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Food and Beverage Chemistry/Biochemistry

Catalytic, computational, and evolutionary analysis of the D-lactate dehydrogenases responsible for D-lactic acid production in lactic acid bacteria Baolei Jia, Zhongji Pu, Ke Tang, Xiaomeng Jia, Kyung Hyun Kim, Xinli Liu, and Che Ok Jeon J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b02454 • Publication Date (Web): 16 Jul 2018 Downloaded from http://pubs.acs.org on July 17, 2018

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Journal of Agricultural and Food Chemistry

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Catalytic, computational, and evolutionary analysis of the D-lactate

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dehydrogenases responsible for D-lactic acid production in lactic acid bacteria

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Baolei Jia1,2, Zhong Ji Pu3, Ke Tang1, Xiaomeng Jia2, Kyung Hyun Kim2, Xinli Liu1,

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and Che Ok Jeon2,*

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1

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Bioengineering, Qilu University of Technology (Shandong Academy of Sciences),

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Jinan, China

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2

Department of Life Science, Chung-Ang University, Seoul 06974, Republic of Korea

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3

School of Life Science and Biotechnology, Dalian University of Technology, Dalian

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116024, China

State Key Laboratory of Biobased Material and Green Papermaking, School of

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Running title: A comprehensive analysis of D-lactate dehydrogenases

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Address for correspondence: Dr. Che Ok Jeon, Department of Life Science, Chung-

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Ang University, Seoul 06974, Republic of Korea

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Tel: +82-2-820-5864; Email: [email protected]

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ACS Paragon Plus Environment


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Abstract

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D-lactate

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+ H+ lactate + NAD+, which is a principal step in the production of D-lactate in

20

lactic acid bacteria. In this study, we identified and characterized the major D-LDH

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(D-LDH1) from three

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extensively used in food processing. A molecular simulation study of D-LDH1 showed

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that the conformation changes during substrate binding. During catalysis, Tyr101 and

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Arg235 bind the substrates by hydrogen bonds and His296 acts as a general acid/base

25

for proton transfer. These residues are also highly conserved and have coevolved.

26

Point mutations proved that the substrate binding sites and catalytic site are crucial for

27

enzyme activity. Network and phylogenetic analyses indicated that D-LDH1 and the

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homologs are widely distributed but are most abundant in bacteria and fungi. This

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study expands the understanding of the functions, catalytic mechanism, and evolution

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of D-LDH.

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Keywords:

32

evolution.

dehydrogenase (D-LDH) catalyzes the reversible reaction pyruvate + NADH

D-lactate

D-LDHs

in Leuconostoc mesenteroides, which has been

dehydrogenases, catalytic mechanism, lactic acid bacteria,

2


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Introduction

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Lactic acid bacteria (LAB) have been used in various fermented foods throughout

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history.1 The LAB encompass a phylogenetically diverse group of species that play

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crucial roles in a variety of food and feed fermentations worldwide. The ability of

37

LAB to produce organic acids and other antimicrobial substances has made them

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indispensable in manufacturing fermented foods such as sauerkraut, cheese, sausage,

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sourdough bread, animal silage, and kimchi.1 LAB can also serve as biofertilizers,

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biocontrols, biostimulants, and bioelicitors in plant production.2 Furthermore, the

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LAB-mediated conversion of carbohydrates into organic acids, especially lactic acid,

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has prompted interest in industrial applications,3 because poly (lactic acid) (PLA)

43

polymers are thermoplastic and biodegradable polyesters with applications ranging

44

from medicine to agriculture and packaging.4

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Lactic acid is the major metabolite of LAB; this acid reduces the pH and prevents

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the risk of proliferation of undesirable microorganisms.5 Lactic acid is chiral, and

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thus, has two optical isomers: L (+)-lactic acid and D (−)-lactic acid. L (+)-lactic acid is

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used in the food industry in many ways, because the human body can only assimilate

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this form of lactic acid.6 On the other hand, Lactobacillus bulgaricus, commonly used

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in the dairy industry to produce yogurt, converts 90% of the pyruvate into D-lactic

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acid and triggers the aggregation of casein micelles.7 The generation of lactic acid

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from pyruvate by lactate dehydrogenase (LDH) is a main step LAB use to regenerate

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NAD+, therefore all LAB harbor a large amount of NAD+-dependent LDH.8 In the

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genome of LAB, stereospecific L- or D-LDHs are encoded that are responsible for

55

producing the corresponding lactic acids.

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because L-lactic acid is widely used in producing food, cosmetics, and medicine.9 D-

L-LDHs

have been thoroughly studied

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LDHs have also been studied, but not as intensely as L-LDHs.10 According to the

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structures of D-LDHs from L. bulgaricus and Lactobacillus jensenii, D-LDHs form a

59

homodimeric structure and the monomer consists of the N-terminal substrate-binding

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domain and the C-terminal NAD+-binding domain.11

61

As an important group of LAB, Leuconostoc mesenteroides and its subspecies are

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key players responsible for fermenting many foods. L. mesenteroides subsp. cremoris

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is a main aroma producer during cheese fermentation.12-13 During coffee fermentation,

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L. mesenteroides can initiate acidification and play a role in solubilizing pectic

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substances.13-15 Especially when used as a fermentation starter for sauerkraut and

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kimchi, L. mesenteroides can help shorten fermentation time by organic acid and

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mannitol production and can improve organoleptic properties.16-17 Fermentation

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involving L. mesenteroides results in the accumulation of organic acids, primarily D-

69

lactic acid and a small amount of L-lactic acid as the major end products of glucose

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and other carbohydrate metabolism.8, 18 L. mesenteroides subsp. mesenteroides J18 is

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one of the most predominant LAB during kimchi fermentation, and the genome of this

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strain contains three genes annotated as

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AET31034, and D-LDH3: AET31236).19 However, it is still unclear whether one of

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these three genes plays the leading role or whether they equally contribute to

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producing D-lactic acid. In this study, we cloned and expressed these three D-LDH

76

genes in Escherichia coli. Further analysis showed that D-LDH1 is the major enzyme

77

responsible for producing D-lactic acid in this strain. The biochemical characteristics

78

and catalytic mechanism were further studied by activity assays and molecular

79

simulation. Finally, a large-scale in silico analysis was performed to study the

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evolution and distribution of this gene.

D-LDH

(D-LDH1: AET30962,

4


D-LDH2:

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Materials and Methods

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Cloning of

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PCR using L. mesenteroides subsp. mesenteroides J18 genomic DNA as a template

84

was performed to isolate

85

Supplementary Table 1. The PCR product and pET28-(a) were digested by the

86

restriction enzymes (BamHI and HindIII) and ligated into the pET28a vector. The

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ligation products were transformed into E. coli BL21 (DE3) by electroporation and

88

were confirmed by sequencing.

89

Expression and purification of

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E. coli BL21 (DE3) containing the pET28a-D-LDHs plasmids were cultured in 2 L of

91

LB broth containing 50 µg/mL kanamycin at 37°C for 3 h. When the OD600 reached

92

0.7, 1mM isopropyl-β-D-thiogalactopyranoside (IPTG) was added to induce protein

93

expression. The cells were cultured in the presence of IPTG for 4 h with shaking,

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harvested by centrifugation at 5,000 ×g for 10 min, then resuspended in lysis buffer

95

containing 50 mM Tris (pH 8.0), 300 mM NaCl, and 20 mM β-mercaptoethanol. The

96

cell suspension was sonicated, then the supernatant was collected by centrifugation

97

and loaded on a Ni-NTA column. After washing th e column with lysis buffer, the

98

proteins were eluted using an imidazole gradient (20 mM, 50 mM, and 250 mM). The

99

purified enzymes were visualized after separation by 12% sodium dodecyl sulfate

100

polyacrylamide gel electrophoresis (SDS-PAGE). After dialysis with 50 mM Tris-HCl

101

buffer (pH 8.0) containing 300 mM NaCl and 20 mM β-mercaptoethanol, the purified

102

proteins were stored at -80°C. Protein concentrations were estimated by the Bradford

103

method using bovine serum albumin (BSA) as a standard.20

D-LDHs

from L. mesenteroides subsp. mesenteroides J18

D-LDHs

using the oligonucleotide primers listed in

D-LDHs

5



104

Cloning, expression, and purification of single amino acid mutants of D-LDH1

105

The primers used for the single amino acid mutants were listed in Supplementary

106

Table 2. PCR was performed by Pfu polymerase using pET28-D-LDH1 as template,

107

and the cycling parameters were: 95°C for 5 min followed by 12 cycles of 95°C for 30

108

s and 68°C for 12 min. After amplification, the PCR mixture was digested with DpnI

109

and transformed into E. coli BL21 (DE3) by electroporation. The mutants were

110

confirmed by DNA sequencing. Expression, purification, and enzyme assays of the

111

mutants followed previously steps for wild type D-LDH1.

112

Enzyme activity assay

113

The activity of D-LDHs was measured in two reactions: pyruvate reduction and lactic

114

acid oxidation. For pyruvate reduction, a reaction volume of 500 µL was prepared in a

115

microfuge tube containing 0.5 µg enzyme, 0.5 mM NADH, and 10 mM pyruvate

116

dissolved in 100 mM Tris-HCl buffer (pH 8.0). For lactic acid oxidation, the same

117

reaction volume of 500 µL was prepared in a microfuge tube contacting 0.5 µg

118

enzyme, 5 mM NAD+, and 500 mM

119

hydroxide (pH 9.0). The activity of D-LDHs was measured by a spectrophotometer at

120

absorbance = 340 nm. One unit of activity was defined as the amount of enzyme

121

needed to oxidize 1 µM NADH per min or reduce 1 µM NAD+ per min. The effect of

122

pH on D-LDH activity was measured using 50 mM sodium acetate buffer (pH 3.0-

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5.0), 50 mM MOPS buffer (pH 5.5-7.5), 50 mM HEPES (pH 8.0-8.5), and 50 mM

124

glycine (pH 9.0-13.0) at 30°C. To test the effect of temperature on D-LDH activity,

125

reactions were performed at a range of temperatures (0–50°C). Michaelis-Menten

D-lactic

acid in 100 mM glycine sodium

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equation was employed to calculate the kinetic parameters. All the enzyme activity

127

experiments were performed in three biological and three technical replicates.

128

Molecular dynamic (MD) simulation

129

The three-dimensional structure of

130

software.21 Structural validation of the enzyme was performed by creating a

131

Ramachandran plot using the PROCHECK server.22 The complexes of D-LDH1 and

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the substrates were built by LeDock (http://www.lephar.com/index.htm). The

133

hydrogen atoms were added to the protein system using the H++ web-server.23 The

134

minimization of five models (apo-LDH, NAD+-LDH, NADH-LDH, NAD+-lactate-

135

LDH, and NADH-pyruvate-LDH) was performed using the combined steepest

136

descent/conjugated gradient in three stages: (1) the enzyme model was frozen, with a

137

force constant of 500 kcal/mol, and free waters and ions; (2) the heavy atoms in the

138

system were kept fixed with a force constant of 500 kcal/mol, and all hydrogens in the

139

system were set free; (3) all the atoms in the system were freely optimized.

D-LDH1

was modeled using Modeller 9

140

To gradually heat the temperature of the system to the standard temperature of

141

300 K at a constant pressure of 1 bar, both models were pre-heated for a total of 100

142

ps in a NVT ensemble. In the first 50 ps, the heating was linear, from 0 K to 300 K; in

143

the 50 ps that followed, the temperature of 300 K was fixed. MD proceeded on a NPT

144

ensemble. A constant temperature of 300 K was maintained using Langevin Dynamics

145

implemented in the AMBER14 package and a fixed pressure of 1 bar was set. Periodic

146

boundary conditions were imposed to the models built to account for long-range

147

interactions. Short- and long-range electrostatic interactions were calculated with

148

Ewald summation methods. The SHAKE algorithm was applied to all bonds

7



149

involving hydrogen atoms for a mechanical relaxation time of 2 ps. To calculate long-

150

range VDW interactions, a cutoff distance of 12 Å was established. The root-mean-

151

square deviation (RMSD) and root-mean square fluctuation (RMSF) for both

152

simulations were calculated with the CPPTRAJ module of AmberTools15. The hybrid

153

quantum mechanical/molecular mechanical (QM/MM)/steered molecular dynamics

154

(SMD) methods were employed to study catalytic mechanisms.24

155

Calculation of binding free energy

156

Binding free energies of cofactors and substrates for D-Lactate dehydrogenase were

157

calculated using the MM/GBSA program in AmberTools15. For each complex, 200

158

snapshots were taken from the last 40 ns of the MD trajectory with an interval of 200

159

ps. In this method, the binding free energy (∆G) can be represented as:

160

∆Gtotal = ∆EMM + ∆Gsol – T∆S

161

where ∆G denotes the binding free energy, ∆EMM denotes the difference in molecular

162

mechanics energy term between the complex and each binding partner in a vacuum,

163

∆Gso denotes the solvation free energy term, and T∆S denotes the entropy change

164

term. ∆EMM can be further divided into two parts:

165

∆EMM = ∆Eele + ∆Evdw

166

where ∆Eele and ∆Evdw denote the electrostatic interaction and van der Waals energy in

167

a vacuum, respectively. In addition, the solvation free energy can also be divided into

168

two parts:

169

∆Gsol = ∆Gploar + ∆Gnp (3),

(1),

(2),

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where ∆Gpolar and ∆Gnp denote the polar and non-polar solvation free energy,

171

respectively. For ∆Gpolar, the dielectric constants of the solute and solvent were set to

172

2.0 and 80.0 in our calculations. For ∆Gnp, the values of γ and β coefficients were set

173

to 0.0054 kcal/mol/A2 and 0.92 kcal/mol, respectively.

174

The entropy changes term (T∆S) arises from changes in the translational, rotational,

175

and vibrational degrees of freedom. Calculating the entropy change term is extremely

176

time-consuming and inaccurate, and for the similar protein-inhibitor complex system,

177

the entropy change is also similar. Therefore, in our study, we ignored the calculation

178

of the entropy change term.

179

The substrate–residue interaction, which is valuable to qualitatively define the binding

180

mechanisms of the substrate to enzyme, was analyzed using a per-residue-based

181

decomposition method and consisted of four energetic terms:

182

∆Gsubstrate-residue = ∆Eele + ∆Evdw + ∆Gpolar+ ∆Gnp (4),

183

Where ∆EMM represents the non-bonded van der Waals interactions and electrostatic

184

interactions between the substrate and each enzyme residue in a vacuum, respectively,

185

and ∆Gpolar and ∆Gnp represent the polar and non-polar contributions to the substrate–

186

residue interaction, respectively.

187

Collection of

D-LDHs,

MSA, and coevolving protein residues

188

A protein BLAST search was performed using D-LDH1 (AET30962) as a query

189

sequence in UniProt with a cut-off e value of 10-80 (> 40% sequence identity).25 MSA

190

of 3,664 protein sequences (Supporting Information Dataset S1) were performed

191

using the ClustalW (version 2) software program.26 Phylogenetic trees were

192

constructed with MEGA7 using the neighbor-joining method and a bootstrap test was 9



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carried out with 1000 iterations.27-28 Analysis of coevolving residues was carried out

194

by calculating MI between two positions in the MSA. MI reflects the extent to which

195

knowing the amino acid at one position can predict the amino acid identity at another

196

position. MI was calculated between pairs of columns in the MSA using the MISTIC

197

approach and a web server.29 Sequence similarity networks (SSNs) of the

198

homologous proteins obtained via BLAST were constructed using the Enzyme

199

Function Initiative-Enzyme Similarity Tool30 and visualized by Cytoscape 3.3.31 Each

200

node in the network indicates a protein and the edge indicates that the two nodes share

201

significant similarity with an e-value less than the selected cutoff.

202

Results

203

Expression, purification, and activity assay of D-LDHs

204

To classify the function of the three genes that were annotated as

205

mesenteroides, the genes were cloned and the proteins were overexpressed in E. coli.

206

The D-LDHs were purified to be homogenous using a Ni-NTA column. The molecular

207

masses of the three purified

208

Information Figure S-1). To investigate the enzymes’ activities, we measured the

209

reduction activity of the three enzymes with different substrates. The results indicated

210

that D-LDH1 showed activity toward pyruvate, 2-ketobutyric acid, and oxaloacetate in

211

a time course. However, D-LDH2 and D-LDH3 only reduced 2-ketobutyric acid and

212

oxaloacetate, but not pyruvate (Fig. 1). All three enzymes could not reduce

213

phenylpyruvate, an aromatic lactate derivative. Furthermore, metatranscriptomic

214

analysis showed that the expression of D-LDH1 was much higher than that of D-LDH2

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and D-LDH3 during kimchi fermentation (Supporting Information Figure S-2A).32 The

D-LDHs

D-LDHs

in L.

were approximately 45 kDa (Supporting

10


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RT-PCR analysis also indicated that the expression of the

217

higher than that of other two genes in L. mesenteroides J18 cells cultivated in

218

chemically defined media (Supporting Information Figure S-2B). This result

219

suggested that D-LDH1 is the major enzyme responsible for D-lactic acid production

220

in L. mesenteroides J18, so this enzyme was used for further study.

221

Oligomerization and Kinetics of D-LDH1

D-LDH1

gene was also

222

To examine the native structure of D-LDH1 and determine whether D-LDH1 can

223

form a stable complex in vitro, the purified protein was analyzed by protein cross-

224

linking (Supporting Information Figure S-3). The cross-linked products were then

225

separated by SDS-PAGE followed by Coomassie Blue staining. The results showed

226

that the subunits of the D-LDH1 proteins are cross-linked to tetramer. Upon prolonged

227

incubation, no additional band appeared. These results suggest that D-LDH1 can form

228

a stable tetramer in its native form, which is similar to the D-LDHs from Leuconostoc

229

mesenteroides ATCC 8293.33

230

To further characterize the enzyme, the relative activity of D-LDH1 to different

231

substrates was measured in two directions. The reduction activities to 2-keto acids

232

were measured using pyruvate, 2-ketobutyric acid, oxaloacetate, and phenylpyruvate

233

as the substrates, respectively. The activity decreased as the carbon number of the

234

substrate increased, and the activity toward phenylpyruvate was negligible. D-lactic

235

acid, 2-hydroxybutyrate,

236

substrates to monitor the oxidation activities to 2-hydroxyacids, respectively. D-LDH1

237

also showed high oxidation activity to

238

hydroxybutyrate and D-malic acid was less than 10% compared to the activity to D-

D-malic

acid, and D-3-phenyllactic acid were used as the

D-lactic

acid and the activity to 2-

11



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lactic acid (Fig. 2A). Potassium slightly increased the enzyme activity, while fructose

240

1,6-bisphosphate and certain divalent metal ions such as Mg2+ did not enhance the

241

activity (Supporting Information Table S-1). As D-LDH1 showed the highest activity

242

to pyruvate and D-lactic acid, the optimal pH and temperature for

243

determined using the two substrates. We found that the optimal pH for pyruvate

244

reduction by D-LDH1 was 9.0, whereas the enzyme activity for lactate oxidation was

245

high at pH 10.0 (Fig. 2B). This result indicated that D-LDH1 is strongly resistant to

246

alkali conditions. This finding agrees with results of previous reports on L.

247

mesenteroides, and the optimum pH for lactate oxidation is usually higher than the

248

optimum pH for the reaction with pyruvate.8, 34 The effect of temperature on D-LDH1

249

activity of the purified enzyme was determined at temperatures ranging from 0 to

250

50°C, and the optimal activity was obtained at 25–30°C for both pyruvate reduction

251

and lactate oxidation (Fig. 2C). This temperature is similar to the optimal growth

252

temperature of L. mesenteroides. Additionally,

253

activity at 0°C. This suggested that the reaction could proceed efficiently when L.

254

mesenteroides was used as starter in food fermentation at low temperature. On the

255

other hand, D-LDH1 is not a heat-stable enzyme; incubation at 35°C for 10 min was

256

sufficient to inactivate the enzyme (Fig. 2D).

257

D-LDH1

D-LDH1

D-LDH1

were

retained almost half of its

catalyzes a reversible reaction with two substrates and two products.

258

The Km values of D-LDH1 were determined by varying one substrate concentration

259

and keeping another constant. The plot of velocity versus NADH substrate

260

concentration showed a typical Michaelis–Menten profile (Table 1, Supporting

261

Information Figure S-4). The Km and Vmax values for pyruvate were 2.66 mM and

262

13,843.31 U/mg when using NADH as the substrate. Meanwhile, Km values for 212


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263

ketobutyric acid and oxaloacetic acid increased to 234.60 mM and 78.81 mM,

264

respectively. The Vmax values for the two substrates also decreased. These results

265

indicated that the enzyme prefers pyruvate as the substrate. On the other hand, the Km

266

value for

267

which is more than 30 times higher than the Km for pyruvate. Furthermore, the Vmax

268

for NADH was almost 8-fold greater than Vmax for NAD+, even though Km values of

269

NADH and NAD+ were similar. These results indicated that the reaction tends to

270

proceed in the direction to produce lactate and NAD+.

271

MD simulation

272

To study the catalytic mechanism of D-LDH1, we constructed a 3D model of D-LDH1

273

using Modeller 9 software based on the crystal structures of D-LDH1 homologs (PDB

274

ID: 4CUK, 1DXY, 2DLD, 1J4A, and 1J49). The stereochemical parameters of

275

modeled proteins were measured using G-factors generated by PROCHECK, which

276

showed that 92.2% of residues were found in the most favored regions of the

277

Ramachandran plot (Supporting Information Figure S-5). Besides, all residues were

278

positioned in allowed regions. The model of D-LDH1 was used for further analyzing

279

substrate binding, the MD simulation, and the calculation of binding free energy.

D-lactic

acid was 87.89 mM when using NAD+ as a cofactor substrate,

280

According to previous studies, D-LDH follows an ordered mechanism.35 In the

281

mechanism, the coenzyme NADH always binds first, with pyruvate binding

282

afterward. During the reaction, pyruvate is reduced to lactate while NADH is oxidized

283

to NAD+ by the enzyme. Lactate is then released first, followed by the release of

284

NAD+.35 Based on this mechanism, the cofactors and substrates were docked to the

285

structure of

D-LDH1

to form the complexes of NADH-LDH1, NADH-pyruvate-

13



286

LDH1, NAD+-lactate-LDH1, and NAD+-LDH1. The docking analyses revealed that

287

D-LDH1

288

NADH-pyruvate-LDH1 complex (Fig. 3A). Among the three amino acids, Tyr101 and

289

Arg235 form hydrogen bonds with the carboxyl group of pyruvate. While the

290

hydrogen bond between pyruvate and His296 is generated by the ketone functional

291

group of pyruvate and imidazole group of histidine. In the NAD+-lactate-LDH1

292

complex, His296 also formed a hydrogen bond with lactate between hydroxyl group

293

(O2) of lactate and the imidazole group of the amino acid. These analyses indicated

294

that His296 plays a central role during catalysis.

binds pyruvate using Tyr101, Arg235, and His296 by hydrogen bonds in the

295

To gain more information about the catalysis, the complexes were used for MD

296

simulations to investigate the conformational changes and protein internal motions

297

using GROMACS software.36 In the simulation, the RMSD is a crucial parameter of

298

convergence in protein structure changes over the course of a simulation. Based on

299

the RMSD analysis, the first 25 ns MD trajectory was deleted and the remaining 25 ns

300

trajectory was used in the analysis (Fig. 3B). The backbone RMSD of apo-LDH1 and

301

NAD+-LDH1 equilibrated around 0.40 nm and 0.41 nm, respectively, after 25 ns of

302

simulation. On the other hand, the backbone RMSDs of NADH-LDH1, NAD+-

303

lactate-LDH1, and NADH-pyruvate-LDH1 equilibrated around 0.22 nm, 0.21 nm, and

304

0.23 nm over the same time frame, respectively, as shown in Fig. 3B. These results

305

suggested that NAD+ binding does not cause significant conformational changes in the

306

protein, while binding of NADH and the two substrates does cause a conformational

307

change in D-LDH1 and decreases protein flexibility.

308

In contrast to RMSD, RMSF can be used to analyze the flexibility of each residue

309

present in the systems. RMSF calculation showed that the five systems showed a 14


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310

similar pattern of fluctuation; the highest RMSF values were obtained between Ser250

311

to Tyr300 near the C terminus where substrate binding occurs. The second highest

312

RMSF values were between Val125 to Val150 where cofactor binding occurs (Fig.

313

3C). The RMSF values suggested that the amino acids that function in binding and

314

catalysis are more flexible than other regions.

315

Free energy analysis for substrate binding

316

As discussed above, differences in the conformations and dynamic behaviors were

317

observed when the enzyme bound pyruvate or lactic acid. Quantifying the average

318

energy of the interaction between lactate/pyruvate-D-LDH1 and the specific residues

319

located in the binding sites could provide further insight about which residues are

320

important for the substrates’ binding. Therefore, ligand-residue interaction

321

decomposition was performed by the molecular mechanic/Poisson–Boltzmann surface

322

area (MM/PBSA) program in AmberTools15 (Fig. 3D).37 The summations of the total

323

interaction free energies (∆Gtotal) were separated into electrostatic interaction (∆Eele),

324

van der Waals energy (∆Evdw), polar binding energy (∆Gpolar), and non-polar solvation

325

free energy (∆Gnp). The energy contributions from these residues are summarized in

326

Fig. 3D and all the residues binding pyruvate or lactate showed favorable ∆Gtotal

327

values. The results showed that the three key residues (Tyr101, Arg235, and His296)

328

have weak electrostatic interactions and polar interactions with both substrates.

329

Among them, His296 showed the most obvious difference for binding pyruvate and

330

lactic acid. The total binding energy between His296 and D-lactate (-7.6 kcal/mol)

331

was 15-fold higher than the binding energy between His296 and pyruvate (-0.5

332

kcal/mol). The contribution of Tyr101 also changed and the calculated ∆Gtotal values

333

of Tyr101 for pyruvate and D-lactate were 0.06 and -2.2 kcal/mol, respectively. Based 15



334

on the above data, the amino acids in the active site have varying contributions to

335

ligand binding and Tyr101/His296 showed an obvious binding energy shift during the

336

catalytic process.

337

Conservation and coevolution of amino acids in D-LDH sequences

338

MD analysis showed that several amino acids played important functions in substrate

339

binding and catalysis. To further study the functions of these amino acids, MSA were

340

performed to detect the conservation and coevolution of the amino acids based on

341

3,664 protein sequences that showed > 40% identity to D-LDH1 from the UniProt

342

database (Fig. 4, Supporting Information Dataset S-1). This threshold was chosen

343

because several studies have shown that sequences that share > 40% identity are very

344

likely to share functional similarity, as judged by Enzyme Commission numbers.38

345

His296 was found to be the most highly conserved amino acid, followed by Tyr101,

346

which function in substrate binding in the active site during the reaction (Fig. 4A&B).

347

The two amino acids together with the neighboring amino acids in the sequences

348

formed two highly conserved clusters. Another conserved cluster contained Gly152,

349

Gly154, Gly157, and Asp175 that formed a GxGxxGx(17)D motif, providing positive

350

charge to bind the pyrophosphate moiety in many dinucleotide binding enzymes.11, 39

351

The coevolution of

352

information (MI) (Fig. 4). MI can be used to estimate the extent of the coevolutionary

353

relationship between two positions in a protein family, and high MI values are

354

suggestive of coevolution.40 If two residues share a high MI score, they are most

355

likely coevolving, meaning that to maintain a given enzymatic function, a mutation of

356

one residue is linked to a specific compensatory mutation of the other residue.41-42 The

357

MI network for 3,664 D-LDH members revealed that higher MI values (the top 10%

D-LDH

amino acids was further investigated using mutual

16


Page 16 of 42

Page 17 of 42


358

of MI values) were distributed across all amino acid positions from the N-terminus to

359

the C-terminus (Fig. 4A). The 30 most conserved residues were chosen for further

360

analysis (Fig. 4B). In the MI network, these residues formed a close connection.

361

These amino acid residues also shared high MI values. Mapping the top coevolving

362

and conserved residues onto the LDH1 structure illustrated the distances between and

363

communication among the amino acids in this network (Fig. 4C). Mapping the

364

conserved amino acids revealed that these amino acid residues formed tow clusters in

365

the structure (Fig. 4C). The first conserved cluster contained Pro99, Tyr101, Arg235,

366

and His296 forming a pyruvate-binding pocket. The second cluster including Gly152,

367

Gly154, Asp175, Tyr188, Pro207, Asp259, Tyr205, and Asn232 is responsible for

368

NADH/NAD+ binding. Thus, we propose here that the conserved and coevolving

369

amino acids in D-LDH play important roles in catalysis and in substrate and cofactor

370

binding.

371

Site-directed mutagenesis of D-LDH1

372

Both the MD simulation and amino acid sequence analysis revealed that Tyr101,

373

Arg235, and His296 play critical roles in both pyruvate and lactate binding. Pro99 and

374

Asp259, which are positioned near the substrates and NAD+ binding pockets,

375

respectively, are highly conserved. Additionally, the GxGxxGx(17)D motif (from

376

Gly153 to Asp175) which is in charge of NADH and NAD+ binding is also conserved.

377

Therefore, Pro99, Tyr101, Asp176, Asp259, and His296 were mutated to confirm their

378

functions. Mutating Y101S, D259R, and H296D resulted in complete loss of enzyme

379

activity, indicating the importance of these two amino acids (Supporting Information

380

Figure S-6). The kinetic parameters of P99A and D176R were then determined and

381

compared with those of the wild type enzyme (Table 2; Supporting Information 17



382

Figure S-7). Mutation of Pro99 to Ala did not affect on the Km values toward NADH

383

but the value to NAD+ was increased. The Kcat values of the mutant to both pyruvate

384

and lactic acid were also decreased. D176R had no remarkable effect on the Km values

385

toward NADH and NAD+, suggesting the mutation has no significant effect on

386

cofactor binding. However, the Kcat values to NADH and NAD+ decreased obviously,

387

which led the Kcat/Km toward the cofactor to decrease by about three-fold. These

388

results indicated that the amino acids binding lactate/pyruvate have critical roles for

389

activity while the amino acids binding cofactors affected enzyme activity to some

390

extent, but not severely. This may be because that a single point mutation will not

391

significantly affect the activity since the enzyme binds NADH and NAD+ via a large

392

cluster.

393

Distribution and evolution of D-LDHs

394

Considering that D-LDHs play essential roles in LAB, and the enzymes’ products

395

are an important building block for chemical production of the green polymer PLA

396

that could be applied in bio-plastics,43 we further analyzed the distribution and

397

evolution of the 3,664 D-LDHs in the biosphere (Supporting Information Dataset S-1)

398

using a sequence similarity network and a phylogenetic tree. Sequence similarity

399

networks (SSNs) for these sequences were constructed using e-value cut-offs of 10−60,

400

at which > 40% sequence identity was required to draw an edge between nodes.44 At

401

this e-value threshold, all the nodes were located in one cluster (Supporting

402

Information Figure S-8). As the e-value threshold stringency was decreased to 10−80

403

(sequence identity required to draw an edge was decreased to > ~60%), the 3,664

404

proteins could be segregated into 11 clusters containing 10 or more members (Fig.

405

5A). To analyze the distribution, the nodes were painted by taxonomic classification 18


Page 18 of 42

Page 19 of 42


406

to explore the occurrence of the enzymes. D-LDHs can be found in archaea, bacteria,

407

and eukaryotes. In archaea, only three proteins are found in the phylum of

408

Euryarchaeota. Members of the enzymes were found abundant in Bacteria (96.58%)

409

and Eukarya (3.32%). At the phylum level, the enzymes were mainly distributed in

410

Proteobacteria (39.93%) and Firmicutes (30.92%), followed by Bacteroidetes

411

(9.03%), Actinobacteria (6.96%), and Cyanobacteria (1.56%) in Bacteria. In Eukarya,

412

D-LDHs

413

Basidiomycota (0.74%). The enzymes from fungi clustered with the proteins from

414

Proteobacteria and Bacteroidetes in cluster 1. In other clusters, the enzymes from

415

Firmicutes were the main members. To provide a more detailed view of the

416

evolutionary relationships across the groups, we performed a phylogenetic analysis

417

using the proteins in the clusters assigned based on sequence comparisons (Fig. 5B).

418

Proteins from the same cluster always clustered together and were well-separated in

419

the phylogenetic tree, except that clusters 4 and 9 were clustered in the same branch.

420

The separation of these groups had a high level of bootstrap support in the

421

phylogenetic tree. Meanwhile, the proteins in cluster 1 from Proteobacteria,

422

Bacteroroidetes, and Actinobacteria were gathered in a clade with the proteins in

423

clusters 5, 6, and 7 from Firmicutes, which had a high level of bootstrap support.

424

Proteins from other clusters formed another clade. Since all these clusters had the

425

same branch length, we can consider them as quite similar from an evolutionary point

426

of view.

are mainly found in fungi, including Ascomycota (2.40%) and

19



Page 20 of 42

427

Discussion

428

In this study, we identified and characterized the

429

producing D-lactic acid in L. mesenteroides. The activity assay showed that D-LDH1

430

prefers short-chain and aliphatic substrates. MD simulation showed that Tyr101 and

431

Arg235 are important residues for substrate binding. Evolutionary and point mutation

432

analyses further confirmed their importance. Finally, we performed a large-scale in

433

silico analysis of

434

abundant in bacteria (96.58%) and distributed in eukaryota (3.32%).

D-LDHs,

D-LDH

that is responsible for

which revealed that homologs of the enzymes were

435

L. mesenteroides members are important LAB that are reported to be mainly

436

responsible for fermentation of various vegetables, such as sauerkraut (pickled

437

cabbage) and kimchi (a Korean fermented vegetable food), under moderate salinity

438

and low-temperature conditions; some L. mesenteroides strains have also been

439

isolated from dairy products such as cheese.32 In L. mesenteroides ATCC 8293, 7

440

genes encode lactate dehydrogenases or related enzymes, but only LEUM_1756 was

441

the major gene responsible for the production of D-lactic acid.33 In the genome of L.

442

mesenteroides J18, there are three genes annotated as LDHs that may be involved in

443

generating D-lactic acid.19 Based on evolution analysis, D-LDH gene is under positive

444

selection, possibly a consequence of long-term domestication.

445

we showed that D-LDH1 is the major enzyme directly responsible for producing D-

446

lactate in L. mesenteroides J18. LEUM_1756 and D-LDH1 showed high identity in

447

sequences and similar catalytic characteristics. For example, the NADH reduction

448

activity was much higher than oxidation activity for both of them at pH values below

449

7. Considering the growth conditions of L. mesenteroides are usually acidic, the

450

reaction would mainly produce lactate and NAD+. The optimal activities of the two 20


45

In the current study,

Page 21 of 42


451

enzymes were obtained at 25–30°C for both pyruvate reduction and lactate oxidation.

452

This temperature is similar to the optimal growth temperature of L. mesenteroides.

453

Both enzymes prefer to use pyruvate as a substrate to produce D-lactate and other

454

kinetics of the two enzymes were also in the same order of magnitude. 33 Additionally,

455

D-LDH1

456

could proceed smoothly if L. mesenteroides is used as fermentation starter at low

457

temperatures. Fructose 1,6-bisphosphate and divalent metal ions did not increase the

458

enzyme activity, indicating that the D-LDH1 did not have the allosteric property like

459

the D-LDHs from Fusobacterium nucleatum and Pseudomonas aeruginosa.46

460

still retained almost half its activity even at 0°C, suggesting the reaction

D-LDHs

prefer to use NADH as a cofactor but not NADPH. 8 Point mutations on

461

three amino acids (D176S, I177R, F178T) of D-LDH from Lactobacillus delbrueckii

462

resulted in the enzyme can use NADH and NADPH efficiently.

463

study can only use NADH but not NADPH (Supporting Information Figure S-9). But

464

D-LDH

465

could use both NADH and NADPH efficiently and with a preference for NADPH as

466

its coenzyme; the Asn174 in GxGxxGx(17)D motif of D-LDH from S. inulinus was

467

critical for NADPH utilization based on structure and mutagenesis assay.

468

Sequence alignments showed that the corresponding amino acid residue in D-LDH1

469

was tyrosine with a large and hydrophobic side chain. The hydrophobic and large side

470

chain of tyrosine may increase the steric hindrance to bind the negatively charged

471

phosphate group of NADPH as proposed by Zhu et. al. 48

47

D-LDH1

in this

from Sporolactobacillus inulinus, a strain producing optically pure D-lactate,

48-49

472

Considering the importance of D-LDH1 in LAB for food fermentation, we further

473

analyzed the catalytic mechanism of the enzyme. In the current MD analysis, His296

474

had an appreciable interaction energy contribution with values of −1 kcal/mol and −8 21



Page 22 of 42

475

kcal/mol to pyruvate and lactate, respectively. Based on the crystal structure of D-

476

LDH from L. bulgaricus, His296 can function as an acid/base catalyst.11 The H296K

477

substitution in L. bulgaricus D-LDH does not significantly change the Kcat or Km in

478

pyruvate reduction, but shifts the optimum pH from 7–7.5 to 6.11, 50 Furthermore, MD

479

analysis showed that the residues Tyr101 and Arg235 also contribute to substrate

480

binding with a binding energy less than -1 kcal/mol. Previous research also showed

481

that Tyr101 is responsible for substrate specificity.11 Substitutions of Arg235 with Lys

482

and Gln in the D-LDH of Lactobacillus plantarum induced drastic decreases in the

483

catalytic efficiency.51 We showed that mutations of substrate-binding amino acids,

484

Y101S and H296D, resulted in complete loss of activity. These results indicated that

485

Tyr101, and Arg235, and His296 are crucial for enzyme activity. Pro99 is also highly

486

conserved and P99A mutant showed 70% activity compared with activity of the wild

487

type protein. Pro99 is positioned in the same loop with Tyr100. We proposed that

488

mutation of Pro99 may affect the position of Tyr101 slightly and not result in a drastic

489

decrease in activity. Meanwhile, we also mutated D176 which binds adenine ribose of

490

NADH/NAD+. The D176R mutant retained almost 60% activity compared with

491

activity of the wild type enzyme. But the Kcat/Km value toward cofactor decreased by

492

about three-fold. We proposed that the coenzyme-binding the GxGxxGx(17)D motif is

493

a large conserved cluster, and mutation of one residue could not obviously affect the

494

binding. The mutation of Asp259 to Arg also lead to loss of activity, because Asp259

495

functions to orient the carboxamide group of coenzymes for electron transfer.47 Based

496

on the current and previous research, the catalytic mechanism of

497

proposed. As can be seen in Figure 6, Tyr101 and Arg235 form hydrogen bonds with

498

the carboxyl group of the substrates. The binding directs the ketone moiety of

499

pyruvate toward His296. The distance between the hydrogen atom on the 22


D-LDH

was

Page 23 of 42


500

nicotinamide ring of the NADH and C2 atom of pyruvate is around 2.5 Å, which is

501

suitable for hydride transfer reaction.52 Detailed bond forming and breaking in the

502

catalytic process from pyruvate to D-lactate and the revered direction were shown in

503

supplementary movies (Supporting Information Movie S-1&S-2).

504

High production of polymer-grade D-lactate is urgently required, particularly for

505

the synthesis of polylactic acid.

506

engineered Saccharomyces cerevisiae with introduction of

507

Leuconostoc mesenteroides subsp. mesenteroides strain NBRC3426.53 The engineered

508

Klebsiella oxytoca PDL-5 strain with D-LDH gene produced 111.9 g/L of D-lactate. 54

509

D-lactic

510

LDH from Thermodesulfatator indicus, which cause a trapping effect on carbon flux

511

redistribution.

512

of D-LDH1 and a total of 3,664 protein sequences that showed > 40% identity to D-

513

LDH1 were retrieved. Most proteins were from bacteria and fungi. Few proteins were

514

also found in algae, fish, and archaea. Some enzymes are from thermophilic

515

organisms, such as the enzyme from Thermosynechococcus elongatus (Uniprot ID:

516

Q8DKY8) and Thermosynechococcus sp. NK55a (Uniprot ID: V5V6P2). Some

517

enzymes may have the halophilic property, such as the enzyme from Halomonas

518

cupida (Uniprot ID: A0A1M7DCP1), and Algoriphagus halophilus (Uniprot ID:

519

A0A1N6EIC2). The extremophilic property of these enzymes implicates they are

520

valuable for various industrial applications.

D-lactate

production could reach 61.5 g/L by the D-LDH

gene from

acid reached the titer of 226.6 g/L at 50°C by introduction a thermophilic D-

55

To explore potential D-lactate producers, we searched the homologs

521

In conclusion, we identified D-LDH from L. mesenteroides and characterized D-

522

LDH1 as the major enzyme directly responsible for producing D-lactic acid among

523

related genes. The evolutionary analysis of D-LDHs revealed that the residues directly 23



Page 24 of 42

524

involved in substrate/cofactor binding and catalytic activity are highly conserved and

525

have coevolved. The MD and biochemical analysis further confirmed their function in

526

catalysis. Finally, the D-LDH and its homologs are most abundant in bacteria and

527

distributed in eukaryotes. This study expands the understanding of evolution,

528

functions, and catalytic mechanisms of D-LDH.

529

AUTHOR INFOMATION

530

Corresponding Author

531

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

532

ORCID

533

Baolei Jia: 0000-0001-6434-5604

534

Zhongji Pu: 0000-0003-1046-7336

535

Kyung Hyun Kim: 0000-0002-7336-920X

536

Che Ok Jeon: 0000-0003-1665-2399

537 538

Acknowledgments

539

This

540

(2017M3C1B5019250, 2018R1A5A1025077) of the Ministry of Science and ICT,

541

Republic of Korea. The MS analysis was carried out at the National Supercomputer

542

Center in LvLiang of China, and the calculations were performed on TianHe-2.

543

Notes

544

The authors declare no competing financial interest.

545

Supporting Information

546

The Supporting Information is available free of charge on the ACS Publications

547

website at DOI: .

work

was

supported

by

the

National

24


Research

Foundation

Page 25 of 42


548

Effects of metal ions and fructose 1,6-bisphosphate on the activity of the D-LDH1

549

(Table S1), purification of enzymes (Figure S1), expression of D-LDH genes(Figure

550

S2), crosslink of

551

validation of protein structure of D-LDH1 (Figure S5), Site-directed mutagenesis of D-

552

LDH1 (Figure S6), kinetics graph of D-LDH1 mutants (Figure S7), protein sequence

553

similarity network of D-LDHs (Figure S8), Oxidation activity to NADH and NADPH

554

(Figure S9) (PDF). The bond forming and breaking in the catalytic process from

555

pyruvate to lactate (Movie S1) and from lactate to pyruvate (Movie S2) (avi). Proteins

556

sequences used for the protein similarity network (data sheet 1) (xlsx).]

557

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558

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Leuconostoc mesenteroides subsp. mesenteroides ATCC 8293. Enzyme Microb.

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Technol. 2012, 51, 274-279.

660

(34) Doelle, H. W., Nicotinamide adenine dinucleotide- dependent and nicotinamide

661

adenine dinucleotide-independent lactate dehydrogenases in homofermentative and

662

heterofermentative lactic acid bacteria. J. Bacteriol. 1971, 108, 1284-1289.

663

(35) Singh, D.; Chaudhury, S., Single-molecule kinetics of an enzyme in the presence

664

of multiple substrates. Chembiochem 2018, Epub ahead of print.

665

(36) Abraham, M. J.; Murtola, T.; Schulz, R.; Páll, S.; Smith, J. C.; Hess, B.; Lindahl,

666

E., GROMACS: High performance molecular simulations through multi-level

667

parallelism from laptops to supercomputers. SoftwareX 2015, 1–2, 19-25.

668

(37) Genheden, S.; Ryde, U., The MM/PBSA and MM/GBSA methods to estimate

669

ligand-binding affinities. Expert Opin. Drug Discov. 2015, 10, 449-461.

670

(38) Pearson, W. R., Pearson, W. R. of referencing In Current protocols in

671

bioinformatics (ed. Andreas, D. B. et al.) Chapter 3, Unit3-1. 2013.

672

(39) Wierenga, R. K.; Terpstra, P.; Hol, W. G. J., Prediction of the occurrence of the

673

ADP-binding beta alpha beta-fold in proteins, using an amino acid sequence

674

fingerprint. J. Mol. Biol.1986, 187, 101-107. 28


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(40) Jia, B.; Zhu, X. F.; Pu, Z. J.; Duan, Y. X.; Hao, L. J.; Zhang, J.; Chen, L.-Q.; Jeon,

676

C. O.; Xuan, Y. H., Integrative view of the diversity and evolution of SWEET and

677

SemiSWEET sugar transporters. Front. Plant Sci. 2017, 8, 2178.

678

(41) Jia, B.; Jia, X.; Kim, K. H.; Jeon, C. O., Integrative view of 2-

679

oxoglutarate/Fe(II)-dependent oxygenase diversity and functions in bacteria. Biochim.

680

Biophys. Acta, Gen. Subj. 2017, 1861, 323-334.

681

(42) Petit, D.; Teppa, E.; Mir, A.-M.; Vicogne, D.; Thisse, C.; Thisse, B.; Filloux, C.;

682

Harduin-Lepers, A., Integrative view of α2,3-sialyltransferases (ST3Gal) molecular

683

and functional evolution in deuterostomes: significance of lineage specific losses. Mol.

684

Biol. Evol. 2015, 32, 906-927.

685

(43) Juturu, V.; Wu, J. C., Microbial production of lactic acid: the latest development.

686

Crit. Rev. Biotechnol. 2016, 36, 967-977.

687

(44) Jia, B.; Tang, K.; Chun, B. H.; Jeon, C. O., Large-scale examination of functional

688

and sequence diversity of 2-oxoglutarate/Fe(II)-dependent oxygenases in Metazoa.

689

Biochim. Biophys. Acta, Gen. Subj. 2017, 1861, 2922-2933.

690

(45) Zhang, J.; Gong, G.; Wang, X.; Zhang, H.; Tian, W., Positive selection on D-

691

lactate dehydrogenases of Lactobacillus delbrueckii subspecies bulgaricus. IET Sys.

692

Biol. 2015, 9, 172-179.

693

(46) Furukawa, N.; Miyanaga, A.; Togawa, M.; Nakajima, M.; Taguchi, H., Diverse

694

allosteric and catalytic functions of tetrameric D-lactate dehydrogenases from three

695

Gram-negative bacteria. AMB Express 2014, 4, 76-76.

696

(47) Meng, H.; Liu, P.; Sun, H.; Cai, Z.; Zhou, J.; Lin, J.; Li, Y., Engineering a D-

697

lactate dehydrogenase that can super-efficiently utilize NADPH and NADH as

698

cofactors. Sci. Rep. 2016, 6, 24887.

699

(48)

700

Lactate Dehydrogenase from Sporolactobacillus inulinus. Appl. Environ. Microbiol.

701

2015, 81, 6294-6301.

702

(49) Wang, L.; Zhao, B.; Li, F.; Xu, K.; Ma, C.; Tao, F.; Li, Q.; Xu, P., Highly efficient

703

production of D-lactate by Sporolactobacillus sp. CASD with simultaneous enzymatic

704

hydrolysis of peanut meal. Appl. Microbiol. Biotechnol. 2011, 89, 1009-1017.

705

(50) Kochhar, S.; Lamzin, V. S.; Razeto, A.; Delle, M.; Hottinger, H.; Germond, J.-E.,

706

Roles of His205, His296, His303 and Asp259 in catalysis by NAD+-specific D-lactate

707

dehydrogenase. Eur. J. Biochem. 2000, 267, 1633-1639.

Zhu, L.; Xu, X.; Wang, L.; Dong, H.; Yu, B.; Ma, Y., NADP+-Preferring D-

29



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708

(51) Taguchi, H.; Ohta, T., Essential role of arginine 235 in the substrate-binding of

709

Lactobacillus plantarum D-lactate dehydrogenase. J. Biochem. 1994, 115, 930-936.

710

(52) Dhoke, G. V.; Loderer, C.; Davari, M. D.; Ansorge-Schumacher, M.;

711

Schwaneberg, U.; Bocola, M., Activity prediction of substrates in NADH-dependent

712

carbonyl

713

parameterization of catalytic zinc environment. J Comput. Aided Mol. Des. 2015, 29,

714

1057-1069.

715

(53) Ishida, N.; Suzuki, T.; Tokuhiro, K.; Nagamori, E.; Onishi, T.; Saitoh, S.;

716

Kitamoto, K.; Takahashi, H., D-lactic acid production by metabolically engineered

717

Saccharomyces cerevisiae. J. Biosci. Bioeng. 2006, 101, 172-177.

718

(54) Xin, B.; Tao, F.; Wang, Y.; Liu, H.; Ma, C.; Xu, P., Coordination of metabolic

719

pathways: Enhanced carbon conservation in 1,3-propanediol production by coupling

720

with optically pure lactate biosynthesis. Metab. Eng. 2017, 41, 102-114.

721

(55) Li, C.; Tao, F.; Xu, P., Carbon flux trapping: highly efficient production of

722

polymer-grade

723

Chembiochem. 2016, 17, 1491-1494.

reductase

by

D-Lactic

docking

requires catalytic

constraints

and

charge

acid with a thermophilic d-lactate dehydrogenase.

724

30


Page 31 of 42


725

Figure captions:

726

Figure 1. Reduction activity of

727

different substrates. The

728

pyruvate (square), phenylpyruvate (diamond), 2-ketobutyric acid (cross), and

729

oxaloacetate (triangle). The inactivated enzyme incubated at 100 °C for 5 min was

730

used as control (circle).

731

Figure 2. Activity assay of D-LDH1. (A) The effect of different substrates on D-LDH1

732

activities. (B) Optimal pH of D-LDH1. Different buffers with pH from 3.0-13.0 were

733

used in this assay. (C) Optimal temperature of D-LDH1. The assay was performed in

734

100 mM Tris buffers (pH 8.0) from 0-50°C. (D) Thermostability of

735

determine the thermostability of

736

indicated temperature for 10 min. Residual activity was measured at 30°C for 5 min,

737

pH 9.0 for reduction, and pH 10.00 for oxidation. The reduction and oxidation

738

activities under the optimal conditions were set to be 100%.

739

Figure 3. Molecular dynamic analysis of D-LDH1. (A) The substrate and cofactor

740

binding sites. The representations of ligand–protein interactions for the

741

pyruvate (a) and

742

deviations are shown for D-LDH1 and D-LDH1 complexes at 300 K. (C) root-mean

743

square fluctuations of the residue positions of D-LDH1 and D-LDH1 complexes at 300

744

K. (D) The decomposition of the binding energy on a per-residue basis in the binding

745

sites of the

746

complex (b) to pyruvate and lactate, respectively.

747

Figure 4. Conserved and coevolved residues in D-LDHs represented using D-LDH1 as

D-LDHs

D-LDH1–lactate

D-LDH1

(A),

D-LDH2

(B), and

D-LDH3

(C) to

activity were measured with different substrates:

D-LDH1,

D-LDH1.

To

the enzyme was pre-incubated at the

D-LDH1–

(b) complexes. (B) Backbone root mean square

D-LDH1–NADH-pyruvate

complex (a) and

31


+

D-LDH1–NAD

-lactate


Page 32 of 42

748

a reference sequence. (A) Network analysis of conserved and coevolving residues.

749

The circular network shows the connectivity of coevolving residues. The outermost

750

circle represents the TMHs (orange), the intrafacial region (yellow), and the

751

extrafacial region (green). The labels in the second circle indicate the alignment

752

positions and amino acids of D-LDH1. The colored square boxes in the second circle

753

indicate MSA position conservation (highly conserved positions are shown in red and

754

less conserved positions in blue). The third and fourth circles show the proximity

755

mutual information (MI) and cumulative MI (cMI) values as histograms facing inward

756

and outward, respectively. In the center of the circle, the edges that connect pairs of

757

positions represent significant MI values (> 6.5), with red lines indicating the highest

758

MI scores (top 5%), black lines indicating midrange scores (between 70% and 95%),

759

and gray lines indicating the lowest scores (the remaining 70%) as defined by

760

MISTIC. (B) The network cMI with a high conservation value. Nodes represent the

761

30 most conserved residues (labeled with position and code) and nodes are colored to

762

indicate conservation, from red (higher) to pink (lower). The length of each edge is

763

inversely proportional to its MI value (the closest nodes have the highest MI values).

764

(C) Ribbon diagram of

765

residues.

766

Figure 5. Taxonomic distribution and evolutionary relationships of

767

Taxonomic

768

Information Dataset S1 were used to generate the network using an e-value of 10−150

769

(> 60% sequence identity). Each node represents one protein. Edges are shown with

770

BLASTP e-values below the indicated cutoff. A cluster was sequentially labeled if

771

there were more than 10 nodes in that cluster. The proteins from bacteria are indicated

distribution

D-LDH1

of

showing the 24 most coevolved and conserved

D-LDHs.

The

proteins

32


listed

in

D-LDHs.

(A)

Supporting

Page 33 of 42


772

by circle nodes and from fungi are represented by triangles. Nodes from the same

773

taxonomic groups in the global network are the same color. The nodes representing

774

enzymes that have been “reviewed” in the Uniprot database are shown with a large

775

size. The colors corresponding to phylum with the top ten distributions and protein

776

percentage in the phylum are listed on the right. The proteins in other phylum are

777

shown by light blue (B) Maximum likelihood phylogenetic tree for 3,664 proteins

778

from bacteria and fungi generated using MEGA. The tree with the highest log

779

likelihood (−325820.9727) is shown. The percentage of replicate trees in which the

780

associated taxa clustered together in the bootstrap test (1000 replicates) is shown next

781

to the branches.

782

Figure 6. Proposed catalytic mechanism of

783

direction, a hydride is transferred from the C4N atom of NADH to C2 atom of

784

pyruvate with protonation of pyruvate by His296. In the

785

direction, a hydride is transfer from the C2 atom of

786

dihydronicotinamide ring of NAD+ and the dehydrogenation of substrate is facilitated

787

by the protonated His296. Tyr101 and Arg235 stabilize the substrates and transition

788

states by hydrogen bonds. The red line indicates hydrogen bond. Blue line represents

789

the bond formed between atoms for catalysis. The arrow strands for electron transfer

790

process.

D-LDH.

In the pyruvate to

33


D-lactate

D-lactate

to pyruvate

D-lactate

to the


Page 34 of 42

Table 1. Substrate specificity of recombinant D-LDH1. Substrate

Km (mM)

Vmax (U/mg)

Kcat (s-1)

Kcat/Km (mM-1s-1)

NADH

0.32

1.64×104

3.26×103

1.02×104

Pyruvate

2.66

1.38×104

2.75×103

1.03×103

2-Ketobutyric acid

234

1.12×104

2.22×103

9.48

Oxaloacetic acid

78.8

0.79×104

1.57×103

19.87

NAD+

0.12

0.21×104

0.34×103

2.82×103

D-Lactate

87.9

0.56×104

0.91×103

10.32

34


Page 35 of 42


Table 2. Kinetic parameters of the wild-type D-LDH1, P99A, and D176R. Protein

Substrate

Km (mM)

Vmax (U/mg)

Kcat (s-1)

Kcat/Km (mM-1s-1)

Wild type

Pyruvate

2.66

1.38×104

2.75×103

1.03×103

NADH

0.32

1.64×104

3.26×103

1.02×104

D-Lactate

87.9

0.56×104

0.91×103

10.32

NAD+

0.12

0.21×104

0.34×103

2.82×103

Pyruvate

3.2

1.13×104

2.15×103

0.67×103

NADH

0.34

1.09×104

2.07×103

0.61×104

D-Lactate

102

0.44×104

0.80×103

7.84

NAD+

0.29

0.98×103

0.17×103

0.59×103

Pyruvate

2.97

0.78×104

1.56×103

0.52×103

NADH

0.19

0.44×104

0.88×103

0.47×104

D-Lactate

60.9

0.15×104

0.30×103

4.87

NAD+

0.17

0.43×103

0.84×102

0.50×103

P99A

D176R

35



Figure 1

36


Page 36 of 42

Page 37 of 42


Figure 2

37



Figure 3

38


Page 38 of 42

Page 39 of 42


Figure 4

39



Figure 5

40


Page 40 of 42

Page 41 of 42


Figure 6

41



Table of Contents Graphic

42


Page 42 of 42

Lactate Dehydrogenases Responsible for - ACS Publications

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