Corynebacterium Glutamicum Mycoredoxin 3 Protects Against Multiple Oxidative Stresses

Advance Publication

Full Paper 1 Corynebacterium glutamicum Mycoredoxin 3 protects against multiple oxidative stresses

2 and displays thioredoxin-like activity

3 (Received September 24, 2019; Accepted October 28, 2019; J-STAGE Advance publication date: October 30, 2020) 4 Tao Su#, Chengchuan Che#, Ping Sun, Xiaona Li, Zhijin Gong, Jinfeng Liu, Ge Yang *

6 College of Life Sciences, Qufu Normal University, Qufu, Shandong 273165, China;

8 Running title: Feature of C. glutamicum mycoredoxin 3

11 # These authors contributed equally to this work.

13 * Corresponding authors:

14 Ge Yang

16 E-mail [email protected]

17 Tel: 86-13953760056

26 27 Abstract

28 Glutaredoxins (Grxs) and thioredoxins (Trxs) play a critical role in resistance to oxidative

29 conditions. However, physiological and biochemical roles of Mycoredoxin 3 (Mrx3) that shared a

30 high amino acid sequence similarity to Grxs remain unknown in Corynebacterium glutamicum.

31 Here we showed that mrx3 deletion strains of C. glutamicum was involved in the protection

32 against oxidative stress. Recombinant Mrx3 not only catalytically reduced the disulfide bonds in

33 ribonucleotide reductase (RNR), insulin and 5, 5’-dithiobis-(2-nitro-benzoicacid) (DTNB), but

34 also reduced the mixed disulphides between mycothiol (MSH) and substrate, which was

35 exclusively linked to the thioredoxin reductase (TrxR) electron transfer pathway by a dithiol

36 mechanism. Site-directed mutagenesis confirmed that the conserved Cys17 and Cys20 in Mrx3

37 were necessary to maintain its activity. The mrx3 deletion mutant showed decreased resistance to

38 various stress, and these sensitive phenotypes were almost fully restored in the complementary

39 strain. The physiological roles of Mrx3 in resistance to various stress were further supported by

40 the induced expression of mrx3 under various stress conditions, directly under the control of the

41 stress-responsive extracytoplasmic function-sigma (ECF-σ) factor SigH. Thus, we presented the

42 first evidence that Mrx3 protected against various oxidative stresses by acting as a disulﬁde

43 oxidoreductase behaving like Trx.

44 Keywords: Glutaredoxin-like proteins; Mycoredoxin 3; enzyme activity; redox active disulﬁde;

45 redox regulation

48 Introduction

49 Adverse environment conditions, such as oxidants, low pH, heavy metal, high temperature,

50 diamide and antibiotic (gentamicin), induced the production of deleterious reactive oxygen species

·- 51 (ROS), including the highly destructive hydroxyl radicals (OH ), hydrogen peroxide (H2O2),

─ ·- 1 52 peroxynitrite (ONOO ), superoxide radical (O2 ), singlet oxygen ( O2), and organic

53 hydroperoxides (OHPs) [Mols and Abee, 2011]. The elevated ROS levels could destroy the

54 intracellular redox state and cause detrimental effects including oxidation of sulfhydryl groups in

55 proteins that led to protein disulfide bond formation, eventually resulting in the loss of function.

56 To protect cells against the damage, cells produced many antioxidant enzymes for continuously 57 monitoring the change in the intracellular redox state and facilitating the proper folding of proteins

58 [Trivedi, 2009; Rietsch and Beckwith 1998].

59 Two small redox active proteins (molecular mass, 9-12 kDa) thioredoxins (Trxs) and

60 glutaredoxins (Grxs) were distributed throughout the field of organisms. They were major

61 thiol-disulphide reductases and involved in the reduction of a wide variety of protein disulfides,

62 such as insulin, 3-phosphoadenosine 5-phosphosulfate (PAPS), ribonucleotide reductase (RNR),

63 thiol peroxidase, and the mixed disulphide [Holmgren, 1985 ].They had also been shown to have a

64 large number of functions in cell growth, such as redox control of transcription factors [Dalton,

65 1999], or defense against oxidative stress and apoptosis [Arnér, 2000; Jordan, 1998]. Glutathione

66 (GSH)-dependent Grxs, heat-stable proteins with a typical Trx-fold structure, catalyzed

67 glutathione-protein mixed disulfides by monothiol mechanism or protein disulfides by a dithiol

68 mechanism [Holmgren, 1985; Herrero, 2007; Eberle, 2018]. During catalysis, the thiolate of the

69 active site nucleophilic cysteine of Grx attacked the disulﬁde bond of a S-glutathiolated protein

70 (P-SSG) or protein (P-S-S-P), releasing the protein thiol in the reduced form while becoming itself

71 glutathiolated or disulﬁde bond. Then, GSH attacked the glutathiolated Grx or disulﬁde

72 bond-containing Grx, releasing reduced Grx and GSSG. However, Trxs reduced protein disulfides

73 by a dithiol mechanism. In reaction, the reduced Trxs transferred reducing equivalents to disulfide

74 bonds on target proteins, allowing modulation of target enzymatic activities and leading to the

75 formation of oxidized Trx, Trx-S2. Oxidized Trxs depended on thioredoxin reductase (TrxR) for

76 reduction [Holmgren, 1985].

77 Corynebacterium glutamicum not only was non-pathogenic Mycobacteria often used as a

78 model to study, but also a widespread Gram-positive bacterium of industrial importance. C.

79 glutamicum produced significant amounts of various L-amino acids, including L-lysine and

80 L-glutamate, and vitamins [Oide, 2015]. However, during culture, C. glutamicum inevitably

81 encountered adverse circumstances, such as high osmotic pressure, low pH, and oxidation

82 [Atichartpongkul, 2001], causing a serious problem-oxidative stress [Lee, 2013]. Oxidative stress

83 caused detrimental effects including oxidation of sulfhydryl groups in proteins that led to loss of

84 function. To conquer this, C. glutamicum hosted several redox active molecules such as the major

85 low-molecular-weight thiol mycothiol (MSH), Trxs, and mycoredoxins (Mrxs), participating in

86 oxidative stress defense and reducing enzymes that were oxidized to form a disulfide [Liu, 2013; 87 Van Laer, 2012]. MSH, present in millimolar concentrations in C. glutamicum, was considered as

88 the equivalent of GSH. MSH played an important role in the defense against several external

89 stresses including oxidative stress, alkylating agents and antibiotics [Liu, 2013]. C. glutamicum

90 Trx reduced protein disulfides by a dithiol mechanism. In C. glutamicum, Mrx1 and Mrx2, two

91 small proteins (both ~10 kDa) with a glutaredoxin-like sequence, were reported [Van Laer, 2012;

92 Ordóñez, 2009]. C. glutamicum Mrx1 and Mrx2 reduced the mixed disulﬁde, in which Mrx1 and

93 Mrx2 acted in combination with mycothiol (MSH) and mycothiol disulﬁde reductase (Mtr) as a

94 biological relevant monothiol reducing system [Van Laer, 2012; Rosado, 2017]. C. glutamicum

95 Mrx2, also named NrdH, has been shown to share structural and functional identity with Trx [Si,

96 2014]. However, the biological and biochemical properties of a Grx-like protein, encoded by the

97 ncgl0401 gene, remained unknown. Here, we focus on the description of the function of Ncgl0401,

98 providing an important understanding toward the intracellular redox network of C. glutamicum.

99 Material and methods

100 Bacterial strains and culture conditions

101 The bacterial strains and plasmids that were in this study listed in Table S1. Luria-Bertani (LB)

102 broth or LB agar plates were used for growing Escherichia coli or C. glutamicum. E. coli and C.

103 glutamicum were cultivated at 37 °C and 30 °C under vigorous agitation (220 rpm) as previously

104 reported, respectively [Shen, 2005]. 0.5 M sorbitol-containing brain-heart broth medium (BHIS)

105 was used for producing and maintaining mutant of a gene in C. glutamicum [Shen, 2005]. To

106 create a mrx3 (ncgl0401) gene deletion in C. glutamicum wild type (WT) strain, the

107 pK18mobsacB-Δmrx3 plasmids were transformed into C. glutamicum WT through electroporation

108 and then integrated into the chromosome of C. glutamicum through homologous recombination to

109 perform single crossover [Shen, 2005]. The transconjugants were selected on LB agar plate

110 containing nalidixic acid and kanamycin. Nalidixic acid and 20% sucrose-containing LB agar

111 plates were used to carry out counter-selection for markerless in-frame deletion [Schäfer, 1994].

112 Strains growing on this plate were detected for kanamycin sensitivity by parallel picking on LB

113 plates with nalidixic acid and kanamycin or nalidixic acid and sucrose. Sucrose-resistant and

114 kanamycin-sensitive strains were detected for in-frame deletion by PCR using the

115 DMrx3-F1/DMrx3-R2 primer pair (Table S2) and verified by DNA sequencing. To complement 116 mrx3 expression in Δmrx3 mutants, the pXMJ19-mrx3 derivatives were transformed into Δmrx3

117 mutants by electroporation, creating complementary strains [Si, 2018a]. 0.5 mM isopropyl

118 β-D-thiogalactopyranoside (IPTG) was added into medium to induce the expression of mrx3 gene

119 on the pXMJ19-mrx3 derivatives in complementary strains. For constructing chromosomal fusion

120 reporter strains, the plasmid pK18mobsacB-Pmrx3::lacZY was transformed into relevant C.

121 glutamicum strains by electroporation. The chromosomal pK18mobsacB-Pmrx3::lacZY fusion

122 reporter strain was selected on LB agar plates with kanamycin and nalidixic acid. All chemicals

123 were of Analytical Reagent Grade purity or higher. Antibiotics were added at the following

124 concentrations: kanamycin, 50 µg ml-1 for E. coli and 25 µg ml-1 for C. glutamicum; nalidixic acid,

125 40 µg ml-1 for C. glutamicum; chloramphenicol, 20 µg ml-1 for E. coli and 10 µg ml-1 for C.

126 glutamicum.

127 Plasmid Construction

128 Primers in this study were listed in Table S2. Primers for the amplification of genes and real-time

129 reverse transcription-PCR (RT-PCR) were synthesized at Sangon Biotech Co., Ltd. (Shanghai,

130 China). For creating expression plasmids, the mrx3 gene region of C. glutamicum was amplified

131 with primer pair OMrx3-F and OMrx3-R from genomic DNA of C. glutamicum RES167 by PCR,

132 and then the resulting fragments cut with BamHI and SalI enzymes were cloned into appropriately

133 digested pET28a to give plasmids pET28a-mrx3.

134 To create the Δmrx3 in-frame deletion mutant, the suicide plasmid pK18mobsacB-Δmrx3 was

135 constructed by overlap PCR [Si, 2018b; Su, 2018]. First, based on DNA sequences of mrx3 gene

136 and its adjacent regions, two oligonucleotide primer pairs namely DMrx3-F1/DMrx3-R1 and

137 DMrx3-F2/DMrx3-R2 listed in Table S2 were made (Sangon Biotech Co., Ltd., Shanghai, China).

138 Primer pair DMrx3-F1/DMrx3-R1 was used to amplify the mrx3′s upstream 850 bp fragment;

139 while primer pair DMrx3-F2/DMrx3-R2 was used to amplify the mrx3′s upstream 699 bp

140 fragment. The upstream and downstream fragments were fused together by overlap PCR with the

141 primer pair DMrx3-F1/DMrx3-R2. The resulting PCR products were cut with BamHI and PstI and

142 then clouded into similar sites of pK18mobsacB vector to generate plasmid pK18mobsacB-Δmrx3.

143 To produce pXMJ19-mrx3, primer pair CMrx3-F/CMrx3-R was designed to amplify the

144 DNA fragments of open reading frames region of mrx3 gene from C. glutamicum genomic DNA.

145 The amplified DNA fragments were cut with SalI and BamHI and then cloned into pXMJ19 vector 146 between SalI and BamHI sites.

147 To obtain the DNA fragment of mutant mrx3:C17S, site-directed mutagenesis was carried out

148 by two rounds of PCR [Si, 2017]. Briefly, two oligonucleotide primer pairs namely

149 DMrx3-F1/Mrx3-C17S-R and Mrx3-C17S-F /DMrx3-R2 listed in Table S2 were designed and

150 synthesized. In the first round of PCR, primer pair DMrx3-F1/Mrx3-C17S-R was used to amplify

151 the 5′ prime region of mrx3 (Fragment I); while primer pair Mrx3-C17S-F/DMrx3-R2 was used to

152 amplify the 3′ prime region of mrx3 (Fragment II). The second round of PCR was performed by

153 using OMrx3-F/Mrx3-R as primer pair and fragment I and fragment II as templates to get the

154 mrx3:C17S fragment. The BamHI and SalI cut mrx3:C17S DNA fragments was cloned in pET28a

155 plasmid digested with similar enzymes to create plasmid pET28a-mrx3:C17S. The mrx3:C20S and

156 mrx3:C17SC20S fragments were obtained using a similar procedure as described above and

157 cloned into pET28a to obtain pET28a-mrx3:20S and pET28a-mrx3:C17SC20S derivatives.

158 For getting the lacZY fusion reporter vector pK18mobsacB-Pmrx3::lacZY, the fusion of mrx3

159 promoter to the lacZY reporter gene by overlap PCR was perform. First, two oligonucleotide

160 primer pairs namely Pmrx3-F/Pmrx3-R and lacZY-F/lacZY-R were designed in the first round of

161 PCR to amplify the 352-bp mrx3 promoter DNA fragments (corresponding to nucleotides +12 to

162 -340 relative to the translational start codon (ATG) of mrx3 gene) and the lacZY DNA fragments,

163 respectively. Second, Pmrx3-F/lacZY-R as primers and the first round PCR products as templates

164 were used to carry out the second round of PCR, and the resulting fragments cut with SmaI and

165 PstI were inserted into pK18mobsacB between SmaI and PstI sites to get the

166 pK18mobsacB-Pmrx3::lacZY fusion construct [Si, 2018b; Su, 2018]. The fidelity of all constructs

167 was confirmed by DNA sequencing (Sangon Biotech, Shanghai, China).

168 Plasmid Construction Overexpression and Purification of Recombinant Protein

169 The overexpression of His6-Mrx3 protein in E. coli BL21(DE3) cells harboring pET28a-mrx3

170 derivatives plasmids and purification of the recombinant protein with the His • Bind Ni-NTA resin

171 (Novagen, Madison, WI) were carried out as described previously [Si, 2018b]. Eluted recombinant

172 His6-Mrx3 proteins were concentrated and loaded onto a Superdex-75 10/300 gel filtration column

173 (GE Healthcare, Piscataway, NJ) with a running condition of 10 mM Tris (pH 7.4), 100 mM NaCl,

174 and 5 mM β-mercaptoethanol. For conducting subsequent enzyme activity experiments, the His6

175 tag in protein was cut in the presence of 10 units of Enterokinase-Max (Invitrogen, Karlruhe, 176 Germany) at 4°C overnight. To remove the cleaved tag and uncleaved protein from the tag-free

177 protein, Ni-NTA agarose was used. All enzymes were purchased from Sigma-Aldrich (St. Louis,

178 MO). The resulting His6-tag-free protein was dialyzed against PBS at 4 °C and concentrated for

179 further experiments (>95% purity as estimated by SDS/PAGE).

180 Sensitivity assays

181 Disk diffusion assays were performed for antibiotics, alkylating agents, and oxidative agents

182 according to Rawat et al. [Rawat, 2004]. Briefly, cells were grown to the mid-log phase and a

183 lawn of cells was plated onto LB plates. After the paper disks were placed into the plates, 100 mM

184 hydrogen peroxide (H2O2), 5.5 mM cumene hydroperoxide (CHP), 0.2 mM sodium hypochlorite

185 (NaOCl), 5 mM diamide, 50 mM 2, 4-dinitrochlorobenzene (DNCB), and 0.6 mM Iodoacetamide

186 (IAM) (10 µl) were added to the disk. The disks were allowed to dry and the plates were incubated

187 for 2 to 3 days.

188 Electrophoretic mobility shift assay (EMSA)

189 The binding of SigH to mrx3 promoters was performed using the method of Si et al. [Si, 2018a].

190 Briefly, different concentration of purified His6-SigH (0-5.0 µg) was incubated with 160 bp mrx3

191 promoter (Pmrx3, 30 nM) that contained the predicted SigH binding site and was amplified from the

192 sequence −160 to -1 bp upstream of the start site of the mrx3 transcript using primer pair

193 EMrx3-F/EMrx3-R (Table S2) in a total volume of 20 µl. A 160 bp fragment from the mrx3

194 coding region amplified with primers Control-F and Control-R instead of Pmrx3 and bovine serum

195 albumin (BSA) instead of SigH were used as negative controls. The binding reaction buffer

196 contained 10 mM Tris-HCl (pH 7.4), 5 mM MgCl2, 50 mM KCl, 5% glycerol, 0.1% Nonidet P 40

197 (NP40), 1 µg poly(dI:dC), 1 mM DTT. The binding reaction mixtures were incubated at room

198 temperature for 30 min and then loaded onto 10% native polyacrylamide gel made with 10 mM

199 Tris buffer containing 50 mM KCl, 5 mM MgCl2 and 10% glycero1 in 0.5×TBE electrophoresis

200 buffer [50 mM Tris, 41.5 mM borate (pH 8.0), 10 mM Na2EDTA.H2O]. Electrophoresis was

201 performed at 4°C temperature and 100V using 1✕TBE (89 mM Trisbase, 89 mM boricacid, 2

202 mM EDTA) as the electrophoresis buffer. The gel was subsequently stained with a 10,000-fold

203 diluted SYBR Gold nucleic acid staining solution (Molecular Probes) for 30 min. The DNA bands

204 were visualized with UV light at 254 nm.

205 Formation and separation of heterodimers 206 The assays of heterodimers were performed based on the method described by Si et al. with minor

207 modifications [Si, 2018a]. 20 µM MPx:C64S and Mrx3 or its variants (final concentration 20 µM)

208 were mixed in TE buffer (30 mM Tris-HCl, pH 8.0, 1 mM EDTA) to a final volume of 20 µl. This

209 reaction mixture was incubated at room temperature for 30 min after the addition of 50 µM H2O2

210 and then subjected to 15% nonreducing SDS-PAGE. Finally, the gels were stained with

211 Coomassie Brilliant Blue R-250.

212 MPx:C64SC79S-SSM (the mixed disulfide between MPx:C64SC79S-SH and MSH) and

213 Prx-SSM (the mixed disulfide between Prx-SH and MSH) were produced according to the method

214 of Chi et al and Si et al [Si, 2018a; Chi, 2014]. To detect the MSH transfer from

215 MPx:C64SC79S-SSM to Mrx3, 10 µM MPx:C64SC79S-SSM was mixed with 30 µM WT Mrx3,

216 Mrx3:C17S, or Mrx3:C20S, and incubated for 30 min at room temperature. After the mixtures

217 were separated on nonreducing SDS-PAGE, Coomassie Brilliant Blue R-250 and MSH-speciﬁc

218 Western blot were performed. Western blot was developed with anti-MSH antibody (1:1000

219 dilution) for 1 h at room temperature followed with an overnight incubation at 4°C with the

220 secondary antibody alkaline phosphatase (AP) conjugate (goat anti-rabbit, Sigma-Aldrich,

221 1:10000 dilution).

222 Steady-state kinetics of oxidized MPx:C64S-S2 and MPx:C64SC79S-SSM by Mrx3/TrxR

223 pathway

224 To determine the kinetics of Mrx3 reduction of the oxidized MPx:C64S-S2, oxidized

225 MPx:C64S-S2 was prepared according to previously described [Van Laer, 2012; Pedre, 2015]. A

226 solution of pre-reduced MPx:C64S was oxidized with a 30-fold molar excess of H2O2 and

227 incubated for 1 min at room temperature. Excessive H2O2 was removed by size exclusion

228 chromatography using a Superdex75 10/300 GL column (GEHealthcare), pre-equilibrated with the

229 reaction buffer solution. Mrx3 was also oxidized using a 10-molar excess of diamide for 30 min at

230 room temperature and puriﬁed on a Superdex75 10/300 GLcolumn (GEHealthcare) equilibrated

231 with 50 mM HEPES/NaOH pH 8.0, 150 mM NaCl according to previously described [Liu, 2013].

232 For the preparation process and the preparation conditions being reliable, Mrx1 was used as a

233 positive control and the formation of MPx:C64S-S2 was detected using 5, 5’-dithio-bis

234 (2-nitrobenzoic acid) (DTNB) based on the method described [Lian, 2012]. For the steady-state

235 kinetic parameters of the Mrx3/TrxR reduction, the assay mixture consists of 50 mM Tris-HCl 236 buffer (pH 7.5), 1 mM EDTA, 5 µM TrxR, 250 µM NADPH, 30 µM Mrx3 (WT or its variants),

237 and varying concentrations of oxidized MPx:C64S-S2 in a ﬁnal volume of 500 µl reaction buffer.

238 The reaction was started by the addition of oxidized MPx:C64S-S2. To determine the kinetics of

239 MPx:C64SC79S-SSM reduction by Mrx3 (WT or its vaiants)/TrxR, the reaction mixture

240 contained 50 mM Tris-HCl buffer (pH 7.5), 1 mM EDTA, 5 µM TrxR, 250 µM NADPH, 30 µM

241 Mrx3 (WT or its variants), and varying concentrations of MPx:C64SC79S-SSM in a ﬁnal volume

242 of 500 µl. In this case, the reaction was started by the addition of MPx:C64SC79S-SSM. NADPH

243 oxidation was monitored as A340. The activity was determined after subtracting the spontaneous

244 reduction rate observed in the absence of MPx:C64SC79S-SSM, and the number of micromoles of

245 NADPH oxidized per second per micromole of enzyme (i.e. turnover number, s−1) was calculated

−1 −1 246 using the molar absorption coefficient of NADPH at 340 nm (ε340) of 6220 M ·cm . Three

247 independent experimental replicates were performed for each analysis. The kcat and Km values

248 were obtained from a non-linear fit with the Michaelis-Menten equation using the program

249 GraphPad Prism 5.

250 Construction of chromosomal fusion reporter strains and β-galactosidase assay

251 The lacZY fusion reporter plasmid pK18mobsacB-Pmrx3::lacZY was transformed into the wild type

252 C. glutamicum WT (wild-type C. glutamicum with empty plasmid pXMJ19), ∆sigH (strains

253 lacking sigH gene contained empty pXMJ19) and ∆sigH+ (ΔsigH was complemented with

254 plasmids pXMJ19 carrying the wild-type sigH gene) by electroporation and the chromosomal

255 fusion reporter strains were selected by plating on LB agar plates supplemented with kanamycin

256 [Si, 2018b 21]. β-galactosidase activities were assayed with O-Nitrophenyl-β-D-galactopyranoside

257 (ONPG) as the substrate [Miller, 1992]. The β-galactosidase data represented the mean of one

258 representative assay performed in triplicate, and error bars represent standard deviation. Statistical

259 analysis was carried out with Student’s t-test.

260 Ribonucleotide reductase reduction assays

261 Ribonucleotide Reductase (RNR) activity was measured based on the method described by Ceylan

262 et al. [Ceylan, 2010] with minor modifications as follow: RNR activity was performed from the

263 conversion of GDP to dGDP by coupling the reaction to NADPH oxidation. The reaction mixture

264 contained 50 mM Hepes, 100 mM KCl, 7 mM MgCl2, pH 7.6, 200 µM NADPH, 500 µM dTTP,

265 1.6 mM ATP, 3 µM NrdE, 18 µM NrdF, 150 milliunits TrxR, and 10 µM different Mrx3 (WT or 266 its variants). The addition of 500 µM GDP was to the mixtures to start the reaction. The NADPH

267 oxidation was monitored at 340 nm. The decreased absorbance values were subtracted from that

268 of the background without GDP, and the RNR activity was calculated from the ΔΔA/min value.

269 As a positive control, Trx1 (10 µM) was used in place of Mrx3 in the same conditions.

270 Insulin reduction assay

271 The ability of proteins to reducing insulin disulfide was measured as described previously [Du,

272 2012]. Briefly, 0.32 mM insulin was added to the assay mixture (0.5 ml) containing 50 mM

273 Tris-HCl (pH 7.5), 1 mM EDTA, 200 µM NADPH, and 10 µM TrxR. The addition of 10 µM

274 different Mrx3 (WT or its variants) was to the mixtures to start the reaction. Changed in turbidity

275 was monitored by measuring the decrease in absorbance at 340 nm during the first hour of

276 reaction every 1 min. As a positive control, Trx1 (10 µM) was used in place of other Mrx3 in the

277 same conditions.

278 DTNB assay

279 DTNB [5, 5-dithiobis-(2-nitrobenzoic acid)] assays were used to measure reduction of DTNB

280 disulfides [Holmgren, 1979]. The assay mixture contained 50 mM Tris-HCl, pH 7.5, 1 mM EDTA,

281 200 µM NADPH, 1 mM DTNB, 10 µM TrxR, and 10 µM different Mrx3 (WT or its variants). The

282 activity was monitored by following the increase at 412 nm during the first 10 minutes due to

283 production of 3-carboxy-4-nitrobenzenethiol (NBT). Keeping all the reaction in the same

284 conditions, Trx1 (10 µM) was used in place of Mrx3 as a positive control.

285 Reductase activity of protein toward MSH mixed disulfides

286 The ability of protein to catalyze reduction of the HED-SSM was measured according to Kim et al.

287 [Kim, 2012]. The mixed disulphide between hydroxyethyl disulphide (HED) and MSH

288 (HED-SSM) was formed by incubating 0.7 mM HED with 1 mM MSH at 30°C for 5 min. 0.25

289 mM HED-SSM as the substrate was added to the electron transfer mixtures in final volume of 200

290 µl with 5 µM TrxR, 200 mM NADPH and 10 µM different Mrx3 (WT or it variants). The

291 reactions were carried out at 25°C for 10 min and the absorption of NADPH at 340 nm was

292 recorded. As a positive control, Trx1 was used in place of Mrx3 in the reaction mixture.

293 MPx:C64SC79S-SSM and Prx-SSM instead of HED-SSM in the assay system were used to detect

294 enzyme activity.

295 Quantitative RT-PCR Analysis 296 Quantitative RT-PCR analysis (7500 Fast Real-Time PCR; Applied Biosystems, Foster City, CA)

297 was performed as described previously [Si, 2018a]. The primers used were listed in Table S2. To

298 obtain standardization of results, the relative abundance of 16S rRNA was used as the internal

299 standard.

300 Statistical analysis

301 GraphPad Prism Software was used to carry out statistical analyses (GraphPad Software, San

302 Diego California USA).

303 Results and discussion

304 Indentification of mrx3 gene from C. glutamicum genome

305 Through running a direct subsequent BLAST search and analyzing the genomic sequence, the

306 gene coding for a putative glutaredoxin protein (NCgl0401) was identified, composing of 249 bp

307 and encoding a protein of 82 amino acids with a theoretical molecular mass of 9 kDa. The

308 NCgl0401 had very high sequence identity with the Grx from other Corynebacteriums (Fig. S1A).

309 Although NCgl0401 also contained the active site motif CXXC as C. glutamicum Trx and Mrx,

310 the intervening residue Gly was similar to that of Trx and different from Pro of Mrx (Fig. S1B).

311 Therefore, we speculated that Mrx3 with a conserved CGXC active site motif behaved like Trx

312 but not Mrx.

313 Mrx3 of C. glutamicum exhibits a phenotype sensitive to oxidative stress

314 To assess the role of Mrx3 in protecting cells against external oxidants, we determined the

315 phenotype of a mrx3 knockout in C. glutamicum under various oxidizing and H2O2-inducing

316 agents [hydrogen peroxide (H2O2), cumene hydroperoxide (CHP), sodium hypochlorite (NaOCl),

317 diamide, 2, 4-dinitrochlorobenzene (DNCB), and Iodoacetamide (IAM)]. As shown in Fig.1, all

318 chemical reagents tested gave a signiﬁcantly larger zone of clearance for Δmrx3 mutant (the

319 mutant lacking mrx3 with the empty plasmid pXMJ19) than for wild-type strain (WT, the

320 wild-type C. glutamicum strain with the empty plasmid pXMJ19). To confirm that the susceptivity

321 to reagents may occur when lacking mrx3, complementation experiment was performed in Δmrx3.

322 As shown in Fig.1, Δmrx3+, corresponding complementary strains containing the C. glutamicum 323 mrx3 genes provided by plasmid pXMJ19 in trans, almost fully complemented the growth defect

324 of the mrx3 null mutants under various reagents. Collectively, the above results indicated the

325 important role of Mrx3 under persistent various stress.

326 Mrx3 reduces the disulfide bond and MSH mixed disulphide bonds using electrons from the

327 TrxR/NADPH pathway

328 As Mrx3 had Cys-X-X-Cys motif that was similar to that of Trxs (Fig. S1), we investigated the

329 possible role of Mrx3 as dithiol-disulfide exchange reaction. We first employed in vitro assay

330 system (see Materials and Methods) by using various disulfide-containing enzymes or substrates,

331 such as RNR, insulin, and DTNB, and the coupled NADPH oxidation by TrxR was monitored at

332 340 nm. As shown in Fig. 2A, Mrx3 functioned as a reductant for the class Ib RNR with TrxR and

333 NADPH as the electronic donor system, displaying comparable activities towards class Ib RNR

334 with that of C. glutamicum Trx1 [Si, 2014]. To assess whether C. glutamicum Mrx3 possessed

335 general thiol-disulfide redox activity as Trx1, we examined the capacity of Mrx3 to catalyze the

336 reduction of insulin. As shown in Fig. 2B, the addition of Mrx3 to the reaction mixture led to be

337 an obviously reduced absorbance at 340 nm in the presence of TrxR/NADPH, similar to Trx1. We

338 further explored the capacity of C. glutamicum Mrx3 to reduce a small molecule artificial

339 disulfide compound DTNB. As shown in Fig. 2C, Mrx3 caused a catalytic relevant reduction of

340 DTNB in the presence of TrxR and NADPH.

341 Apart from reducing the disulfide bond, Trx could also take the role of Mrx1 in reducing a

342 cysteine-MSH mixed disulfide [Pedre, 2015]. Therefore, we tested whether Mrx3 reduced

343 cysteine-MSH mixed disulfide. In a MSH-coupled hydroxyethyl disulphide (HED) assay,

344 although the reduction ability of Mrx3 linked to TrxR/NADPH pathway was lower than

345 Mrx1/Mtr/MSH/NADPH pathway, Mrx3 could reduce the mixed disulphide between MSH and

346 HED (HED-SSM) via TrxR/NADPH pathway to a certain extent (Fig. 3A). Similarly, Mrx3 also

347 reduced MPx:C64SC79S-SSM, in agreement with the results of Pedre et al. reported for Trx1 (Fig.

348 3B) [Pedre, 2015]. In the next stage, we evaluated the possibility of cross-talk between the Mrx3

349 and the MSH/Mtr pathways. To test this hypothesis, we used a coupled enzyme assay based on

350 NADPH consumption. As shown in Fig. 3C and d, Mrx3 did not reduce HED-SSM and

351 MPx:C64SC79S-SSM via coupling with MSH/Mtr reductive pathway. Together, Mrx3 showed 352 almost equal function of Trx1 in vitro.

353 Both cysteines of the CXXC motif in Mrx3 are required to maintain its activity

354 To check whether Mrx3 used one active site cysteine or two active site cysteines in the reaction

355 process, we mutated the first, the second, and both cysteines of the CXXC motif to serines. The

356 functionalities of Mrx3:CXXS, Mrx3:SXXC, and Mrx3:SXXS variants to reduce RNR, insulin,

357 DTNB, HED-SSM, MPx:C64SC79S-SSM, and Prx-SSM were tested in progress curves by

358 following the oxidation of NADPH in the presence of the TrxR/NADPH system. As shown in Fig.

359 4, electron transfer was almost the same as back ground levels when Mrx3:CXXS, Mrx3:SXXC or

360 Mrx3:SXXS was present, indicating that both cysteines in the CXXC motif of Mrx3 were

361 essential for reducing disulfide bond and MSH mixed disulphide bonds. As such, Mrx3 was

362 functioning as a dithiol reductase with essential N-terminal cysteine and C-terminal cysteine.

363 Mrx3 reduces disulfide bond and MSH mixed disulphide bonds via a dithiol mechanism

364 To further conﬁrm that Mrx3 operated with CXXC active site motif through a dithiol mechanism,

365 reduction activity of oxidized MPx:C64S-S2 with the Mrx3/TrxR, Mrx3:C17S/TrxR and

366 Mrx3:C20S/TrxR electron transfer pathway was studies. As shown in Fig. 5A, the enzyme activity

367 confirmed that oxidized MPx:C64S-S2 could be reduced with the Mrx3/TrxR electron transfer

368 pathway but not Mrx3:C17S/TrxR or Mrx3:C20S/TrxR. Only the sample with oxidized

369 MPx:C64S-S2 coupled to the Mrx3/TrxR electron transfer pathway showed consumption of

370 NADPH, but no NADPH consumption was observed when Mrx3:C17S/Mrx1:C20S was added

371 into the assay instead of Mrx3 WT. After determining the ability of disulﬁde bond reduction, we

372 investigated the rate of disulﬁde bond reduction in a coupled enzyme assay, in which different

373 concentration of oxidized MPx:C64S-S2 was used as substrate by being linked to C. glutamicum

374 Mrx3 WT, TrxR and NADPH (Fig. 5B). From the Michaelis–Menten kinetic plot, a kcat of

−1 375 0.077±0.003 s and a Km of 0.753 ±0.095 µM were obtained, resulting in a speciﬁcity constant

4 −1 −1 376 (kcat/Km) of 10.2×10 M s , compared with C. glutamicum oxidized MPx:C64S-S2 with Trx1

377 (8.4 × 104 M−1 s−1) [Pedre, 2015 26]. However, the rate constant in the absence of Mrx3 was only

378 8.9± 0.3 M−1 s−1 (Fig. 5C). On nonreducing SDS-PAGE gel, we observed that, in the presence of

379 H2O2, the intermediate complex about 35 kDa was formed only between MPx:C64S and

380 Mrx3:C20S (Fig. 5D). However, no heterodimer was detected between Mrx3:C17S and

381 MPx:C64S. The phenomenon was in agreement with previous studies that the reduction of H2O2 382 by MPx:C64S in the presence of Trx:C35S resulted in the formation of a mixed disulﬁde between

383 Trx and MPx:C64S [Si, 2018a; Pedre, 2015]. Together, Mrx3 with its N-terminal cysteine first

384 attacked the oxidized MPx to form disulfide bond, and then reduced the disulfide bond with its

385 C-terminal cysteine to release reduced MPx and simultaneously form oxidized Mrx3. Finally,

386 TrxR/NADPH provided electron for oxidized Mrx3.

387 Next, we investigated the reduction activity of MPx:C67SC79S-SSM with the WT

388 Mrx3/TrxR, Mrx3:C17S/TrxR and Mrx3:C20S/TrxR electron transfer pathway. As shown in Fig.

389 6A, the enzyme activity showed that the MPx:C67SC79S-SSM could be reduced with the WT

390 Mrx3/TrxR electron transfer pathway but not Mrx3:C17S/TrxR or Mrx3:C20S/TrxR. In the next

391 step, we aimed at determining the rate constant of MPx:C67SC79S-SSM de-mycothiolation by

392 WT Mrx3. From a Michaelis–Menten plot, the kinetic parameters of MPx:C67SC79S-SSM

−1 393 de-mycothiolation by WT Mrx3 were obtained: a kcat of 0.029±0.001 s and a km of 31.87 ±4.786

2 −1 −1 394 µM, resulting in a speciﬁcity constant (kcat/km) of 9.1 × 10 M s (Fig. 6B upper panel). We also

395 obtained an estimate of the de-mycothiolation rate constant by MSH in the absence of Mrx3,

396 which was only 7.2 ± 0.2 M−1 s−1 (Fig. 6B lower panel). We conﬁrmed this ﬁnding using an

397 anti-MSH Western blot (Fig. 6C lower panel). Although MPx:C64SC79S, MPx:C64SC79S-SSM,

398 WT Mrx3, Mrx3:C20S, and Mrx3:C17S were detected on nonreducing 15% SDS-PAGE (Fig. 6C

399 upper panel) , the signal corresponding to MPx:C64SC79S-SSM disappeared from the Western

400 blot after addition of WT Mrx3. Moreover, after addition of Mrx3:C20S, Mrx3:C20S-SSM was

401 observed (Fig. 6C lower panel). However, after addition of Mrx3:C17S, MPx:C64SC79S-SSM

402 still existed but Mrx3:C17S-SSM did not appear. All together, these results indicated that Mrx3

403 could indeed reduce the mixed disulﬁde, attacking the sulfur of MSH, and Mrx3 used a dithiol

404 mechanism for this reduction step.

405 Oxidized Mrx3-S2 is speciﬁcally reduced with electrons from the TrxR/NADPH pathway

406 First, we identiﬁed the electron transfer pathway coupled to Mrx3. To do so, the

407 TrxR/NADPH and MSH/Mtr/NADPH electron transfer pathway, two alternative physiological

408 reducing systems in C. glutamicum, were used. Oxidized Mrx3-S2 with a single disulphide

409 between its active site cysteines was added as substrate for this pathway (Fig. S2). By monitoring

410 the reduction in the absorption at 340 nm due to NADPH consumption, we found that electrons

411 were transferred by the TrxR/NADPH but not MSH/Mtr/NADPH pathway to oxidized Mrx3. 412 mrx3 expression is up-regulated by SigH

413 To know whether mrx3 responded to stress, we decided to monitor the stress response of

414 mrx3 from C. glutamicum RES167 strain after various reagents treatment. Expectedly, lacZY

415 activity and real time RT-PCR data presented in Fig. 7A and B indicated that mrx3 responded to

416 stresses.

417 In C. glutamicum, SigH, the stress-responsive extracytoplasmic function-sigma (ECF-σ)

418 factor, was found to control the expression of multiple resistance genes, including the thioredoxin

419 system (trx and trxR), NADPH-dependent mycothiol reductase (mtr) and

420 1-D-myo-inosityl-2-amino-2-deoxy-alphaD-glucopyranoside-L-cysteine ligase (mshC) of the

421 mycothiol system, mycothiol S-conjugate amidase (mca) [Busche, 2012]. Thus, mrx3 regulation

422 was investigated by chromosomal Pmrx3::lacZ fusion reporter and quantitative real-time RT-PCR

423 (qRT-PCR) analysis. As shown in Fig. 7A, deletion of sigH significantly decreased the lacZY

424 activity of the mrx3 promoter, fully recovered by introducing a plasmid expressing SigH

425 (pXMJ19-sigH). These results clearly demonstrated that C. glutamicum Mrx3 was positively

426 regulated by SigH. The positive regulation of mrx3 by SigH was also confirmed by qRT-PCR,

427 with the observation that the mRNA levels of mrx3 were reduced in the ΔsigH mutant and

428 restored to the wild-type level in the complemented strain (Fig. 7B). To further determine whether

429 SigH regulated mrx3 expression directly, we examined the interaction between SigH and the mrx3

430 promoter using electrophoretic mobility shift assay (EMSA). Incubation of a 160 bp DNA element

431 containing the mrx3 promoter (Pmrx3) sequence with His6-SigH led to the formation of DNA–

432 protein complexes, and the abundance of such complexes depended on the amount of SigH (Fig.

433 7C). However, both a 160 bp control DNA fragment amplified from the mrx3 coding open reading

434 frame region and BSA instead of His6-SigH showed no detectable binding (Fig. 7C, lane ). Thus,

435 SigH directly activated the expression of mrx3 by specifically recognizing an operator within the

436 mrx3 promoter region.

437 Conclusion

438 We have demonstrated that the physiological and biochemical function of C. glutamicum

439 Mrx3 were more identical with Trx but not with Mrx1. Our data revealed that C. glutamicum

440 Mrx3 (a protein of 82 amino acid residues) was a substrate for thioredoxin reductase (TrxR) and

441 catalyzed NADPH-dependent disulfide and mixed disulfide reduction by dithiol mechanism. 442 Site-directed mutagenesis confirmed that the conserved Cys17 and Cys20 in Mrx3 were necessary

443 to maintain its activity. The expression of Mrx3 was induced by various stress conditions, and it

444 was directly under the control of the stress-responsive extracytoplasmic function-sigma (ECF-σ)

445 factor SigH, similar to other previously characterized trx1 genes. Thus, C. glutamicum Mrx3 was

446 characterized by a glutaredoxin-like amino acid sequence but a thioredoxin-like activity proﬁle.

447 Acknowledgements

448 This work was supported by the National Natural Science Foundation of China (31500087), Scien

449 ce and Technology Plan Project of Shandong Higher Education Institutions (J16LE03). 450

451 Supporting Material

452 Supplementary Table S1-Bacterial strains, plasmids and primers used in this study.

453 Supplementary Table S2- primers used in this study.

454 Supplementary Fig. S1- Sequence alignment. Sequence alignment of C. glutamicum NCgl0401

455 and Grx from other Corynebacteriums.

456 Supplementary Fig. S2- Oxidized Mrx3-S2 is reduced via the the TrxR/NADPH pathway.

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550

551 Figure Legends

552 Fig. 1 The Δmrx3 strains of C. glutamicum were more sensitive to various stress. The zone

553 diameter of clearance (mm) of C. glutamicum wild type (WT, the wild-type C. glutamicum strain

554 with the empty plasmid pXMJ19), Δmrx3 mutant (the mutant lacking mrx3 with the empty

555 plasmid pXMJ19) and Δmrx3+ (the Δmrx3 mutant expressed the wild-type mrx3 gene with a

556 shuttle vector pXMJ19) caused by the paper disks (Ø = 5 mm) with stress inducing several agents.

557 The dot plot showed the mean and standard error of the 3 samples sets for each agent.

558 Fig. 2 Mrx3 reduced disulfide bonds by the TrxR/NADPH pathway but not the

559 Mtr/MSH/NADPH pathway. Reduction of RNR (A), insulin (B) and DTNB (C) by Mrx3 (10 µM)

560 coupled to the TrxR/NADPH regeneration system and the MSH/Mtr/NADPH regeneration system.

561 Trx1 (10 µΜ) was used as a positive control. Negative control was the omission of Mrx3 in the

562 presence of TrxR or MSH/Mtr. The reduction of RNR (A) was calculated from the conversion of

563 GDP to dGDP by coupling the reaction to NADPH oxidation at 340 nm. The reduction of insulin

564 (B) was recorded by measuring the decrease of NADPH oxidation at 340 nm. The reduction of

565 DTNB was recorded as an increase in absorption at 412 nm (C). 566 Fig. 3 Mrx3 reduced MSH mixed disulphide bonds by the TrxR/NADPH pathway but not the

567 Mtr/MSH/NADPH pathway. The mixed disulphide between HED and MSH (HED-SSM) (A and

568 C) and MPx:C64SC79S-SSM (B and D) were used as substrates in the reaction pathways and the

569 consumption of NADPH due to the reduction of substrates was monitored by A340 nm decrease. The

570 assay was measured by using the TrxR/NADPH (A and B) and the Mtr/MSH/NADPH pathway (C

571 and D). Trx1 (10 µΜ) and Mrx1/MSH/Mtr/NADPH pathway was used as a positive control.

572 Negative control was the omission of both Mrx3 and Mrx1 in the presence of TrxR or Mtr/MSH.

573 Fig. 4 The active site cysteines of Mrx3 were essential for reduction of disulfide bonds and MSH

574 mixed disulphide bonds. RNR (A), insulin (B), DTNB (C), HED-SSM (D), MPx:C64SC79S-SSM

575 (E), and Prx-SSM (F) reduction were measured in the presence of 10 µM of each Mrx3 variants

576 by using the TrxR/NADPH regeneration system as indicated in Fig 2 and 3.

577 Fig. 5 Mrx3 reduced disulfide bond via a dithiol mechanism. (A) MPx:C64S-S2 could be

578 regenerated by Mrx3 electron transfer pathway in vitro. Negative control was the omission of

579 Mrx3 WT and its variants in the presence of TrxR. The data were represented as mean±SD of

580 three independent experiments. (B) The Mrx3 reduction of the intramolecular disulﬁde of

581 MPx:C64S followed Michaelis–Menten steady-state kinetics. Different concentrations of oxidized

582 MPx:C64S-S2 were mixed with a pre-incubated mixture of Mrx3, TrxR and NADPH. The

583 decrease in A340 nm, due to NADPH oxidation, was monitored in function of time. (C) Negative

584 control was the omission of Mrx3 WT in the presence of TrxR. The data were represented as

585 mean±SD of three independent experiments. (D) Validation the interaction between MPx:C64S

586 and Mrx3 in vitro. Disulfide bond-containing MPx:C64S created stable association with

587 Mrx3:C20S. Nonreducing 15% SDS-PAGE showed MPx:C64S incubated with each Mrx3 (WT,

588 Mrx3:C17S and Mrx3:C20S) in the presence of H2O2. MPx:C64Sox and MPx:C64Sred expressed

589 oxidized and reduced MPx:C64S, respectively.

590 Fig. 6 Mrx3 reduced MSH mixed disulphide bonds via a dithiol mechanism. (A)

591 MPx:C67SC79S-SSM could be regenerated by Mrx3 electron transfer pathway in vitro. The data

592 were represented as mean±SD of three independent experiments. (B) The Mrx3 reduction of

593 MPx:C67SC79S-SSM followed Michaelis-Menten steady-state kinetics. Different concentrations

594 of MPx:C67SC79S-SSM were mixed with a pre-incubated mixture of Mrx3, TrxR and NADPH.

595 The decrease in A340 nm, due to NADPH oxidation, was monitored in function of time (upper). The 596 data were represented as mean±SD of three independent experiments. Negative control was the

597 omission of Mrx3 WT in the presence of TrxR (lower). (C) MPx:C67SC79S-SSM was

598 de-mycothiolated by Mrx3 by a dithiol mechanism. Nonreducing 15% SDS-PAGE stained by

599 Coomassie Brilliant Blue R-250 (upper) and anti-MSH Western blot (lower) of protein mixtures

600 containing reduced/S-mycothiolated MPx:C67SC79S with Mrx3 WT, Mrx3:C17S, or Mrx3:C20S

601 were shown.

602 Fig. 7 Positive regulation of C. glutamicum mrx3 expression by SigH. (A) β-Galactosidase

603 analysis of the mrx3 promoter activity was performed using the transcriptional Pmrx3::lacZY

604 chromosomal fusion reporter expressed in the wild type WT(pXMJ19) strain, ΔsigH (pXMJ19)

605 mutant, and the complementary ΔsigH(pXMJ19-sigH) strain. β-Galactosidase activity was

606 assayed as described in “Materials and Methods”. Mean values with standard deviations (error bar)

607 from at least three repeats were shown. (B) qRT-PCR assay was performed to analyze the

608 expression of mrx3. Exponentially growing C. glutamicum cells were exposed to different toxic

609 agents at indicated concentrations for 30 min. The mRNA levels were presented relative to the

610 value obtained from wild type cells without treatment. The values represent the mean results from

611 three independent cultivations, with standard errors. (C) Interaction between SigH and the mrx3

612 promoter was analyzed by EMSA. The increasing amounts of SigH used were 0, 0.5, 1.5, 2.5, and

613 3.0 µg (lane1, 2, 3, 4, and 5, respectively). As negative controls, a 160 bp fragment from the mrx3

614 coding region amplified with primer pair Control-F and Control-R instead of the 160 bp mrx3

615 promoter was incubated with 3.0 µg His6-SigH (lanel 6) or BSA instead of His6-SigH was

616 incubated with the 160 bp mrx3 promoter (lane 7) in the binding assay. 617

618 619 Fig. 1

Fig. 2

Fig. 3 Fig. 4

Fig. 5 Fig. 6

Fig. 7