A novel D-2-hydroxy acid dehydrogenase with high substrate preference for phenylpyruvate originating from lactic acid bacteria: Structural analysis on the substrate specificity

Article abstract of DOI:10.1016/j.enzmictec.2019.02.008

2-Hydroxy acid dehydrogenases (2-HADHs) have been implicated in the synthesis of 2-hydroxy acids from 2-oxo acids that are used in wide areas of industry. D-lactate dehydrogenases (D-LDHs), a subfamily of 2-HADH, have been utilized to this purpose, yet they exhibited relatively low catalytic activity to the 2-oxo acids with large functional groups at C₃. In this report, four putative 2-HADHs from Oenococcus oeni, Weissella confusa, Weissella koreensis and Pediococcus claussenii were examined for activity on phenylpyruvate (PPA), a substrate to 3-phenyllactic acid (PLA) with a C₃ phenyl group. The 2-HADH from P. claussenii was found to have the highest k_cat/K_m on PPA with 1,348.03 s^?1 mM^?1 among the four enzymes with higher substrate preference for PPA than pyruvate. Sequential, structural and mutational analysis of the enzyme revealed that it belonged to the D-LDH family, and phenylalanine at the position 51 was the key residue for the PPA binding to the active site via hydrophobic interaction, whereas in the 2-HADHs from O. oeni and W. confusa the hydrophilic tyrosine undermined the interaction. Because phenyllactate is a potential precursor for pharmaceutical compounds, antibiotics and biopolymers, the enzyme could increase the efficiency of bio-production of valuable chemicals. This study suggests a structural basis for the high substrate preference of the 2-HADH, and further engineering possibilities to synthesize versatile 2-hydroxy acids.

Full text of DOI:10.1016/j.enzmictec.2019.02.008

Accepted Manuscript
Title: A novel D-2-hydroxy acid dehydrogenase with high
substrate preference for phenylpyruvate originating from lactic
acid bacteria: structural analysis on the substrate specificity
Authors: Hoe-Suk Lee, Jisu Park, Young Je Yoo, Young Joo
Yeon
PII:
S0141-0229(19)30030-4
DOI:
Reference:
https://doi.org/10.1016/j.enzmictec.2019.02.008
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Revised date:
Accepted date:
21 December 2018
15 February 2019
22 February 2019
Please cite this article as: Lee H-Suk, Park J, Yoo YJ, Yeon YJ, A novel D-2-hydroxy
acid dehydrogenase with high substrate preference for phenylpyruvate originating
from lactic acid bacteria: structural analysis on the substrate specificity, Enzyme and
Microbial Technology (2019), https://doi.org/10.1016/j.enzmictec.2019.02.008
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A novel D-2-hydroxy acid dehydrogenase with high substrate preference for
phenylpyruvate originating from lactic acid bacteria: structural analysis on the
substrate specificity
Hoe-Suk Leea, Jisu Parkb, Young Je Yooa,c*, Young Joo Yeonb*
a
Program of Bioengineering, Seoul National University, 1 Gwanak-ro, Gwanak-gu, Seoul, 08826, Republic of
Korea
b Department of Biochemical Engineering, Gangneung-Wonju National University, 7, Jukheon-gil, Gangneung-
si, Gangwon-do, 25457, Republic of Korea
c
School of Chemical and Biological Engineering, Seoul National University, 1 Gwanak-ro, Gwanak-gu, Seoul,
08826, Republic of Korea
∗Corresponding authors
E-mail addresses: hsl0828@snu.ac.kr (HS. Lee), jspark@gwnu.ac.kr (J. Park), yjyoo@snu.ac.kr (YJ. Yoo),
yjyeon@gwnu.ac.kr (YJ. Yeon)
Highlights
 A novel D-LDH with high substrate preference for phenylpyruvate (PPA) was discovered.
 The catalytic efficiency of D-LDH from P. claussenii with PPA was 1,348 (s-1 mM-1).
 Biochemical, phylogenetic and structural analysis for the enzymes were investigated.
 Phenyl ring of PPA interacts hydrophobically with phenylalanine in the active site.
 Structural analysis provides new engineering possibilities for D-LDHs.
Abstract
2-Hydroxy acid dehydrogenases (2-HADHs) have been implicated in the synthesis of 2-hydroxy acids from 2-
oxo acids that are used in wide areas of industry. D-lactate dehydrogenases (D-LDHs), a subfamily of 2-HADH,
have been utilized to this purpose, yet they exhibited relatively low catalytic activity to the 2-oxo acids with
large functional groups at C3. In this report, four putative 2-HADHs from Oenococcus oeni, Weissella confusa,
1

Weissella koreensis and Pediococcus claussenii were examined for activity on phenylpyruvate (PPA), a substrate
to 3-phenyllactic acid (PLA) with a C3 phenyl group. The 2-HADH from P. claussenii was found to have the
highest kcat/Km on PPA with 1348.03 s-1 mM-1 among the four enzymes with higher substrate preference for PPA
than pyruvate. Sequential, structural and mutational analysis of the enzyme revealed that it belonged to the D-
LDH family, and phenylalanine at the position 51 was the key residue for the PPA binding to the active site via
hydrophobic interaction, whereas in the 2-HADHs from O. oeni and W. confusa the hydrophilic tyrosine
undermined the interaction. Because phenyllactate is a potential precursor for pharmaceutical compounds,
antibiotics and biopolymers, the enzyme could increase the efficiency of bio-production of valuable chemicals.
This study suggests a structural basis for the high substrate preference of the 2-HADH, and further engineering
possibilities to synthesize versatile 2-hydroxy acids.
Keywords
D-2-hydroxy acid dehydrogenase; Phenyllactic acid; Substrate specificity; Structural analysis
1. Introduction
D-2-hydroxy acids are versatile building block compounds used in pharmaceutical, cosmetic and organic
synthetic industries, including D-2-hydroxybutanoate, D-2-hydroxy-4-phenylbutyrate and D-phenyllactic acid
(D-2-hydroxy-3-phenylpropanoic acid, D-PLA) [1-4]. Recently, special attention has been focused on the
synthesis of D-PLA for its antimicrobial activity against various deleterious bacteria and fungi [5-9] as well as a
precursor to Danshensu (3,4-dihydroxyphenyllactic acid), a drug for cardiovascular diseases [10]. Potent uses as
a monomeric substrate of poly(PLA) has also been reported [11]. D-PLA was discovered as a metabolite of
lactic acid bacteria (LAB) [6, 12], and many D-lactate dehydrogenase (D-LDH, EC. 1.1.1.28) genes were cloned
from various species of LAB strains for the synthesis of D-PLA from phenylpyruvate (PPA) [4, 9, 13-18]. D-
2

lactate dehydrogenase is a member of D-isomer-specific 2-hydroxy acid dehydrogenase (D-2-HADH) family
with typical two Rossmann-fold motifs each constituting a catalytic domain and a cofactor binding domain [19-
23]. Many structural studies with pyruvate, the primary substrate, show that D-LDHs are allosteric dimers with
the active site located in the interface of the two domains [19, 21]. The cofactor binding domain side of the
active site comprises of a bound NADH and a H296 whose pKa is modified by E264, for a hydride transfer to
the pyruvate C2 and 2-oxo group, respectively [19, 21, 22, 24]. R235 is known to stabilize the carboxylic group
of pyruvate from face-on or at an angle. N77, V78 and G79 from the catalytic domain side of the active site
were also proposed to stabilize the carboxylic group in a productive binding mode. Cofactor and substrate
binding enables a motion of the two domains so that pyruvate is tightly held by the residues from both side.
However, much less is known for the PPA binding to these D-LDHs compared to the detailed structural analysis
with respect to pyruvate.
In this study, four putative 2-hydroxy acid dehydrogenases (2-HADH) based on the homologous amino acid
sequences in four LAB species, Oenococcus oeni, Weissella confusa, Weissella koreensis, and Pediococcus
claussenii (oo, wc, wk and pcHADH, henceforth), were examined for the enzymatic synthesis of D-PLA from
PPA. Phylogenetic analysis indicated oo, wc and pcHADH belong to the D-LDH subfamily, while wkHADH is
a member of glyoxylate-hydroxypyruvate subfamily of the D-2-HADH family. A wild-type enzyme that belongs
to the D-LDH family but with higher substrate specificity for PPA than pyruvate was found, whereas wild-types
of other members of this family generally display pyruvate preference, although several engineering attempts
succeeded in creating PPA-specific enzymes [4, 13, 25, 26]. Structural analysis with PPA bound to the active site
of the enzymes was carried out to explain their substrate specificities. The enzyme with higher preference for
PPA and its structural analysis will help developing biocatalysts capable of synthesizing α-2-hydroxy acid with
larger substituent (phenyl, 4-hydroxyphenyl or aliphatic chains with a length of 4 ~ 6 carbon) at C3 position.
2. Materials & methods
2.1 Materials
The ooHADH, wcHADH, wkHADH and pcHADH genes were synthesized from GenScript (New Jersey, USA)
3

with additional C-terminal His6-tag and codon optimization for E. coli [27, 28]. Their NCBI accession numbers
are as follows; ooHADH (MH920337), wcHADH (MH920336), wkHADH (MH920338) and pcHADH
(MH920335). They were cloned in pET22b(+) vectors using NdeI and XhoI restriction sites. Oligonucleotide
synthesis and mutation sequencing were performed by Cosmogenetech (Seoul, Korea). E. coli strains were
purchased from Invitrogen (Carlsbad, CA). Sodium phenylpyruvate was purchased from Tokyo Chemical
Industry (Tokyo, Japan). All other chemicals were purchased from Sigma-Aldrich (St Louis, USA).
2.2 Mutagenesis, expression and purification of 2-HADHs
The 2-HADH genes were transformed into E. coli DH5α for cloning and mutation and E. coli BL21 (DE3) for
expression. The pcHADH gene was mutated with the primers described in the Supplemental material S1
following the QuikChange II Site-Directed Mutagenesis protocol (Agilent, Santa Clara, USA). The cells
transformed with wild-type ooHADH, wcHADH, wkHADH, pcHADH and mutant pcHADH were grown for 16
h in lysogeny broth (LB) medium supplemented with 100 μg/ml ampicillin, at 37 °C and 200 rpm under aerobic
conditions. For protein expression purposes, 50 ml of BL21 cell culture were grown to reach the optical density
at 600 nm (OD600) of 0.6, followed by induction with 0.8 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) at
20 °C, 200 rpm for 24 h. The induced cell cultures were harvested by centrifugation at 3200 rpm, 4 °C for 20
minutes. Harvested cells were lysed by Bugbuster® (Merck Millipore, Billerica, USA), centrifuged (8000 rpm,
4 °C, 20 min) for cell debris removal and subjected to Ni-affinity chromatography (Qiagen, Hilden, Germany)
for elution with 250 mM imidazole. Purification of the enzymes to homogeneity was confirmed by SDS-PAGE.
Protein concentration was determined by the Bradford method using bovine serum albumin as a standard [13,
29].
2.3 Enzyme activity assays with the purified 2-HADHs
In vitro enzyme activity was assayed at 30 °C in 1ml cuvette containing 50 mM sodium acetate buffer (pH 5.5),
0.2 mM NADH, up to 20 mM pyruvate or PPA as a substrate, and 0.5-5.4 μg of the purified enzyme. The rate of
NADH decrease was determined by absorbance change at 340 nm, with molar extinction coefficient of 6.22
mM-1cm-1 in a spectrophotometer (Molecular Devices, San Jose, USA). One unit of enzyme activity was defined
as the amount of enzyme that oxidizes 1 μmol NADH per minute. The specific enzyme activity was measured as
4

units per milligram of protein [14]. Apparent kcat and Km were calculated by measuring activities on varying
degree of substrate concentrations (0.04 ~ 40 mM) and fitting the data to the Michaelis-Menten equation. All
measurements were carried out in triplicates.
2.4 In vitro enzymatic conversion of PPA into PLA
Conversion of PPA into PLA was analyzed via HPLC. The reaction mixture consisted of 8.59 nM purified 2-
HADH, 20 mM PPA, 1.5 mM NADH, 30 mM sodium formate, 1 unit of formate dehydrogenase from Candida
boidinii (Sigma Aldrich, St Louis, USA) and 50 mM sodium acetate buffer (pH 6.5) in total volume of 3 ml. The
mixture was sampled at 0, 0.5, 1, 2, 4, 8 and 12 h, heat-inactivated at 99 °C, centrifuged for 5 min (13000 rpm),
and filtered by 0.2 μm Whatman PVDF filter (GE Healthcare, Pittsburgh, USA) before being subjected into
YL9100 HPLC system (YL instruments, Anyang, Korea) installed with Aminex® HPX-87H Ion exclusion
Column (300 mm x 7.8 mm) from Bio-Rad (Hercules, USA). 5 mM H2SO4 was used as a mobile phase at a flow
rate of 0.6 ml min-1 and the peaks for PPA and PLA were observed at 19.42 min and 41.28 min respectively, at
37 °C by a UV detector at 210 nm [26]. For a chiral detection of the enantiomeric PLA product, Chiralcel OJ-H
column (250 mm x 0.46mm, 5 μm) from Daicel (Tokyo, Japan) was used with a mobile phase of hexane, 2-
propanol, and trifluoroacetic acid in 90:10:0.1 (v:v), and UV detection at 261 nm. PPA was detected at 38 min,
D-PLA at 30 min and L-PLA at 34 min.
2.5 Phylogenetic analysis and multiple sequence alignment
Phylogenetic trees were constructed using the four 2-HADHs and seven other D-LDHs previously reported to
have PPA reductase activity, along with 365 sequences that belong to the D-2-HADH family. The D-LDH
sequences from following species were retrieved from NCBI database, with their accession numbers in
parenthesis: P. pentosaceus ATCC25745 D-LDH (CP000422), P. acidilactici DSM20284 D-LDH (X70925), L.
plantarum SK002 D-LDH (FJ712707), L. pentosus JCM1558 D-LDH (P26298), B. coagulans SDM D-LDH
(HQ148710), L. bulgaricus ATCC11842 D-LDH (CR954253) and S. inulinus CASD (MG579976). All other
sequences were obtained from PROSITE database (ID: PDOC00063) [30]. A neighbor-joining unrooted tree
was generated with MEGA-X 10.0.5 software using 100 iterations and a 60% threshold value for bootstraps [31,
5

32]. Multiple sequence alignment was carried out using Clustal Omega [33].
2.6 Homology modeling and molecular docking
Homology models of ooHADH, wcHADH and pcHADH were constructed based on the X-ray crystal structures
of D-lactate dehydrogenase from Lactobacillus helveticus and Lactobacillus bulgaricus (PDB: 2dld and 1j49
respectively) [21]. The templates were determined by searching each sequence against the Protein Data Bank
database using the position specific iterated-BLAST algorithm [34, 35]. The secondary structures were aligned
between the templates and the query sequences and homology modeling was carried out by the Prime homology
modeling program from Schrödinger [36]. The crystal-bound NAD was manually changed to NADH and energy
minimized. The stereochemical quality of the models were validated by Ramachandran Plot in Rampage server
[37]. Glide program from Schrödinger was used for the docking of the substrate into the homology modeled
structures with OPLS3 forcefield [38, 39]. The grid-generation module from Glide was used to generate grids
comprising the active sites of the 2-HADHs and the nicotinamide group of NADH. Pyruvate and PPA as ligands
were created with all possible ionization states via LigPrep [40]. Standard Precision mode of Glide was used to
produce docking poses.
3. Results & discussion
3.1 Activity screening of 2-HADHs
The four 2-HADHs were heterologously expressed in E. coli BL21 (DE3) and purified up to 5 mg, and their in
vitro enzymatic activities toward pyruvate and PPA were determined (Fig. 1). For pyruvate, wcHADH showed
the highest activity with 428 U mg-1. pcHADH and ooHADH had 0.67 and 0.55 fold lower activities than the
wcHADH, respectively. For PPA, however, pcHADH was found with the highest activity of 591 U mg-1.
Activities of ooHADH and wcHADH were much lower than pcHADH, 0.03 and 0.15 fold respectively.
wkHADH had no significant activities against either pyruvate or PPA, even though Bradford assay showed all
four 2-HADHs were expressed in similar quantities.
3.2 Kinetic parameters of 2-HADHs
6

The kinetic parameters of the 2-HADHs for pyruvate and PPA were determined using NADH as the cofactor
(Table 1). Apparent Km (PPA) was 14 ~ 16 fold higher than Km (pyruvate) in ooHADH and wcHADH, indicating low
binding affinity with PPA in these enzymes. On the other hand, pcHADH maintained low Km for both pyruvate
and PPA, notably slightly lower Km (PPA). In terms of kcat (pyruvate), wcHADH showed 2 ~ 3 fold higher value than
oo and pcHADH. kcat (PPA) was the highest with pcHADH, closely followed by wcHADH, which were 6 fold
higher than that of ooHADH. The overall catalytic efficiency, kcat (pyruvate)/Km (pyruvate), was the highest with
wcHADH (1124 s-1 mM-1), and kcat (PPA)/Km (PPA) was the highest with pcHADH (1348 s-1 mM-1). This suggests
pcHADH as a promising biocatalyst for biosynthesis of D-PLA, as it shows an inversion of substrate specificity
for PPA over pyruvate, unlike many other D-LDH wild-types from previous studies (Table 2).
In addition, enzymatic conversion of PPA into PLA was confirmed by HPLC analysis of in vitro batch reaction
with 20 mM of PPA (Fig. 2). In correlation with the kinetic parameters, the pcHADH was able to convert over
80 % of the PPA within 2 h, with productivity of 1.4 g-1 L-1 hr-1. The oo and wcHADH did not reach 100 %
conversion within the timescale of the experiment. Chiral column HPLC detected the peaks for the D-isomer but
no L-isomer of PLA in the reaction by oo, wc and pcHADH. Unreacted remnant of PPA was also detected in the
reaction by oo and wcHADH (Supplemental material S2).
3.3 Phylogenetic analysis and sequence alignment
A phylogenetic analysis on the 2-HADHs examined in this study as well as 365 members of D-2-HADH family
with non-redundant sequences revealed the ooHADH, wcHADH and pcHADH belonged to the D-LDH
subfamily, whereas wkHADH belonged to D-3-phosphoglycerate dehydrogenase subfamily which may explain
its negligible activity with the pyruvate or PPA (Fig. 3a). All four 2-HADHs had a conserved sequence of G-X-
G-X-X-G (where X represents any of the 20 amino acids) for a dinucleotide binding motif at the position 151 ~
163, similar to the D-2-HADHs characteristic described by Taguchi and Ohta, compared to L-isomer specific 2-
HADHs with the same motif at the position 28-33 [23]. Furthermore, ooHADH, wcHADH and pcHADH had
Y174 or equivalent that was found to be crucial for NADH-dependency over NADPH, as reported by Zhu et al.,
in the study of NADP+ preferred D-LDH from S. inulinus [41].
The four 2-HADHs and seven D-LDHs previously reported to have PPA reductive activity were further
7

analyzed for phylogenetic relationship (Fig. 3b). The sequence of pcHADH revealed a close relationship with P.
acidilactici DSM 20284 D-LDH and P. pentosaceus D-LDH, while the ooHADH and the wcHADH were closer
to L. bulgaricus ATCC 11842 D-LDH. Both D-LDHs from P. acidilactici DSM 20284 and P. pentosaceus have
presented relatively high catalytic efficiencies of 105 and 100 s-1 mM-1 for PPA [9, 14]. However, it is notable
that D-LDHs from P. acidilactici and P, pentosaceus have substrate specificity for pyruvate over PPA, yet
pcHADH with a closely related sequence have inverted substrate preference. L. bulgaricus ATCC 11842 D-LDH
had only kcat/Km of 1.0 s-1 mM-1 [4], showing similar catalytic efficiency with ooHADH and wcHADH.
Multiple sequence alignment via Clustal Omega shows highly conserved residues in the active site including the
R234, N76, V77 and G78 known to stabilize the carboxylate and 2-oxo functional groups of the substrates via
hydrogen bonding, H295 as a proton donor for reduction of the oxo group, and E263 modulating the pKa of the
H295 (Fig. 4, corresponding residues are numbered based on those of pcHADH henceforth) [21]. In B.
coagulans D-LDH and S. inulinus D-LDH several different amino acid residues at the position 51, 76-77, 100,
105 and 298 in the active site may have led to their comparatively low kcat/Km of 3.9 and 1.49 s-1 mM-1,
respectively [13, 16]. Notably, the pcHADH with the highest kcat (PPA)/Km (PPA) among wild-type D-LDHs has the
same residues in the active site, but with phenylalanine at the position 51 instead of tyrosine such as most other
D-LDHs. The corresponding position has been recognized as a recognition site for the side chain of 2-oxo
acids and subjected to engineering in many D-LDHs [4, 13, 25, 26, 42].
3.4 Mutation study of pcHADH
The pcHADH was subjected to mutation to examine the effect of the F51 on its activity. The position was
mutated into aliphatic residues or aromatic residues as well as hydrophilic serine for variance in steric size and
hydrophobicity. The wild-type pcHADH had the highest specific activity for PPA of 611 U mg-1 followed by 95 %
relative activity with F51G mutation, but mostly with much lower activities (Fig. 5). Furthermore, mutation of
F51 into tyrosine as in most wild-types of D-LDHs decreased up to 4% relative activity on phenylpyruvate, but
retained 99% relative activity on pyruvate (data not shown). Mutation into leucine that had predominant activity
improvement from previous studies in L. pentosus and L. bulgaricus D-LDH decreased relative activity to 10%
[4, 25, 26, 42]. In these studies, mutations into aliphatic residues such as leucine, alanine and valine were carried
out based on the work of Tokuda et al., where a sequence alignment of D-LDHs with L. casei D-
8

hydroxyisocaproate dehydrogenase revealed a possibility of leucine to accommodate 2-oxo acid substrates with
larger C3 substituents [42]. From our work, phenyalanine at the position 51 is a plausible mutation candidate for
a high PPA activity, including but not limiting to pcHADH considering the similarities in the folds and the active
site structures of many D-2-HADHs and D-LDHs [23, 43].
3.5 Structural analysis of the 2-HADHs
For structural modeling of oo, wc and pcHADHs that showed the activities toward both pyruvate and PPA, they
were homology modeled based on structural templates from D-LDHs of L. helveticus (PDB: 2dld) and L.
bulgaricus (PDB: 1j49) which showed the highest sequence identity (ooHADH, 69%; wcHADH, 68%;
pcHADH, 50%). The validity of the homology models was confirmed by the Rampage server, which provided
Ramachandran plot data for ooHADH, wcHADH and pcHADH as 96.4%, 97.0% and 99.1% of the residues in
favored/allowed region, respectively. This is comparable to the 95.8% for 2dld and 99.5% for 1j49 whose
structures are from X-ray crystallography.
Pyruvate and PPA were then docked as a substrate in the active site of the 2-HADHs to explain the activity (Fig.
6). Productive binding modes of the substrates are in reference to the oxamate analog in 2dld, and the
configuration of pyruvate modeled in the active site of 1j49 [21]. The three 2-HADHs revealed a similar active
site environment, except F51 for pcHADH, yet there were deviations to the binding of the substrates in all three
enzymes. In oo and pcHADH, the carboxylate groups of the pyruvate and PPA were stabilized by hydrogen
bonding with the N76-V77-G78 backbone and Y100 side chain in the catalytic domain. In wcHADH, PPA
followed similar configuration, but pyruvate was rotated for the carboxylate to face stabilization by R235, which
may account for its high pyruvate activity. Both mechanisms had been proposed previously where these
configurations were regarded to enable the required electron transfer from the donors, NADH and H295, to
substrates for reductive catalysis [21].
The hydride transfer distances between the NADH and the C2 of the substrate (d1) and between H295 and
substrate 2-oxo group (d2), which affect the activation free energy barrier are presented in Table 3 [44]. For PPA,
pcHADH showed d1(PPA) and d2(PPA) values of 4.1 Å and 3.3 Å. The wcHADH with similar kcat (PPA) as pcHADH
presented increased d1(PPA) but decreased d2(PPA) (5.7 Å and 2.7 Å), while the ooHADH with 6 fold reduced
9

kcat(PPA) had similar d2(PPA) as pcHADH but longer d1(PPA) (6.3 Å). The pcHADH had similar kcat (pyruvate) as kcat
(PPA), in correlation with longer d1(pyruvate) (5.1 Å) but shorter d2(pyruvate) (2.8 Å) than those for PPA.
In order to see the effect of residue 51 on the substrate recognition, the distances from the centroid of the phenyl
substituent of PPA or C3 of pyruvate to the centroid of the phenyl ring of the residue 51 (d3) as well as to the
hydroxyl group of tyrosine in oo and wcHADH (d4) were measured. The d3(PPA) and d4(PPA) showed that
pcHADH (4.2 Å) had a stable hydrophobic contact with the phenyl substituent while in oo and wcHADH the
residue was farther away from the substituent (7.4 Å for d3 and 6.1 ~7.0 Å for d4), an indication of relatively
unfavorable interaction with the hydrophilic tyrosine. It was shown that the phenyl substituent is steered by the
tyrosine in oo and wcHADH, distorting the binding form of PPA so that the d1 becomes larger in the Fig. 6.
Steric hindrance effect is also expected due to the additional hydroxyl groups in the tyrosine. For pyruvate, the
tyrosine has less steric and hydrophilic effect on the small C3, whereas hydrophobic phenylalanine in pcHADH
could be drawing the C3, resulting in longer d1(pyruvate) and lower kcat(pyruvate) than those for PPA.
In terms of binding free energy, more stable binding of PPA marked by ΔG
were in line with lower
binding(PPA)
apparent Km. ΔG binding(pyruvate) showed similar values for all three enzymes, with respect to similar apparent Km.
However, there is no correlation between ΔG of pyruvate and PPA with respect to Km, because the large phenyl
group of PPA contributes more in the binding energy calculation as indicated by 2 fold lower ΔG binding(PPA) than
ΔG binding(pyruvate)
.
Modeling of PPA with pcHADH mutants was also carried out to examine the mutation effect (Fig. 7 and Table
4). The carboxylate group of PPA was directed away from the N76-V77-G78 loop in F51L mutant, marked by
the longer distance to the backbone nitrogen of G78 residue (d6). The d1 increased, as opposed to d2 in F51L in
comparison to the wild-type. The ΔG binding(PPA) indicated less binding affinity in the mutants, which could be due
to less hydrophobic effect exerted by a longer distance to the leucine than phenylalanine. The pcHADH F51G
and F51S mutants, which are expected to have less hydrophobic effect than F51L due to the longer d3, were
more stabilized by the shorter d6 than the F51L mutant in correlation with their rather high relative specific
activities.
10

4. Conclusion
Screening of dehydrogenases capable of synthesizing versatile 2-hydroxy acids is important for industrial
applications. Many previous studies have reported enzymes to improve the activity on PPA, with focuses on
Y52L in various D-LDHs. In this study, four putative 2-HADHs were investigated for the PPA activity, of which
pcHADH presented the highest catalytic efficiency and more substrate preference for PPA than pyruvate.
Phylogenetic analysis revealed it belonged to a D-LDH family, and a detailed structural analysis of the active
site with both reducing cofactor and PPA brought phenylalanine residue at the position 51 of pcHADH to
attention. Phenylalanine at the position was not engineered in other D-LDHs previously to our knowledge, and it
seems to make a strong hydrophobic interaction with PPA in pcHADH. Further engineering possibilities on
other D-LDHs and 2-HADHs based on our structural analysis are open for synthesis of 2-hydroxy acids with
large functional substituents at C3.
Conflict of interest statement
The authors declare that there are no conflicts of interest.
Acknowledgement
This research was supported by the Technology Development Program to Solve Climate Changes of the
National Research Foundation (NRF) funded by the Ministry of Science and ICT (Grant Number:
2017M1A2A2087630).
Appendix A. Supplementary Material
The following is supplementary data to this article:
11

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16

Figure Captions
Fig. 1 Relative specific activities of 2-HADHs on (a) pyruvate and (b) PPA. Specific activities of wcHADH for
pyruvate and pcHADH for PPA corresponding to 100 % relative activity were 428 ± 27.4 and 591 ± 35.7 U mg-1,
respectively
17

Fig. 2 In vitro enzymatic conversion of PPA into PLA by 2-HADHs. Circles, triangles and squares represent
conversion by ooHADH, wcHADH and pcHADH, respectively. 100 % concentration corresponds to 20 mM of
PLA
Fig. 3 Phylogenetic analysis presenting (a) unrooted neighbor-joining tree of the 2-HADHs investigated in this
study and 365 non-redundant members of D-isomer specific 2-hydroxy acid dehydrogenase family. Proteins are
grouped by their characterized functions. GHPR, glyoxylate and hydroxypyruvate reductase; SERA, D-3-
phosphoglycerate dehydrogenase; CTBP, C-terminal binding protein; FDH, formate dehydrogenase; D-LDH, D-
18

lactate dehydrogenase; PDXB, erythronate-4-phosphate dehydrogenase. ⓐ, ⓑ, ⓒ and ⓓ represents oo, wc,
pc and wkHADH, respectively. Scale bar indicates 0.5 amino acid substitution per position. (b) unrooted
neighbor-joining tree the 2-HADHs investigated in this study and other D-LDHs with known PPA reductase
activity. Bootstrap value was >60 %. Accession numbers from the NCBI database are given in brackets. Scale
bar indicates 0.2 amino acid substitution per position.
Fig. 4 Sequence alignment of the 2-HADHs and the D-LDHs with PPA activity. Active site residues are shaded
in gray, with their residue numbers corresponding to those of pcHADH above.
Fig. 5 Relative specific activities of pcHADH wild-type and F51 mutants for PPA. The specific activity of the
wild-type corresponding to 100 % relative activity was 525 ± 58.9 U mg-1
19

Fig. 6 The active site of the 2-HADHs docked with substrates: (a), ooHADH with pyruvate; (b), ooHADH with
PPA; (c), wcHADH with pyruvate; (d), wcHADH with PPA; (e), pcHADH with pyruvate; (f), pcHADH with
PPA. The carbon atoms of NADH, substrates docking poses and active site residues are colored in yellow, green
and gray, respectively with the residue numbers. The distances are indicated in reference to the Table 3: d1,
between NADH and the substrate C2 atom; d2, between the H295 NƐ atom equivalent and the substrate 2-oxo
group; d3, between the centroid of phenyl ring of the F51 (Y52 in oo and wcHADH) and C3 atom in pyruvate
(or the centroid of phenyl group in PPA); d4, between the hydroxyl group of Y52 and substrate C3 atom in
pyruvate (or the centroid of phenyl group in PPA)
20

Fig. 7 Comparison of the active site of pcHADH docked with PPA. Wild-type, (a); F51L, (b); F51G, (c); F51S,
(d). The distances are indicated in reference to the Table 4: d1, between NADH and the PPA C2 atom; d2,
between the H295 NƐ atom and the substrate 2-oxo group; d3, between the centroid of phenyl group in PPA and
the closest heavy atom of the residue 51 side chain; d5, between the carboxylate of PPA and the backbone N of
V77; d6, between the carboxylate of PPA and the backbone N of G78.
21

Table 1 Kinetic parameters of 2-HADHs investigated in this study
Kinetic parameters for substrates
푘cat/퐾m (PPA)
푘cat/퐾m (Pyruvate)
Pyruvate
Km (mM)
1.12 ± 0.04
PPA
2-HADH
× 100
kcat/Km
kcat/Km
kcat (s-1)
kcat (s-1)
Km (mM)
(s-1 mM-1)
(s-1 mM-1)
(%)
ooHADH
wcHADH
pcHADH
674 ± 22
1434 ± 240
459 ± 30
602 ± 1.43
77.8 ± 5.36
457 ± 15.1
472 ± 33.8
15.6 ± 0.53
20.3 ± 0.60
5.00 ± 0.18
22.5 ± 0.07
0.83
2.00
165
1.28 ± 0.24 1124 ± 23.6
0.56 ± 0.01 813 ± 46.1
0.35 ± 0.05 1348 ± 99.2
Table 2 Comparative analysis of activity of wild-type 2-HADHs and D-LDHs reported in the literatures
푘cat/퐾m (PPA)
Pyruvate
PPA
푘cat/퐾m (Pyruvate)
Bacterial origin of
enzymes
kcat/Km
kcat/Km
Reference
× 100
kcat
Km
(mM)
kcat
Km
(mM)
(s-1 mM-
(s-1 mM-
(s-1)
(s-1)
1
1
)
)
(%)
22

L. bulgaricus ATCC11842
P. pentosaceus ATCC25745
S. inulinus CASD
NR
320
8.85
287
NR
NR
0.49
3.40
0.09
0.06
2.20
NR
NR
658
22.60
3157
NR
11.3
173
4.94
305
NR
11.4
1.73
3.32
2.92
5.40
4.40
1.68
1.00
100
1.49
105
NR
-
[4]
[9]
15.2
6.59
3.33
-
[13]
[14]
[15]
[16]
[17]
P. acidilactici DSM20284
L. plantarum SK002
B. coagulans SDM
23.6
NR
11
16.5
123
3.90
73.0
35.5
-
L. fermentum JN248
NR
Lactobacillus sp. CGMCC
9967
NR
NR
NR
47.3
0.82
57.7
-
[18]
W. fluorescens TK1
NR
321
NR
NR
150
40
0.40
0.80
380
-
[29]
[41]
L. pentosus JCM1558
0.12
2675
50.0
1.87
NR: Not reported
Table 3 Binding free energy and the key distances in the modeling of the substrate in 2-HADHs
Pyruvate
PPA
Enzyme
ΔG binding
d1
(Å)
d2
(Å)
d3
(Å)
d4
(Å)
ΔG binding
d1
(Å)
d2
(Å)
3.2
d3
(Å)
d4
(Å)
7.0.
(kcal mol-1)
(kcal mol-1)
ooHADH
-3.32
4.4
2.9
6.9
4.8
-5.31
6.3
7.4
wcHADH
pcHADH
-3.39
-3.45
4.6
5.1
2.9
2.8
4.8
4.6
3.5
-5.09
-6.53
5.7
4.1
2.7
3.3
7.4
4.2
6.1
N/A
N/A
*N/A: Not applicable
ΔG binding, binding free energy; d1, between NADH and the substrate C2 atom; d2, between the H295 NƐ atom equivalent and
the substrate 2-oxo group; d3, between the centroid of phenyl ring of the F51 (Y52 in oo and wcHADH) and C3 atom in
pyruvate (or the centroid of phenyl group in PPA); d4, between the hydroxyl group of Y52 and substrate C3 atom in pyruvate
(or the centroid of phenyl group in PPA)
Table 4 Binding free energy and the key distances in the modeling of PPA in pcHADH wild-type and F51L mutant
ΔG binding (PPA)
d1
(Å)
d2
(Å)
d3
(Å)
d5
(Å)
d6
(Å)
pcHADH
(kcal mol-1)
Wild type
F51L
-6.53
-6.08
-6.36
-6.19
4.1
4.6
4.4
4.2
3.3 3.4
2.8 4.2
2.8 5.4
2.7 5.3
2.8
2.8
2.8
2.6
3.2
4.8
3.4
3.6
F51G
F51S
ΔG binding, binding free energy; d1, between NADH and the substrate C2 atom; d2, between the H295 NƐ atom and the
substrate 2-oxo group; d3, between the centroid of phenyl group in PPA and the closest heavy atom of the residue 51 side
chain; d5, between the carboxylate of PPA and the backbone N of V77; d6, between the carboxylate of PPA and the
backbone N of G78
23