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Corynebacterium Glutamicum Mycoredoxin 3 Protects Against Multiple Oxidative Stresses

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Corynebacterium Glutamicum Mycoredoxin 3 Protects Against Multiple Oxidative Stresses Advance Publication J. Gen. Appl. Microbiol. doi 10.2323/jgam.2019.10.003 ©2020 Applied Microbiology, Molecular and Cellular Biosciences Research Foundation 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 * 5 6 College of Life Sciences, Qufu Normal University, Qufu, Shandong 273165, China; 7 8 Running title: Feature of C. glutamicum mycoredoxin 3 9 10 11 # These authors contributed equally to this work. 12 13 * Corresponding authors: 14 Ge Yang 15 16 E-mail [email protected] 17 Tel: 86-13953760056 18 19 20 21 22 23 24 25 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 46 47 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,
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