ20
n.d.
25ꢁ2
6500ꢁ500
1500ꢁ200
3300ꢁ900
0.29ꢁ0.13
5200ꢁ1100
23ꢁ2
48ꢁ0
H20A
N28A
S86A
2.2ꢁ0.2
16ꢁ4
42.5ꢁ0.9
46.5ꢁ0
49.5ꢁ0
44ꢁ2
21ꢁ9
M87A
K90A
0.18ꢁ0.08
n.d.
94ꢁ42
1.5ꢁ0.3
1.0ꢁ0.1
2.8ꢁ0.5
4.5ꢁ0.4
23ꢁ3
n.d.
41.5ꢁ0.9
43.5ꢁ0
43.5ꢁ0
45.5ꢁ0.9
42ꢁ0
D110A
Y116A
R120A
H121A
F158A
Y209A
H235A
W236A
R120K
Y209F
5ꢁ1
0.43ꢁ0.10
8.8ꢁ1.9
58ꢁ6
57ꢁ10
5ꢁ1
3300ꢁ300
1600ꢁ200
2200ꢁ600
5300ꢁ300
88ꢁ21
3.2ꢁ0.3
7.3ꢁ0.9
6.2ꢁ0.4
1.9ꢁ0.1
19ꢁ4
For a variety of substrates, in-
cluding a/b unsaturated, terti-
ary, and fluorinated esters, Vc-
ybfF had low but measurable
activity (Figure 2). This low ac-
tivity is common to the majority
of bacterial hydrolases, as only
a small class of enzymes readily
catalyzes the hydrolysis of fluo-
rinated esters,[11] and tertiary
esters are often used as chal-
lenging substrates in directed
evolution studies.[12] Given the
84ꢁ12
16ꢁ4
140ꢁ10
0.50ꢁ0.03
0.074ꢁ0.020
0.085ꢁ0.30
130ꢁ10
170ꢁ20
13ꢁ0.8
3.9ꢁ0.9
0.5ꢁ0.2
5ꢁ1
45ꢁ0
45.5ꢁ0.9
44ꢁ0.9
39ꢁ0
58ꢁ23
27ꢁ77
2.1ꢁ0.1
1.8ꢁ0.2
12ꢁ2
12ꢁ0.7
6.9ꢁ0.8
4500ꢁ300
5000ꢁ600
45ꢁ0
45ꢁ0
[a] Kinetic constants for substrates 3 and 6 were determined by measuring the increase in fluorogenic enzyme
substrate fluorescence over time.[10a] Data were fitted to a standard Michaelis–Menten equation to determine
values for kcat, KM, and kcat/KM. Kinetic measurements for each substrate were repeated three times; values are
given as the meanꢁSEM, [b] n.d.: measurement below the detection limit. [c] Values for Tm were determined
by following the change in SYPRO Orange fluorescence with increasing temperature. Melting curve experi-
ments were repeated three times for each variant, and the Tm values are reported as the meanꢁSEM.
unusual binding pocket structure of ybfF and its known ability
to catalyze commercially relevant ester hydrolysis reactions,[7b]
these low-activity substrates could be used in future experi-
ments to test the adaptability of the ybfF binding pocket to
new substrates and reactions through directed-evolution stud-
ies.
Structural determinants of ybfF enzymatic activity
The global picture of the substrate specificity of ybfF shows
that it prefers short, polar esters. Given the sizes of each cleft
of the ybfF binding pocket, this substrate-specificity profile
does not reveal which cleft of the ybfF binding pocket is inter-
acting with the substrates. To differentiate the roles of each
cleft of the Vc-ybfF binding pocket, comprehensive site-direct-
ed mutagenesis was performed on each of cleft. As an initial
assessment of their contributions to the substrate selectivity,
13 amino acid residues surrounding the binding pocket were
substituted with alanine to scan the binding surface for group-
ings of residues that make significant contributions to the en-
zymatic activity of ybfF (Figure 3A).
These 13 residues were chosen based upon their proximity
to the binding pocket, their potential roles in catalysis, their
distribution across both clefts of the binding pocket, and their
conservation across species (Figure 3A and B). The majority of
the 13 residues are highly conserved across ybfF homologues
from bacteria to fungi to humans, with only highly conserva-
tive substitutions across homologues (Figure 3B). Amongst the
13 residues, the least-conserved binding-pocket residues are
proposed to be involved in substrate recognition (Tyr116,
Arg120, and His121) where diverse residues are present at
these positions in mammalian homologues of ybfF (Figure 3B
and Table S3).
Figure 2. Kinetic characterization of Vc-ybfF with the library of fluorogenic
ester substrates. A) Catalytic specificity (kcat/KM) of Vc-ybfF with each of the
21 substrates (Scheme 1). To illustrate patterns within classes of substrates,
important subclasses are compared: B) alkyl esters, C) alkyl ether ester, D) cy-
cloalkyl ester, and E) p-nitrophenyl esters. Detailed kinetic results for each
substrate are provided in Table S1.
found for the cycloalkyl esters: substrates with rings of four or
five carbons (cyclobutyl (11), cyclopentyl (12), and furanyl (14))
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Figure 3. Substrate-binding pocket residues important to the catalytic activity and thermal stability of Vc-ybfF. A) Residues (ball and stick) were substituted in-
dividually with alanine, and the effects on catalytic activity and thermal stability were determined. B) Conservation of substrate binding residues across bacte-
rial and mammalian homologues of ybfF. Sequences were aligned with ClustalW,[25b] and relative weightings were evaluated with Weblogo.[25a] (Detailed se-
quence analysis in Table S3 and Figure S4.) C) Relative catalytic activity of Vc-ybfF variants with substrates 3 and 6. D) Thermal stability of Vc-ybfF variants, de-
termined by measuring the increase in SYPRO Orange fluorescence in response to increasing temperature. Results are shown with standard error values. (De-
tailed kinetic and thermal stability analysis in Table 1.) E) Relative catalytic activity and F) thermal stability of Y116X variants. Experiments conducted as for (C)
and (D). (Detailed kinetic and thermal stability analysis in Table S2.)
Vc-ybfF variants were then constructed with single alanine
substitutions of each of the 13 amino acids by site-directed
mutagenesis, and purified to homogeneity (Figure S1 and
Table S4). Each of the 13 Vc-ybfF variants was tested for enzy-
matic activity with two of the highly active fluorogenic sub-
strates (3 and 6) from the general screen (Figure 3C and
Table 1). With one important exception (Tyr116), the relative ac-
tivity differences between the two substrates for the 13 Vc-
ybfF variants were minor (within experimental error; Fig-
ure 3C). Given the similarity in activity with the two substrates,
comparisons of relative enzymatic activity between the bind-
ing pocket variants were conducted from the results with 6.
Many of the residues significantly affecting the catalytic ac-
tivity were predicted based on the structure of Ec-ybfF, includ-
ing complete loss of activity upon substitution of the nucleo-
philic serine (Ser86) and the general acid-base (His235; Fig-
ure 3C). The third amino acid in the catalytic triad (Asp110),
which forms hydrogen bonds with His235, had a lesser role in
catalysis. The decreased importance of the acidic amino acid in
the catalytic triad has been observed in other hydrolases, and
was predicted for Ec-ybfF based on the shifted orientation of
Asp110 in relation to His235.[7a,13] Serine hydrolases also utilize
an oxyanion hole (in addition to the catalytic triad) to stabilize
the negative charge on the tetrahedral transition state.[2,13b]
The oxyanion hole in Ec-ybfF was proposed to be formed by
the backbone nitrogens of Leu22 and Met87 (Vc-ybfF number-
ing), and this was supported by modeling of bound IPG sub-
strates.[7a,9] Although Leu22 was not tested in the present
study, substitution of Met87 with alanine did significantly
reduce the overall catalytic efficiency of ybfF, thus confirming
its important role in catalysis.
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In addition to substitution of the expected catalytic triad res-
idues, substitution of four additional residues (His20, Lys90,
Tyr209, and Trp236) in Vc-ybfF led to complete loss of catalytic
activity with both fluorogenic substrates. Two of these residues
(Lys90 and Tyr209) interact directly with the bound malonate
in Ec-ybfF, and this suggests direct roles for Lys90 and Tyr209
in substrate positioning.[7a] In spite of the interaction with mal-
onate in Ec-ybfF, substitutions of Arg120 and His121 with ala-
nine produced nearly identical (three- to fourfold) changes in
catalytic efficiency (Figure 3C). More conservative substitutions
(Arg120 and Tyr209 with lysine and phenylalanine, respective-
ly) resulted in near wild-type activity, thus suggesting that size
and charge are more important than hydrogen bonding for
these amino acids in Vc-ybfF (Figure 3E). Of the other two resi-
dues essential for catalytic activity, Trp236 was predicted to
play an important role in ybfF based on its position at the in-
terface of the helical cap and hydrolase domain; this was con-
firmed by the complete loss of activity in W236A Vc-ybfF.[7a]
Substitution of the analogous tryptophan residue in Ec-ybfF
also led to the greatest increase in enantioselectivity toward R-
IPG esters.[9] The His20 residue, although completely con-
served, was not however previously predicted to play an im-
portant role in catalysis by ybfF.
have differences in the adjacent amino acids (Arg120 and
His121), and concerted substitution of these residues might be
required to fully shift the substrate specificity of the bacterial
and human homologues.
Structural determinants of ybfF stability
Definitive explanation of the role of each binding pocket cleft
of Vc-ybfF requires consideration of its contribution to the cat-
alytic activity and structural stability; large structural changes
upon substitution would shift the catalytic activity of the
enzyme. Thermal stability measurements assess the role of
each binding-pocket residue in maintaining the folded struc-
ture of ybfF. The thermal denaturation of each Vc-ybfF variant
was measured, and Tm values for the mid-point of the two
state transition are shown in Figure 3D. A large shift in Tm be-
tween wild-type and a particular Vc-ybfF variant suggests an
important role for the substituted residue in maintaining the
folded structure of Vc-ybfF.
Significant changes in folded stability (>58C) were observed
for the H20A, K90A, H121A, and W236A variants (Figure 3D).
Apart from H121A, each showed near-zero catalytic activity,
thus confirming the connection between folded stability and
catalytic efficiency (Figure 3C). The change in stability for
W236A (98C) was especially dramatic and indicates an essential
role for Trp236 in maintaining stability (Figure 3D). Based on
the connected nature of the stability and catalytic activity of
this group of substitutions, a “stability threshold” can be iden-
tified: residue changes that result in a Tm less than 428C drasti-
cally affect the stability and hence catalytic activity of ybfF.
Substitution of the catalytic serine (S86A) is the only Vc-ybfF
variant that showed increased protein stability. This is likely
due to the observed distorted geometry adopted by Ser86.[2]
As ybfF is a member of the hydrolase-fold family, it contains
a conserved GxSxG nucleophilic elbow, which adopts a tight
turn and thus strained bond angles.[2] Removal of this strain
upon alanine substitution led to elevated thermal stability. This
phenomena is, however, unique to Ser86, as substitution of
the neighboring amino acid (Met87) resulted in a protein that
was slightly less stable than wild-type Vc-ybfF (Figure 3D).
For Tyr116, thermal stability differences do not explain the
observed shifts in catalytic activity (Figure 3E and F). Alanine
substitution decreased the thermal stability (Tm =43.58C), but
similar changes in thermal stability were seen for substitution
with serine (Tm =438C) and threonine (Tm =448C). The phenyl-
alanine variant exhibited near-wild-type stability (Tm =478C);
none of the Tyr116 variants had stabilities below the 428C
threshold (that defines the instability mark for Vc-ybfF). The
thermal stability curves for the Y116D and Y116V variants also
failed to show a folding transition, thereby confirming improp-
er folding of these Tyr116 variants. With the lack of major ther-
mal stability changes, the shifts in catalytic activity for the
Tyr116 variants can be directly related to differences in sub-
strate interaction. This further supports the argument that
Tyr116 is essential to the substrate selectivity profile of Vc-ybfF.
Amongst the 13 alanine mutants, the Y116A variant showed
the greatest difference in activity between substrates 3 and 6.
With the alkyl substrate (3), catalytic activity was significantly
decreased, whereas with 6, it showed only a twofold decrease
in catalytic activity (Figure 3C). The difference in the activity of
Y116A Vc-ybfF between 6 and 3 was also ten times greater
than the difference measured for wild-type ybfF (Figure 2). In-
triguingly, the residue in eukaryotic organisms homologous to
Tyr116 is either serine or threonine (Figure 3B), thus suggest-
ing that the residue at position 116 might act as a substrate-
selectivity filter for different ybfF homologues.
To confirm this hypothesis, Tyr116 was substituted with vari-
ous residues (phenylalanine, serine, threonine, valine, and as-
partate), and each of the resulting Vc-ybfF variants was charac-
terized for activity with 3 and 6 (Figure 3D). Even with multiple
attempts at growth and purification by a variety of expression
strategies, two of the Tyr116 variants (Y116V and Y116D) pro-
duced only limited soluble, well-folded protein, thus kinetic
characterization was impossible. Phenylalanine substitution,
however, showed the opposite pattern to Y116A: the activity
toward 3 was near wild-type levels, whereas the activity
toward 6 was significantly lower. The enzymatic efficiency
values for the serine and threonine variants were somewhere
in between, with slightly decreased activity toward 3, and
wild-type activity with 6. Thus, single substitutions of Tyr116
with serine or threonine likely provide eukaryotic-type homo-
logues, with greater selectivity toward polar substrates that are
not evident in bacterial homologues. The selectivity in mam-
malian homologues with serine or threonine at position 116 is
not, however, as large as is possible with an alanine substitu-
tion, which interestingly was not observed in any of the homo-
logues (Figure 3B). Full elucidation of the role of Tyr116 in con-
trolling the substrate specificity of ybfF will require more de-
tailed investigation, as eukaryotic homologues of ybfF also
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of evolutionary necessity.[16] To refine the structural factors de-
termining the catalytic activity and substrate specificity of ybfF,
we wanted to assign a role to each cleft of the unusual bifur-
cated binding pocket of ybfF in its activity. The ability of Vc-
ybfF to accommodate chains larger than four atoms and to
catalyze reactions with bulky aromatic groups indicates that
the larger binding cleft is the major substrate-binding cleft in
ybfF, as the smaller cleft does not have sufficient space to ac-
commodate these larger substrates (Scheme 1 and Figure 4).
The mutagenesis results support this conclusion, as the majori-
ty of residues essential (or highly important) to the catalytic ac-
tivity of Vc-ybfF (with the exception of Tyr236) are located
around the larger binding cleft (Figure 4A–C). Additionally, the
proposed selectivity residue (Tyr116), which differs between
bacterial and mammalian homologues of ybfF, is at the
bottom of the larger binding cleft, thus suggesting full immer-
sion of the substrates into the large binding cleft (Figures 3A
and 4B, C). The identification of the larger binding cleft as the
major substrate-recognition region also reinforces the modeled
binding of IPG esters to ybfF.[7b,9]
Discussion
Serine hydrolases constitute a diverse enzyme family, and cata-
lyze a range of biological functions by using the same basic
structural fold.[1a,2,14] Large variations in the structure of the
binding pocket and the addition of secondary domains (lipase
lids and helical caps) within serine hydrolases allow family
members to recognize diverse intracellular substrates.[1a,2] This
diversity of substrate selectivity and general adaptability of the
a/b hydrolase fold has made serine hydrolases common tar-
gets for directed evolution and now for inhibitor design.[3b,c,14b]
Within the diverse serine hydrolase superfamily, the ybfF pro-
tein family shows an unusual architecture of its substrate-bind-
ing pocket and a unique helical cap (Figure 1).[7a] With only lim-
ited sequence similarity to other hydrolases, limited enzymatic
characterization, and the known ability to catalyze commercial-
ly applicable reactions, the ybfF protein family provides an in-
teresting opportunity to understand the plasticity of substrate
binding and reactivity in serine hydrolases.
The overall substrate specificity landscape of ybfF places it
in the carboxylesterase family, with strict substrate selectivity
for smaller, only slightly branched substrates (Figure 2). The
preference for polarity close to the ester bond reinforces the
proposed biological assignment as malonyl-CoA or succinyl-
CoA esterase.[7a] Yet like many bacterial esterases where limited
numbers of hydrolases are present in the proteome,[15] ybfF
has the ability to accept a range of substrates, especially a vari-
ety of cycloalkyl substrates, as ambiguous substrates in cases
The role of the smaller binding lobe in the ybfF binding
pocket is less well-defined, as the presence of clearly posi-
tioned water molecules in the structure of Ec-ybfF led to the
hypothesis that this cleft positions the water molecules for hy-
drolysis of the acyl–enzyme intermediate (Figure 1).[7a] Substitu-
tion of residues around the narrow cleft did not, however, lead
to significant changes in catalytic activity, as would be predict-
ed if the cleft played this key role in catalysis (Figure 3 and 4).
Figure 4. Substrate-binding residue relevance to Vc-ybfF catalytic activity and thermal stability. Surface representations of A) and B) exterior and C) interior of
the binding pocket showing relative contributions of residues to catalytic activity with substrate 6. Colors represent relative changes in catalytic activity (kcat
/
KM) upon substitution to alanine: red (>1000-fold); orange (>400-fold); yellow (>20-fold); green (<20-fold). D–F) Relative contributions or residues to ther-
mal stability (Tm): red (>5.58C), orange (>4.58C), yellow (>2.58C), and green (<2.58C).
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Instead, residues surrounding the narrow cleft seemed more
directly associated with thermal stability and proper folding of
the Vc-ybfF structure, as this region is enriched in residues im-
portant for the stability of Vc-ybfF, including His20, Phe158,
His235, and Trp236 (Figure 4D–F). Our results cannot, however,
discount the modeled role for the narrow binding cleft in fit-
ting short alkyl chains bound to IPG esters, as the difunctional-
ized fluorescein is likely too large to fit within the larger bind-
ing pocket of ybfF, as has been proposed for the IPG
groups.[7b,9] Our results do, however, suggest that this role for
the narrow binding pocket is likely minor in standard sub-
strate-binding and catalytic activity.
riphery of the binding pocket. The divergent roles for the large
and narrow binding cleft will also allow future substitution
analysis to decouple the effect of a substitution on the catalyt-
ic activity and on thermal stability of ybfF. Future mutagenesis
studies will now focus on concerted saturation mutagenesis of
selectivity residues and seek to determine if concerted substi-
tutions at these residues can redirect the substrate selectivity
of Vc-ybfF.
In addition to their potential roles as novel biocatalysts,
members of the ybfF enzyme family have also been implicated
as potential therapeutic targets. Abhydrolase 11 (ABHD11), the
human homologue of ybfF, was recently identified as a bio-
marker for aggressive lung adenocarcinoma and was previous-
ly linked to the development of Williams syndrome, a complex
neurodevelopmental disorder caused by a large deletion in
chromosome 21.[14a,19] Although no explicit studies of the activ-
ity or function of ABHD11 have been conducted to date, two
current inhibitors of ABHD11 have been identified in activity-
based protein-profiling screens with triazole and carbamate
core structures.[14b,20] We note that these inhibitor structures
parallel the substrate specificity profile of Vc-ybfF, as each in-
hibitor contains a slightly bulky cyclic side chain connected to
a polar alkyl chain of less than four carbons.[14b] This dual func-
tionality of the ABHD11 inhibitors not only matches the sub-
strate specificity profile of Vc-ybfF, but also the structure of the
substrate-binding pocket of ybfF (Figure 4). Given the structur-
al similarity between ABHD11 inhibitors and the substrate-se-
lectivity profile of ybfF (Figure 2), the current ABHD11 inhibi-
tors could be used as primary leads in designing ybfF-specific
inhibitors or could be used as in cellulo modulators of ybfF ac-
tivity to better assign the biological function of ybfF.
In addition to surrounding the narrow binding cleft, key resi-
dues for the stability of ybfF (His121 and Trp236) are at the en-
trance to the binding pocket and at the interface of the helical
cap (Figure 4D–F). Two potential explanations for the position-
ing of these key stability residues at this position are: 1) rigid
structural positioning is required to maintain an open entrance
to the tunnel or 2) these residues are required for movement
of the entrance tunnel to accommodate larger substrates.
Movement of lid domains in lipases is required for their full
catalytic activity and to provide additional regulation.[17] Al-
though ybfF is not a lipase, with its specificity for shorter ester
chains (Figure 2), the predicted location of the coenzyme A
molecule along the interface could provide a similar mecha-
nism for substrate-induced activation in ybfF.[7a] Movement be-
tween the helical cap and hydrolase domain in ybfF could also
explain the necessity for a unique helical cap and the place-
ment of the buried binding pocket at the interface of these
domains. Similar flexibility was observed in the loop connect-
ing the lid and hydrolase domains in the structure of the ybfF
homologue, Rv0045c.[8b] Confirmation of this proposed move-
ment will require further substitution and kinetics analysis with
longer substrates.
Conclusions
The broad substrate selectivity of ybfF and the high enantio-
selectivity of ybfF with IPG esters make ybfF a potential new
commercial biocatalyst.[7b,9] An initial study on activity en-
hancement of Ec-ybfF toward IPG esters focused on four
amino acids (Leu22, His121, Ile186, and Trp236; Vc-ybfF num-
bering) that were in direct contact with the modeled IPG
esters.[9] Only substitutions at Ile186 and Trp236 made signifi-
cant improvements to the activity of Ec-ybfF, with nonpolar
substitutions at Tr236 providing the largest increase in enantio-
selectivity toward R-IPG esters.[9] These results confirm the po-
tential for improving the biocatalyst properties of ybfF, but fur-
ther development of ybfF as a biocatalyst will require broader
directed-evolution studies. For these studies, the assignment
of key residues involved in activity, stability, and substrate se-
lectivity can be used to decrease the size of the random library
and to increase the probability of finding a successful var-
iant.[3c,18] Our results have determined both potential selectivity
residues (Tyr116, Arg120, Tyr209) and residues to avoid in
future substitution studies (His20, His121, His235, Trp236) be-
cause of their essential roles in stability or activity. The map of
each residues role in the catalytic activity and stability of ybfF
(Figure 4) also highlights hot spots for residues not varied in
this study and second sphere residues positioned on the pe-
The ybfF protein family exhibits unusual structural, functional,
and kinetic characteristics, and presents potential avenues for
biocatalyst development. The structural architecture of the bi-
furcated binding pocket of ybfF allows it to recognize a range
of short, bulky, and slightly polar ester substrates. This sub-
strate selectivity is mainly provided by the larger substrate
binding cleft and by key substrate-selectivity residues (Tyr116,
Arg120, and Tyr209). The narrow binding pocket, which lacks
a defined role in the catalytic activity of Vc-ybfF but is essential
to the stability, presents an intriguing location for future bio-
catalyst design. The distinct roles of the large and narrow bind-
ing clefts in catalytic activity and thermal stability can now be
exploited to decouple these two important functions when de-
signing future biocatalysts. The combined contributions of
each cleft of this unusual binding pocket to the catalytic activi-
ty and substrate selectivity of ybfF also provides a new mecha-
nism for serine hydrolases to differentiate between their di-
verse biological substrates.
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Fluorescein bis(((3-methoxy)propionyloxy)methyl ether) (7).
Data for chloromethyl 3-methoxypropanoate: 0.40 g, 32%; H NMR
Experimental Section
1
Synthesis of fluorogenic ester substrates:
(400 MHz, CDCl3): d=5.72 (s, 2H), 3.68 (t, J=6.2 Hz, 2H), 3.36 (s,
3H), 2.66 ppm (t, J=6.2 Hz, 2H). Data for 7: 199 mg, 13%; H NMR
1
General chemical synthesis: Compounds 1–5, 9, 11, 17, and 18
were synthesized as described previously.[10a,b,c] All reagents were
of the highest grade available and obtained from Sigma–Aldrich or
Fisher Scientific. Preactivated, powdered 4 ꢁ molecular sieves
(Sigma–Aldrich) were used as received. Anhydrous solvents were
drawn from Sigma–Aldrich Sure-Seal bottles. Thin-layer chromatog-
raphy was performed with aluminum-backed plates coated with
silica gel containing F254 phosphor (Sigma–Aldrich) and visualized
by UV illumination, charring, or staining with I2, ceric ammonium
molybdate, or phosphomolybdic acid.
(400 MHz, CDCl3): d=8.03 (d, J=7.3 Hz, 1H), 7.68 (ddd, J=7.5, 7.4,
1.4 Hz, 1H), 7.63 (dd, J=7.2, 7.2 Hz, 1H), 7.15 (d, J=7.4 Hz, 1H),
6.99–6.93 (m, 2H), 6.76–6.71 (m, 4H), 5.81 (s, 4H), 3.67 (t, J=6.2 Hz,
4H), 3.32 (s, 6H), 2.65 ppm (t, J=6.2 Hz, 4H).
Fluorescein bis((2-(2-methoxyethoxy)acetoxy)methyl ether) (8).
Data for chloromethyl 2-(2-methoxyethoxy)acetate: 0.57 g, 39%;
1H NMR (400 MHz, CDCl3): d=5.76 (s, 2H), 4.24 (s, 2H), 3.78–3.72
(m, 2H), 3.63–3.56 (m, 1H), 3.39 ppm (s, 3H). Data for 8: 32 mg,
7%; 1H NMR (400 MHz, CDCl3): d=8.03 (d, J=7.3 Hz, 1H), 7.68
(ddd, J=7.5, 7.3, 1.2 Hz, 1H), 7.64 (ddd, J=7.4, 7.4, 1.2 Hz, 1H),
7.15 (d, J=7.5 Hz, 1H), 6.97–6.93 (m, 2H), 6.77–6.69 (m, 4H), 5.86
(s, 4H), 4.22 (s, 4H), 3.76–3.69 (m, 4H), 3.59–3.54 ppm (m, 4H), 3.36
(s, 6H).
Flash chromatography was performed on an Isolera 4 system with
SNAP columns (Biotage, Uppsala, Sweden). Because of closely elut-
ing byproducts in the alkylation reaction, several chromatography
runs were sometimes required to obtain pure material, thus result-
ing in low isolated yields. The term “concentrated in vacuo”
(below) refers to removal of solvents and other volatile materials
by using a rotary evaporator or speedvac system at variable pres-
sure (controlled diaphragm pump; ꢂ0.5 mm Hg) while maintain-
ing the water-bath or chamber temperature below 408C. NMR
spectra were obtained with a 400 MHz Avance II+ spectrometer
(Bruker) at the Janelia Farm Research Campus (Ashburn, VA).
Fluorescein bis((acryloyloxy)methyl ether) (10). Data for chloro-
methyl acrylate: 0.30 g, 31%; 1H NMR (400 MHz, CDCl3): d=6.54
(dd, J=17.3, 1.2 Hz, 1H), 6.16 (dd, J=17.3, 10.5 Hz, 1H), 5.98 (dd,
J=10.5, 1.2 Hz, 1H), 5.80 ppm (s, 2H). Data for 10: 110 mg, 35%;
1H NMR (400 MHz, CDCl3): d=8.03 (ddd, J=7.3, 1.4, 0.8 Hz, 1H),
7.67 (ddd, J=7.4, 7.4, 1.4 Hz, 1H), 7.63 (ddd, J=7.4, 7.4, 1.2 Hz,
1H), 7.18–7.13 (m, 1H), 7.00–6.96 (m, 2H), 6.78–6.71 (m, 4H), 6.51
(dd, J=17.3, 1.3 Hz, 2H), 6.16 (dd, J=17.3, 10.5 Hz, 2H), 5.94 (dd,
J=10.5, 1.3 Hz, 2H), 5.88 ppm (s, 4H).
Fluorescein bis((2-methoxy)acetoxymethyl ether) (6). The follow-
ing protocol is representative. Methoxyacetic acid (730 mg,
8.08 mmol, 1.0 equiv) was added to a solution of tetrabutylammo-
nium hydrogensulfate (274 mg, 0.808 mmol, 0.1 equiv) and potassi-
um carbonate (4.47 g, 32.3 mmol, 4.0 equiv) in H2O (10 mL) and
CH2Cl2 (10 mL). A solution of chloromethyl chlorosulfate (1.23 mL,
12.12 mmol, 1.5 equiv) in CH2Cl2 (5 mL) was then added dropwise,
and the reaction was stirred vigorously (~600 rpm) at room tem-
perature under N2 for 4 h. The mixture was then diluted in H2O
(30 mL) and CH2Cl2 (50 mL), and the layers were separated. The
aqueous layer was extracted with CH2Cl2 (2ꢂ 50 mL), the organic
layers were combined and dried over MgSO4, filtered, and concen-
trated in vacuo (note: volatile). The residue was filtered through
a plug of silica gel, washed with CH2Cl2 (100 mL), and carefully con-
centrated in vacuo (note: volatile), to afford chloromethyl 2-me-
Fluorescein bis(((cyclopentanecarbonyl)oxy)methyl ether) (12).
Data for chloromethyl cyclopentanecarboxylate: 0.99 g, 75%;
1H NMR (400 MHz, CDCl3): d=5.71 (s, 2H), 2.80 (tt, J=8.6, 7.3 Hz,
1H), 1.99–1.53 ppm (m, 8H). Data for 12: 354 mg, 40%; 1H NMR
(400 MHz, CDCl3): d=8.06–8.00 (m, 1H), 7.68 (ddd, J=7.4, 7.4,
1.4 Hz, 1H), 7.63 (ddd, J=7.4, 7.4, 1.2 Hz, 1H), 7.17 (dd, J=7.6,
1.0 Hz, 1H), 7.01–6.92 (m, 2H), 6.79–6.68 (m, 4H), 5.78 (s, 5H), 2.80
(tt, J=8.6, 7.4 Hz, 2H), 2.03–1.44 ppm (m, 16H).
Fluorescein bis(((cyclohexanecarbonyl)oxy)methyl ether) (13).
Data for chloromethyl cyclohexanecarboxylate: 1.29 g, 90%;
1H NMR (400 MHz, CDCl3): d=5.71 (s, 2H), 2.37 (tt, J=11.2, 3.7 Hz,
1H), 2.02–1.84 (m, 4H), 1.83–1.15 ppm (m, 6H). Data for 13:
1
211 mg, 19%; H NMR (400 MHz, CDCl3): d=8.03 (ddd, J=7.3, 1.2,
1
1.0 Hz, 1H), 7.68 (ddd, J=7.4, 7.4, 1.4 Hz, 1H), 7.63 (ddd, J=7.4,
7.4, 1.2 Hz, 1H), 7.17 (ddd, J=7.6, 1.3, 1.0 Hz, 1H), 6.98–6.92 (m,
2H), 6.76–6.70 (m, 4H), 5.78 (s, 4H), 2.37 (tt, J=11.3, 3.7 Hz, 2H),
1.97–1.86 (m, 4H), 1.83–1.12 ppm (m, 16H).
thoxyacetate as a colorless liquid (0.22 g, 20%). H NMR (400 MHz,
CDCl3): d=5.77 (s, 2H), 4.12 (s, 2H), 3.48 ppm (s, 3H).
Chloromethyl 2-methoxyacetate (220 mg, 1.59 mmol, 4.0 equiv)
was dissolved in HPLC-grade acetone (4.0 mL). NaI (241 mg,
1.59 mmol, 4.0 equiv) was added, and the mixture was stirred for
24 h at room temperature. The reaction mixture was adsorbed
onto celite and purified by column chromatography (silica gel,
CH2Cl2 (4!34% v/v in hexanes). Removal of solvent gave a color-
less oil (note: volatile), which was taken up in anhydrous CH3CN
(7.0 mL) under N2. Fluorescein (0.13 g, 0.40 mmol, 1.0 equiv), pow-
dered 4 ꢁ molecular sieves (100 mg), and anhydrous Ag2O (0.23 g,
1.0 mmol, 2.5 equiv) were added, then the mixture was stirred for
72 h at room temperature, diluted in CH2Cl2 (100 mL), and filtered
through a pad of celite. The resulting solution was concentrated in
vacuo to give a yellow-brown oil, and purified by column chroma-
tography (silica gel, EtOAc (10!80% v/v) in hexanes containing
CH2Cl2 (30% v/v) as cosolvent) to afford 6 as a white solid
(56.5 mg, 27%). 1H NMR (400 MHz, CDCl3): d=8.03 (d, J=7.2 Hz,
1H), 7.68 (ddd, J=7.4, 7.4, 1.1 Hz, 1H), 7.63 (ddd, J=7.4, 7.1,
0.6 Hz, 1H), 7.15 (d, J=7.6 Hz, 1H), 6.96 (m, 2H), 6.74 (m, 4H), 5.87
(s, 4H), 4.10 (s, 4H), 3.46 ppm (s, 6H).
Fluorescein bis((furan-2-carboxy)methyl ether) (14). Data for
chloromethyl furan-2-carboxylate: 1.02 g, 79%; 1H NMR (400 MHz,
CDCl3): d=7.65 (dd, J=1.7, 0.8 Hz, 1H), 7.31 (dd, J=3.5, 0.9 Hz,
1H), 6.56 (dd, J=3.5, 1.7 Hz, 1H), 5.92 ppm (s, 2H). Data for 14:
1
309 mg, 33%; H NMR (400 MHz, CDCl3): d=8.02 (ddd, J=7.4, 1.4,
0.9 Hz, 1H), 7.67 (ddd, J=7.4, 7.4, 1.5 Hz, 1H), 7.64–7.59 (m, 3H),
7.28 (dd, J=3.5, 0.9 Hz, 2H), 7.15 (ddd, J=7.4, 1.4, 0.9 Hz, 1H), 7.04
(d, J=2.4 Hz, 2H), 6.80 (dd, J=8.8, 2.5 Hz, 2H), 6.75 (d, J=8.8 Hz,
2H), 6.53 (dd, J=3.6, 1.7 Hz, 2H), 6.08–5.91 ppm (m, 4H).
Fluorescein bis((benzoyloxy)methyl ether) (15). Data for chloro-
1
methyl benzoate: 1.06 g, 77%; H NMR (400 MHz, CDCl3): d=8.13–
8.04 (m, 2H), 7.68–7.55 (m, 1H), 7.52–7.43 (m, 2H), 5.96 ppm (s,
1
2H). Data for 15: 68 mg, 7%; H NMR (400 MHz, CDCl3): d=8.13–
8.05 (m, 4H), 8.02 (ddd, J=7.3, 1.5, 0.8 Hz, 1H), 7.66 (ddd, J=7.4,
7.4, 1.5 Hz, 2H), 7.63–7.54 (m, 3H), 7.52–7.38 (m, 4H), 7.20–7.11 (m,
1H), 7.05 (d, J=2.4 Hz, 2H), 6.81 (dd, J=8.8, 2.4 Hz, 2H), 6.75 (d,
J=8.8 Hz, 2H), 6.28–5.79 ppm (m, 4H).
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Fluorescein bis((2-phenylacetoxy)methyl ether) (16). Data for
chloromethyl 2-phenylacetate: 0.803 g, 54%; 1H NMR (400 MHz,
CDCl3): d=7.56–7.06 (m, 5H), 5.70 (s, 2H), 3.70 ppm (s, 2H). Data
for 16: 25.4 mg, 4%; 1H NMR (400 MHz, CDCl3): d=8.07–8.02 (m,
1H), 7.69 (ddd, J=7.5, 7.4, 1.4 Hz, 2H), 7.64 (ddd, J=7.4, 7.4,
1.2 Hz, 1H), 7.36–7.20 (m, 10H), 7.18–7.12 (m, 1H), 6.88 (d, J=
2.3 Hz, 2H), 6.70 (d, J=8.7 Hz, 4H), 6.67 (dd, J=8.8, 2.3 Hz, 2H),
5.79 (s, 4H), 3.69 ppm (s, 4H).
added to the soluble fraction, and the mixture was incubated (48C,
15 min). The resin was washed with ice-cold PBS (3ꢂ1 mL) contain-
ing increasing concentrations of imidazole (10, 25, and 50 mm),
and collected by centrifugation (2000g, 2 min, 258C). Vc-ybfF was
eluted in PBS (1000 mL) containing imidazole (250 mm), and dia-
lyzed against PBS overnight at 48C with constant stirring (10 K
MWCO; Pierce/Thermo Scientific).
The purity of Vc-ybfF was confirmed by SDS-PAGE on a 4–20% gra-
dient gel (purity >95%, Figure S1). The concentration of Vc-ybfF
was determined by measuring the absorbance at 280 nm and by
calculating the extinction coefficient (e280 =23950mꢀ1 sꢀ1 with all
free cysteines) by using ProtParam (http://web.expasy.org/protpar-
am/)
Fluorescein bis(((3,3,3-trifluoropropanoyl)oxy)methyl ether) (19).
Data for chloromethyl 3,3,3-trifluoropropanoate: 0.37 g, 26%;
1H NMR (400 MHz, CDCl3): d=5.75 (s, 2H), 3.28 ppm (q, J=9.8 Hz,
1
2H). Data for 19: 35.2 mg, 11%; H NMR (400 MHz, CDCl3): d=7.95
(ddd, J=7.3, 1.3, 0.8 Hz, 1H), 7.60 (ddd, J=7.4, 7.4, 1.4 Hz, 1H),
7.56 (ddd, J=7.4, 7.4, 1.2 Hz, 1H), 7.08 (ddd, J=7.6, 1.4, 0.8 Hz,
1H), 6.87 (dd, J=2.2, 0.6 Hz, 2H), 6.68 (dd, J=8.8, 0.6 Hz, 2H), 6.65
(dd, J=8.8, 2.2 Hz, 2H), 5.81–5.74 (m, 4H), 3.18 ppm (q, J=9.9 Hz,
4H).
Site-directed mutagenesis and purification: Variants of Vc-ybfF
were created by site-directed mutagenesis by using Quikchange II
(Agilent, Santa Clara, CA) according to the manufacturer’s suggest-
ed procedure (mutagenesis primers in Table S4). Correct mutations
in Vc-ybfF DNA were confirmed by DNA sequencing (Genewiz).
Plasmids with Vc-ybfF variants were transformed into E. coli BL21
(DE3) RIPL, and variants of Vc-ybfF were purified by the procedure
for wild-type Vc-ybfF (Figure S1). For variants of Vc-ybfF with tyro-
sine and tryptophan substitutions, specific extinction coefficients
Fluorescein bis((perfluorobenzoyloxy)methyl ether) (20). Data
for chloromethyl perfluorobenzoate: 1.66 g, 79%; 1H NMR
(400 MHz, CDCl3): d=5.93 ppm (s, 2H). Data for 20: 62.2 mg, 5%;
1H NMR (400 MHz, CDCl3): d=8.03 (ddd, J=7.4, 1.5, 0.8 Hz, 1H),
7.68 (ddd, J=7.5, 7.4, 1.5 Hz, 2H), 7.64 (ddd, J=7.4, 7.4, 1.2 Hz,
1H), 7.16 (dt, J=7.5, 1.2, 0.8 Hz, 1H), 7.05–6.98 (m, 2H), 6.82–6.74
(m, 4H), 6.08–5.98 ppm (m, 4H).
were obtained from ProtParam (Tyr e280 =22460mꢀ1 sꢀ1; Trp e280
18450mꢀ1 ꢀ1).
=
s
Kinetic measurements with fluorogenic ester substrates: The en-
zymatic activities of Vc-ybfF and variants were measured with the
fluorogenic ester substrates (Scheme 1) in a 96-well microplate
assay.[10c] Fluorogenic substrates were prepared as stock solutions
in DMSO (10 mm) and were diluted in PBS containing acetylated
BSA (PBS–BSA; 0.1 mgmLꢀ1) depending on the Km of Vc-ybfF for
the substrate (10 mm (1–5, 9–18), 100 mm (6–8), 1000 mm (19–21)).
Eight serial dilutions in PBS/BSA (1:2; 180 mL each) were made for
each substrate. Fluorogenic substrate dilutions (95 mL) were then
transferred to wells of a black 96-well microplate (Corning, Lowell,
MA).
Fluorescein bis((2-(pentafluorophenyl)acetoxy)methyl ether)
(21). Data for chloromethyl 2-(perfluorophenyl)acetate: 1.58 g,
1
71%; H NMR (400 MHz, CDCl3): d=5.73 (s, 2H), 3.84–3.79 ppm (m,
1
2H). Data for 21: 29.8 mg, 3%; H NMR (400 MHz, CDCl3): d=8.05
(ddd, J=7.4, 1.1, 0.8 Hz, 1H), 7.70 (ddd, J=7.4, 7.4, 1.3 Hz, 1H),
7.65 (ddd, J=7.4, 7.4, 1.1 Hz, 1H), 7.18 (ddd, J=7.4, 1.3, 0.9 Hz,
1H), 6.89 (d, J=2.3 Hz, 2H), 6.76 (d, J=8.8 Hz, 2H), 6.71 (dd, J=
8.8, 2.4 Hz, 2H), 5.88–5.78 (m, 4H), 3.84–3.78 ppm (m, 4H).
Purification of Vc-ybfF from V. cholerae: A bacterial expression
plasmid (pANT-GST) containing the VC2097 gene from V. cholerae
O1 biovar El Tor strain N16961 (GenBank ID: NP_231729.1) was ob-
tained from the Harvard Plasmid Repository (Clone ID:
VcCD00035533). The VC2097 gene was subcloned into a pET-22b
(+) plasmid (Merck Millipore) between the NdeI and XhoI restric-
tion sites and with an in-frame C-terminal His6 tag, by using pri-
mers VC2097 Forward and VC2097 Reverse (Table S4). Correct se-
quence insertion was confirmed by DNA sequencing (Genewiz Inc,
South Plainfield, NJ).
Wild-type Vc-ybfF (5 mL final concentration 7.5 mgmLꢀ1) or variant
(5 mL, final concentration 15 mgmLꢀ1) was added to the diluted flu-
orogenic substrates in the 96-well microplate (100 mL final volume),
and the fluorescence (lex =485 nm, lem =528 nm) was measured
over 4 min at 258C on a Synergy 2 fluorescence plate reader
(Biotek Instruments; Winooski, VT). Fluorescence values were mea-
sured simultaneously and converted to molar concentration by
using a fluorescein standard curve (30 nm–0.23 nm (1–5, 9–18),
300 nm–2.3 nm (6–8, 19–21)). The initial rates of the reactions
were measured in triplicate and plotted against fluorogenic sub-
strate concentration. The saturation enzyme kinetic traces were
fitted to a standard Michaelis–Menten equation with Origin 6.1
(OriginLab Corp., Northhampton, MA), and values for kcat, KM, and
kcat/KM were calculated. The background fluorescence was deter-
mined by measuring the kinetics of fluorogenic substrate hydroly-
sis by the S86A variant of Vc-ybfF (Table 1).
This resulting plasmid (pET-22b-Vc2097) was transformed into
E. coli BL21 (DE3) RIPL cells (Agilent). A saturated overnight culture
in lysogeny broth (LB) containing ampicillin (100 mgmLꢀ1) and
chloramphenicol (30 mgmLꢀ1) was used to inoculate LB (250 mL)
containing ampicillin (100 mgmLꢀ1
)
and chloramphenicol
(30 mgmLꢀ1), and the bacterial culture was grown with shaking
(225 rpm) at 378C. When OD600 reached 0.6–0.8, the temperature
of the culture was decreased to 168C, and isopropyl b-d-1-thioga-
lactopyranoside (IPTG) was added (final concentration 0.5 mm) for
protein induction. After 16–20 h at 168C, cells were collected by
centrifugation (6000g, 10 min, 48C), and the pellet was resuspend-
ed in PBS (2 mL) and stored at ꢀ208C.
Kinetics measurements for p-nitrophenyl substrates with Vc-
ybfF: Stock solutions (p-nitrophenyl acetate (2m), p-nitrophenyl
butyrate (2m), p-nitrophenyl octanoate (200 mm), p-nitrophenyl
laurate (200 mm)) in acetonitrile were diluted in PBS–BSA
(0.1 mgmLꢀ1) to starting concentrations of 20 mm (acetate) or
2 mm (butyrate, octanoate, and laurate). Eight serial dilutions (1:1,
total volume 220 mL; 20 mm–156 mm (acetate) and 2 mm–15.6 mm
(butyrate, octanoate, and laurate)) were made in PBS–BSA contain-
ing acetonitrile (1%). Substrate dilutions (95 mL) were transferred
to a clear 96-well microplate, and Vc-ybfF (5 mL; final concentration
To disrupt the bacterial cell wall, lysozyme (50 mg) was added to
the thawed cell pellet, and the mixture was set on ice for 30 min.
Bug Buster solution (250 mL, 10ꢂ; EMD Millipore) was added, and
cell lysis proceeded on an orbital shaker for 1 h at 48C. To remove
insoluble cell material, lysed cells were centrifuged (18500g,
10 min, 48C). Ni-NTA agarose (750 mL; Qiagen, Valencia, CA) was
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ChemBioChem 2013, 14, 1134 – 1144 1143

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15 mgmLꢀ1) was added to start the reaction. The absorbance at
410 nm was measured in a Synergy 2 plate reader (Biotek) over
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1.034 mmꢀ1 cmꢀ1).[22] Initial rates of the reactions were measured in
triplicate, and kinetics parameters were determined as for the fluo-
rogenic assay.
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Acknowledgements
We thank Nathan Clarke, Erica Couch, Grace Douglas, Laurel
Heckman, Lin Liu, Andrew Lutkewitte, Samantha Minnette,
Nathan Tavenor, and Ben Trefilek for assistance with preliminary
enzyme purification and enzymatic analysis, Courtney Walker
and Ann Yoon for help with size-exclusion chromatography,
Paula LeBlanc and Michael Slack for help with pH activity meas-
urements, and Kelly McKenna for help with cloning Vc-ybfF. E.E.E.
was supported by a Butler Summer Institute Scholarship. R.J.J.
was supported by start-up funds and a Holcomb grant from
Butler University, and a Senior Research Grant from the Indiana
Academy of Sciences. C.T.A. and L.D.L. were supported by the
Howard Hughes Medical Institute.
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Keywords: biocatalysts
hydrolases · substrate specificity · Vibrio cholerae
· fluorogenic enzyme substrates ·
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Published online on May 13, 2013
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ChemBioChem 2013, 14, 1134 – 1144 1144