Directed evolution of a pyruvate aldolase to recognize a long chain acyl substrate

Article abstract of DOI:10.1016/j.bmc.2011.08.056

The use of biological catalysts for industrial scale synthetic chemistry is highly attractive, given their cost effectiveness, high specificity that obviates the need for protecting group chemistry, and the environmentally benign nature of enzymatic procedures. Here we evolve the naturally occurring 2-keto-3-deoxy-6-phosphogluconate (KDPG) aldolases from Thermatoga maritima and Escherichia coli, into enzymes that recognize a nonfunctionalized electrophilic substrate, 2-keto-4-hydroxyoctonoate (KHO). Using an in vivo selection based on pyruvate auxotrophy, mutations were identified that lower the K_M value up to 100-fold in E. coli KDPG aldolase, and that enhance the efficiency of retro-aldol cleavage of KHO by increasing the value of k_cat/K _M up to 25-fold in T. maritima KDPG aldolase. These data indicate that numerous mutations distal from the active site contribute to enhanced 'uniform binding' of the substrates, which is the first step in the evolution of novel catalytic activity.

Full text of DOI:10.1016/j.bmc.2011.08.056

Bioorganic & Medicinal Chemistry 19 (2011) 6447–6453
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Directed evolution of a pyruvate aldolase to recognize a long chain
acyl substrate
Manoj Cheriyan , Matthew J. Walters , Brian D. Kang , Laura L. Anzaldi , Eric J. Toone b,c,
Carol A. Fierke
a
a
b
a
c
a,d,
⇑
Department of Chemistry, University of Michigan, Ann Arbor, MI 48109, USA
Department of Biochemistry, Duke University Medical Center, Durham, NC 27710, USA
Department of Chemistry, Duke University, Durham, NC 27708, USA
b
c
d
Department of Biological Chemistry, University of Michigan, Ann Arbor, MI 48109, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
The use of biological catalysts for industrial scale synthetic chemistry is highly attractive, given their cost
effectiveness, high specificity that obviates the need for protecting group chemistry, and the environmen-
tally benign nature of enzymatic procedures. Here we evolve the naturally occurring 2-keto-3-deoxy-6-
phosphogluconate (KDPG) aldolases from Thermatoga maritima and Escherichia coli, into enzymes that
recognize a nonfunctionalized electrophilic substrate, 2-keto-4-hydroxyoctonoate (KHO). Using an
Received 23 June 2011
Revised 19 August 2011
Accepted 26 August 2011
Available online 30 August 2011
in vivo selection based on pyruvate auxotrophy, mutations were identified that lower the K
to 100-fold in E. coli KDPG aldolase, and that enhance the efficiency of retro-aldol cleavage of KHO by
increasing the value of kcat/K up to 25-fold in T. maritima KDPG aldolase. These data indicate that
numerous mutations distal from the active site contribute to enhanced ‘uniform binding’ of the sub-
strates, which is the first step in the evolution of novel catalytic activity.
Ó 2011 Elsevier Ltd. All rights reserved.
M
value up
Keywords:
Biocatalysis
Protein engineering
Thermostable enzyme
Substrate specificity
Random mutagenesis
M
1
. Introduction
aldolase from a number of species includes a variety of 3 and 4 car-
bon sugars such as erythrose and lactaldehyde with a preference for
6
,7
Protein engineering is a rapidly growing field of study where
phosphorylated substrates. These enzymes also accept 2-, 3-, and
4-pyridine carboxaldehyde as electrophilic substrates with low effi-
naturally occurring enzymes are altered to improve activity for
industrial and laboratory applications. The advantages of biocata-
lysts over traditional chemical synthesis methodologies are their
exquisite chemo-, stereo- and regio-selectivity, high rates of catal-
6
,7
ciency, but do not catalyze aldol addition reactions with unfunc-
tionalized aldehydes such as butyraldehyde and valeraldehyde.
Since the synthesis of many valuable natural products require car-
bon–carbon bond formation using unfunctionalized aldehydes (e.g.,
1
ysis, and reactivity under mild and chemically benign conditions.
1
0
11
The former properties are highly desirable for pharmaceutical
applications where the production of enantiomerically pure com-
pounds is critical, while the latter property offers cost savings for
large-scale industrial applications.
syrigomycin
and lyngbyabellin ), and since phosphorylated
12
compounds are inconvenient for most synthetic schemes, we
sought to develop enzymes with improved catalytic activity to-
wards long chain acyl substrates.
Much research has focused on the development of aldolases as
synthetic reagents because of the centrality of carbon–carbon bond
formation in synthetic chemistry.2 We have focused our efforts on
evolving Escherichia coli (EC) 2-keto-3-deoxy-6-phosphogluconate
To broaden the substrate tolerance of the KDPG aldolases, we em-
ployed directed evolution to selectively enhance the ability of the
enzyme to catalyze aldol addition with 2-keto-4-hydroxyoctonoate
(KHO), an unnatural electrophile that represents a first step towards
aldol synthesis with long chain acyl aldehydes. As previously
reported, our selection uses a pyruvate kinase (pykA and pykF)
–4
(
KDPG) aldolase and a closely related isoform from the thermo-
philic bacteria Thermatoga maritima (TM). These aldolases preferen-
tially catalyze si-stereo facial aldol addition reactions between
pyruvate and a range of aldehydic electrophiles5 (Fig. 1). The
types of structures afforded by the enzyme have proven useful in
the synthesis of nikkomycins.8 The substrate tolerance of KDPG
–7
,9
⇑
Corresponding author.
E-mail address: fierke@umich.edu (C.A. Fierke).
Figure 1. KDPG aldolase-catalyzed reversible aldol reaction.
0
968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.08.056

6
448
M. Cheriyan et al. / Bioorg. Med. Chem. 19 (2011) 6447–6453
deficient cell line that is unable to grow on minimal media supple-
mented with ribose as the sole carbon source13,14 (Fig. S1).
Addition of exogenous pyruvate rescues the auxotrophic phenotype.
Furthermore, we have previously demonstrated that cleavage
generated by error prone PCR, allowing for rapid incorporation of
one to five amino acid changes over the 0.6 kilobase gene, resulting
1
4
in an overall library complexity of 10 . Although it is not
currently feasible to exhaustively screen such a large library using
in vivo selection methodologies, literature examples suggest that
meaningful improvements in catalytic activity can be obtained
0
of an unnatural substrate, 2-keto-4-hydroxy-4-(2 -pyridyl)butyrate
(
KHPB), catalyzed by a KDPG aldolase mutant can produce enough
1
4
3
5
17,18
pyruvate to rescue cell growth, validating our selection approach.
by screening libraries of 10 –10 clones.
To examine the hypothesis that higher protein stability would
allow for sampling of a wider range of sequence space while
maintaining a properly folded structure, we carried out directed
evolution experiments on both the T. maritima and E. coli KDPG
aldolase enzymes. The E. coli and T. maritima KDPG aldolases have
2.1. Directed evolution and selectionofT. maritima KDPGaldolase
Libraries of T. maritima aldolase variants were generated by er-
ror-prone PCR, using a commercially available low fidelity poly-
merase (Mutazyme II, Stratagene). Compared to mutagenesis
3
4% identity (pairwise alignment at http://blast.ncbi.nlm.nih.gov);
2
+
the sequence differences afford the TM KDPG aldolase both thermo-
methods using Taq DNA polymerase with Mn , Mutazyme II pro-
6
19
stability and minor differences in substrate specificity. Using ran-
vides a more even distribution of mutations at all nucleotides.
dom mutagenesis and selection we identified KDPG aldolase
mutants that improve the efficiency for cleavage (kcat/K ) of KHO
M
by up to 25-fold. The majority of mutations identified are located
far from the active site, suggesting that subtle long-range remodel-
ing of the active site is the mechanism of adaptation, consistent with
Albery and Knowles’ model of ‘uniform binding’.15
Plasmids that confer growth of PB25 cells on ribose were identified
by plating on ribose/KHO minimal media at 34 °C (optimal growth
temperature for PB25 cells). Colonies that grew within 120 h of
plating were isolated. Based on transformation controls, the
approximate size of the total number of variants screened in this
7
selection was 4 ꢁ 10 .
Plasmid DNA from colonies recovered in the first round of selec-
tion was isolated and the mutant eda genes were sequenced
2
. Results
(
shown in Table 1, TM 1–3). These plasmids were retransformed
According to the Haldane relationship,16 the efficiency for catal-
into PB25 cells and subjected again to the selection conditions to
confirm that the growth phenotype was dependent on the plasmid
encoded eda gene. Only plasmids that engendered growth were
analyzed further. These isolated mutants contain 2–9 altered
nucleotides in the KDPG gene leading to 2–7 amino acid changes
M
ysis (kcat/K ) of the forward and reverse reactions is related by the
overall equilibrium constant for the reaction, Keq. Thus changes in
the efficiency of retro-aldol cleavage for a given substrate also re-
flect the efficiency of the enzyme to perform aldol addition reac-
tions. Similarly, since Keq is unchanged by mutation of the
enzyme, enhancements in the efficiency of retro-aldol cleavage re-
flect a comparable improvement in the synthetic direction. So the
steady-state kinetic parameters for retro-aldol cleavage of KHO
catalyzed by wild-type KDPG aldolase were measured to provide
a baseline for evaluation of improvements in catalytic efficiency
(Table 1). Unexpectedly, one selected plasmid contained a TM
eda gene with a frameshift mutation (TM 3) in the 2nd codon of
the gene. Expression of nearly full-length protein is still observed
for this mutant since the third codon is a redundant start. However,
a Western blot of whole cell lysates of each first round mutant
indicated that the selected mutations decrease expression of the
mutants up to 10-fold as compared to the wild-type enzyme
made during mutagenesis. The kinetic parameters for kcat/K
the cleavage of KHO catalyzed by the T. maritima and E. coli KDPG
aldolases are 2.2 ± 0.3 M
M
for
(Fig. 2A, Table 1).
ꢀ
1
ꢀ1
ꢀ1 ꢀ1
To examine whether the selection procedure identified KDPG
s
(34 °C) and 14 ± 4 M
s
(25 °C),
aldolases with increased catalytic efficiency, we analyzed the ki-
netic parameters for KHO cleavage catalyzed by these proteins.
Each KDPG aldolase mutant was expressed in PB25 cells grown
on rich media and purified using a C-terminal 6 ꢁ His-tag. Proteins
of approximately 24 kDa, as determined by SDS–PAGE analysis,
were obtained for all TM mutants. The degree of fitness towards
cleavage of the KHO substrate was analyzed by measuring the stea-
respectively. Compared to the cleavage of the natural substrate,
KDPG, the catalytic efficiency for cleaving KHO is decreased by
4
6
1
0 –10 -fold, indicating that KHO is a very poor substrate for these
enzymes, presumably due in part to unfavorable interactions be-
tween the hydrophobic chain of KHO and the hydrophilic phos-
phate binding site of the enzyme.9 Furthermore, the initial
velocity has a nearly linear dependence on the concentration of
KHO up to 50 mM for both enzymes, indicating that the value of
M
K is significantly larger than 50 mM. (Concentrations of KHO
above 50 mM lead to noticeable precipitation of the coupling en-
zyme.) A fit of the Michaelis–Menten equation to these data pro-
Table 1
T. maritima KDPG aldolase mutants selected for enhanced catalysis of KHO cleavage
ꢀ1
vided estimated values for
k
cat of 0.4 ± 0.05 s
of 200 ± 30 mM (TM) and 150 ± 50 mM
are a lower limit and could reflect the sol-
ubility of the substrate. Although K frequently includes multiple
(TM) and
Name
Mutations
Relative expression level
ꢀ1
2
.0 ± 0.6 s (EC), and K
M
TM 1
TM 2
TM 3
G34R/V172A/R190I
K9T/G83E
++++
+++
++
(
EC). These values for K
M
c
Deletion of first two AA,
M
L16P/V31A/S82C/G83V/F99L/W167R
V136M
A30V
I52F/V135M/T185A
P109S
terms and does not simply reflect binding affinity, the high value
of this parameter coupled with the low turnover number suggests
that wild-type KDPG aldolase binds KHO in a productive conforma-
tion with low affinity. Furthermore, the low value of kcat suggests
that KHO bound to KDPG aldolase is not positioned to maximize
catalytic efficiency. With such low catalytic activity, overexpres-
sion of either T. maritima or E. coli KDPG aldolase is not sufficient
a
TM 4
++
++
+
+
+
a
TM 5
TM 6b
b
TM 7
TM 8b
N150I
+
+++ 100%;+++50%;++10%;+ 5% expression relativeto wild-type TMAinTMEDA-pUC
plasmid.
1
4
a
to rescue growth of the PB25 cell line on ribose/KHO media.
These proteins were selected in a pUC plasmid where the expression of wild-
type TMA is 10% of the expression in the TMEDA-pUC plasmid.
To identify mutants of KDPG aldolase with enhanced catalytic
activity towards retro-aldol cleavage of KHO, we prepared libraries
of T. maritima and E. coli KDPG aldolases by random mutagenesis
and selected for cells that showed growth on KHO. Libraries were
b
These proteins were selected in a pUC plasmid where the expression of wild-
type TMA is ꢂ2% of the expression in the TMEDA-pUC plasmid.
c
Signifies a frameshift mutation where expression of the gene is initiated at the
third amino acid.

M. Cheriyan et al. / Bioorg. Med. Chem. 19 (2011) 6447–6453
6449
Figure 2. (A) Western blot of His-tagged KDPG aldolase mutants after the first round of selection probing with an anti-His antibody and; (B) dependence of the initial velocity
for pyruvate formation on the concentration of KHO catalyzed by wild-type T. maritima KDPG aldolase (open circle) and mutants TM 5 (open square) and TM 8 (filled
diamond), measured using a lactate dehydrogenase coupled assay at 34 °C.
Table 2
region between the ꢀ10 and ꢀ35 region of the promoter decreases
Kinetic parameters for KHO cleavage catalyzed by selected TM KDPG aldolase
mutantsa
protein expression ꢂ50-fold. Using these plasmids, libraries of TM
KDPG aldolase genes were generated by error-prone PCR amplifi-
cat (sꢀ1)
K
(mM)
k
cat/K
(M
ꢀ1
s
ꢀ1
)
Relative kcat/K
M
Name
k
M
M
7
cation. A library of 1 ꢁ 10 plasmids was selected for conferring
wt-TMA
0.40 ± 0.05
0.07 ± 0.01
0.5 ± 0.1
0.14 ± 0.10
0.36 ± 0.02
0.75 ± 0.10
0.13 ± 0.02
1.2 ± 0.3
200 ± 30
9 ± 2
2.2 ± 0.3
9.3 ± 1.8
5.5 ± 1.0
7 ± 4
16 ± 2
54 ± 10
17 ± 6
1
4
2.5
3
7
25
8
11
growth on ribose/KHO minimal media in PB25 cells and a total of
TM 1
TM 2
TM 3
TM 4
TM 5
TM 6
TM 8
5
plasmids that allow for growth within 120 h were identified (Ta-
90 ± 20
20 ± 14
23 ± 4
14 ± 5
8 ± 3
ble 1, TM 4–8). In this case, expression of the selected mutant pro-
teins is comparable to the reduced level observed for the wild-type
enzyme in these plasmids.
Mutant KDPG aldolase genes were subcloned into a pET expres-
sion plasmid and the protein was expressed in XL1-Blue cells
grown in rich media with IPTG induction. Mutant TM 7 precipi-
tated during purification so kinetic data could not be obtained
for this mutant. The kinetic parameters for catalysis of KHO cleav-
age for the other four mutants (Table 2) demonstrate that the value
49 ± 19
25 ± 5
a
Initial velocity for pyruvate formation was measured using a lactate dehydro-
genase coupled assay at 34 °C. Steady state kinetic parameters were determined by
fitting of the Michaelis–Menten equation to the initial velocity as a function of the
substrate concentration.
of kcat/K
ments in K
M
increases up to 25-fold, from a combination of improve-
(4 to 25-fold), as well as changes in the turnover num-
M
ber (0.3–3-fold). The activity of TM 5 showed the greatest
improvement (Fig. 2B), due primarily to a >14-fold decrease in
dy-state kinetic parameters at the selection growth temperature,
3
4 °C. Values of kcat/K
M
for KHO cleavage increased for all three
the value of K
substrate contacts occur in this mutant. TM 8 shows a threefold
increase in kcat in addition to a fourfold decrease in K (Fig. 2B)
M
for KHO, suggesting that new, beneficial protein-
mutants by 2–4-fold (Table 2).
The most improved mutants, TM 3 and TM 1, increase the value
M
of kcat/K
M
by three- and fourfold and decrease the value of K
M
by at
making it one of the most promising mutants for biocatalysis,
where maximal turnover under saturating conditions is critical.
least 10 and 22-fold, respectively, compared to the corresponding
parameters for KHO cleavage catalyzed by wild-type enzyme.
Interestingly, kcat decreases up to sixfold for these mutants, imply-
ing that the selective pressure in vivo is for increased catalytic effi-
2.3. Directed evolution and selection of E. coli KDPG aldolase
ciency (kcat/K
M
) rather than turnover (kcat). This result suggests
For comparison, we constructed a library of E. coli KDPG aldol-
ase variants and selected for enhanced cleavage of KHO. Mutagen-
esis was limited to two to three mutations per KDPG aldolase gene
and the library was incorporated into a pUC plasmid that contains
a deletion at the ꢀ35 RNA polymerase binding region (TTTACA to
TTACA) to decrease protein expression and enhance the selection
stringency. Plasmids were selected for conferring growth of PB25
cells within 120 h on minimal ribose/KHO plates at 34 °C. Based
on transformation controls, the approximate size of the library
that the in vivo concentration of KHO under selective conditions
is low, a conclusion consistent with the nature of KHO, a charged
molecule that cannot easily pass through a lipid bilayer by simple
diffusion. Based on these data we presume that the cellular con-
centration of KHO is in the
conditions.
lM to nM range under the selection
2
.2. Selection of T. maritima KDPG aldolase mutants under
7
increased stringency
screened in each case was ꢂ10 members. Plasmids from recov-
ered colonies were isolated and sequenced, and used to template
subsequent rounds of error-prone PCR without extensive kinetic
analysis. After five sequential rounds of mutagenesis and selection,
the mutant aldolase proteins from the final round of selection were
purified and assayed for catalysis of cleavage of KHO.
To both increase the selection pressure and to mitigate any
cellular strain created by overexpression of KDPG aldolase, we con-
structed plasmids that confer decreased KDPG aldolase expression
by making mutations in the RNA polymerase binding region of the
lac promoter (between ꢀ10 and ꢀ35) that have previously been
shown to significantly decrease gene expression.20 A TMEDA-pUC
plasmid containing an A to C mutation at the ꢀ35 RNA polymerase
binding region decreases TM KDPG aldolase expression about 10-
fold, while a single guanosine nucleotide insertion into the 18 bp
Unfortunately, 2 of the 3 selected mutant proteins lost substan-
tial activity during purification and did not yield reproducible ki-
netic results. The remaining mutant, EC 11, also showed limited
stability. This mutant contained substitutions both proximal and
distal to the active site that improved the value of K
M
for KHO

6
450
M. Cheriyan et al. / Bioorg. Med. Chem. 19 (2011) 6447–6453
Table 3
Kinetic parameters for KHO cleavage catalyzed by selected EC KDPG aldolase
mutantsa
Name
Mutations
k
cat (sꢀ1)
K
M
(mM)
kcat/K
M
ꢀ
1 ꢀ1
(
M
s )
Name
EC 9
None
V17L/E51K/P94L/
G124M/L173M
2 ± 0.6
—
150 ± 50
—
14 ± 4
>0.1
EC 10
EC 11
V17L/V23I/E51K/
P94L/F134I/L173M/D190E
V17L/A53T/P94L/
V118L/A137T/L173M
—
—
>0.2
0.01 ± 0.005
1.5 ± 0.4
10 ± 3
a
Initial velocity for pyruvate formation was measured using a lactate dehydro-
genase coupled assay at 25 °C. Steady state kinetic parameters were determined by
fitting of the Michaelis–Menten equation to the initial velocity as a function of the
substrate concentration.
cleavage by 100-fold to 1.5 mM. However, the turnover number
also decreased by a similar amount resulting in a mutant with a
Figure 3. Schematic diagram of KHO binding to TM KDPG aldolase.
k
M
cat/K value comparable to that of wild-type enzyme (Table 3).
structural changes to the neighboring b-sheets that line the active
site. The most remarkable of these mutants is TM 5, in which a sin-
gle A30V mutation results in a 25-fold improvement in the value of
These measurements may underestimate the value of kcat due to
the instability of the protein.
M
kcat/K . A30 is located on a-helix 1 with the side chain of this res-
3
. Discussion
idue directed towards the interface with helix 8 (Fig. 4A). Helix 1
attaches to the loop containing the pyruvate-binding residue
R17, while helix 8 is attached to the loop containing the phosphate
binding site residue S179 (equivalent position to EC S184). Substi-
tuting alanine with valine at this site may increase the distance be-
tween these two helices and alter the position of the residues on
the adjoining loops to enhance catalysis of KHO cleavage (Fig. 4A).
Several recovered mutations are near the active site (L16P,
P109S, and N150I) and may directly affect catalytic residues and/
or directly contact the substrate. Of note is mutation L16P (TM
3), located at the C-terminal end of b-sheet 1 (Fig. 4A) adjacent
to R17, the arginine that interacts with the carboxylate moiety of
KHO. Another interesting mutation is P109S (TM 7), which was
identified as a single mutation in the selection. Proline 109 is lo-
cated on the inward face of b-sheet 5, and causes a kink in the
sheet adjacent to the active site lysine 129. P109 is located 4 Å
away from the catalytic lysine; mutation at this site may reposition
or altering the mobility of the K129 side chain. Unfortunately this
mutation also significantly diminishes enzyme stability, compli-
cating analysis of catalytic properties. Additional rounds of muta-
genesis might identify secondary mutations that enhance protein
stability.
Directed evolution is a powerful tool for altering the catalytic
properties of enzymes where diversity libraries are created by error
prone PCR or DNA shuffling methods and then subjected to iterative
rounds of screening for the desired property.2
1,22
More recently,
semi-rational methods of library generation have helped minimize
library complexity by focusing mutations on active site positions or
2
3
mutational hotspots, and have yielded significant success. Here
we used random whole gene mutagenesis of T. maritima and
E. coli KDPG aldolase to identify mutations both proximal and distal
to the active site which significantly alter the efficiency for catalysis
of KHO cleavage. Selections in both isoforms clearly demonstrate
that improvements in K
mal number of mutations at sites that would not have been pre-
dicted a priori based on protein structural data.
The KDPG aldolases are TIM barrel proteins that catalyze the
retro-aldol cleavage of deoxy-sugars to pyruvate and the corre-
sponding aldehyde via a Schiff base-dependent mechanism. The
active site is located at the top of the pore created by the barrel
structure and contains the important catalytic residues K129,
M M
and kcat/K can be achieved with a mini-
2
4,25
E40, and R17 (Fig. 3) (K133, E45 and R49 in the E. coli protein).
Lysine 129 forms a Schiff base with the substrate, while E40 serves
as an acid/base catalyst, and R49 forms critical charge–charge
In contrast to the generally robust mutant proteins identified
from the TM KDPG aldolase selections, recovered E. coli KDPG aldol-
ase mutants showed marked instability. As a result the observed
2
5,26
interactions with the carboxylate of the pyruvyl- substrate.
Part of the electrophile binding pocket is created by serine 179,
equivalent to S184 in the E. coli protein), a position that is impor-
kcat and kcat/K
M
values reported for these mutants represent a lower
(
limit for these parameters; however, the K value is independent of
M
tant in EC KDPG aldolase for recognition of phosphorylated sub-
strates. The rest of the phosphate binding pocket consists of the
the active protein concentration and should reflect a genuine char-
acteristic of the isolated proteins. The selected E. coli KDPG aldolase
9
amide nitrogen of G157, G158, and V159 (Fig. 3). Since KHO is
not phosphorylated, it is likely to make poor contacts with the res-
idues that line this pocket. Surprisingly, we did not select any mu-
tants with alterations in these residues that enhance catalytic
activity for cleavage of KHO.
M
mutations primarily lower the value of K for KHO, likely reflecting
enhancements in the binding affinity for this substrate. The most
active mutant after five rounds of mutagenesis (EC 11) improves
the value of K
a maximum of 200-fold, resulting in a mutant with a kcat/K
parable to that of the wild-type enzyme. (Mutants EC 9 and 10 also
showed lower values for K in the order of 10- to 20-fold but are not
M
by 100-fold; however, the value of kcat decreases
M
com-
In the T. maritima selections, many (32%) of the observed muta-
tions are located 12–16 Å from the active site lysine, on solvent ex-
M
posed
a
-helices and loops. Mutations on solvent exposed surfaces
reported due to rapid inactivation of the proteins in vitro.) Three
mutations are common to each of the selected EC KDPG aldolase
mutants: V17L, P94V, and L173M. (Fig. 4B) Residue P94 lines the
pyruvate binding cavity and is located ꢂ4.6 Å from the catalytic
lysine (K133). The conversion of proline 94 to valine likely alters
the structure of the substrate binding pocket, enhancing interac-
tions with KHO. V17L and L173M are located adjacent to each other,
with their side chains pointing towards each other in the buried
include K9T, S82C, G83V, F99L, V172A, and T185A. The proteins
containing these mutations also contain additional mutations bur-
ied within the protein, and these surface mutations may constitute
neutral drift. Although, we did not test these mutations individu-
ally, it is possible that some may improve protein stability. Sev-
eral mutations (A30V, V31A, I52F, and W167R) are located in
buried portions of the helices and may therefore communicate
2
7

M. Cheriyan et al. / Bioorg. Med. Chem. 19 (2011) 6447–6453
6451
Figure 4. Buried mutations in T. maritima and E. coli KDPG aldolases. (A) Structure of T. maritima KDPG aldolase (PDB:1WA325. Mutant TM 5 contains a single mutation A30V.
The side chain of A30 (in red) points between the helices 1 and 8. TM 3 contained numerous mutations but only L16P is buried. The side chain of L16 is shown in red. Helices
are labeled as H1-8. The side chains of K129, R17 and a bound sulfate are shown for reference. (B) Structure of E. coli KDPG aldolase (PDB:1EUA24). All final round EC mutants
possess V17L, L173M, and P94V (in red). The side chains of V17 and L173 point towards each other at the interface between b-sheets 1 and 8, and a-helix 8. P94 lines the
pyruvate binding pocket. Residues K133, R49 and a bound sulfate are shown for reference.
interface between b-sheets 1 and 8, and
a
-helix 8. Similar to the
applications: (i) when the concentration of the substrate is limit-
ing, most improvements in catalysis are attributed to enhance-
ments in the apparent substrate affinity rather than improving
turnover at saturating substrate, and (ii) enhancements observed
under in vivo conditions may not translate to the in vitro condi-
tions used for biocatalytic transformations; and (iii) selection
may lead to proteins with decreased stability. To achieve selective
pressure required to increase turnover numbers, the in vivo sub-
strate concentration must be comparable to or higher than the va-
A30V mutation observed in T. maritima aldolase, the V17L and
L173M pair likely reposition helices 1 and 8, thereby affecting sub-
strate recognition. The surprising correlation in the location of
mutations observed in the two related aldolases may imply that
the interface between helices 1 and 8 is important for defining
the structure of the substrate binding pocket of KDPG aldolases.
The function of the additional mutations in EC 11 (A53T, V118L,
and A137T) is unclear, although they may alter protein stability.
The mutants isolated from TM and EC KDPG aldolase libraries
M
lue of K . For biocatalysis applications it is desirable that the
all show considerable improvements in the K
modest, if any, increases in kcat. This observation suggests that
the pressure applied by this selection is for the value kcat/K , con-
ferring a growth advantage to the cellular host. A relatively small
concentration of KHO (2.5 mM) is used in the media; the intracel-
lular concentration is presumably much lower. Therefore, it is not
M
parameter with
binding affinity be weak and values of kcat be large. In this case,
the selections must be carried out under conditions where the cel-
lular substrate concentration is high. Unfortunately the KHO sub-
strate is toxic to bacterial cells at concentrations above 10 mM,
preventing such experiments for this application.
Our results also suggest that thermophilic, rather than the mes-
ophilic, scaffolds may be superior for directed evolution, due to an
enhanced initial stability that facilitates recovery of catalytically
beneficial yet destabilizing mutations. In our hands, it was far eas-
ier to achieve an improvement in the T. maritima aldolase than the
E. coli aldolase. Two rounds of mutagenesis yielded up to a 25-fold
improvement in catalysis of KHO using the thermophilic aldolase;
however after multiple rounds of mutagenesis, mutations in the
M
surprising that selections led to improvements in the K
given that the cellular concentration of KHO is likely far lower than
the wild-type K value. The subtle remodeling of the binding pock-
M
parameter
M
et leads to enhanced binding of the substrate and enhances reactiv-
ity with the low concentration of the available substrate. However
these mutations have a similar stabilizing effect on both the
ground and transition states of the rate-limiting step leaving the
value of kcat unchanged. This type of enhancement, characterized
M
E. coli enzyme improved the value of K but had negligible impact
1
5
as ‘uniform binding’, is proposed as a first step in the evolution
of novel enzymatic activity. Alterations in numerous interactions
on the value of kcat/K . Moreover, the E. coli mutants proved to be
M
difficult to handle in vitro, frustrating efforts to accurately analyze
their kinetic parameters. The additional stability of the T. maritima
scaffold presumably allows it to sample a greater number of muta-
tions while still remaining properly folded, thereby making the
screening effort more effective. A similar result was reported in
(
hydrogen bonding, hydrophobic interactions, electrostatic effects,
etc.) that are often distant from the active site can lead to uniform
binding enhancements, increasing the likelihood that this form of
enhancement is identified when screening libraries of modest size.
2
8
Once the value of K
M
approaches the cellular concentration of the
the case of subtilisin.
substrate, additional enhancements in catalytic efficiency can only
be achieved by ‘differential binding’ where the transition states are
stabilized relative to the substrate-bound ground states and spe-
cific catalysis of elemental steps.15
Our findings reveal significant limitations with in vivo selec-
tion methodologies to obtain enzymes suitable for biocatalytic
4. Conclusions
Our data demonstrate that selection using a pyruvate auxotro-
phic strain can generate enzymes with enhanced kinetic efficiency
towards cleavage of substrates with unnatural electrophiles. In

6
452
M. Cheriyan et al. / Bioorg. Med. Chem. 19 (2011) 6447–6453
theory, this method could select for reaction of aldolases with any
unnatural aldehyde within the limits of solubility, chemical stabil-
ity, competing reactivity, and acute toxicity to the cell. It is clear
The PB25 strain is an E. coli K12 JM101-derived strain where the
pyruvate kinase genes, pykA and pykF, have been disrupted [supE
r
r
thi
were made according to the protocol described by Hanahan et al.
except that 0.2% glucose was added to the growth media to en-
hance growth of the auxtrophic cell line and stored in 80 L ali-
quots at ꢀ80 °C for up to 3 months. Electroporation of up to 1
of DNA was done using a BIORAD Gene Pulser in 0.1 mm cuvettes
D(lac-proAB) pykA::kan pykF::cat ]. Electrocompetent PB25 cells
31
M M
that significant improvements in K and kcat/K can be achieved
with a minimal number of mutations at sites that would not have
been identified a priori from protein structural data. However,
in vivo selections may not be optimal for generating improvements
in the turnover number (kcat) of an enzyme, as high substrate con-
centrations may not be readily achieved inside the cell. These find-
ings also point to one of the challenges of in vivo selections; the
overall fitness of the organism is a function of careful tuning of
expression levels and activity of multiple proteins.
l
l
g
3
1
under standard E. coli transformation conditions. The cells were
immediately resuspended in a total of 4 mL of SOC media and al-
lowed to recover for 1 h at 34 °C and 250 rpm. The cells were pel-
leted and resuspended in 4 mL of M9 media twice to remove trace
amounts of glucose before plating on selective minimal media.
Most of the selected mutants improve KHO catalysis by lower-
ing the K
concentration of KHO is low. As a result, these in vivo selections
mainly identify improvements in K until the value of K is com-
parable to the cellular concentration of the substrate. Beyond this
point further improvements in K will not increase the catalytic
M
parameter, suggesting that the cellular steady-state
5.2. Low expression KDPG aldolase vectors
M
M
Mutations were made in the lac promoter of the pUC plasmid
using site-directed mutagenesis. The ꢀ35 region was changed from
TTTACA to TTTCCA by whole plasmid PCR amplification using the
M
efficiency of the enzyme or viability of the cells; additional
improvements must derive from an increase in the value of the
turnover number. For biocatalyst development, the value of kcat
is arguably the most important kinetic parameter, so developing
methods to engineer enzymes with improved kcat is desirable.
The intracellular concentration of KHO is difficult to control; how-
ever the periplasmic influx of small molecules is generally unregu-
0
following primer and its reverse complement: 5 -GCACCCCAGGCTT
0
TCCACTTTATGC-3 . A single guanosine nucleotide was introduced
in the spacer region between ꢀ10 and ꢀ35 using the following primer
0
and its reverse complement: 5 -CACTTTATGCTGTCCGGCTCGTAT
0
GTTG-3 . A point deletion was made in the ꢀ35 region of the lac pro-
moter (TTTACA to TTACA) using the following primer and its reverse
2
9
0
0
lated and KHO concentrations in the periplasm should parallel
those in the medium. Therefore, localization of the selected en-
zyme to the periplasm may circumvent issues with substrate con-
centration. A key advantage of this approach would be that the
concentration of KHO can be directly controlled by the growth con-
ditions, presumably allowing for the rapid engineering of aldolases
complement; 5 -GCACCCCAGGCTTACACTTTATGC-3 . The PCR products
were transformed into SmartCells (Genlantis) using the standard heat
shock methods. Following overnight growth on LB agar plates con-
taining 100 lg/mL ampicillin (LB/AMP), a single colony was grown
up and the plasmid DNA was purified and sequenced.
The level of protein expression achieved by various plasmids
was determined by isolating PB25 cells that were transformed with
each plasmid. Ten large colonies were picked from LB/AMP and
with improved values of kcat
.
resuspended in 100
l
L
H
2
O
to
a
final concentration of
5
5
. Materials and methods
.1. Library creation
OD600 = 1.25. The cells were lysed by heating for 10 min at 95 °C
after addition of SDS loading dye. The cell debris was pelleted
and the supernatant was fractionated on a 12% polyacylamide
gel. The proteins were transferred electrophoretically to nitrocellu-
lose paper and blotted according to the manufacturer’s procedures
using Mouse anti-His antibody (Novagen) followed by Goat anti-
Mouse AP-conjugate secondary antibody (Novagen). The presence
of bound antibody was visualized by staining with 5-bromo-
Error prone PCR amplification of the T. maritima eda genes was
conducted using the Genemorph mutagenesis kit (Stratagene).
Primers containing SacI and XhoI restriction sites were used
0
(
forward 5 -GGAAACAGCTATGACCATGATTACGAATTCGAGCTCTAC
0
0
CATG-3 and reverse 5 -CTCAGTGGTGGTGGTGGTGGTGCTCGACT
TC -3 ). The eda genes were encoded on pUC derived plasmid TME-
0
0
4-chloro-3 -indolyphosphate p-toluidine and nitro-blue tetrazo-
DA-pUC as previously reported.14 Following PCR amplification, the
DNA was fractionated on a 1% agarose gel and extracted using
Microcon Ultrafree DA spin filters. The plasmid was simultaneously
digested with SacI and XhoI in NEB Buffer 4 and BSA (New England
BioLabs) and the DNA encoding the eda gene was purified on a 1%
agarose gel and extracted with phenol/chloroform followed by pre-
cipitation with ethanol using standard protocols.30 The vector for
ligation reactions was made by digestion of the original TMEDA-
pUC plasmid, (or reduced expression vectors described below) with
SacI and XhoI and purification on a gel followed by phenol:chloro-
form extraction. Ligations were run using a 1:1.2 molar excess of in-
lium chloride to detect phosphatase activity. The blot was
quantified using the Gel-Pro Analyzer software (Media Cybernet-
ics) to determine the level of expression.
5
.3. Selection conditions
Transformed cells were plated on selective M9 minimal media
that contained the following: 0.2% ribose, 1
g/mL proline, 50 g/mL carbenecillin, and 2.5 mM KHO. The
plates were further supplemented with 1 M FeCl , 50 M ZnSO
.1 M CaCl , 40 g/mL each adenosine, guanosine, thymidine,
cytosine and uracil, and 1 g/mL each of -biotin, cyanocobalamin,
folic acid, niacinamide, -pantothenate, pyridoxal hydrochloride
lg/mL thiamine,
4
0
l
l
l
3
l
4
,
0
l
2
l
l
D
sert to vector at 125 ng/lL total DNA concentration. T4 DNA Ligase
D
(
New England BioLabs) was used with the supplied buffer and
and riboflavin. These supplements improve the growth of the
PB25 strain on minimal plates without negating the pyruvate aux-
otroph phenotype. The library size was estimated by counting the
number of recovered colonies on LB plates containing carbenicillin.
Selection plates were grown at 34 °C.
1
0 mM ATP. Ligation reactions were incubated on ice overnight,
allowing the reaction to slowly reach room temperature. The prod-
uct DNA was extracted with phenol:chloroform and precipitated
with ethanol before resuspending in water to a final concentration
of 625 ng/
lL. The E.coli KDPG aldolase library was constructed
As controls, plasmids containing the wild-type aldolase genes
were transformed into PB25 cells and plated on selective media
alongside the libraries. No significant growth was seen on this
plate within 120 h. All colonies that grew in the first 120 h on
similarly to the TM library using the following primers: forward
0
0
5
-GAAAACAGCTATGACCATGATTACGAATTCGAGCTCGCTCATG-3 ;
0
0
reverse 5 -ATCTCAGTGGTGGTGGTGGTGGTGCTCGAGTT-3 and the
pUC-ECEDA derived plasmid.14

M. Cheriyan et al. / Bioorg. Med. Chem. 19 (2011) 6447–6453
6453
selection plates were picked and restreaked on selective media and
References and notes
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Supplementary data associated with this article can be found in
the online version at doi:10.1016/j.bmc.2011.08.056.