The structural basis for the narrow substrate specificity of an acetyl esterase from Thermotoga maritima

Article abstract of DOI:10.1016/j.bbapap.2012.05.009

Acetyl esterases from carbohydrate esterase family 7 exhibit unusual substrate specificity. These proteins catalyze the cleavage of disparate acetate esters with high efficiency, but are unreactive to larger acyl groups. The structural basis for this distinct selectivity profile is unknown. Here, we investigate a thermostable acetyl esterase (TM0077) from Thermotoga maritima using evolutionary relationships, structural information, fluorescent kinetic measurements, and site directed mutagenesis. We measured the kinetic and structural determinants for this specificity using a diverse series of small molecule enzyme substrates, including novel fluorogenic esters. These experiments identified two hydrophobic plasticity residues (Pro228, and Ile276) surrounding the nucleophilic serine that impart this specificity of TM0077 for small, straight-chain esters. Substitution of these residues with alanine imparts broader specificity to TM0077 for the hydrolysis of longer and bulkier esters. Our results suggest the specificity of acetyl esterases have been finely tuned by evolution to catalyze the removal of acetate groups from diverse substrates, but can be modified by focused amino acid substitutions to yield enzymes capable of cleaving larger ester functionalities.

Full text of DOI:10.1016/j.bbapap.2012.05.009

Biochimica et Biophysica Acta 1824 (2012) 1024–1030
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Biochimica et Biophysica Acta
journal homepage: www.elsevier.com/locate/bbapap
The structural basis for the narrow substrate specificity of an acetyl esterase from
Thermotoga maritima
Matthew K. Hedge a, Alexandra M. Gehring a, Chinessa T. Adkins b, Leigh A. Weston a,
Luke D. Lavis b, R. Jeremy Johnson a,
⁎
a
Department of Chemistry, Butler University, 4600 Sunset Ave, Indianapolis, IN 46208, USA
Janelia Farm Research Campus, The Howard Hughes Medical Institute, 19700 Helix Dr, Ashburn, VA 20147, USA
b
a r t i c l e i n f o
a b s t r a c t
Article history:
Acetyl esterases from carbohydrate esterase family 7 exhibit unusual substrate specificity. These proteins
catalyze the cleavage of disparate acetate esters with high efficiency, but are unreactive to larger acyl groups.
The structural basis for this distinct selectivity profile is unknown. Here, we investigate a thermostable acetyl
esterase (TM0077) from Thermotoga maritima using evolutionary relationships, structural information, fluores-
cent kinetic measurements, and site directed mutagenesis. We measured the kinetic and structural determinants
for this specificity using a diverse series of small molecule enzyme substrates, including novel fluorogenic esters.
These experiments identified two hydrophobic plasticity residues (Pro228, and Ile276) surrounding the nucleo-
philic serine that impart this specificity of TM0077 for small, straight-chain esters. Substitution of these residues
with alanine imparts broader specificity to TM0077 for the hydrolysis of longer and bulkier esters. Our results
suggest the specificity of acetyl esterases have been finely tuned by evolution to catalyze the removal of acetate
groups from diverse substrates, but can be modified by focused amino acid substitutions to yield enzymes
capable of cleaving larger ester functionalities.
Received 11 April 2012
Received in revised form 9 May 2012
Accepted 22 May 2012
Available online 1 June 2012
Keywords:
Acetyl esterase
Cephalosporin-C deacetylase
Fluorogenic enzyme substrates
Hydrolases
Substrate specificity
© 2012 Elsevier B.V. All rights reserved.
1. Introduction
including cephalosporins, glucose pentaacetate, p-nitrophenyl acetate,
4-methylumbelliferyl acetate, and α/β-naphthyl acetate (Fig. 1A) and
The α/β hydrolases together with the oxidoreductases and trans-
ferases represent 75% of the known enzymes encoded by all genomes
[1]. Biochemists and chemists have long appreciated the expansive
reaction and substrate scope contained within the α/β hydrolase su-
perfamily [2–4]. This broad specificity originates from the same α/β
hydrolase protein fold and utilizes the same catalytic triad, suggesting
great plasticity and generality in this privileged structure [3,5]. This
broad specificity, however, complicates the prediction of endogenous
substrates and biological functions of α/β hydrolases [1,2,5].
Among α/β hydrolases, the acetyl esterase subclass from carbo-
hydrate esterase family 7 (CE7) (EC 3.1.1.6) exhibits a distinct combi-
nation of narrow substrate specificity for acetate esters, but broad
substrate specificity for disparate alcohol moieties [6–8]. The
broad substrate specificity for alcohols is highlighted by the range of
acetylated substrates that are hydrolyzed by CE7 acetyl esterases,
by their position independence to hydrolysis of β-D-xylopyranoside
monoacetates [8–11]. The three-dimensional structures of CE7 acetyl
esterases from Bacillus pumilus (PDB ID: 2XLB), Bacillus subtilis (PDB
ID: 1ODS), and Thermotoga maritima (PDB ID: 1VLQ; 3M81; 3M82;
3M83) show the presence of a cavernous substrate-binding pocket
lined with large hydrophobic amino acids [6,8,12]. This large
substrate-binding pocket contains sufficient space to accommodate
diverse substrates and provides an explanation for the broad substrate
specificity of acetyl esterases to a range of alcohols [6,8,12]. Current
structural or enzymatic data do not, however, account for the distinc-
tively narrow substrate specificity of CE7 acetyl esterases for acetate
esters and the inability of acetyl esterases to catalyze reactions besides
deacetylations.
In this study, we measure the precise reactivity of TM0077, a cyto-
plasmic CE7 acetyl esterase from T. maritima, toward libraries of
disparate alcohol- and ester-containing substrates. We synthesize a
series of chemically stable diacyloxymethyl ether fluorescein deriva-
tives to measure the precise substrate specificity of TM0077 for
diverse esters. Utilizing the kinetics of these reactions, the three-
dimensional structures of TM0077, and the evolutionary conservation
of binding pocket residues, we identify the structural and enzymatic
features of acetyl esterases that maintain their unique combination
of broad substrate specificity for alcohols, but narrow substrate spec-
ificity for acetate esters.
Abbreviations: 7-ACA, 7-aminocephalosporanic acid; BTB, bromothymol blue; CE7,
carbohydrate esterase family 7; DSF, differential scanning fluorimetry; LB, Luria–
Bertani broth; MWCO, molecular weight cut-off; Ni-NTA, nickel-nitrilotriacetic acid;
NPA, 4-nitrophenol acetate; PDB, protein data bank; TM0077, acetyl esterase from
Thermotoga maritima
⁎
Corresponding author at: Butler University, 4600 Sunset Ave., Indianapolis, IN,
46208, USA. Tel.: +1 317 940 9062; fax: +1 317 940 9519.
E-mail address: rjjohns1@butler.edu (R.J. Johnson).
1570-9639/$ – see front matter © 2012 Elsevier B.V. All rights reserved.
doi:10.1016/j.bbapap.2012.05.009

M.K. Hedge et al. / Biochimica et Biophysica Acta 1824 (2012) 1024–1030
1025
Chloromethyl butyrate (540 mg, 3.95 mmol, 4.0 eq) was dissolved in
HPLC-grade acetone (4.0 mL). NaI (592 mg, 3.95 mmol, 4.0 eq) was
added and the reaction mixture was stirred for 24 h at ambient temper-
ature under N2(g). The white precipitate was removed via filtration (cel-
ite) and the resulting yellow solution was concentrated in vacuo. The
dark yellow viscous oil was purified via column chromatography (silica
gel, 4→34% v/v EtOAc in hexanes containing constant 20% v/v CH2Cl2
as cosolvent) to yield a pale yellow oil. This material was taken up in an-
hydrous CH3CN (6.0 mL) under N2(g). Fluorescein (328 mg, 0.988 mmol,
1.0 eq), powdered 4-Å molecular sieves (300 mg), and anhydrous Ag2O
(572 mg, 2.46 mmol, 2.5 eq) were added, and the reaction mixture was
stirred for 48 h at ambient temperature. The reaction mixture was then
diluted with CH2Cl2 (100 mL) and filtered (celite). The resulting solution
was concentrated in vacuo to give a yellow-brown oil and purified via
column chromatography (silica gel, 3→28% v/v EtOAc in hexanes con-
taining constant 20% v/v CH2Cl2 as cosolvent) affording 3 as a white
solid (186 mg, 35%). 1H NMR (400 MHz, CDCl3) δ 8.03 (d, J=7.5 Hz,
1H), 7.68 (dd, J=7.2, 7.0 Hz, 1H), 7.63 (dd, J=7.3, 7.3 Hz, 1H), 7.16 (d,
J=7.6 Hz, 1H), 7.01–6.90 (m, 2H), 6.80–6.65 (m, 4H), 5.79 (s, 4H), 2.36
(t, J=7.4 Hz, 4H), 1.77–1.60 (m, 4H), 0.95 (t, J=7.4 Hz, 6H). 13C NMR
(101 MHz, CDCl3) δ 172.41 (C), 169.22 (C), 158.44 (C), 152.97 (C),
152.24 (C), 135.10 (CH), 129.88 (CH), 129.36 (CH), 126.67 (C), 125.17
(CH), 123.88 (CH), 113.28 (C), 112.74 (CH), 103.55 (CH), 84.83 (CH2),
82.46 (C), 36.01 (CH2), 18.17 (CH2), 13.55 (CH3).
Fig. 1. Compounds used to measure substrate specificity: A) Broad specificity. Three
model substrates (1: 7-amino cephalosporanic acid, 2: p-nitrophenyl acetate, and 3:
fluorescein di(acetoxymethyl ether) were used to determine the substrate specificity
of TM0077 for different alcohol substituents. B) Narrow specificity. Fluorogenic ester-
ase substrates (substrates 3–11) were used to characterize the specificity of TM0077
for esters, including its ability to accept varying acyl chains (substrates 3–7) and
increased steric bulk and steric hindrance (substrates 8–11). All substrates have
fluorescein and an acyloxymethyl ether with varying R groups.
2. Materials and methods
2.3. Fluorescein divaleryloxymethyl ether (6)
2.1. General synthesis
Data for chloromethyl valerate: (70%, colorless oil). 1H NMR
(400 MHz, CDCl3) δ 5.70 (s, 2H), 2.39 (t, J=7.5 Hz, 2H), 1.73–1.58
(m, 2H), 1.46–1.28 (m, 2H), 0.93 (t, J=7.3 Hz, 3H). 13C NMR (101 MHz,
CDCl3) δ 171.51 (C), 68.30 (CH2), 33.44 (CH2), 26.32 (CH2), 21.81 (CH2),
13.36 (CH3). Data for 4: (44%, white solid). 1H NMR (400 MHz, CDCl3) δ
8.07–7.99 (m, 1H), 7.68 (ddd, J=7.5, 7.4, 1.4 Hz, 1H), 7.63 (ddd, J=7.4,
7.3, 1.2 Hz, 1H), 7.19–7.11 (m, 1H), 7.01–6.90 (m, 2H), 6.80–6.65
(m, 4H), 5.79 (s, 4H), 2.38 (t, J=7.5 Hz, 4H), 1.70–1.57 (m, 4H),
1.41–1.29 (m, 4H), 0.89 (t, J=7.3 Hz, 6H). 13C NMR (101 MHz, CDCl3)
δ 172.58 (C), 169.21 (C), 158.43 (C), 152.98 (C), 152.24 (C), 135.09
(CH), 129.87 (CH), 129.36 (CH), 126.66 (C), 125.17 (CH), 123.87 (CH),
113.27 (C), 112.74 (CH), 103.55 (CH), 84.83 (CH2), 82.44 (C), 33.87
(CH2), 26.69 (CH2), 22.14 (CH2), 13.64 (CH3).
Fluorogenic enzyme substrates 3, 4, and 8–11 were synthesized as
previously described [13]. All reagents were the highest grade available
and obtained from Sigma–Aldrich or Fisher Scientific. Preactivated,
powdered 4-Å molecular sieves from Sigma–Aldrich were used as
received. Anhydrous solvents were drawn from Aldrich Sure-Seal
bottles. Thin-layer chromatography was performed by using aluminum-
backed plates coated with silica gel containing F254 phosphor and visual-
ized by UV-illumination, charring, or staining with I2, ceric ammonium
molybdate, or phosphomolybdic acid. Column chromatography was
performed on an Isolera 4 system with SNAP columns (Biotage). The
term “concentrated in vacuo” refers to the removal of solvents and
other volatile materials by using a rotary evaporator with a controlled
diaphragm pump (≥1 mm Hg) while maintaining the water-bath
temperature below 40 °C. NMR spectra were obtained with a Bruker
400 MHz Avance-II+ spectrometer at the Janelia Farm Research Campus.
1H and 13C NMR spectra were referenced to tetramethylsilane or residual
solvent peaks. 13C NMR spectra hydrogen multiplicity (C, CH, CH2, CH3)
information was obtained from DEPT spectra.
2.4. Fluorescein dicaproyloxymethyl ether (7)
Data for chloromethyl caproate: (76%, colorless oil). 1H NMR
(400 MHz, CDCl3) δ 5.70 (s, 2H), 2.38 (t, J=7.5 Hz, 2H), 1.75–1.59
(m, 2H), 1.45–1.21 (m, 4H), 1.03–0.80 (m, 3H). 13C NMR (101 MHz,
CDCl3) δ 171.77 (C), 68.55 (CH2), 33.94 (CH2), 31.07 (CH2), 24.20 (CH2),
22.22 (CH2), 13.82 (CH3). Data for 5: (13%, white solid). 1H NMR
(400 MHz, CDCl3) δ 8.07–7.97 (m, 1H), 7.67 (ddd, J=7.5, 7.4, 1.4 Hz,
1H), 7.63 (ddd, J=7.4, 7.3, 1.2 Hz, 1H), 7.19–7.09 (m, 1H), 7.02–6.88
(m, 2H), 6.81–6.65 (m, 4H), 5.79 (s, 4H), 2.37 (t, J=7.5 Hz, 4H),
1.74–1.57 (m, 4H), 1.38–1.19 (m, 8H), 0.96–0.77 (m, 6H). 13C NMR
(101 MHz, CDCl3) δ 172.59 (C), 169.20 (C), 158.42 (C), 152.98 (C),
152.23 (C), 135.08 (CH), 129.87 (CH), 129.36 (CH), 126.66 (C), 125.17
(CH), 123.86 (CH), 113.27 (C), 112.74 (CH), 103.52 (C), 84.81 (CH2),
82.43 (C), 34.13 (CH2), 31.15 (CH2), 24.35 (CH2), 22.26 (CH2), 13.85
(CH3).
2.2. Fluorescein dibutyloxymethyl ether (5)
The following procedure for 5 is representative. To a solution of
tetrabutylammonium hydrogensulfate (274 mg, 0.808 mmol, 0.1 equiv)
and potassium carbonate (4.47 g, 32.3 mmol, 4.0 equiv) in 10 mL
H2O and 10 mL CH2Cl2 was added butyric acid (740 μL, 8.08 mmol,
1.0 equiv) and a solution of chloromethyl chlorosulfate (1.23 mL,
12.12 mmol, 1.5 equiv) in 5 mL CH2Cl2. The reaction was stirred
vigorously (~600 RPM) at room temperature under N2(g) for 4 h.
The mixture was then diluted to 30 mL H2O and 50 mL CH2Cl2 and
the layers were separated. The aqueous layer was extracted with
2×50 mL CH2Cl2, the organic layers were combined and dried over
MgSO4, filtered, and concentrated in vacuo. Note: material is extremely
volatile. The residue was filtered through a plug of silica gel, washed
with 100 mL CH2Cl2, and concentrated in vacuo, affording chloromethyl
butyrate as a colorless liquid (637 mg, 58%). 1H NMR (400 MHz, CDCl3)
δ 5.71 (s, 2H), 2.37 (t, J=7.4 Hz, 2H), 1.77–1.62 (m, 2H), 0.97
(t, J=7.4 Hz, 3H). 13C NMR (101 MHz, CDCl3) δ 171.59 (C), 68.53
(CH2), 35.81 (CH2), 18.06 (CH2), 13.46 (CH3).
2.5. TM0077 purification
A bacterial expression plasmid (pMH1) containing the TM0077
gene from T. maritima (UniprotKB: Q9WXT2; protein name
TM0077) was obtained from the Harvard Plasmid Repository
(Clone ID: TmCD00083900). This bacterial plasmid was transformed
into Escherichia coli BL21 (DE3) RIPL cells (Agilent, La Jolla, CA). A
saturated overnight culture of E. coli BL21 (DE3) RIPL (pMH1-

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M.K. Hedge et al. / Biochimica et Biophysica Acta 1824 (2012) 1024–1030
TM0077) in LB media containing ampicillin (100 μg/mL) and chloram-
phenicol (30 μg/mL) was used to inoculate LB-media (100 mL) con-
taining ampicillin (100 μg/mL) and chloramphenicol (30 μg/mL) and
the bacterial culture was grown with constant shaking (225 rpm) at
37 °C. When the OD600 reached 0.6–0.8, L-arabinose (Alfa-Aesar, Ward
Hill, MA) was added to 0.2% w/v and protein induction proceeded for
3 h. Bacterial cultures were collected by centrifugation at 6000 ×g for
10 min at 4 °C. The bacterial cell pellet was resuspended in PBS (2 mL)
and stored at −20 °C.
To disrupt the bacterial cell wall, lysozyme (50 mg; Sigma-Aldrich,
St. Louis, MO) was added and incubated with the cell pellet for 30 min
at 25 °C. The disrupted cell pellet was transferred to a 65 °C water
bath and incubated for 60 min. Precipitated proteins and cell material
were removed by centrifugation at 16,000 ×g for 10 min at 25 °C. Ni-
NTA agarose (500 μL; Qiagen, Valencia, CA) was added to the soluble
fraction and allowed to incubate at 25 °C for 15 min. The resin was
washed three times with PBS containing increasing concentrations
of imidazole (1 mL each of PBS containing 10 mM imidazole, 25 mM
imidazole, or 50 mM imidazole) and recollected by centrifugation at
1000×g for 1 min at 25 °C. TM0077 was eluted in PBS containing
250 mM imidazole (600 μL) and dialyzed against PBS overnight at
4 °C with constant stirring (10 K MWCO; Pierce, Rockford, IL).
The purity of TM0077 was confirmed by SDS–PAGE on a 4–20%
gradient gel and the purity was shown to be greater than 95%
(Supplementary Fig. 1). The concentration of TM0077 was determined
by measuring the absorbance at 280 nm and by calculating the ex-
tinction coefficient (ε280=55810 M−1 s−1 with all free cysteines)
using the modified Edelhoch and Gill/Von Hippel methods on Expasy
[14–16].
Corp., Northhampton, MA) and values for kcat, KM and kcat/KM calculated.
The background fluorescence of substrates 3–11 was determined by
measuring the kinetics of fluorogenic substrate hydrolysis by an active
site knockout of TM0077 (S188A).
2.8. Enzymatic activity against substrate 1 (7-aminocephalosporanic
acid)
The enzymatic activity of TM0077 toward substrate 1 (7-amino
cephalosporanic acid; 7-ACA; Sigma-Aldrich, St. Louis, MO; Fig. 1A)
was determined by following a previously published method [10]. Brief-
ly, the deacetylation of substrate 1 by TM0077 was determined by mea-
suring the change in pH over time using the pH indicator bromothymol
blue (BTB; Sigma-Aldrich, St. Louis, MO). Serial dilutions (1:2) of sub-
strate 1 (300 mM–0.14 mM final concentrations) were made in 5 mM
phosphate buffer pH 7.3 containing 0.01% BTB and the serial dilutions
transferred to a 96-well clear microplate (95 μL). To start the reaction,
TM0077 or variants of TM0077 (5 μL of a 2 mg/mL solution) were
added to a final concentration of 0.1 mg/mL. Plates were preshaken
for 30 s to insure complete mixing and the change in absorbance at
630 nm was measured for 5 min at 25 °C in a Biotek Synergy 2 fluores-
cent plate reader (Biotek Instruments; Winooski, VT). The initial veloc-
ity of the reaction was calculated using the following equation:
M= min ¼ ðdA=dTÞ ꢀ ððCB þ CInÞ=CInÞ ꢀ ð1=ðΔε ꢀ lÞ
where CB is the molar concentration of buffer (0.005 M), CIn is the molar
concentration of indicator (16 nM), Δε is the difference in the extinction
coefficient between protonated and deprotonated BTB (Δε630
=
15,700 M−1 cm−1), and l is the pathlength (1 cm). Values for kcat, KM,
and kcat/KM were determined by fitting the hyperbolic curve of sub-
strate 1 concentrations versus initial velocity to a standard Michaelis–
Menten equation using Origin 6.1.
2.6. Site-directed mutagenesis and purification
Variants of TM0077 were created by Quikchange II site-directed
mutagenesis using the manufacturer's suggested procedure (Agilent,
Santa Clara, CA) and the mutagenesis primers outlined in Supplementary
Table 1. Proper mutations in the TM0077 DNA sequence were confirmed
by DNA sequencing (Genewiz, South Plainfield, NJ). Plasmids with
TM0077 variants were transformed into E. coli BL21 (DE3) RIPL cells
and variants of TM0077 were purified using the same procedure as for
wild-type TM0077 (Supplemental Fig. 1). For variants of TM0077
with tyrosine and tryptophan substitutions, the extinction coeffi-
cients were adjusted to correct for the loss of a chromophore
(Tyr ε280=54320 M−1 s−1; Trp ε280=50310 M−1 s−1) [14–16].
2.9. Enzymatic activity against substrate 2 (p-nitrophenol acetate)
The enzymatic activity of TM0077 was measured against substrate 2
(p-nitrophenol acetate; NPA; Sigma–Aldrich; St. Louis, MO; Fig. 1B)
using a 96-well microplate assay [11]. Substrate 2 was prepared as
stock solutions in acetonitrile (2 M) and diluted into PBS containing
BSA (PBS–BSA; 0.1 mg/mL) to a starting concentration of 20 mM.
Eight serial dilutions (1:1; 110 μL into 220 μL total volume; 20 mM–
156 μM final concentrations) were made using PBS–BSA containing 1%
acetonitrile. Substrate 2 dilutions (95 μL) were transferred to a clear
96-well microplate (Corning, Lowell, MA).
2.7. Fluorescent enzymatic assays
TM0077 or variants of TM0077 (5 μL of 30 μg/mL) were added to the
diluted substrate 2 in the 96-well microplate (100 μL final volume and
final TM0077 concentration=1.5 μg/mL) and the absorbance change
(412 nm) was measured for 4 min at 25 °C on a Biotek Synergy 2 fluores-
cent plate reader (Biotek Instruments; Winooski, VT). The absorbance
change was converted to molar concentrations using the extinction coef-
ficient of NPA (Δε412 =1.4 mM−1 cm−1). The initial rates of the reac-
tions were measured in triplicate and plotted versus substrate 2
concentration. The saturation enzyme kinetic traces were fitted to a stan-
dard Michaelis–Menten equation using Origin 6.1 (OriginLab Corp.,
Northhampton, MA) and values for kcat, KM and kcat/KM calculated.
The enzymatic activity of TM0077 was measured against
flurogenic substrates (Fig. 1) using a 96-well microplate assay [13].
Fluorogenic substrates were prepared as stock solutions in DMSO
(10 mM) were diluted into PBS containing BSA (PBS–BSA; 0.1 mg/mL)
to a starting concentration of 10 μM. Eight serial dilutions (1:2; 60 μL
into 180 μL total volume) of each substrate (10 μM–4.6 nM final
concentrations) were made using PBS–BSA. Fluorogenic substrate
dilutions (95 μL) were transferred to a black 96-well microplate
(Corning, Lowell, MA).
TM0077 or variants of TM0077 (5 μL of 30 μg/mL) were added to the
diluted fluorogenic substrates in the 96-well microplate (100 μL final vol-
ume and final TM0077 concentration=1.5 μg/mL) and the fluorescence
change (λex =485 nm, λem =528 nm) was measured for 4 min at 25 °C
on a Biotek Synergy 2 fluorescent plate reader (Biotek Instruments;
Winooski, VT). The fluorescence change was converted to molar concen-
trations using a fluorescein standard curve (30 nM–0.23 nM), whose
fluorescence was determined simultaneously. The initial rates of the reac-
tions were measured in triplicate and plotted versus fluorogenic enzyme
substrate concentration. The saturation enzyme kinetic traces were fitted
to a standard Michaelis–Menten equation using Origin 6.1 (OriginLab
2.10. Thermal stability measurements
Similar to previously published methods [17,18], the thermal stability
of TM0077 and variants of TM0077 was determined using differential
scanning fluorimetry (DSF). Wild-type TM0077 and variants of TM0077
(0.5 mg/mL) were diluted in triplicate in PBS containing a 1:1000 dilution
of Sypro Ruby (Invitrogen, Carlsbad, CA). The samples was heated
from 50 °C to 100 °C at 2 °C/min in a thermocycler (Bio-rad C1000
Thermocylcer with CFX96 Real-time System, Hercules, CA) and the

M.K. Hedge et al. / Biochimica et Biophysica Acta 1824 (2012) 1024–1030
1027
Table 1
change in Sypro Ruby fluorescence followed over time ((λex
=
450–490 nm, λem =610–650 nm). The melting temperature (Tm) was
determined by plotting the first derivative of fluorescence versus temper-
ature and finding the temperature at the midpoint of the transition. As
in previous analyses [17], all graphs were normalized so that mini-
mum fluorescence was set to 0 and maximum fluorescence set to 1
(Supplemental Fig. 4).
Kinetic values for ester hydrolysis of substrates 1–11 by TM0077.
Substrate
Km (μM)
kcat/Km (M−1 s−1
)
1a
2b
3c
4
5
6
20800 350
760 100
3.7 0.2
0.97 0.2
0.41 0.1
1.0 0.2
830 150
23000 3200
14100 800
6400 1400
15
4
1.5 0.3
2.11. Sequence alignment
7
2.6 0.7
0.40 0.11
140 10
8
9
0.51 0.04
0.96 0.2
0.54 0.07
N/A
23
5
The sequence alignment of bacterial acetyl xylan esterases was
completed using the standard parameters for ClustalW 2.0.12 [19]. The
sequences used in the alignment were from T. maritima (UniprotKB:
Q9WXT2), Thermotoga neapolitana (UniprotKB: B9K758), Streptomyces
coelicolor (UniprotKB: Q9FC27), B. subtilis (UniprotKB: P94388),
B. pumilus (UniprotKB: Q9K5F2), Thermoanaerobacterium (UniprotKB:
O30361), Enterococcus faecalis (UniprotKB: Q835Y2), Clostridium
perfringens (UniprotKB: Q8XK07), Streptococcus pneumoniae (UniprotKB:
Q97PE0), and Leptospira interrogans (UniprotKB: Q8F524).
10
4.6 1.4
N/A
11d
a
Kinetic constants for substrate 1 (7-ACA) were determined by measuring the
change in pH due to acetate release during substrate 1 hydrolysis. Kinetic measure-
ments for each substrate were repeated three times and the values are given SE.
Data were fitted to a standard Michaelis–Menten equation to determine the values
for kcat, KM, and kcat/KM
.
b
Kinetic constants for substrate 2 (NPA) were determined by measuring the change
in A412 due to acetate release.
c
Kinetic constants for substrates 3–11 were determined by measuring the increase
in fluorogenic substrate fluorescence over time [13].
d
3. Results and discussion
Kinetic constant values for the cleavage of substrate 11 by TM0077 were below the
detection limit.
3.1. Determination of substrate specificity
Most bacterial esterases and lipases show broad substrate specific-
ity, highlighted by their ability to catalyze the hydrolysis of aliphatic
esters with varying structures [2,20,21]. Acetyl esterases also display
broad specificity and catalyze the deacetylation of a broad range of
substrates irrespective of the alcohol scaffold [8–11]. Yet, based on
the binding of the irreversible suicide inhibitors paraoxon and
phenylmethylsulfonyl fluoride and enzymatic activity measurements
using p-nitrophenyl propionate and p-nitrophenyl butyrate, acetyl es-
terases were known to be highly specific for acetate substrates, not
possessing the structural space available to accommodate larger
ester substrates [8,12,22].
To provide a comprehensive evaluation of the specificity of the
thermostable acetyl esterase TM0077, we utilized two distinct groups of
substrates (Fig. 1). To confirm the broad substrate specificity of TM0077
for the alcohols substituents, ester hydrolysis by TM0077 was measured
against three acetate-based substrates: 7-aminocephalosporonic
acid (7-ACA, 1), p-nitrophenyl acetate (NPA, 2), and fluorescein
di(acetoxymethyl ether) (3) (Fig. 1A). This group of substrates consists
of alcoholic functionalities with diverse complexity and steric hindrance
that closely mimic the size and polarity of its natural sugar substrates
[8]. To probe the narrow substrate selectivity of TM0077 for esters, we
synthesized a focused series of esterase substrates all based on the
fluorescein scaffold (Fig. 1B). These nine fluorogenic enzyme substrates
examined the ability of TM0077 to adapt to slight differences in carbon
chain length (substrates 4–7), to accommodate steric bulk and changes
in electronic structure (substrates 8 and 9), and to cleave esters with
quaternary α‐carbons (substrates 10 and 11). Compared to common
ester substrates, including p‐nitrophenyl esters and fluorescein diacetate,
the diacyloxymethyl ether fluorescein substrates have significantly
improved chemical stability, which increases the sensitivity of the
enzymatic measurements and permits measurement of even very low
enzymatic rates (Table 1 and Fig. 2A) [13].
(substrate 3) decreases the catalytic rate by 2.2-fold; the catalytic
activity continues to drop substantially with each additional carbon
(substrates 5–7). Fig. 2B clearly illustrates the very narrow substrate
specificity of TM0077 even for similar straight chain esters. Although
the KM values shift slightly with carbon chain length, the majority of
the decreased catalytic efficiency is caused by a substantial decrease in
substrate turnover (kcat) (Fig. 2C). As the steric hindrance surrounding
the ester carbonyl increases from substrate 8 to substrate 11, the enzy-
matic efficiency also decreases significantly, again caused by decreased
substrate turnover with the hydrolysis of substrate 11 falling below the
detection limit of the experiment (Table 1). Substitution of Ser188 with
alanine completely abolished the activity of TM0077 against substrate 3
and defined a background level of substrate hydrolysis for each
substrate (Supplemental Fig. 2). These results confirm the proposed
specificity of TM0077, as it is able to catalyze the rapid hydrolysis of
a range of substrates independently of the alcohol moiety, but with
narrow selectivity for acetate esters.
The substrate turnover (kcat) value measured for the hydrolysis of
substrate 3 by TM0077 is slower than the turnover of substrates 1 and
2 and likely reflects the pKa value of the hemiacetal leaving group on
substrate 3, estimated to be similar to formaldehyde hydrate
(pKa =12.78) [13]. Thus, hydrolysis of the acyl-enzyme intermediate
formed between TM0077 and substrate 3 would be much slower than
the hydrolysis of the intermediate formed with p-nitrophenol acetate
(pKa=7.18) [6,13]. This stability in the ester bond is necessary to main-
tain the extremely low background hydrolysis of these fluorogenic
substrates [13,23]. The lower KM value for substrate 3 may also signify
that the hydrolysis of the acyl-enzyme intermediate is the rate-
determining step in the reaction, instead of the formation of the covalent
acyl-enzyme intermediate [6]. Although the relative values for kcat and
KM for TM0077 hydrolysis of substrate 3 shifts from substrates 1 and 2,
the overall ratio of enzymatic catalysis kcat/KM remains fairly constant
for acetate esters (Table 1).
Kinetic measurements show TM0077 is active against all three
acetyl substrates (1–3; Fig. 1A) with kcat/KM values that vary by only
three-fold (830 M−1 s−1 for substrate 1 to 2.3×104 M−1 s−1 for
substrate 2; Table 1), confirming its broad specificity irrespective of
the alcohol scaffold. Experiments using a series of diverse ester
substrates (compounds 3–11; Fig. 1B) show that TM0077 displays a
strong preference for acetate groups. Nevertheless, the high chemical
3.2. Structural basis for substrate specificity
To determine the substrate binding residues that contribute to the
unusual substrate specificity of TM0077, the positioning and sequence
conservation of potential substrate-binding residues were characterized.
Crystal structures of TM0077 from T. maritima (PDB ID: 1VLQ; 3M81;
3M82; 3M83) depict a deep and well-defined substrate-binding pocket
where the catalytic triad is found within a sheltered, smaller gorge
stability of the fluorescein substrates enabled determination of kcat
/
KM values for each substrate (Table 1; Fig. 2A). Increasing the carbon
chain by a single carbon (substrate 4) from the acetate ester

1028
M.K. Hedge et al. / Biochimica et Biophysica Acta 1824 (2012) 1024–1030
(Fig. 4A). This minor affect of multiple nonconservative substitutions
on the catalytic activity of TM0077 suggests that the substrate-binding
surface has been evolutionarily tuned to provide robust substrate recog-
nition and catalytic activity [24]. Similar large, hydrophobic binding
pockets with a minimal number of hydrogen bonding groups are
known to facilitate broad substrate specificity [25,26].
The large binding pocket of TM0077 then narrows at one end to a
small gorge defined by the side-chain atoms on Tyr92, Phe213, Pro228
and Ile276 (Fig. 3) where irreversible inhibitors and transition state
mimics are known to bind [8,12]. Among these amino acids, proper
substrate positioning by Pro228 and Ile276, whose side-chains define
the sides of the catalytic gorge, are most important to the catalytic activ-
ity of TM0077 (Fig. 3C and D). Substitution of either Pro228 or Ile276
significantly reduced (>10-fold) the catalytic efficiency of TM0077
toward all three acetate substrates (Fig. 4A). Thus, the large surface
area of the binding pocket determines the broad specificity of TM0077
toward diverse alcohol scaffolds, but only Pro228 and Ile276 appear to
play a major role in controlling the narrow specificity of TM0077 toward
acetate containing substrates.
As further confirmation of their roles in the deacetylation reaction,
these amino acid substitutions (Y92A, F213A, P228A, and I276A)
were combined into double alanine-substituted variants and the
catalytic activity of the variants measured against substrate 3 (Y92A
P228A TM0077, F213A P228A TM0077, or P228A I276A TM0077).
Each of the double alanine variants further decreased the catalytic
activity of TM0077 toward substrate 3 (Supplemental Table 3) and all
combinations of double alanine variants were worse than any single
substitution individually. Overall, the substrate specificity of TM0077
was defined by only a few key substrate-binding residues.
3.3. Structural basis for the narrow substrate specificity for esters
To understand the structural features that control the narrow
substrate specificity of TM0077 for acetate esters, we searched for
amino acid substitutions that could broaden the substrate specificity
[24], and focused on potential plasticity residues near the active site
[27]. Similar to the reaction with diverse alcohols, Pro228 and
Ile276 had the largest affect on the hydrolytic rate and the greatest
control of the narrow substrate specificity of TM0077 (Fig. 4B and
Supplemental Table 6). Whereas, wild-type TM0077 shows fairly
narrow specificity toward acetyl and propyl esters with steep drops in
catalytic activity toward sterically bulky or hindered substrates like
substrates 8 and 9 (Table 1 and Fig. 1B), the P228A and I276A variants
of TM0077 retained much higher relative catalytic activities toward
substrates 8 and 9 (Fig. 4B and Supplemental Table 6). For instance,
P228A decreased the activity of TM0077 against substrate 3 by 33-
fold, but it only lowered the activity against substrate 8 by 8-fold. For
substrate 9, the P228A variant had 4-fold higher enzymatic efficiency
Fig. 2. Kinetic measurements for the substrate specificity of TM0077: A) hydrolysis of
fluorogenic esterase substrates by TM0077. Kinetic values are shown SE and the
values are fitted to the standard Michaelis–Menten equation. The substrates are
shown with the following symbols: substrate
3 (○); substrate 4 (●); substrate
8 (□); substrate 9 (■); and substrate 10 (△). Data fitted using Origin 6.1 (OriginLab
Corp., Northampton, MA). B) Substrate specificity of TM0077 based on carbon chain
length. Overall substrate specificity of TM0077 plotted versus carbon chain length
(substrates 3–7) where the carbonyl carbon is defined as carbon 1. Constants deter-
mined as in Fig. 2A. C) Individual rate constants for the hydrolysis of substrates 3–7
versus the carbon chain length. Values for kcat shown with (□) and scale given on
the left y-axis. Values for Km shown with (♦) and scale given on the right y-axis. All
values are shown SE.
(Fig. 3A–C) [8]. By combining the structural information about the
binding pocket residues and their amino acid conservation, eight amino
acids residues (Asn88, Tyr92, Trp102, Trp105, Trp124, Phe213, Pro228,
and Ile276) were chosen for this study that define the walls of the larger
substrate binding pocket, are located near the nucleophilic serine, and
show reasonable sequence conservation (Table 2; Fig. 3). These eight
amino acids were hypothesized to regulate the unusual substrate speci-
ficity of TM0077. Each of these amino acids was then converted separate-
ly to alanine and the kinetics of enzymatic catalysis by these variants
of TM0077 and combinatorial double alanine variants of TM0077
were measured against substrates 1–3 to determine their importance to
alcohol recognition and to substrates 8 and 9 to determine their impor-
tance to ester recognition (Fig. 4). Substrates 8 and 9 were chosen for
further analysis of ester recognition because they contain branched
ester moieties and displayed low, but measurable, catalytic activity with
the wild-type enzyme (Table 1).
(kcat/KM=95 M−1 s−1
) than wild-type TM0077 and the double
alanine variants (Y92A/P228A, F213A/P228A, and I276A/P228A) had
only small differences in kcat/KM values (0.8-fold to 1.6-fold) from wild-
type TM0077 and these differences were smaller than either of the single
mutations independently.
Residues Ile276 and especially Pro228 can thus be classified as plas-
ticity residues, as single substitutions shifted the substrate specificity of
TM0077 [27]. Although the P228A and I276A variants are trading low
substrate turnover (low kcat) for broadened specificity, such trade-offs
are common in plasticity residues near the active site that make direct
contact with the substrate [28]. Pro228 is an unusual plasticity residue
due to its proximity to the active site and its lack of conformational
flexibility [24]. Pro228 is in the rigid cis conformation and its γ and δ
carbons define the surface area, directly opposite the nucleophilic
serine (Figs. 3B and C). Thus, the rigid substrate-binding surface of
Pro228 may correctly position the substrate for reaction with Ser188.
Substitution of Pro228 likely removed steric hindrance in the binding
pocket to allow the bulkier substrates to access the active site gorge
Kinetic measurement with the TM0077 variants shows that the
large hydrophobic section of the TM0077 binding pocket contributes
little to the catalytic activity of TM0077, as substitution of Asn88,
Trp102, Trp105, and Trp124 with alanine changed the catalytic effi-
ciency toward the three acetate substrates 1–3 by less than 10-fold

M.K. Hedge et al. / Biochimica et Biophysica Acta 1824 (2012) 1024–1030
1029
Fig. 3. Substrate recognition by TM0077: A) monomer of TM0077. Surface representations of one monomer of the hexameric structure of TM0077. B) Substrate binding pocket of
TM0077. Surface representation of the binding pocket of TM0077 with the catalytic serine nucleophile (Ser188) highlighted in tan. C) Ball and stick representation of the substrate
binding pocket of TM0077. TM0077 is oriented identically to part (A). Residues shown in sticks were substituted with alanine and in tan are the catalytic triad residues. Residues
colored based on their affect on the enzymatic efficiency of TM0077 hydrolysis of substrate 3. The residues highlighted in red had kcat/Km value changes from wild-type greater than
ten-fold, in orange had a greater than two-fold change, and in purple had less than a 2-fold change. D) Surface representation of relative kinetic results. Coloration is identical to
panel C. Figures made using PDB ID: 1VLQ and PyMol.
and increased the structural plasticity of TM0077 [29]. The γ2 carbon on
Ile276 then defines the remaining surface area of the binding pocket
and packs against the catalytic histidine (His303). Although, isoleucine
is not fully conserved at this position in CE7 acetyl esterases, the
branched methyl group that forms the binding surface is completed
conserved (Table 2).
changes at Pro228 and Ile276 present potential starting points for
future directed evolution studies, as enzymes with broad specificity
increase the likelihood of uncovering novel enzyme functionalities
[27,30]. With its broad substrate specificity to diverse alcohol scaffolds
and robustness to substitution, this broadened specificity toward steri-
cally hindered ester bonds could make TM0077 a valuable biocatalyst.
4. Conclusion
Acknowledgments
Together these precise enzyme kinetic measurements combined
with the structural and mutagenesis results confirm the unusual
substrate specificity of acetyl esterases from CE7 and suggest strict
evolutionary selection for the broad substrate specificity for alcohol
substituents and for the narrow substrate specificity for ester substit-
uents. Only limited substitutions to the substrate-binding pocket
significantly affected this substrate specificity profile and only two
plasticity residues, Pro228 and Ile276, control the narrow substrate
specificity of TM0077 for acetate esters. Variants of TM0077 with
We thank Andrea Bolen, Ali Donovan, Jessi Grostefon, Erin
Hemmelgarn, Stephanie Knezz, Brent Kochert, Alicia Phelps, Bri Richine,
and Ariel Tyring for assistance with preliminary enzyme purification
and enzymatic analysis. M.A.H. was supported by a Butler Summer
Institute Scholarship. A.M.G. was supported by a Holcomb grant from
Butler University. R.J.J. was supported by start-up funds and a Holcomb
grant from Butler University and a Senior Research Grant from the Indi-
ana Academy of Sciences. C.T.A. and L.D.L. were supported by the How-
ard Hughes Medical Institute.
Table 2
Amino acid conservation among acetyl esterases from CE7 based on ClustalW alignment to TM0077 from Thermotoga maritima.
Amino acid residue number in TM0077a
Identity (%)
88
92
102
105
124
145
186–192
213
228
229
276
Thermotoga maritima
Thermotoga neapolitana
Streptomyces coelicolor
Bacillus subtilis
Q
Q
E
K
R
R
D
E
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
W
W
H
M
I
K
F
N
M
I
W
W
W
W
W
Y
W
Y
F
W
W
W
–
M
M
M
M
M
I
M
I
I
GGSQGGG
GGSQGGG
GASQGGG
GGSQGGG
GGSQGGA
GPSQGGG
GGSQGGG
GGSQGGA
GASQGGA
GKSMGAS
F
F
F
Y
Y
F
S
F
F
N
P
P
P
P
P
N
G
D
E
S
Y
Y
Y
Y
Y
Y
Y
Y
Y
M
I
I
90
54
42
41
33
31
30
30
25
T
V
I
V
I
I
V
I
Bacillus pumilus
–
Thermoanaerobacterium
Enterococcus faecalis
Clostridium perfringens
Streptococcus pneumoniae
Leptospira interrogans
–
–
–
H
Y
–
L
L
F
a
The amino acid sequence of TM0077 was aligned using ClustalW to nine other acetyl xylan esterases [19]. The sequence identity of the amino acids in the TM0077 binding pocket
is highlighted. The amino acid numbering corresponds to the amino acid numbering in TM0077. The full sequence alignment is given in Supplemental Fig. 3.

1030
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