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Cite This: J. Agric. Food Chem. 2018, 66, 8371−8381

Catalytic, Computational, and Evolutionary Analysis of the D‑Lactate Dehydrogenases Responsible for D‑Lactic Acid Production in Lactic Acid Bacteria Baolei Jia,†,‡ Zhong Ji Pu,§ Ke Tang,† Xiaomeng Jia,‡ Kyung Hyun Kim,‡ Xinli Liu,† and Che Ok Jeon*,‡

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†

State Key Laboratory of Biobased Material and Green Papermaking, School of Bioengineering, Qilu University of Technology (Shandong Academy of Sciences), Jinan 250353, China ‡ Department of Life Science, Chung-Ang University, Seoul 06974, Republic of Korea § School of Life Science and Biotechnology, Dalian University of Technology, Dalian 116024, China S Supporting Information *

ABSTRACT: D-Lactate dehydrogenase (D-LDH) catalyzes the reversible reaction pyruvate + NADH + H+ ↔ lactate + NAD+, which is a principal step in the production of D-lactate in lactic acid bacteria. In this study, we identified and characterized the major D-LDH (D-LDH1) from three D-LDHs in Leuconostoc mesenteroides, which has been extensively used in food processing. A molecular simulation study of D-LDH1 showed that the conformation changes during substrate binding. During catalysis, Tyr101 and Arg235 bind the substrates by hydrogen bonds and His296 acts as a general acid/base for proton transfer. These residues are also highly conserved and have coevolved. Point mutations proved that the substrate binding sites and catalytic site are crucial for enzyme activity. Network and phylogenetic analyses indicated that D-LDH1 and the homologues are widely distributed but are most abundant in bacteria and fungi. This study expands the understanding of the functions, catalytic mechanism, and evolution of D-LDH. KEYWORDS: D-lactate dehydrogenases, catalytic mechanism, lactic acid bacteria, evolution

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dependent LDH.8 In the genome of LAB, stereospecific L- or D-LDHs are encoded that are responsible for producing the corresponding lactic acids. L-LDHs have been thoroughly studied because L-lactic acid is widely used in producing food, cosmetics, and medicine.9 D-LDHs have also been studied but not as intensely as L-LDHs.10 According to the structures of DLDHs from L. bulgaricus and Lactobacillus jensenii, D-LDHs form a homodimeric structure, and the monomer consists of the N-terminal substrate-binding domain and the C-terminal NAD+-binding domain.11 As an important group of LAB, Leuconostoc mesenteroides and its subspecies are key players responsible for fermenting many foods. L. mesenteroides ssp. cremoris is a main aroma producer during cheese fermentation.12,13 During coffee fermentation, L. mesenteroides can initiate acidification and play a role in solubilizing pectic substances.13−15 Especially when used as a fermentation starter for sauerkraut and kimchi, L. mesenteroides can help shorten fermentation time by organic acid and mannitol production and can improve organoleptic properties.16,17 Fermentation involving L. mesenteroides results in the accumulation of organic acids, primarily D-lactic acid and a small amount of L-lactic acid as the major end products of glucose and other carbohydrate metabolism.8,18 L. mesenter-

INTRODUCTION Lactic acid bacteria (LAB) have been used in various fermented foods throughout history.1 The LAB encompass a phylogenetically diverse group of species that play crucial roles in a variety of food and feed fermentations worldwide. The ability of LAB to produce organic acids and other antimicrobial substances has made them indispensable in manufacturing fermented foods such as sauerkraut, cheese, sausage, sourdough bread, animal silage, and kimchi.1 LAB can also serve as biofertilizers, biocontrols, biostimulants, and bioelicitors in plant production.2 Furthermore, the LAB-mediated conversion of carbohydrates into organic acids, especially lactic acid, has prompted interest in industrial applications3 because poly(lactic acid) (PLA) polymers are thermoplastic and biodegradable polyesters with applications ranging from medicine to agriculture and packaging.4 Lactic acid is the major metabolite of LAB; this acid reduces the pH and prevents the risk of proliferation of undesirable microorganisms.5 Lactic acid is chiral and thus has two optical isomers: L(+)-lactic acid and D(−)-lactic acid. L(+)-Lactic acid is used in the food industry in many ways because the human body can only assimilate this form of lactic acid.6 On the contrary, Lactobacillus bulgaricus, commonly used in the dairy industry to produce yogurt, converts 90% of the pyruvate into 7 D-lactic acid and triggers the aggregation of casein micelles. The generation of lactic acid from pyruvate by lactate dehydrogenase (LDH) is a main step LAB use to regenerate NAD+; therefore, all LAB harbor a large amount of NAD+© 2018 American Chemical Society

Received: Revised: Accepted: Published: 8371

May 9, 2018 July 13, 2018 July 16, 2018 July 16, 2018 DOI: 10.1021/acs.jafc.8b02454 J. Agric. Food Chem. 2018, 66, 8371−8381

Article

Journal of Agricultural and Food Chemistry

(0−50 °C). The Michaelis−Menten equation was employed to calculate the kinetic parameters. All enzyme activity experiments were performed in three biological and three technical replicates. Molecular Dynamics Simulation. The 3D structure of D-LDH1 was modeled using Modeler 9 software.21 Structural validation of the enzyme was performed by creating a Ramachandran plot using the PROCHECK server.22 The complexes of D-LDH1 and the substrates were built by LeDock (http://www.lephar.com/index.htm). The hydrogen atoms were added to the protein system using the H++ Web server.23 The minimization of five models (apo-LDH, NAD+− LDH, NADH−LDH, NAD+−lactate−LDH, and NADH−pyruvate− LDH) was performed using the combined steepest descent/ conjugated gradient in three stages: (1) The enzyme model was frozen with a force constant of 500 kcal/mol and free waters and ions; (2) the heavy atoms in the system were kept fixed with a force constant of 500 kcal/mol, and all hydrogens in the system were set free; and (3) all of the atoms in the system were freely optimized. To gradually heat the temperature of the system to the standard temperature of 300 K at a constant pressure of 1 bar, both models were preheated for a total of 100 ps in a NVT ensemble. In the first 50 ps, the heating was linear, from 0 to 300 K; in the 50 ps that followed, the temperature of 300 K was fixed. Molecular dynamics (MD) proceeded on an NPT ensemble. A constant temperature of 300 K was maintained using Langevin dynamics implemented in the AMBER14 package, and a fixed pressure of 1 bar was set. Periodic boundary conditions were imposed to the models built to account for long-range interactions. Short- and long-range electrostatic interactions were calculated with Ewald summation methods. The SHAKE algorithm was applied to all bonds involving hydrogen atoms for a mechanical relaxation time of 2 ps. To calculate long-range van der Waals interactions, a cutoff distance of 12 Å was established. The root-mean-square deviation (RMSD) and root-mean square fluctuation (RMSF) for both simulations were calculated with the CPPTRAJ module of AmberTools15. The hybrid quantum mechanical/ molecular mechanical (QM/MM)/steered molecular dynamics (SMD) methods were employed to study catalytic mechanisms.24 Calculation of Binding Free Energy. Binding free energies of cofactors and substrates for D-lactate dehydrogenase were calculated using the MM/GBSA program in AmberTools15. For each complex, 200 snapshots were taken from the last 40 ns of the MD trajectory with an interval of 200 ps. In this method, the binding free energy (ΔG) can be represented as

oides ssp. mesenteroides J18 is one of the most predominant LAB during kimchi fermentation, and the genome of this strain contains three genes annotated as D -LDH ( D -LDH1: AET30962, D-LDH2: AET31034, and D-LDH3: AET31236).19 However, it is still unclear whether one of these three genes plays the leading role or whether they equally contribute to producing D-lactic acid. In this study, we cloned and expressed these three D-LDH genes in Escherichia coli. Further analysis showed that D-LDH1 is the major enzyme responsible for producing D-lactic acid in this strain. The biochemical characteristics and catalytic mechanism were further studied by activity assays and molecular simulation. Finally, a large-scale in silico analysis was performed to study the evolution and distribution of this gene.

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MATERIALS AND METHODS

Cloning of D-LDHs from L. mesenteroides ssp. mesenteroides J18. PCR using L. mesenteroides ssp. mesenteroides J18 genomic DNA as a template was performed to isolate D-LDHs using the oligonucleotide primers listed in Supplementary Table 1. The PCR product and pET28-(a) were digested by the restriction enzymes (BamHI and HindIII) and ligated into the pET28a vector. The ligation products were transformed into E. coli BL21 (DE3) by electroporation and were confirmed by sequencing. Expression and Purification of D-LDHs. E. coli BL21 (DE3) containing the pET28a-D-LDHs plasmids were cultured in 2 L of LB broth containing 50 μg/mL kanamycin at 37 °C for 3 h. When the OD600 reached 0.7, 1 mM isopropyl-β-D-thiogalactopyranoside (IPTG) was added to induce protein expression. The cells were cultured in the presence of IPTG for 4 h with shaking, harvested by centrifugation at 5000g for 10 min, then resuspended in lysis buffer containing 50 mM Tris (pH 8.0), 300 mM NaCl, and 20 mM βmercaptoethanol. The cell suspension was sonicated; then, the supernatant was collected by centrifugation and loaded on a Ni-NTA column. After washing the column with lysis buffer, the proteins were eluted using an imidazole gradient (20, 50, and 250 mM). The purified enzymes were visualized after separation by 12% sodium dodecyl sulfate−polyacrylamide gel electrophoresis (SDS-PAGE). After dialysis with 50 mM Tris-HCl buffer (pH 8.0) containing 300 mM NaCl and 20 mM β-mercaptoethanol, the purified proteins were stored at −80 °C. Protein concentrations were estimated by the Bradford method using bovine serum albumin (BSA) as a standard.20 Cloning, Expression, and Purification of Single Amino Acid Mutants of D-LDH1. The primers used for the single amino acid mutants are listed in Supplementary Table 2. PCR was performed by Pfu polymerase using pET28-D-LDH1 as a template, and the cycling parameters were: 95 °C for 5 min, followed by 12 cycles of 95 °C for 30 s and 68 °C for 12 min. After amplification, the PCR mixture was digested with DpnI and transformed into E. coli BL21 (DE3) by electroporation. The mutants were confirmed by DNA sequencing. Expression, purification, and enzyme assays of the mutants followed the previous steps for wild type D-LDH1. Enzyme Activity Assay. The activity of D-LDHs was measured in two reactions: pyruvate reduction and lactic acid oxidation. For pyruvate reduction, a reaction volume of 500 μL was prepared in a microfuge tube containing 0.5 μg enzyme, 0.5 mM NADH, and 10 mM pyruvate dissolved in 100 mM Tris-HCl buffer (pH 8.0). For lactic acid oxidation, the same reaction volume of 500 μL was prepared in a microfuge tube contacting 0.5 μg enzyme, 5 mM NAD+, and 500 mM D-lactic acid in 100 mM glycine sodium hydroxide (pH 9.0). The activity of D-LDHs was measured by a spectrophotometer at absorbance of 340 nm. One unit of activity was defined as the amount of enzyme needed to oxidize 1 μM NADH per min or reduce 1 μM NAD+ per min. The effect of pH on D-LDH activity was measured using 50 mM sodium acetate buffer (pH 3.0−5.0), 50 mM MOPS buffer (pH 5.5−7.5), 50 mM HEPES (pH 8.0 to 8.5), and 50 mM glycine (pH 9.0−13.0) at 30 °C. To test the effect of temperature on D-LDH activity, reactions were performed at a range of temperatures

ΔGtotal = ΔEMM + ΔGsol − T ΔS

(1)

where ΔG denotes the binding free energy, ΔEMM denotes the difference in molecular mechanics energy term between the complex and each binding partner in a vacuum, ΔGso denotes the solvation free energy term, and TΔS denotes the entropy change term. ΔEMM can be further divided into two parts ΔEMM = ΔEele + ΔEvdw

(2)

where ΔEele and ΔEvdw denote the electrostatic interaction and van der Waals energy in a vacuum, respectively. In addition, the solvation free energy can also be divided into two parts ΔGsol = ΔGpolar + ΔGnp

(3)

where ΔGpolar and ΔGnp denote the polar and nonpolar solvation free energy, respectively. For ΔGpolar, the dielectric constants of the solute and solvent were set to 2.0 and 80.0 in our calculations. For ΔGnp, the values of γ and β coefficients were set to 0.0054 kcal/mol/A2 and 0.92 kcal/mol, respectively. The entropy changes term (TΔS) arises from changes in the translational, rotational, and vibrational degrees of freedom. Calculating the entropy change term is extremely time-consuming and inaccurate, and for the similar protein−inhibitor complex system, the entropy change is also similar. Therefore, in our study, we ignored the calculation of the entropy change term. The substrate−residue interaction, which is valuable to qualitatively define the binding mechanisms of the substrate to enzyme, was 8372

DOI: 10.1021/acs.jafc.8b02454 J. Agric. Food Chem. 2018, 66, 8371−8381

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Figure 1. Reduction activity of D-LDH1 (A), D-LDH2 (B), and D-LDH3 (C) to different substrates. The D-LDHs’ activities were measured with different substrates: pyruvate (square), phenylpyruvate (diamond), 2-ketobutyric acid (cross), and oxaloacetate (triangle). The inactivated enzyme incubated at 100 °C for 5 min was used as control (circle).

Figure 2. Activity assay of D-LDH1. (A) Effect of different substrates on D-LDH1 activities. (B) Optimal pH of D-LDH1. Different buffers with pH from 3.0 to 13.0 were used in this assay. (C) Optimal temperature of D-LDH1. The assay was performed in 100 mM Tris buffers (pH 8.0) from 0 to 50 °C. (D) Thermostability of D-LDH1. To determine the thermostability of D-LDH1, the enzyme was preincubated at the indicated temperature for 10 min. Residual activity was measured at 30 °C for 5 min, pH 9.0 for reduction, and pH 10.00 for oxidation. The reduction and oxidation activities under the optimal conditions were set to be 100%. (SSNs) of the homologous proteins obtained via BLAST were constructed using the Enzyme Function Initiative-Enzyme Similarity Tool30 and visualized by Cytoscape 3.3.31 Each node in the network indicates a protein, and the edge indicates that the two nodes share significant similarity with an e value less than the selected cutoff.

analyzed using a per-residue-based decomposition method and consisted of four energetic terms ΔGsubstrate − residue = ΔEele + ΔE vdw + ΔGpolar + ΔGnp

(4)

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where ΔEvdw and ΔEele represent the nonbonded van der Waals interactions and electrostatic interactions between the substrate and each enzyme residue in a vacuum, respectively, and ΔGpolar and ΔGnp represent the polar and nonpolar contributions to the substrate− residue interaction, respectively. Collection of D-LDHs, Multiple Sequence Alignment, and Coevolving Protein Residues. A protein BLAST search was performed using D-LDH1 (AET30962) as a query sequence in UniProt with a cutoff e value of 10−80 (>40% sequence identity).25 Multiple sequence alignment (MSA) of 3664 protein sequences (Supplementary Dataset 1) was performed using the ClustalW (version 2) software program.26 Phylogenetic trees were constructed with MEGA7 using the neighbor-joining method, and a bootstrap test was carried out with 1000 iterations.27,28 The analysis of coevolving residues was carried out by calculating MI between two positions in the MSA. MI reflects the extent to which knowing the amino acid at one position can predict the amino acid identity at another position. MI was calculated between pairs of columns in the MSA using the MISTIC approach and a web server.29 Sequence similarity networks

RESULTS

Expression, Purification, and Activity Assay of DLDHs. To classify the function of the three genes that were annotated as D-LDHs in L. mesenteroides, the genes were cloned and the proteins were overexpressed in E. coli. The DLDHs were purified to be homogeneous using a Ni-NTA column. The molecular masses of the three purified D-LDHs were ∼45 kDa (Figure S1). To investigate the enzymes’ activities, we measured the reduction activity of the three enzymes with different substrates. The results indicated that DLDH1 showed activity toward pyruvate, 2-ketobutyric acid, and oxaloacetate in a time course. However, D-LDH2 and DLDH3 only reduced 2-ketobutyric acid and oxaloacetate but not pyruvate (Figure 1). All three enzymes could not reduce phenylpyruvate, an aromatic lactate derivative. Furthermore, 8373

DOI: 10.1021/acs.jafc.8b02454 J. Agric. Food Chem. 2018, 66, 8371−8381

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Journal of Agricultural and Food Chemistry metatranscriptomic analysis showed that the expression of DLDH1 was much higher than that of D-LDH2 and D-LDH3 during kimchi fermentation (Figure S2A).32 The RT-PCR analysis also indicated that the expression of the D-LDH1 gene was also higher than that of other two genes in L. mesenteroides J18 cells cultivated in chemically defined media (Figure S2B). This result suggested that D-LDH1 is the major enzyme responsible for D-lactic acid production in L. mesenteroides J18, so this enzyme was used for further study. Oligomerization and Kinetics of D-LDH1. To examine the native structure of D-LDH1 and determine whether DLDH1 can form a stable complex in vitro, the purified protein was analyzed by protein cross-linking (Figure S3). The crosslinked products were then separated by SDS-PAGE, followed by Coomassie Blue staining. The results showed that the subunits of the D-LDH1 proteins are cross-linked to tetramer. Upon prolonged incubation, no additional band appeared. These results suggest that D-LDH1 can form a stable tetramer in its native form, which is similar to the D-LDHs from Leuconostoc mesenteroides ATCC 8293.33 To further characterize the enzyme, the relative activity of DLDH1 to different substrates was measured in two directions. The reduction activities to 2-keto acids were measured using pyruvate, 2-ketobutyric acid, oxaloacetate, and phenylpyruvate as the substrates, respectively. The activity decreased as the carbon number of the substrate increased, and the activity toward phenylpyruvate was negligible. D-Lactic acid, 2hydroxybutyrate, D-malic acid, and D-3-phenyllactic acid were used as the substrates to monitor the oxidation activities to 2hydroxyacids, respectively. D -LDH1 also showed high oxidation activity to D-lactic acid, and the activity to 2hydroxybutyrate and D-malic acid was 6.5), with red lines indicating the highest MI scores (top 5%), black lines indicating midrange scores (between 70 and 95%), and gray lines indicating the lowest scores (the remaining 70%) as defined by MISTIC. (B) Network cMI with a high conservation value. Nodes represent the 30 most conserved residues (labeled with position and code) and nodes are colored to indicate conservation, from red (higher) to pink (lower). The length of each edge is inversely proportional to its MI value (the closest nodes have the highest MI values). (C) Ribbon diagram of D-LDH1 showing the 24 most coevolved and conserved residues.

Table 2. Kinetic Parameters of the Wild-Type D-LDH1, P99A, and D175R protein wild type

P99A

D175R

substrate pyruvate NADH D-lactate NAD+ pyruvate NADH D-lactate NAD+ pyruvate NADH D-lactate NAD+

Km (mM)

Vmax (U/mg)

Kcat (s−1)

2.66 0.32 87.9 0.12 3.2 0.34 102 0.29 2.97 0.19 60.9 0.17

× × × × × × × × × × × ×

× × × × × × × × × × × ×

1.38 1.64 0.56 0.21 1.13 1.09 0.44 0.98 0.78 0.44 0.15 0.43

4

10 104 104 104 104 104 104 103 104 104 104 103

2.75 3.26 0.91 0.34 2.15 2.07 0.80 0.17 1.56 0.88 0.30 0.84

3

10 103 103 103 103 103 103 103 103 103 103 102

Kcat/Km (mM−1 s−1) 1.03 × 1.02 × 10.32 2.82 × 0.67 × 0.61 × 7.84 0.59 × 0.52 × 0.47 × 4.87 0.50 ×

103 104 103 103 104 103 103 104 103

The two amino acids together with the neighboring amino acids in the sequences formed two highly conserved clusters. Another conserved cluster contained Gly152, Gly154, Gly157, and Asp175 that formed a GxGxxGx(17)D motif, providing positive charge to bind the pyrophosphate moiety in many dinucleotide binding enzymes.11,39 The coevolution of D-LDH amino acids was further investigated using mutual information (MI) (Figure 4). MI can be used to estimate the extent of the coevolutionary relationship between two positions in a protein family, and high MI values are suggestive of coevolution.40 If two residues share a high MI score, then they are most likely coevolving, meaning that to maintain a given enzymatic function a mutation of one residue is linked to a specific compensatory mutation of the other residue.41,42 The MI network for 3664 D-LDH members revealed that higher MI values (the top 10% of MI values) were distributed across all amino acid positions from the N-terminus to the C-terminus (Figure 4A). The 30 most conserved residues were chosen for further analysis (Figure 4B). In the MI network, these residues formed a close connection. These amino acid residues also shared high MI values. Mapping the top coevolving and conserved residues onto the LDH1 structure illustrated the

higher than the binding energy between His296 and pyruvate (−0.5 kcal/mol). The contribution of Tyr101 also changed, and the calculated ΔGtotal values of Tyr101 for pyruvate and Dlactate were 0.06 and −2.2 kcal/mol, respectively. On the basis of the above data, the amino acids in the active site have varying contributions to ligand binding, and Tyr101/His296 showed an obvious binding energy shift during the catalytic process. Conservation and Coevolution of Amino Acids in DLDH Sequences. MD analysis showed that several amino acids played important functions in substrate binding and catalysis. To further study the functions of these amino acids, MSA was performed to detect the conservation and coevolution of the amino acids based on 3664 protein sequences that showed >40% identity to D-LDH1 from the UniProt database (Figure 4, Supplementary Dataset 1). This threshold was chosen because several studies have shown that sequences that share >40% identity are very likely to share functional similarity, as judged by Enzyme Commission numbers.38 His296 was found to be the most highly conserved amino acid, followed by Tyr101, which function in substrate binding in the active site during the reaction (Figure 4A,B). 8376

DOI: 10.1021/acs.jafc.8b02454 J. Agric. Food Chem. 2018, 66, 8371−8381

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Figure 5. Taxonomic distribution and evolutionary relationships of D-LDHs. (A) Taxonomic distribution of D-LDHs. The proteins listed in Supplementary Dataset 1 were used to generate the network using an e value of 10−80 (>60% sequence identity). Each node represents one protein. Edges are shown with BLASTP e values below the indicated cutoff. A cluster was sequentially labeled if there were more than 10 nodes in that cluster. The proteins from bacteria are indicated by circle nodes, and those from fungi are represented by triangles. Nodes from the same taxonomic groups in the global network are the same color. The nodes representing enzymes that have been “reviewed” in the UniProt database are shown with a large size. The colors corresponding to phylum with the top 10 distributions and protein percentage in the phylum are listed on the right. The proteins in other phylum are shown by light blue (B) Maximum likelihood phylogenetic tree for 3664 proteins from bacteria and fungi generated using MEGA. The tree with the highest log likelihood (−325 820.9727) is shown. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) is shown next to the branches.

Km toward the cofactor to decrease by about three-fold. These results indicated that the amino acids binding lactate/pyruvate have critical roles for activity while the amino acids binding cofactors affected enzyme activity to some extent, but not severely. This may be because a single point mutation will not significantly affect the activity because the enzyme binds NADH and NAD+ via a large cluster. Distribution and Evolution of D-LDHs. Considering that D-LDHs play essential roles in LAB and the enzymes’ products are an important building block for chemical production of the green polymer PLA that could be applied in bioplastics,43 we further analyzed the distribution and evolution of the 3664 DLDHs in the biosphere (Supplementary Dataset 1) using a sequence similarity network and a phylogenetic tree. Sequence similarity networks (SSNs) for these sequences were constructed using e-value cut-offs of 10−60, at which >40% sequence identity was required to draw an edge between nodes.44 At this e-value threshold, all of the nodes were located in one cluster (Figure S8). As the e-value threshold stringency was decreased to 10−80 (sequence identity required to draw an edge was decreased to >∼60%), the 3664 proteins could be segregated into 11 clusters containing 10 or more members (Figure 5A). To analyze the distribution, the nodes were painted by taxonomic classification to explore the occurrence of the enzymes. D-LDHs can be found in archaea, bacteria, and eukaryotes. In archaea, only three proteins are found in the phylum of Euryarchaeota. Members of the enzymes were found to be abundant in Bacteria (96.58%) and Eukarya (3.32%). At the phylum level, the enzymes were mainly distributed in Proteobacteria (39.93%) and Firmicutes (30.92%), followed by Bacteroidetes (9.03%), Actinobacteria (6.96%), and Cyanobacteria (2.13%) in Bacteria. In Eukarya,

distances between and communication among the amino acids in this network (Figure 4C). Mapping the conserved amino acids revealed that these amino acid residues formed tow clusters in the structure (Figure 4C). The first conserved cluster contained Pro99, Tyr101, Arg235, and His296 forming a pyruvate-binding pocket. The second cluster including Gly152, Gly154, Asp175, Tyr188, Pro207, Asp259, Tyr205, and Asn232 is responsible for NADH/NAD+ binding. Thus we propose here that the conserved and coevolving amino acids in D-LDH play important roles in catalysis and in substrate and cofactor binding. Site-Directed Mutagenesis of D-LDH1. Both the MD simulation and amino acid sequence analysis revealed that Tyr101, Arg235, and His296 play critical roles in both pyruvate and lactate binding. Pro99 and Asp259, which are positioned near the substrates and NAD+ binding pockets, respectively, are highly conserved. Additionally, the GxGxxGx(17)D motif (from Gly153 to Asp175), which is in charge of NADH and NAD+ binding, is also conserved. Therefore, Pro99, Tyr101, Asp175, Asp259, and His296 were mutated to confirm their functions. Mutating Y101S, D259R, and H296D resulted in a complete loss of enzyme activity, indicating the importance of these two amino acids (Figure S6). The kinetic parameters of P99A and D175R were then determined and compared with those of the wild-type enzyme (Table 2; Figure S7). Mutation of Pro99 to Ala did not effect the Km values toward NADH, but the value to NAD+ was increased. The Kcat values of the mutant to both pyruvate and lactic acid were also decreased. D175R had no remarkable effect on the Km values toward NADH and NAD+, suggesting that the mutation has no significant effect on cofactor binding. However, the Kcat values to NADH and NAD+ decreased obviously, which led the Kcat/ 8377

DOI: 10.1021/acs.jafc.8b02454 J. Agric. Food Chem. 2018, 66, 8371−8381

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Journal of Agricultural and Food Chemistry

Figure 6. Proposed catalytic mechanism of D-LDH. In the pyruvate to D-lactate direction, a hydride is transferred from the C4N atom of NADH to C2 atom of pyruvate with protonation of pyruvate by His296. In the D-lactate to pyruvate direction, a hydride is transferred from the C2 atom of Dlactate to the dihydronicotinamide ring of NAD+ and the dehydrogenation of substrate is facilitated by the protonated His296. Tyr101 and Arg235 stabilize the substrates and transition states by hydrogen bonds. The red line indicates the hydrogen bond. The blue line represents the bond formed between atoms for catalysis. The arrow stands for electron-transfer process.

consequence of long-term domestication.45 In the current study, we showed that D-LDH1 is the major enzyme directly responsible for producing D-lactate in L. mesenteroides J18. LEUM_1756 and D-LDH1 showed high identity in sequences and similar catalytic characteristics. For example, the NADH reduction activity was much higher than oxidation activity for both of them at pH values below 7. Considering that the growth conditions of L. mesenteroides are usually acidic, the reaction would mainly produce lactate and NAD+. The optimal activities of the two enzymes were obtained at 25−30 °C for both pyruvate reduction and lactate oxidation. This temperature is similar to the optimal growth temperature of L. mesenteroides. Both enzymes prefer to use pyruvate as a substrate to produce D-lactate, and other kinetics of the two enzymes were also on the same order of magnitude.33 Additionally, D-LDH1 still retained almost half its activity even at 0 °C, suggesting that the reaction could proceed smoothly if L. mesenteroides is used as fermentation starter at low temperatures. Fructose 1,6-bisphosphate and divalent metal ions did not increase the enzyme activity, indicating that the D-LDH1 did not have the allosteric property like the DLDHs from Fusobacterium nucleatum and Pseudomonas aeruginosa.46 D-LDHs prefer to use NADH as a cofactor but not NADPH.8 Point mutations on three amino acids (D176S, I177R, F178T) of D-LDH from Lactobacillus delbrueckii resulted in the enzyme using NADH and NADPH efficiently.47 D-LDH1 in this study can only use NADH but not NADPH (Figure S9). But D-LDH from Sporolactobacillus inulinus, a strain producing optically pure D-lactate, could use both NADH and NADPH efficiently and with a preference for NADPH as its coenzyme; the Asn174 in GxGxxGx(17)D motif of D-LDH from S. inulinus was critical for NADPH utilization based on structure and mutagenesis assay.48,49 Sequence alignments showed that the corresponding amino acid residue in D-LDH1 was tyrosine with a large and hydrophobic side chain. The hydrophobic and large side chain of tyrosine may increase the steric hindrance to bind the negatively charged phosphate group of NADPH, as proposed by Zhu et al.48 Considering the importance of D-LDH1 in LAB for food fermentation, we further analyzed the catalytic mechanism of the enzyme. In the current MD analysis, His296 had an appreciable interaction energy contribution with values of −1 and −8 kcal/mol to pyruvate and lactate, respectively. On the basis of the crystal structure of D-LDH from L. bulgaricus,

D-LDHs

are mainly found in fungi, including Ascomycota (2.40%) and Basidiomycota (0.74%). The enzymes from fungi clustered with the proteins from Proteobacteria and Bacteroidetes in cluster 1. In other clusters, the enzymes from Firmicutes were the main members. To provide a more detailed view of the evolutionary relationships across the groups, we performed a phylogenetic analysis using the proteins in the clusters assigned based on sequence comparisons (Figure 5B). Proteins from the same cluster always clustered together and were well-separated in the phylogenetic tree, except that clusters 4 and 9 were clustered in the same branch. The separation of these groups had a high level of bootstrap support in the phylogenetic tree. Meanwhile, the proteins in cluster 1 from Proteobacteria, Bacteroroidetes, and Actinobacteria were gathered in a clade with the proteins in clusters 5, 6, and 7 from Firmicutes, which had a high level of bootstrap support. Proteins from other clusters formed another clade. Because all of these clusters had the same branch length, we can consider them to be quite similar from an evolutionary point of view.

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DISCUSSION In this study, we identified and characterized the D-LDH that is responsible for producing D-lactic acid in L. mesenteroides. The activity assay showed that D-LDH1 prefers short-chain and aliphatic substrates. MD simulation showed that Tyr101 and Arg235 are important residues for substrate binding. Evolutionary and point mutation analyses further confirmed their importance. Finally, we performed a large-scale in silico analysis of D-LDHs, which revealed that homologues of the enzymes were abundant in bacteria (96.58%) and distributed in eukaryota (3.32%). L. mesenteroides members are important LAB that are reported to be mainly responsible for the fermentation of various vegetables, such as sauerkraut (pickled cabbage) and kimchi (a Korean fermented vegetable food), under moderate salinity and low-temperature conditions; some L. mesenteroides strains have also been isolated from dairy products such as cheese.32 In L. mesenteroides ATCC 8293, seven genes encode lactate dehydrogenases or related enzymes, but only LEUM_1756 was the major gene responsible for the production of D-lactic acid.33 In the genome of L. mesenteroides J18, there are three genes annotated as LDHs that may be involved in generating D-lactic acid.19 On the basis of evolution analysis, D-LDH gene is under positive selection, possibly a 8378

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Journal of Agricultural and Food Chemistry His296 can function as an acid/base catalyst.11 The H296 K substitution in L. bulgaricus D-LDH does not significantly change the Kcat or Km in pyruvate reduction but shifts the optimum pH from 7−7.5 to 6.11,50 Furthermore, MD analysis showed that the residues Tyr101 and Arg235 also contribute to substrate binding with a binding energy less than −1 kcal/mol. Previous research also showed that Tyr101 is responsible for substrate specificity.11 Substitutions of Arg235 with Lys and Gln in the D-LDH of Lactobacillus plantarum induced drastic decreases in the catalytic efficiency.51 We showed that mutations of substrate-binding amino acids, Y101S and H296D, resulted in complete loss of activity. These results indicated that Tyr101, Arg235, and His296 are crucial for enzyme activity. Pro99 is also highly conserved, and P99A mutant showed 70% activity compared with activity of the wild-type protein. Pro99 is positioned in the same loop with Tyr100. We proposed that the mutation of Pro99 may affect the position of Tyr101 slightly and not result in a drastic decrease in activity. Meanwhile, we also mutated D175, which binds adenine ribose of NADH/NAD+. The D175R mutant retained almost 60% activity compared with activity of the wild-type enzyme, but the Kcat/Km value toward cofactor decreased by about three-fold. We proposed that the coenzyme binding the GxGxxGx(17)D motif is a large conserved cluster, and mutation of one residue could not obviously affect the binding. The mutation of Asp259 to Arg also lead to loss of activity because Asp259 functions to orient the carboxamide group of coenzymes for electron transfer.47 On the basis of the current and previous research, the catalytic mechanism of DLDH was proposed. As can be seen in Figure 6, Tyr101 and Arg235 form hydrogen bonds with the carboxyl group of the substrates. The binding directs the ketone moiety of pyruvate toward His296. The distance between the hydrogen atom on the nicotinamide ring of the NADH and C2 atom of pyruvate is ∼2.5 Å, which is suitable for hydride transfer reaction.52 Detailed bond forming and breaking in the catalytic process from pyruvate to D-lactate and the revered direction are shown in supplementary movies (Movies S1 and S2). High production of polymer-grade D-lactate is urgently required, particularly for the synthesis of polylactic acid. DLactate production could reach 61.5 g/L by the engineered Saccharomyces cerevisiae with the introduction of D-LDH gene from Leuconostoc mesenteroides ssp. mesenteroides strain NBRC3426.53 The engineered Klebsiella oxytoca PDL-5 strain with D-LDH gene produced 111.9 g/L of D-lactate.54 D-lactic acid reached the titer of 226.6 g/L at 50 °C by the introduction a thermophilic D-LDH from Thermodesulfatator indicus, which causes a trapping effect on carbon flux redistribution.55 To explore potential D-lactate producers, we searched the homologues of D-LDH1, and a total of 3664 protein sequences that showed >40% identity to D-LDH1 were retrieved. Most proteins were from bacteria and fungi. Few proteins were also found in algae, fish, and archaea. Some enzymes are from thermophilic organisms, such as the enzyme from Thermosynechococcus elongatus (UniProt ID: Q8DKY8) and Thermosynechococcus sp. NK55a (UniProt ID: V5V6P2). Some enzymes may have the halophilic property, such as the enzyme from Halomonas cupida (UniProt ID: A0A1M7DCP1) and Algoriphagus halophilus (UniProt ID: A0A1N6EIC2). The extremophilic property of these enzymes implicates that they are valuable for various industrial applications. In conclusion, we identified D-LDH from L. mesenteroides and characterized D-LDH1 as the major enzyme directly

responsible for producing D-lactic acid among related genes. The evolutionary analysis of D-LDHs revealed that the residues directly involved in substrate/cofactor binding and catalytic activity are highly conserved and have coevolved. The MD and biochemical analysis further confirmed their function in catalysis. Finally, the D-LDH and its homologues are most abundant in bacteria and distributed in eukaryotes. This study expands the understanding of evolution, functions, and catalytic mechanisms of D-LDH.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.8b02454. Supplementary methods, primers used in this study (Supplementary Table 1), effects of metal ions and fructose 1,6-bisphosphate on the activity of the D-LDH1 (Supplementary Table 2), purification of enzymes (Figure S1), expression of D-LDH genes (Figure S2), cross-link of D-LDH1 (Figure S3), kinetics graph of DLDH1 (Figure S4), validation of protein structure of DLDH1 (Figure S5), site-directed mutagenesis of DLDH1 (Figure S6), kinetics graph of D-LDH1 mutants (Figure S7), protein sequence similarity network of DLDHs (Figure S8), and oxidation activity to NADH and NADPH (Figure S9) (PDF) Bond forming and breaking in the catalytic process from pyruvate to lactate (Movie S1) (AVI) Bond forming and breaking in the catalytic process from lactate to pyruvate (Movie S2) (AVI) Proteins sequences used for the protein similarity network (Supplementary Dataset 1) (XLSX)

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AUTHOR INFORMATION

Corresponding Author

*Tel: +82-2-820-5864. E-mail: [email protected]. ORCID

Baolei Jia: 0000-0001-6434-5604 Che Ok Jeon: 0000-0003-1665-2399 Funding

This work was supported by the National Research Foundation (2017M3C1B5019250, 2018R1A5A1025077) of the Ministry of Science and ICT, Republic of Korea. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The MS analysis was carried out at the National Supercomputer Center in LvLiang of China, and the calculations were performed on TianHe-2.

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REFERENCES

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