Rational engineering of 2-deoxyribose-5-phosphate aldolases for the biosynthesis of (R)-1,3-butanediol

Article abstract of DOI:10.1074/jbc.RA119.011363

Carbon– carbon bond formation is one of the most important reactions in biocatalysis and organic chemistry. In nature, aldolases catalyze the reversible stereoselective aldol addition between two carbonyl compounds, making them attractive catalysts for the synthesis of various chemicals. In this work, we identified several 2-deoxyribose-5-phosphate aldolases (DERAs) having acetaldehyde condensation activity, which can be used for the biosynthesis of (R)-1,3-butanediol (1,3BDO) in combination with aldo-keto reductases (AKRs). Enzymatic screening of 20 purified DERAs revealed the presence of significant acetaldehyde condensation activity in 12 of the enzymes, with the highest activities in BH1352 from Bacillus halodurans, TM1559 from Thermotoga maritima, and DeoC from Escherichia coli. The crystal structures of BH1352 and TM1559 at 1.40 –2.50 ? resolution are the first full-length DERA structures revealing the presence of the C-terminal Tyr (Tyr²²⁴ in BH1352). The results from structure-based site-directed mutagenesis of BH1352 indicated a key role for the catalytic Lys¹⁵⁵ and other active-site residues in the 2-deoxyribose-5-phosphate cleavage and acetaldehyde condensation reactions. These experiments also revealed a 2.5-fold increase in acetaldehyde transformation to 1,3BDO (in combination with AKR) in the BH1352 F160Y and F160Y/M173I variants. The replacement of the WT BH1352 by the F160Y or F160Y/M173I variants in E. coli cells expressing the DERA + AKR pathway increased the production of 1,3BDO from glucose five and six times, respectively. Thus, our work provides detailed insights into the molecular mechanisms of substrate selectivity and activity of DERAs and identifies two DERA variants with enhanced activity for in vitro and in vivo 1,3BDO biosynthesis.

Full text of DOI:10.1074/jbc.RA119.011363

JBC Papers in Press. Published on December 5, 2019 as Manuscript RA119.011363
The latest version is at http://www.jbc.org/cgi/doi/10.1074/jbc.RA119.011363
Rational engineering of 2-deoxyribose-5-phosphate aldolases for the biosynthesis of (R)-1,3-
butanediol
a,e
a
a
a
a
Taeho Kim , Peter J. Stogios , Anna N. Khusnutdinova , Kayla Nemr , Tatiana Skarina ,
a
b
a
a,c
Robert Flick , Jeong Chan Joo , Radhakrishnan Mahadevan , Alexei Savchenko , and
a,d
Alexander F. Yakunin *
a
Department of Chemical Engineering and Applied Chemistry, University of Toronto, Toronto,
Ontario M5S 3E5, Canada
b
Center for Bio-Based Chemistry, Division of Convergence Chemistry, Korea Research Institute
of Chemical Technology, Yuseong-gu, Daejeon, 34114, Republic of Korea
c
Department of Microbiology, Immunology, and Infectious Diseases, University of Calgary,
Calgary, Alberta, T2N 1N4, Canada
d
Centre for Environmental Biotechnology, School of Natural Sciences, Bangor University,
Deiniol Road, LL57 2UW Bangor, UK
e
Future Technology Center, LG Chem, Gangseo-gu, Seoul, Korea
Running title: Engineering DERAs for the biosynthesis of 1,3-BDO
*
Corresponding author: E-mail a.iakounine@utoronto.ca
Department of Chemical Engineering and Applied Chemistry, University of Toronto
00 College Street, Toronto, Ontario, Canada; Phone: (416) 978-4013; Fax: (416) 978-8528
2
Keywords: 2-deoxyribose-5-phosphate aldolase (DERA), BH1352, aldo-keto reductase, (R)-1,3-
butanediol, C-C bond formation, acetaldehyde condensation, protein engineering, fermentation,
E. coli, biotechnology
---------------------------------------------------------------------------------------------------------------------
Abstract
Thermotoga maritima, and DeoC from
Escherichia coli. The crystal structures of
BH1352 and TM1559 at 1.40-2.50 Å
resolution are the first full-length DERA
structures revealing the presence of the C-
terminal Tyr (Tyr224 in BH1352). Results
from structure-based site-directed mutagenesis
of BH1352 indicated a key role of the catalytic
Lys155 and other active site residues in the
DRP cleavage and acetaldehyde condensation
reactions. These experiments also revealed a
Carbon-carbon bond formation is one of the
most important reactions in biocatalysis and
organic chemistry. In nature, aldolases
catalyze the reversible stereo-selective aldol
addition between two carbonyl compounds,
making them attractive catalysts for the
synthesis of various chemicals. In this work,
we identified several 2-deoxyribose-5-
phosphate (DRP) aldolases (DERAs) having
acetaldehyde condensation activity, which can
be used for the biosynthesis of (R)-1,3-
butanediol (1,3BDO) in combination with
aldo-keto reductases (AKRs). Enzymatic
screening of 20 purified DERAs revealed the
2
.5-fold
increase
in
acetaldehyde
transformation to 1,3BDO (in combination
with AKR) in the BH1352 F160Y and
F160Y/M173I variants. The replacement of
the wild type BH1352 by the F160Y or
F160Y/M173I variants in E. coli cells
presence
of
significant
acetaldehyde
condensation activity in 12 of the enzymes,
with the highest activities in BH1352 from
Bacillus
expressing
the
DERA+AKR
pathway
increased the production of 1,3BDO from
halodurans,
TM1559
from
1
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glucose five and six times, respectively. Thus, phosphate and acetaldehyde, which enter
our work provides detailed insights into the glycolysis and the Krebs cycle, respectively
molecular mechanisms of substrate selectivity (12). The DERA reaction is reversible, as it
and activity of DERAs and identifies two also catalyzes the aldol condensation between
DERA variants with enhanced activity for in acetaldehyde (the donor molecule) and D-
vitro and in vivo 1,3BDO biosynthesis.
glyceraldehyde-3-phosphate (the acceptor
molecule) producing DRP (Scheme 1) (13).
This class of aldolases is unique in that it can
catalyze the aldol condensation of two
aldehydes and do not require a ketone
substrate, whereas other aldolases use ketones
as aldol donors and aldehydes as acceptors
Introduction
The formation of carbon-carbon bonds
via aldol condensation of two carbonyl
compounds is indispensable in biological
systems and organic chemistry (1-3). Aldol
condensation reactions generate a new β-
hydroxy carbonyl compound, which is a
valuable precursor in the construction of
complex organic molecules due to the
formation of up to two new stereogenic centers
(
(
10,14). It activates the donor molecule
acetaldehyde) via the catalytic Lys residue
forming a covalent Schiff base intermediate
enamine) followed by the carboligation
(
between the acceptor (D-glyceraldehyde-3-
phosphate or second acetaldehyde) and the
Schiff base (14,15). The crystal structure of
the E. coli DERA (DeoC) adopts the
ubiquitous TIM (Triosephosphate Isomerase)
(4). Using aldehydes as donor substrates in
aldol reactions is particularly of interest since
this provides the opportunity for sequential
aldol condensation reactions to synthesize
more complex molecules (5,6). In biological
systems, aldolase enzymes catalyze the
reversible and stereoselective aldol addition of
a nucleophilic donor onto an electrophilic
aldehyde acceptor (7). The formation of a new
C-C bond is accompanied by the generation of
a new stereocenter making aldolases attractive
tools in the synthesis of chiral compounds and
bioactive molecules. Therefore, aldolases have
emerged as a promising alternative in the
biocatalytic synthesis of rare sugars and sugar
derivatives, such as statins, iminocyclitols,
epothilones, and sialic acids (8-10).
8
barrel (α/β) fold with the catalytic Lys167
(the Schiff base-forming residue) located on
strand β6 (16). A proton relay system
composed of Asp102, Lys201, and a water
molecule is involved in shuffling a proton
between C2 of the acetaldehyde imine and
enamine and subsequent C3 hydroxyl
protonation. In addition, several biochemical
studies suggested that the C-terminal Tyr259
of the E. coli DeoC is crucial for enzyme
activity (16-18). However, all published
crystal structures of DERA show the absence
of electron density for the last eight C-terminal
residues including Tyr (16,19-22). Recently,
using a combination of NMR spectroscopy and
molecular dynamics simulations it has been
shown that the C-terminal Tyr259 of the E.
coli DeoC enters the active site in catalytically
relevant closed states and is required for
efficiency of the proton abstraction step of the
DERA catalytic reaction (18).
2
-Deoxyribose-5-phosphate aldolases
(DERA, E.C. 4.1.2.4) are found in all
kingdoms of life and represent the major
aldolase group. One of the best characterized
DERAs is the Escherichia coli DeoC, which
belongs to the class I (metal-independent)
aldolases (9,10). The E. coli deoC is part of the
deo operon (deoABCD) involved in the
utilization
of
extracellular
Scheme 1. Reversible retro-aldol reaction
catalyzed by DERA
deoxyribonucleotides as energy sources (11).
It transforms the D-2-deoxyribose-5-phosphate
(DRP) intermediate into D-glyceraldehyde-3-
2
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DERA-based pathway for 1,3BDO production
involves three heterologous enzymes: pyruvate
decarboxylase (PDC, producing acetaldehyde
from pyruvate), DERA (catalyzing aldol
condensation of two acetaldehyde molecules
to 3-hydroxybutanal), and aldo-keto reductase
The acetaldehyde-active DERAs are also (AKR), which reduces 3-hydroxybutanal (3HB)
distinguished by their ability to ligate three to 1,3BDO (Scheme 2). The heterologous
aldehyde molecules in a sequential and stereo- expression of this pathway in E. coli resulted
selective manner, making them attractive in the production of 0.3 g of 1,3BDO/liter
biocatalysts for synthetic organic chemistry from glucose (11.2 mg/g of glucose) (33).
(23,24). It has been shown that DERA Using a systems metabolic engineering
catalyzes a sequential tandem aldol reaction of approach, the 1,3BDO titer was increased to
chloroacetaldehyde and two acetaldehyde 2.4 g/liter and yield to 56 mg/g of glucose
molecules, forming (3R,5S)-6-chloro-2,4,6- further highlighting the potential of aldolases
trideoxyhexapyranoside (CTHP). This product for synthesis of valuable products. This study
can be further used as a lactone moiety for the also suggested that the rate-limiting step of the
synthesis
of
3-hydroxy-3-methylglutaryl proposed 1,3BDO pathway is the DERA-
(HMG)-CoA reductase inhibitors, cholesterol- catalyzed aldol condensation of acetaldehyde
lowering statin drugs (e.g. atorvastatin and to 3HB (33).
rosuvastatin) (25-31). Thus, DERA enzymes
offer the great potential by providing an Scheme 2. DERA-based pathway for 1,3BDO
effective and simplified route for their production from pyruvate. PDC, pyruvate
production.
decarboxylase; AKR, aldo-keto reductase.
Recently, we established a novel
pathway to produce (R)-1,3-butanediol
1,3BDO) from acetaldehyde using DERA as
the key enzyme (32,33). The non-natural diol
,3BDO is used as a building block for the
production of synthetic polymers, pheromones,
fragrances, insecticides, and antibiotics (34- great potential of DERAs for biocatalytic
9). Presently, 1,3BDO has been produced conversion of acetaldehyde to 1,3BDO (33),
(
1
Although our recent studies revealed
3
mainly from petroleum-based feedstocks using this activity (acetaldehyde condensation) has
chemical processes, which require harsh not been examined in depth. The scarcity of
reaction conditions and release toxic data on DERAs limits our efforts on increasing
intermediates and by-products (38). Therefore, the acetaldehyde condensation activity of these
the development of biocatalytic processes for enzymes, which represents the rate-limiting
the production of 1,3BDO from renewable step in the biocatalytic synthesis of 1,3BDO
feedstocks is of increasing importance (39,40). and potentially statin drugs. In this work, after
The recently proposed artificial biosynthetic screening 20 purified microbial DERAs we
approach for 1,3BDO production from glucose identified BH1352 from the alkaliphilic
is based on a reversed fatty acid β -oxidation bacterium Bacillus halodurans, as well as
pathway, which includes four heterologous TM1559 from Thermotoga maritima and E.
enzymes and requires three NADPH and one coli DeoC as the most active aldolases in the
CoA molecules per molecule of 1,3BDO DERA-AKR coupled production of 1,3BDO
produced (41,42). In contrast, the proposed from acetaldehyde. The crystal structures of
3
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these enzymes were determined including the Screening of purified DERAs for biosynthesis
first full-length DERA structure (BH1352) and of 1,3BDO from acetaldehyde - In our
revealed the catalytic residues and substrate- previous work, we identified several aldo-keto
binding sites. Using structure-based site- reductases (AKRs) with significant activity in
directed mutagenesis, we identified the reducing 3-hydroxybutanal to 1,3BDO
BH1352 residues critical for acetaldehyde (Scheme 2) (32). From these proteins, PA1127
condensation and designed several DERA from Pseudomonas aeruginosa was found to
variants with higher activity in the production exhibit negligible activity against acetaldehyde
of 1,3BDO both in vitro (from acetaldehyde) making it suitable for coupling of the DERA-
and in vivo (from glucose). We demonstrated catalyzed condensation of acetaldehyde (to 3-
that E. coli cells expressing the DERA-AKR hydroxybutanal) with the AKR-catalyzed
pathway with engineered DERA variants reduction of 3-hydroxybutanal to 1,3BDO
produced 5-6 times more 1,3BDO from (Scheme 2). Using PA1127, we established a
glucose compared to cells with the wild-type coupled enzyme system (DERA+PA1127)
BH1352.
(Fig. S2) and screened 20 purified DERAs for
transformation of acetaldehyde to 1,3BDO.
These screens revealed significant production
of 1,3BDO in the presence of 12 DERAs with
the highest activity observed in TM1559 from
Thermotoga maritima (DERA group-1), E.
coli DeoC (DERA group-4), and BH1352
from Bacillus halodurans (DERA group-2)
Result and Discussion
Phylogenetic analysis of DERA sequences - To
provide insight into the phylogenetic diversity
of DERAs, over 2,500 sequences (2,553) of
putative DERAs were extracted from the
KEGG Orthology (KO) database using the KO
identifier K01619 for the E. coli DERA
(Fig. 1). Since BH1352 was found to support
the highest production of 1,3BDO by E. coli
cells expressing different DERAs (including
TM1559 and DeoC) (33), this protein was
(
DeoC), which is the best characterized DERA
enzyme (23). Initially, this pool of putative
DERA proteins included over 2,500 sequences
selected
for
detailed
structural
and
biochemical studies of the transformation of
acetaldehyde to 1,3BDO.
(
1
1
2,281 from bacteria, 120 from archaea, and
52 from eukaryotes), but it was reduced to
,974 proteins after removing redundant
Since B. halodurans is an alkaliphilic
bacterium (grows well at pH >9.0), we
determined the optimal pH range for BH1352
using the retro-aldol DRP cleavage reaction
coupled with glyceraldehyde-3-phosphate
dehydrogenase and triosephosphate isomerase
sequences. This phylogenetic analysis revealed
the presence of five major clusters of DERA
proteins including one Bacterial domain, one
Firmicutes (Bacilli and Clostridia), one mostly
Proteobacteria, and two mixed clusters (Fig.
(16,24,43,44). These assays revealed a broad
1
A). To screen DERAs for the bioconversion
pH range with the maximal activity of
BH1352 at pH 7.2-9.5 (Fig. S3), whereas the
previously reported DERA enzymes from
other bacteria showed the highest activity at
pH 6.0-7.5 (24,28). At optimal pH, Vmax of
BH1352 with DRP as substrate was calculated
to be 52-67 µmol/min/mg protein, which is
lower than that for the DERA from
Lactobacillus brevis (102 µmol/min/mg
protein), but higher than other DERAs (0.25 -
of acetaldehyde to 1,3BDO, we selected 20
DERA proteins from different phylogenetic
groups, which were found to be soluble when
expressed in E. coli (Fig. S1, Table S1 in
Supplementary Information). Based on the
phylogenetic analysis, 17 selected DERAs
belong to the five large clusters (1-5), whereas
the remaining three proteins were from non-
classified sequences.
4
ꢀ
ꢀ

1
.00 µmol/min/mg protein) (24,28). The L. BH1352 structure, the interfaces between
brevis DERA has also been reported to exhibit monomers in each adjacent dimer involve 13
2
high resistance to aldehydes, but this enzyme hydrogen bonds and buries 1,288 Å surface
showed low activity in the AKR-based screen area. The dimerization interface of BH1352 is
for 1,3BDO synthesis (Fig. 1B) (28). Steady- composed mainly of hydrophobic interactions
state kinetic parameters of BH1352 and its including the BH1352 loops containing Pro16,
variants were also determined using the DRP Phe66, Pro67, Leu68, Ile97, and Phe160
cleavage
reaction
(Table
2).
These (Table S2), while the weak dimerization
2
experiments revealed that BH1352 exhibits interface of DeoC (573 Å ) is comprised of a
typical Michaelis-Menten kinetics with the single hydrogen bond and two salt bridges in
apparent K
for E. coli DeoC and more than 10 times lower (Fig. S6) (21,24,45). On the other hand, the
than that for the L. brevis DERA (3.3 mM) TM1559 structure exhibits stronger
24). dimerization interactions with the interface
m
= 0.22 mM, which is close to that between α3 and α4 helices of each protomer
(
2
1
,464
Å
between TM1559 protomers
Crystal structures of DERAs: overall fold and including 14 hydrogen bonds and two salt
active site - The crystal structures of BH1352 bridges along with many hydrophobic contacts,
(PDB codes 6D33 and 6MSW) and E. coli which is in line with higher structural stability
DeoC (PDB code 1KTN) were determined to of this protein (Table S2) (24).
2
.50 Å and 1.40 Å resolution, respectively,
using the sitting-drop vapor diffusion method
Table 1), whereas the unpublished crystal
The monomeric structures of BH1352,
TM1559, and DeoC displayed a classical
(
(
8
α/β) fold (TIM barrel), which is one of the
structure of TM1559 is available from Protein
Data Bank (PDB codes 3R12 and 3R13, Joint
Center for Structural Genomics). In contrast to
E. coli DeoC, the structures of BH1352 and
TM1559 revealed the presence of electron
density for the C-terminal Tyr residues
most common protein folds catalyzing diverse
enzymatic reactions (46). Interestingly, the
AKR enzyme PA1127 used in combination
with DERAs (EC 4.1.2.4) for 1,3BDO
synthesis also has a TIM barrel fold, but
catalyzes the NADPH-dependent reduction of
(Tyr224 and Tyr246, respectively) making
3
HB and other aldehydes (EC 1.1.1.X) (32). A
them the first full length DERA structures. In
BH1352, Tyr224 is located on a flexible strand,
whereas the TM1559 Tyr246 is located on the
C-terminal α-helix (Fig. 2, Fig. S4). Analysis
of crystal contacts of BH1352 using the
quaternary prediction PDBePISA server
Dali search for structural homologues of
BH1352 identified several DERA structures as
the best matches, including the Entamoeba
histolytica DeoC (PDB code 3NGJ; Z score
3
9.0, root mean square deviation [RMSD], 0.9
Å; 63% sequence identity), Streptococcus suis
DERA (5DBU, Z score 38.5, RMSD 0.6 Å,
(
http://www.ebi.ac.uk /pdbe/pisa/) predicted a
dimeric state (Fig. 2A). This was supported by
the result of size-exclusion chromatography
suggesting that this protein exists as a dimer in
solution (observed molecular mass 51.7 kDa;
predicted mass of monomer molecule of 24.2
kDa) (Fig. S5). It is similar to the dimeric state
of hyperthermophilic and L. brevis DERAs,
but is different from the E. coli DeoC, which
was found to exist in a monomer-dimer
equilibrium (Fig. S6) (21,24). Based on the
6
6%), and L. brevis DERA E78K mutant
(4XBS, Z score 38.1, RMSD 0.9 Å, 53%).
Based on the BH1352 structure, its
active site is located inside of the β-barrel, near
its C-terminal side (Fig. 2B). The active site
entrance is formed by the several loops
connecting β-strands (β1, β6, and β7) with α-
helices (α1, α6, and α7), containing highly or
semi-conserved residues including Thr12,
5
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Leu14, Lys15, Phe66, Ile128, Phe160, Ser186, BH1352 dimer (Fig. 4). In the BH1352 active
and Ser209. The side chains of these residues site, the Tyr224 side chain showed two
create a narrow channel providing access of orientations, with the hydroxyl group pointing
substrates to the catalytic Lys155, located on toward the catalytic Lys155 (2.7 Å) or toward
the β6 strand (Fig. 3A). In the best the β1-α2 loop backbone (near conserved
characterized DERA from E. coli, the catalytic Leu14 and Lys15, 2.9-3.0 Å) (Fig. 4A). In the
Lys167 is in close proximity to the side chains second orientation, the aromatic ring of
of conserved Lys137 and Lys201, and the Tyr224 is part of the hydrophobic wall of the
three Lys residues form salt bridges with the active site (with conserved Leu14, Val63,
side chain oxygens of conserved Asp102 Phe66, Ile128, and Phe160) (Fig. 4B). Thus, in
(
16,21). During the DERA-catalyzed synthesis line with a recent work on molecular dynamics
of 2-deoxyribose-5-phosphate (DRP) (Scheme simulation with DeoC (18), the BH1352
), the uncharged nucleophilic Lys167 of E. crystal structure provides the structural
1
coli DeoC attacks the acetaldehyde carbonyl indication that the C-terminal Tyr residue of
forming a carbinolamine and then a Schiff DERAs might be involved directly in substrate
base, which subsequently tautomerizes to an binding and/or catalytic mechanism of this
enamine group and attacks glyceraldehyde 3- enzyme.
phosphate (16,43). Finally, hydrolysis of the
aldol condensation intermediate produces the Probing the active site of BH1352 using site-
free enzyme and DRP.
directed mutagenesis - Since the catalytic
Previous biochemical studies with the residues of E. coli DeoC are conserved in
E. coli DeoC also suggested an important role BH1352 and TM1559, the same catalytic
for the highly conserved C-terminal Tyr259, mechanism can be applied to aldol
because its replacement by Phe (Y259F) condensation of two acetaldehyde molecules
resulted in a ~100-fold reduction of the DRP catalyzed by these enzymes. In the BH1352
cleavage activity (16,18). Interestingly, the active site, the side chain of conserved Asp92
deletion of Tyr259 (ΔY259) significantly (Lys179 in TM1559) forms salt bridges with
increased the DeoC activity in the the conserved Lys126 (2.7 Å from Asp92),
condensation reaction between acetaldehyde Lys155 (3.1 Å), and Lys184 (3.0 Å). We
and chloroacetaldehyde (27). Using
a
propose that the catalytic Lys155 forms a
combination of NMR and molecular dynamics Schiff base with the acetaldehyde carbonyl,
simulations, it has been shown that the DeoC whereas Asp92 and Lys184 are part of the
C-terminal tail is intrinsically disordered with BH1352 proton relay system involved in imine
the equilibrium between open and catalytically deprotonation to form an enamine (Fig. S7).
relevant closed states, where Tyr259 is This is consistent with the results of alanine
inserted into the active site close to the replacement mutagenesis of BH1352 with the
catalytic Lys167 (~ 6 Å) (18). Remarkably, the respective mutant proteins (D92A, K126A,
structures of both BH1352 and TM1559 K155A, and K184A) showing very low or no
revealed the presence of electron density for catalytic activity both in the DRP cleavage and
the C-terminal Tyr224 and Tyr246, acetaldehyde condensation reactions (Fig. 5).
respectively. In TM1559, Tyr246 is positioned This is also supported by the crystal structure
on the C-terminal α-helix with the side chain of TM1559 in complex with citrate and
exposed to solvent (Fig. S4), whereas Tyr224 glycerol (PDB codes 3R12) indicating that its
in BH1352 is located on the flexible C- active site includes Asp117, Lys150, Lys179
terminal tail with side chain stabilized through (catalytic), and Lys208 (Fig. S8). Another
interactions with the active site of the other crystal structure of TM1559 (PDB code 3R13)
6
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also revealed the presence of additional (Fig. 5), indicating that this residue plays an
electron density in the active site representing important role in catalytic activity of this
an unknown ligand covalently bound to the enzyme.
catalytic Lys179 (likely representing one of
the reaction intermediates).
Another notable feature of the BH1352
and TM1559 structures is the presence of a
To identify other BH1352 residues cluster of hydrophobic residues near the
involved in substrate binding, we modeled catalytic (Lys155 in BH1352) including four
DRP into the BH1352 active site using the residues conserved in all DERAs (Leu14,
structure of the DeoC-DRP complex of DeoC Val63, Phe66, and Ile128 in BH1352; Leu40,
from E. coli (Fig. 3B) (16,43). The produced Val88, Phe91, Ile152, and Phe184 in TM1559)
model of DRP binding in the BH1352 active (Fig. 6C, Fig. S8, Table S3). In the BH1352
site predicts that the side chain of Thr12 structure, the side chains of Leu14, Phe66, and
appears to be involved in substrate Ile128 are oriented towards the α-carbon of
coordination via hydrogen bonding with the β- aldol products (Fig. 6), suggesting that these
hydroxyl group of DRP, as well as with residues provide hydrophobic contacts for
2
6
Lys184 and a water molecule (W , Fig. 3B). ligand binding, and that they might be
This is consistent with the results of site- essential for enzyme activity. This was
directed mutagenesis, which revealed that Ala supported by the results of alanine replacement
replacement of Thr12 resulted in a catalytic mutagenesis of these residues, which produced
impairment in the DRP cleavage reaction (Fig. mutant proteins with a greatly reduced activity
5
A). However, the Thr12Ala mutant protein in both reactions (the L14A protein was found
exhibited acetaldehyde condensation activity to be insoluble) (Fig. 5). Another hydrophobic
comparable to that of the wild type BH1352, cluster comprising of three valine residues
suggesting that this residue is not critical for (Val154, Val177, and Val183), Ile170, and
acetaldehyde condensation (Fig. 5B).
Met173 is located between the two β-strands
The DRP binding model also suggested (β6 and β7) and α6 helix (in BH1352) (Fig. 6).
that the DeoC Lys172 (interacting with the It was previously reported that this cluster may
DRP phosphate and γ -hydroxyl groups) is contribute to the sequential aldol condensation,
replaced by Phe160 in BH1352 (Fig. 6, Fig. 7). as revealed by the DeoC mutations F200I and
Moreover, it proposes that the highly M185V (equivalent to Val183 and Met173 in
conserved BH1352 Arg190 and DeoC Arg207 BH1352) resulting in enhanced condensation
(located close to the conserved phosphate- of acetaldehyde and chloroacetaldehyde (27)
binding Gly-rich loop) might also be involved (27). Also, the DeoC Phe200 is replaced by
in the coordination of the DRP phosphate Val in DERAs from T. maritima and
through the bound water molecule (like Pyrobaculum aerophilum, both of which show
Lys172 in DeoC). In addition, the BH1352 higher sequential aldol condensation of
Lys15 appears to interact with the phosphate acetaldehyde (24). These results suggest that
and γ-hydroxyl groups of DRP. Based on the reducing the size of hydrophobic side chains in
DERA sequence alignment, the BH1352 this cluster might contribute to higher aldol
Lys15 is conserved in DERAs from Bacilli condensation activity.
(group 2), whereas the proteobacterial DERAs
Recently, it has been shown that the C-
(group 4) contain an Asn residue at this terminal Tyr259 of the E. coli DeoC is
position (Asn21 in DeoC) (Fig. 1, Fig. 7). Ala required for the efficient proton abstraction
replacement mutagenesis of Lys15 rendered step in the DRP cleavage reaction (18),
BH1352 completely inactive in both retro- whereas the previous work with the truncated
aldol and acetaldehyde condensation reactions DeoC ΔY259 protein (Tyr259 deleted)
7
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demonstrated an enhanced activity in condensation reactions, because the F160A
acetaldehyde condensation with mutation had no significant effect on both
chloroacetaldehyde (27). Since the BH1352 reactions (Fig. 5). However, the DRP cleavage
structure suggested that the C-terminal Tyr224 activity of BH1352 was negatively affected
might directly contribute to substrate binding when Phe160 was mutated to Glu, Gln, Lys,
or activity of this enzyme (Fig. 4), site- Met, Trp, or His, and slightly stimulated by
directed mutagenesis was also used to mutation to Tyr (~23%) (Fig. 5A).
ascertain the role of this residue. We designed Interestingly, the acetaldehyde condensation
and purified four Tyr224 mutant proteins via BH1352 increased almost three times in
including Y224A, Y224F, ΔY224 (Tyr224 the F160Y protein and was also increased in
deleted), and ΔS223/Y224 (Ser223 and the F160E (72%) and F160H (44 %) proteins
Tyr224 deleted) and tested their catalytic (Fig. 5B, S10). In contrast, the replacement of
activities in the DRP cleavage and Phe160 by Lys, Gln, Met, or Trp had no
acetaldehyde
condensation
(1,3BDO significant effect on this activity. These results
production) reactions. Interestingly, while the suggest that similar to LbDERA (30), the
acetaldehyde condensation reactions of these substitution of Phe160 by Tyr in BH1352
mutant proteins were not affected, their retro- enhances acetaldehyde binding and/or
aldol activity was greatly reduced (especially condensation, but has no effect on DRP
in Y224F), indicating that Tyr224 is essential cleavage (Fig. S9). Based on the BH1352
for DRP cleavage, but not for acetaldehyde crystal structure, the hydroxyl group of Tyr160
condensation (Fig. 5). Thus, the crystal (in F160Y) might interact with the main chain
structures of BH1352 and other DERAs from amide of conserved Lys15 (3.3 Å) located on
different phylogenetic groups revealed the β1-α1 loop (Leu13-Thr19) near the
significant
differences
in
substrate absolutely conserved Leu14 (Fig. S9C). Our
coordination and catalysis of DRP cleavage mutagenesis studies demonstrated that Lys15
and acetaldehyde condensation.
is critical for catalytic activity of BH1352,
whereas Leu14 is part of the hydrophobic
Structure-based engineering of BH1352 for cluster near the catalytic Lys155 (L14A
enhanced production of 1,3BDO - The crystal mutant protein was found to be insoluble) (Fig.
structures of BH1352 and TM1559 revealed 5, Fig. 6). We propose that the hydroxyl group
that their substrate-binding pockets also of Tyr160 provides a stabilizing effect on the
include the side chain of a semi-conserved Phe conformation of both Leu14 and Lys15 in the
(
(
Phe160 in BH1352 and Phe184 in TM1559) BH1352 active site resulting in increased
Fig. 6, S9). This residue is conserved in most acetaldehyde condensation activity of this
DERAs from clusters 1 (Mixed group) and 2 enzyme.
Firmicutes), but it is replaced by a Lys
(
We also mutated the semi-conserved
residues Ile170 and Met173 of BH1352,
located near the catalytic Lys155 (Fig. 6D), to
examine if the reduction or increase of their
hydrophobic side chains will affect the
catalytic activity of BH1352 and improve
acetaldehyde condensation. Our coupled
DERA-AKR assays (1,3BDO production)
revealed 40-50% increase in the production of
residue in Proteobacterial DERAs (cluster 4)
including E. coli DeoC (Fig. 7). In the
Lactobacillus brevis DERA (LbDERA), the
replacement of the homologous Phe163 by Tyr
has been shown to result in enhanced
sequential condensation of acetaldehyde and
chloroacetaldehyde, probably by promoting
substrate access (30). We found that the
BH1352 Phe160 was not essential both for the
retro-aldol (DRP cleavage) and acetaldehyde
1
,3BDO by the purified mutant proteins I170V,
M173I, M173L, and M173V compared to the
8
ꢀ
ꢀ

wild-type BH1352, whereas I170A showed performed by the addition of extra glucose
reduced activity (Fig. 5). In contrast, the retro- (3%) after the induction of 1,3BDO pathway
aldol (DRP cleavage) activity of BH1352 was expression by IPTG addition (1 mM). After
not significantly affected by these mutations. the cultivation, the BDO-0 strain (wild-type
Interestingly, the replacement of Met173 by a BH1352) produced 0.2 g /l of 1,3BDO with a
polar residue (Thr) had a strong negative effect yield of 4 mg/g of glucose (Fig. 8). Under the
on both BH1352 activities indicating that same experimental conditions, BDO-1
retaining hydrophobicity at this position is (BH1352 F160Y) produced 0.9 g/l of 1,3BDO
critical for catalytic activity of this enzyme. with a yield of 18 mg/g of glucose
We also designed the BH1352 double mutant representing a 4.5-fold increase both in the
protein F160Y/M173I, which showed 1,3BDO 1,3BDO titer and yield (Fig. 8). Even higher
formation activity comparable to that of 1,3BDO production was observed in the BDO-
F160Y suggesting that these two mutations are 2 strain, which produced up to 1.1 g/l of
not synergistic in acetaldehyde condensation 1,3BDO with a yield of 28 mg/g of glucose (a
(Fig. 5). Thus, both BH1352 F160Y and 5.5 and 7-fold increase, respectively) (Fig. 8).
F160Y/M173I proteins exhibit enhanced Different titers of 1,3BDO produced by the
activity in acetaldehyde condensation reaction BDO-1 and BDO-2 strains might be due to
and can be used for in vitro and in vivo slightly different expression levels of the
production of 1,3BDO.
BH1352 F160A and F160A/M173I proteins in
E. coli cells. Thus, using structure-based
protein engineering we have identified two
BH1352 mutant proteins supporting enhanced
biosynthesis of 1,3BDO both in vitro (from
acetaldehyde) and in vivo (from glucose).
Application of engineered BH1352 variants
for in vivo production of 1,3BDO in E. coli -
Recently, we demonstrated the production of
1,3BDO by engineered E. coli cells expressing
a
heterologous, aldolase-based pathway
containing the wild-type BH1352, PA1127, Conclusions
and pyruvate decarboxylase (PDC) from Using a combination of purified DERAs and
Zymomonas mobilis (33). Since this work an aldo-keto reductase (PA1127), we have
suggested that 1,3BDO production is limited identified three microbial DERAs with high
by the activity of BH1352, we designed two activity in the transformation of acetaldehyde
novel E. coli strains expressing either the to 1,3BDO. The crystal structure and site-
BH1352 F160Y (BDO-1) or F160Y/M173I directed mutagenesis of BH1352 provided
(
BDO-2) variants and compared 1,3BDO insights into the molecular mechanisms of
production with the original strain expressing substrate selectivity and acetaldehyde
the wild-type BH1352 (BDO-0). In these condensation activity of DERAs. By targeting
experiments, we used the E. coli strain hydrophobic residues near the catalytic
LMSE51C with several non-essential genes Lys155 of BH1352, we generated two variants
deleted including pyruvate-formate lyase of this enzyme (F160Y and F160Y/M173I)
(
pflB),
lactate
dehydrogenase
synthase (ilvB),
(ldhA), with enhanced activity in acetaldehyde
and condensation and 1,3BDO production. E. coli
acetolactate
aldehyde/alcohol dehydrogenase (adhE) (with cells expressing these BH1352 variants as part
the goal of increasing the carbon flux to of the DERA+AKR pathway produced 5-6
1
,3BDO and reducing byproduct formation) times more 1,3BDO from glucose compared to
(
(
Fig. S11) (33). The three E. coli strains cells with the wild-type BH1352. The
BDO-0, BDO-1, and BDO-2) were grown in designed BH1352 variants can be used as a
a bioreactor with pH control (maintained at 7.0) starting material for future protein engineering
using
a
fed-batch aerobic fermentation efforts aimed at improving the activity of
9
ꢀ
ꢀ

DERAs and their performance in the at 100 K at a Rigaku home source Micromax-
biotechnological production of 1,3BDO and 007 with R-AXIS IV++ detector. Diffraction
other chemicals.
data were processed using HKL3000
53). The structure was solved by molecular
(
Experimental Procedures
replacement using Phenix phaser and the
structure of a putative aldolase (PDB 3NGJ)
Phylogenetic and sequence analyses - The
phylogenetic tree was generated by retrieving
(
54). Model building and refinement were
performed using Phenix.refine and Coot
54,55). TLS parameterization was utilized,
and B-factors were refined as
2
,553 sequences from UniProt using KEGG
(
Orthology (KO) K01619, which represents
DERAs (EC 4.1.2.4) involved in the pentose
phosphate pathway. The original dataset was
reduced to 1,974 sequences by removing
redundant sequences and increasing gap-free
sites using CD-HIT and MaxAlign using the
isotropic. Structure geometry and validation
were performed using the Phenix Molprobity
tools. Data collection and refinement statistics
are summarized in Table 1.
Enzyme assays - Purified DERAs were
initially screened using a DERA-AKR coupled
assay with 50 mM acetaldehyde as substrate in
the following reaction mixture (0.2 ml): 100
mM triethanolamine (TEA) buffer (pH 7.5), 10
mM NADPH, DERA (250 µg/ml), and AKR
MAFFT
online
alignment
(
https://mafft.cbrc.jp/alignment/server/) (47-
4
9). The tree was built using FastTree 2.1.5
and visualized by iTOL (Interactive tree of life,
http://itol.embl.de/) (50,51). The DERA
sequence alignment and phylogenetic analysis
were conducted as described in our previous
study (32). Structural images of BH1352 were
prepared using PyMOL Molecular Graphics
System, version 1.8 (Schrödinger, LLC).
Gene cloning, protein purification, and
mutagenesis - The DERA genes studied in this
work (Table S1 in Supplementary materials)
were cloned, overexpressed in E. coli, and
affinity purified (Fig. S1) as described
previously (52). Site-directed mutagenesis of
BH1352 was performed using the Phusion®
High-Fidelity DNA Polymerase (New England
BioLabs) accordingly to the manufacturer’s
protocol.
(PA1127, 250 µg/ml). The production of
1
2
,3BDO was measured using HPLC, following
h incubation at room temperature (used in
DERA-catalyzed reactions) (22). The reaction
samples were filtered through centrifugal filter
device (10K cut-off, VWR) to remove
enzymes and dried to get rid of residual
acetaldehyde from the samples using a vacuum
concentrator. The dry samples were dissolved
in the same volume of ddH O and analyzed
2
using HPLC (Dionex Ultimate 3000, Thermo
Scientific) equipped with an Aminex HPX-
8
7H column, equilibrated with 5 mM H
as an eluent with a flow rate of 0.6 ml/min at
0°C. 1,3BDO was detected using a refractive
2 4
SO
5
Protein
crystallization
and
structure
index detector (Shodex RI-101). Assay
conditions were optimized by varying
concentrations of DERA, AKR, and NADPH,
and the optimal conditions included 100 µg/ml
each of DERA and AKR, and 10 mM NADPH
determination
-
Purified BH1352 was
crystallized at room temperature using the
sitting-drop vapor diffusion method using
protein concentration of 10 mg/mL and
reservoir solution of 0.1 M Tris-HCl (pH 8.5),
(
Fig. S2).
The kinetic parameters of purified
DERA were determined using 2-deoxyribose-
-phosphate (DRP) cleavage reaction using a
0
3
.2 M magnesium chloride, 25% (w/v) PEG
350, and 10 mM acetaldehyde. The crystal
was cryoprotected in the same buffer
supplemented with 2% PEG 200 and flash
frozen in liquid nitrogen. Diffraction data for
the BH1352 apoenzyme crystal was collected
5
glyceraldehyde-3-phosphate
triosephosphate isomerase
dehydrogenase
(GDH/TPI)-
/
1
0ꢀ
ꢀ

coupled assay. The DERA-catalyzed retro- ampicillin from a single colony and grown at
aldol reaction produces acetaldehyde and D- 37°C. 50 ml of modified M9 media
glyceraldehyde-3-phosphate,
which
is (supplemented with 100 µg/ml of ampicillin
converted into dihydroxyacetone phosphate by and 0.5 mg/ml of thiamine and containing 0.1
TPI and further reduced by GDH consuming M MOPS at pH 7.3) was inoculated with the
NADH. The detailed assay conditions were as first seed culture, in a 250 ml baffled flask,
follows: 100 mM TEA buffer (pH 8.5), 0.5 and grown at 37°C and 200 rpm for 16 h. The
mM NADH, wild-type or mutant DERA (1 second seed culture was then used to inoculate
µg/ml), TPI (11 U/ml), GDH (1 U/ml), and 300 ml of modified M9 media (without MOPS)
DRP (from 4 µM to 4 mM) in a 200 µl and supplemented with 100 µg/ ml of
reaction mixture at 30°C. The kinetic carbenicillin in the bioreactors. The pH was
parameters were calculated by a nonlinear controlled at 7.0 by the addition of 10%
regression analysis of raw data fit to the NH
sigmoidal function using GraphPad Prism temperature at 37°C and air flow rate at 1.5
software (version 5.04 for Windows). vvm. When the culture density reached OD600
4
OH, stirrer speed at 1,500 rpm,
For the analysis of DERA resistance nm between 7 and 8, protein expression was
against acetaldehyde, a freshly prepared induced by the addition of 1mM IPTG. After
acetaldehyde solution (final concentration 100 30 min, the air flow rate was reduced to 0.37
mM) was added to the incubation mixture vvm (25% of the initial vvm) to reduce
containing 2 mg/ml of purified BH1352 (wild- dissolved oxygen (dO) and 3 % glucose was
type or mutant proteins). The incubation additionally supplemented.
solution aliquots were taken and diluted for
further use in a DRP cleavage assay (1 mM
Acknowledgements: We thank all members
DRP). The activity of DERA samples was
of the BioZone Centre for Applied Bioscience
analyzed immediately after acetaldehyde
and Bioengineering for help in conducting
addition and then at regular time intervals. The
experiments. This research was supported by
residual DERA activity was calculated by
the Natural Sciences and Engineering
comparison with control samples without
Research Council (NSERC) Strategic Network
grant IBN, Ontario Research Fund (ORF
acetaldehyde (containing enzymes and buffer).
Strains and plasmids - The strains and
plasmids used in this study were adopted from
the previous work and are listed in Table S4
Research
Excellence)
grant
BioCeB
(Biochemicals from Cellulosic Biomass), and
a grant from the Genome Canada, Genomics
Applied Partnership Program (GAPP).
Conflict of interest: The authors declare that
they have no conflicts of interest with the
content of this article.
(33) (34). Expression of pBD1 (pTrC99A
harboring BH1352, PA1127, and PDC from Z.
mobilis) in LMSE51C was used as the wild-
type control (BDO-0) to demonstrate the in
vivo effect of the mutations in BH1352.
E. coli cultivation in mini-bioreactors - For in
vivo studies on 1,3BDO production, the
designed E. coli strains were cultivated in 500
ml bioreactors (Applikon Biotechnology Inc.)
equipped with Rushton impellers and
electrodes for pH and dissolved oxygen
essentially as described previously (33).ꢀ The
first seed culture was prepared by inoculating
Abbreviations. 1,3BDO, (R)-1,3-butanediol;
3
HB, 3-hydroxybutanal; AKR, aldo-keto
reductase; CTHP, (3R, 5S)-6-chloro-2,4,6-
trideoxyhexapyranoside; DRP, 2-deoxyribose-
5
-phosphate;
DERA,
2-deoxyribose-5-
phosphate aldolase; GDH, glyceraldehyde-3-
phosphate dehydrogenase; HMG, 3-hydroxy-
3
-methylglutaryl;
PDC,
pyruvate
decarboxylase; TEA, triethanolamine; TPI,
triosephosphate isomerase.
1
0 ml of LB supplemented with 100 µg/ml of
1
1ꢀ
ꢀ

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alignment program to large data-reexamination of the usefulness of chained guide trees.
Bioinformatics 32, 3246-3251
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1
5ꢀ
ꢀ

Table 1. Crystallographic data collection and model refinement statistics for the crystal
structures of BH1352 (wild-type and K184L) and E. coli DeoC (EcDERA).
Structure
PDB Code
apo BH1352
apo BH1352 K184L
EcDERA
1KTN
6D33
6MSW
Data collection
Space group
Cell dimensions
a, b, c (Å)
C2
C2
C2
240.91, 55.52, 177.71
90, 128.02, 90
30.0 – 2.50
0.049 (0.654)
0.027 (0.392)
0.750
240.78, 55.13, 176.30 62.57, 53.56, 81.36
α, β, γ (°)
90, 127.71, 90
30.0 – 2.17
0.077 (0.734)
0.042 (0.405)
0.815
90, 109.97, 90
50.0 – 1.40
Resolution, Å
a
R
R
CC1/2
merge
0.078 (0.309)
e
pim
--
--
c
e
I / σ(I)
Completeness, %
Redundancy
34.2 (2.56)
98.9 (97.5)
3.9 (3.5)
17.8 (2.11)
100 (100)
4.2 (4.2)
20.0 (2.44)
99.5 (98.2)
6.0 (4.5)
Refinement
Resolution, Å
No. of unique reflections:
working, test
30.02 – 2.50
63680, 1995
29.98 – 2.17
97396, 1997
38.65 – 1.40
88731, 4458
d
R-factor/free R-factor
20.4/25.0 (29.7/32.9)
19.3/21.1 (28.8, 30.0) 18.6/20.5 (18.7/19.9)
No. of refined atoms, molecules
Protein
Solvent
Water
9557, 6
116, 18
461
9514, 6
102, 17
843
3763, 2
N/A
631
B-factors
Protein
Solvent
74.70
97.53
64.25
56.58
85.32
54.92
8.54
N/A
20.50
Water
r.m.s.d.
Bond lengths, Å
Bond angles, °
0.004
0.632
0.004
0.618
0.004
1.300
a
R
h i i h i i i
sym = Σ Σ (h) - 〈I(h)〉/Σ Σ I (h), where I (h) and 〈I(h)〉 are the ith and mean measurement of the
|I
intensity of reflection h.
b
Figures in parentheses indicate the values for the outer shells of the data.
Value refers to the outer shells of the data.
c
d
obs
calc
obs
obs
calc
R = Σ|F
p
– F
p
|/ΣF
p
, where F
p
and F
p
are the observed and calculated structure factor
amplitudes, respectively.
e
Value not measured.
1
6ꢀ
ꢀ

Table 2. Kinetic parameters of the wild type and mutant BH1352 proteins in the DRP retro-aldol
cleavage reaction.
-
1
-1 -1
Protein
WT
K
M
[mM]
V
max [U/mg]
k
cat [s ]
M
kcat/K [mM s ]
0.22 ± 0.02
0.50 ± 0.05
0.45 ± 0.04
0.06 ± 0.01
0.17 ± 0.01
0.22 ± 0.01
0.18 ± 0.01
0.17 ± 0.02
0.21 ± 0.02
0.18 ± 0.02
0.25 ± 0.02
34.1 ± 1.0
47.8 ± 1.6
32.9 ± 1.1
9.1 ± 0.4
22.6 ± 0.5
19.1 ± 0.4
38.2 ± 0.5
30.1 ± 0.8
32.8 ± 1.1
25.4 ± 0.9
27.9 ± 0.7
13.3 ± 0.4
18.6 ± 0.6
12.8 ± 0.4
3.5 ± 0.2
8.8 ± 0.2
7.5 ± 0.2
14.9 ± 0.2
11.7 ± 0.2
12.8 ± 0.4
9.9 ± 0.3
10.9 ± 0.3
60.4
37.2
28.5
58.8
51.7
33.9
82.5
68.9
60.8
54.9
43.5
F160Y
F160H
F160K
F160M
F160W
I170V
M173I
M173L
M173V
F160Y/M173I
1
7ꢀ
ꢀ

Figure 1. Phylogenetic analysis of DERAs and screening of purified proteins for 1,3BDO
formation. (A), Phylogenetic analysis of the DERA family: unrooted phylogenetic tree of 2,553
1
8ꢀ
ꢀ

DERA sequences showing the presence of five main clusters (1-5) and non-clustered sequences.
Black circles indicate the 20 DERA proteins from different clusters selected for activity
screening (with organism names and UniProt codes). BH1352 from B. halodurans characterized
in this work is indicated by the red rectangle. (B), Screening of 20 purified DERAs for the
production of 1,3BDO from acetaldehyde in the presence of PA1127. The graph bars represent
the final concentration of 1,3BDO produced after 2 h of incubation with 10 mM NADPH and 50
mM acetaldehyde (see Materials and Methods for experimental details).
Figure 2. Crystal structures of DERAs: BH1352. (A), Dimeric structure of BH1352. (B), Overall
view of BH1352 protomer. The α helices and β strand structures that compose the TIM barrel are
indicated and labeled. The catalytic triad is displayed with sticks.
1
9ꢀ
ꢀ

Figure 3. Active site of BH1352. (A), Close-up view of the BH1352 active site. The catalytic
residues and the key residues involved in substrate binding are shown as sticks with green
carbons and labeled. (B), Diagram showing the predicted binding mode of 2-deoxyribose-5-
phosphate (DRP) in the active site of BH1352. The model was generated using structural
superimposition of crystal structures of BH1352 and the EcDERA-DRP complex (PDB code
1
JCL) (16). This revealed the BH1352 active site residues and suggested their role in substrate
binding and catalysis.
2
0ꢀ
ꢀ

Figure 4. Crystal structure of BH1352: two orientations of the C-terminal Tyr224 (red boxes in
A and B), each displayed in detail on the right.
2
1ꢀ
ꢀ

Figure 5. Site-directed mutagenesis of BH1352: catalytic activity of purified wild-type and
mutant proteins in the retro-aldol (DRP cleavage) and acetaldehyde condensation reactions. (A),
retro-aldolase activity of purified proteins with 1 mM DRP as substrate. (B), acetaldehyde
condensation reaction of purified proteins measured as the formation of 1,3BDO in the presence
of PA1127 (DERA-AKR ratio 1:1). Experimental details are described in Materials and Methods.
2
2ꢀ
ꢀ

Figure 6. Substrate entrance regions of BH1352 (A) and EcDERA (B, PDB 1JCL, DRP shown
as a green stick). The residues constituting the substrate entrance are displayed with stick model
and labeled; (C, D), The clusters of hydrophobic amino acids (green stick model, the predicted
hydrophobic contact between protein and ligand shown with a dashed arch), one near the
catalytic Lys155 (C) and the other in between the β-strand barrel and the α-helix barrel (D).
2
3ꢀ
ꢀ

Figure 7. Structure-based sequence alignment of DERAs active in 1,3BDO production: six
DERAs from Bacilli including BH1352 (light red background), six proteobacterial DERAs (light
blue background), and TM1559 (the center row). The secondary structure elements derived from
the structures of BH1352 and E. coli DeoC are shown above and below the alignment,
respectively. Residues conserved in all proteins are shown in white font on a red background.
The columns with red residues indicate the presence of more than 70% of biochemically similar
residues. The catalytic residues are indicated by cyan boxes with red residue numbers, whereas
the columns with black boxes and residue numbers indicate the substrate entrance residues. The
residues of the hydrophobic amino acid clusters are labeled with black circles.
2
4ꢀ
ꢀ

Figure 8. Production of 1,3BDO from glucose by E. coli cells expressing the aldolase-based
,3BDO pathway with the wild-type and mutant BH1352 (shown on the top). The E. coli strains
1
used were BDO-0 (wild-type BH1352), BDO-1 (BH1352 F160Y), and BDO-2 (F160Y/M173I).
The white bars show the production of 1,3BDO (g/L) by corresponding strains, whereas the gray
bars represent the corresponding yield of 1,3BDO (mg/g glucose). The results are shown as
means (± S.D.) from duplicate experiments. Experimental details are described in Materials and
Methods.
2
5ꢀ
ꢀ

Rational engineering of 2-deoxyribose-5-phosphate aldolases for the biosynthesis of (R
-1,3-butanediol
)
Taeho Kim, Peter J. Stogios, Anna N. Khusnutdinova, Kayla Nemr, Tatiana Skarina, Robert
Flick, Jeong Chan Joo, Radhakrishnan Mahadevan, Alexei Savchenko and Alexander F.
Yakunin
J. Biol. Chem. published online December 5, 2019
Access the most updated version of this article at doi: 10.1074/jbc.RA119.011363
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