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Special Issue on Industrial Biotechnology-Made in Germany: The path from policies to sustainable energy, commodity and specialty products Edited by: Dr. Thomas Brück Professor of Industrial Biocatalysis, Dept. of Chemistry, Technische Universität München (TUM), Germany Research Article *Corresponding author Prof.Dr. Volker Sieber, Chair of Chemistry of Biogenic Resources, Technische Universität München, A Novel Natural NADH and Schulgasse 16, 94315 Straubing, Germany, Tel: 4909421187301; Email: Submitted: 16 April 2014 NADPH Dependent Accepted: 12 May 2014 Published: 14 May 2014 Reductase as Tool in ISSN: 2333-7117 Copyright Biotechnological Applications © 2014 Sieber et al. OPEN ACCESS Reiter, J.1,2, Pick, A.1, Wiemann, L.O.2, Schieder, D.1 and Sieber, V.1,2* Keywords • 1 Chair of Chemistry of Biogenic Resources, Technische Universität München, Germany • NADH 2 Fraunhofer IGB, Projektgruppe BioCat, Germany • Allochromatium vinosum

Abstract The glutathione (GSH) is an important reducing agent in physiology. Glutathione reductases (GR) of humans and higher organisms convert oxidized glutathione (GSSG) to two reduced GSH under consumption of the co-factor NADPH. GSH acts as an antioxidant eliminating reactive species in the cell. We found a novel GR being able to accept both NADPH and much cheaper NADH for GSSG reduction. For the first time we produced it in E. coli and purified

active GR from Allochromatium vinosum, determined its Km-values for NADH (0.026 mM) and NADPH (0.309 mM), as well as its temperature optimum (20 °C) and pH optimum (pH 8). Since numerous bio-diagnostic assays and enzymatic processes are dependent on GRs the possibility to use a cheaper co-substrate could help to overcome cost limitations in future.

ABBREVIATIONS Moreover, several novel and highly sensitive detection methods for analyzing in vivo levels of glutathione and glutathione GR: Glutathione Reductase; GSH: Reduced Glutathione; GSSG: Oxidized Glutathione important for pharmacology as they allow the monitoring of INTRODUCTION oxidativedisulfide havestress, been or to developed study cellular [10,11]. responses These towards methods drugs are and toxic compounds. Rahman setup a high throughput detection All organisms have developed certain mechanisms to cope method for the GSH level by reducing GSH with 5,5’-dithio-bis with cellular stress caused by , pathogens, (2-nitrobenzoic acid) (i.e. Ellman’s reagent) yielding highly unfavorable temperature and environmental conditions or heavy an electrochemical detection method for glutathione [11]. Both of reactive oxygen species or degradation of xenobiotics in living assayschromophoric are based 5-thio-2-nitrobenzoic on the reduction of GSSG acid [10]by a eukaryoticwhile Noh appliedGR that metal–contaminations to name only a few [1-5]. Detoxification cells, for example, often involves reduced glutathione (GSH). For only accepts NADPH. However, commercially available reduced instance the ascorbate-glutathione cycle is part of the glycin tripeptide) has a mid-point redox potential of -318 mV. β-Nicotinamide adenine dinucleotide is approximately ten times degradationIn addition, glutathione process [5-8]. plays The important GSH (γ-L-Glutamyl-L-cysteinyl- roles in the cells’ iron cheaper than reduced β-Nicotinamide adenine dinucleotide metabolism, DNA and protein synthesis as well as 2′-phosphate. Therefore, switching to NADH dependent assays activation. could significantly decrease costs. , EC 1.8.1.7.] have been described in various , Furthermore, reduced glutathione is the substrate for various ,Glutathione fungi, reeducates and [NAD(P)H: [12-17]. glutathione However, until now most GR’s have been described as being strictly NADPH dependent. Though GRs from human erythrocyte and from Spinach were found However,cellular metabolic the most critical pathways process that in finally living oxidize cells is glutathione,to maintain to also utilize NADH, but the maximal reaction rates achieved here thewhere balance two between GSH molecules reduced are and linked oxidized by glutathione a disulfide (GSSG) bond. and that is permanently adjusted by glutathione reductases (GR) though both are able to use NADH their main their main [5]. were only ca. 20 % of those obtained with NADPH [18-19]. So even

Glutathione reductases have found important applications activity still lies with NADPH [20]. in industrial as well as medical biotechnology. For example, Allochromatium vinosum glutathione reductase was recently used in an enzymatic as AboutChromatium 40 years vinosum ago a glutathione reductase from multistep cascade for the deoxygenation of vicinal diol (reclassified, formerly known derivatives, by a hydrogen borrowing mechanism (Figure 1) [9]. Chromatiaceae represent a) very had rudimentary been purified bacterial and group partially and This cascade has high potential for the defunctionalization of itcharacterized is assumed that and this shown group to bedeveloped highly specificits NADPH for dependency NADH [21]. polyhydroxy compounds. later than its NADH-dependency [22]. Till date, there has been

Cite this article: Reiter J, Pick A, Wiemann LO, Schieder D, Sieber V (2014) A Novel Natural NADH and NADPH Dependent Glutathione Reductase as Tool in Biotechnological Applications. JSM Biotechnol Bioeng 2(1): 1028. Sieber et al. (2014) Email: Central no report about other natural glutathione reductases exhibiting of elution fractions were tested by SDS-PAGE and Coomassie a preference for NADH. Since just recently the genome of staining and protein concentrations were measured by Bradford Allochromatium vinosum (Roti Nanoquant, Carl Roth, Germany) [23]. The enzyme was time were able to clone and express an E. coli codon-optimized avGR and to purify has and been characterize sequenced we the now corresponding for the first protein to make it available for potential applications in proteinstored inconcentration batch samples were ingently the thawed elution directly buffer before (20% ,starting diagnostics and biotechnology where reduced costs are 500 mM imidazole, pH 6.5) at -20°C, single fractions of 3700 µg/mL of advantage. an activity assay, diluted 1:20,000. A slight activity loss of 10 to 20 % MATERIALS AND METHODS occurredAssay conditions on refreezing. Else, the enzyme was stable at 20°C. Activity of the enzyme was measured photometrically at Chemicals, strains and gene synthesis All chemicals in this paper were purchased from Sigma- Aldrich (Germany). The Allochromatium vinosum DSM 180 NADPH340 nm (Carlin 96 Roth, well plates. Germany) The in assay a citrate-borate-phosphate was started by adding (CBP)7 mM universalGSSG (Sigma pH buffer Aldrich, (Carl Germany) Roth, Germany) and 0.5 tomM a solution NADH orof codon optimization for Escherichia coli avGR in the same buffer. Temperature and pH dependence were GmbHglutathione (Germany) reductase in a pUC18 YP_003443292 derivative. The was open synthesized reading frame with measured in a universal pH 4-12 CBP buffer system. of the avGR gene fragment was cut out by of this Life plasmid Technologies with For determining K and k for NADH and NADPH activity BsaI and PsiI and cloned with a C-terminal His-tag in a pET28b m cat derivative (pCBR-C-His with a I and AI restriction site. Bsa Bfu GSSG (7 mM) were performed. The concentration of NADH and Predigested with Bsa measurements at optimal conditions (pH 8, 20 °C) and and constant k for fragment. In the next step the 5´ end was cut with BfuAI, creating m cat I, 3´sticky ends were filled with klenow GSSG was determined in the presence of 1 mM NADH. Calculation a BsaI cloning site) (Novagen, Germany; NEB, U.S.). This plasmid NADPH was varied between 0.3 µM and 1 mM. K was sequenced and used to transform Escherichia coli (DE3) (Merck KGaA, Germany). The protein was expressed in of1 mM the were kinetic diluted constants before was measurement. performed with Sigma Plot 11.0. Escherichia coli BL21 Samples which contained NADH/NADPH concentrations above by Carl Roth (Germany). Inhibition tests BL21 (DE3). NADH and NADPH were purchased Expression and purification (CarlTests Roth, with Germany) flavin adenineor 7 mM dinucleotide GSSG (Sigma (FAD) Aldrich, or GSSGGermany) pre- induced at OD followingincubation the prior above to thestandard assay assaywere conductedprotocol with with CBP-buffer 0.5 mM FAD pH After expression in LB medium with 50 µg/mL kanamycin 600 = 0.8 by 1 mM IPTG at 37°C for 4 h the cells were 2SO4 and Na3PO4 following the above harvested (4500 g, 4°C, 30 min) and applied to a cell disruptor 8 standard at 20 °C. protocol Different under ion concentrationsoptimum conditions. on GR were tested with (Constant Systems Ltd., U.S.) in binding buffer 2+ (Tris/HCl-NTA column pH 5 mM and 10 mM NaCl, Na 7.2, 10% (v/v) glycerol, 20 mM imidazole). The cell extract was Structure modeling centrifuged (25,000 g, 4°C, 20 min), applied to a Ni A structure model for avGR was created using Phyre2 [24]. (4 mL, GE, U.S.) and then fractionated in elution buffer (Tris/ HCl pH 7.2, 10% (v/v) glycerol, 500 mM imidazole). The purity

Figure 1 Enzymatic multistep cascade for the enzymatic deoxygenation of vicinal diol derivatives by a hydrogen borrowing mechanism [9].

JSM Biotechnol Bioeng 2(1): 1028 (2014) 3/8 Sieber et al. (2014) Email: Central

40 avGR Activity A

35

30

25

20 U/mg

15

10

5

0 3 4 5 6 7 8 9 10 11 12 pH

Figure 2 Reaction of av in CBP-buffer pH 4-11. (n=3). B GR at 340 nm with 0.5 mM NADH and 7 mM glutathione

avGR Activity 40

35

30

25

20 U/mg

15

10

5

0 20 30 40 50 60 70 80 90 Figure 4 (A) avGR kinetic under variation of NADH concentrations, T [°C] as triplicates. Figure 3 Temperature-related av and (B) oxidized glutathione concentrations in CBP-buffer pH 8, 20 °C GR activity with 0.5 mM NADH and 7 mM glutathione in CBP-buffer pH8 at 340 nm, measured between 20 °C and 90 °C (n=3). Table 1: Activity of avGR towards different substrates compared to

K = 100% 5 mM 10 mM control experiment (%). (n=3). NaCl Na SO 2 4 40.4 ± 1.9 63.3 ± 16.8 Na PO 3 4 106.7 ± 13.3 111.1 ± 10.2 Abbreviations: GR glutathione reductase; av Allochromatium97.8 ± 10.2 vinosum 117.8 ± 7.7 NaCl Sodium chloride; Na2SO4 Sodium sulfate; Na3PO4 Sodium phosphate

Table 2: Activity of av glutathione in CBP-buffer pH 8 after pre-incubation of the enzyme with FAD or GSSG. (n=3). GR with 0.5 mM NADH, 0.5 mM oxidized avGR avGR +FAD avGR + GSSG

Abbreviations: avGR Allochromatium vinosum glutathione reductase; FAD % 100 194.2 ± 14.2 105.4 ± 8.1 Figure 5 avGR kinetic under variation of NADPH in CBP-buffer pH 8, Flavin adenine dinucleotide; GSSG oxidized glutathione; NADH Nicotine adenine dinucleotide; CBP Citrate borate phosphate buffer. 20 °C (n=3). JSM Biotechnol Bioeng 2(1): 1028 (2014) 4/8 Sieber et al. (2014) Email: Central

av integrate FAD and NADH into the structure [25]. Out of a set of above the published pH of 7 for the native enzyme (Figure 2). Subsequently, the derived model was used for 3DLigandSite to GR was determined to be 35.8 ± 0.3 U/mg at pH 8, thus being candidate was chosen and the remaining removed from the model. different FAD and NADH/NADPH molecules respectively one all use slightly alkaline conditions. The temperature optimum for av This is important as earlier described applications [9,10,11] glutathione reductase from E. coli (PDB: 1 GER and 1 GEU) as well as In a final step the model was compared to crystal structures of a variant thereof with improved acceptance of NADH. GR was investigated in the range from 20 °C to 90 °C at pH 8 andPrevious was determined studies showedto be 34.3 an ± inhibition 1.5 U/mg orat activation20 °C (Figure of native 3). RESULTS AND DISCUSSION avGR by salts like phosphate [21]. We tested the inhibition of We were able to annotate the gene function to a glutathione recombinant avGR activity at its optimal conditions by addition of reductase in Allochromatium vinosum DSM 180. We postulated different concentrations of NaCl, Na2SO4, and Na3PO4. The activity reductase of the sequenced genome of Allochromatium vinosum decreased activity of av the gene YP_003443292 coding for a 458aa predicted glutathione was measured photometrically at 340 nm. Whereas 5 mM NaCl DSM 180 3PO4. This result stands in GR by ca. 60 %, activity in phosphate ago in this organism [21]. The gene was codon optimized for E. contrast to those obtained previously for the native enzyme and increased 17.8 ± 7.7 % at 10 mM Na coli and cloned to be responsible with a His-tag. for theSoluble GR activity protein described was expressed 40 years by it is important when considering its potential applications in E. coli BL21 (DE3) commonly used phosphate buffers (Table 1). column (GE, U.S.). Protein content of the elution peak fractions in LB-media and purified via a metal-chelate Chung observed an inhibition of the Allochromatium vinosum reductase by pre-bound GSSG and proposed to alleviate this by showed clear single bands at 48.8 kDa (data not shown). pre-incubation of GR with FAD. We found almost no difference varied between 3700 µg/mL and 4700 µg/mL and the SDS-PAGE av We were able to express the soluble avGR enzyme with codon by GSSG pre-incubation, but an almost-twofold increase of optimization in E.coli and were able to purify the His-tagged protein in an active form as well. In all assays avGR was pre- 2) [21]. activity with FAD pre-incubation to about 194.2 ± 14.2 % (Table incubated for 5 min with FAD. The external addition of FAD is Km and kcat were determined photometrically following the decline of NADH at different concentrations. The concentrations of avGR and GSSG (7 mM) were kept constant and reactions supposed to replenish potentially lost flavin adenine dinucleotide for NADH and NADPH were from the enzymes active center during the purification process. m The pH dependence of avGR activity was tested in citrate-borate- ± ± The reaction was started with 0.5 mM NADH and 7 mM GSSG. phosphate buffer (CBP) ranging from pH 4-12. The optimum for were run at pH 8 and 20 °C. The K ‑1 respectively. (Figure 5) The kcat s and identified as 0.026 0.004 mM (Figure 4A) and 0.309 0.030 mM values were 33.23 ± 1.23

Figure 6 Alignment of avGR and ecGR with glycine 178 and glutamate 198 (red boxes) of the NADH binding sites of pyridine dinucleotide

(Basic Logarithmic Alignment Search Tool, BLAST). JSM Biotechnol Bioeng 2(1): 1028 (2014) 5/8 Sieber et al. (2014) Email: Central

-1 s respectively. Km and kcat for GSSG with constant well as the sequence was compared to characterized ± -1 glutathione reductases. The characteristic glycine residue (AS 40.02(Figure ± 4B).1.46 178) found for enzymes preferring NADH as well as a negatively NADH at 0.5 mM were 0.033 0.007 mM and 5.34 ± 1.47 s Intensive research of glutathione reductases has been in av conducted not only to elucidate its role in cell physiology but thecharged binding residue of NADPH at the by end decreasing of the β-strand the space (AS for 198) the can 2’phosphate be found also to understand the mechanism of cofactor acceptance. A and preventingGR. Glutamate stabilization. at position (Figure 198 is 7)the main factor influencing comparison of Homo sapiens GR and GR (pf pfGR and hsGR towards These structural features correspond to the measured kinetic

NADH and a lower Km value of pfGR for NADPH compared to hsGR parameters and the observed preference for NADH. Glutathione [26]. Moreover,GR) showed wild up typeto 5 %Escherichia activity of coli GR (ecGR) shows a reductase from Allochromatium vinosum is still superior against

the variant of ecGR in context of Km (three times lower) and also -1 -1 of the crystal structure for hsGR and the high homology towards for kcat m which is with 76.6 min three times higher. The stronglyec decreased activity of around 5 % with NADH. As a result NADH attachment site is within a larger FAD binding domain. ecGR /K µM GR first mutagenesis studies were carried out. Scrutton were human glutathione reductase with the following three steps, (1) successfulfamily glutathione in the redesign reductase of the shares coenzyme some specificity typical features of Huber and Brandt (1980)-1) (2 ) calculated hydride transfer glutathione to FAD reduction (153 s-1) and in found[27]. As in a member enzymes of interactingthe flavoprotein with disulphide pyrophosphate oxidoreductase moieties (3 -1) [31]. In a binding study for (Figure 6) [26]. NADH,NADPH Y197 binding played (>300 an s important role in the catalytic mechanism ) disulfide reduction by FAD (68 s it is also conserved in av hsGR transfer the by opening and blocking the binding pocket of NADH/NADPH and thatThe the differentiation unique βαβ-fold between responsible glycine for and the ADP in binding the third of are closer to NADH thanGR. K66 K66 nitrogen and E201 atoms, of implicating that positionthe cofactor is correlatedcarries a GxGxxG/A with the motif preference [28]. Scrutton towards suspected NADH or thehydride carboxyl by forming group seemsan ion topair be whereasmore important. oxygen atomsBoth sitesof E201 are NADPH [27]. But a more intense study by Carugo and Argos on basis of crystal structures of different enzymes did not reveal any and mercury reductases or liponamide dehydrogenases [31-36]. However,conserved only in the crystallization NADH/NADPH and binding detailed pockets binding of studies glutathione could to distinguish between the two pyridine nucleotide cofactors is significant correlation for this feature [29]. A clear characteristic clearly elucidate the exact mechanism of avGR as NADH and NADPH dependent glutathione reductase. negatively charged when a hydrogen bond to the 2’-hydroxyl groupthe residue of NADH at the is required C-terminal and end in of most the cases second hydrophobic β-strand. It for is CONCLUSION The availability of enzymes that are using the cheapest possible al. for ecGR a change in these characteristic residues allowed a cofactor is essential for cost effective biocatalysis. In making this thecomplete 2’-phosphate switch towardsof NADPH NADH [30]. byWithin lowering the study the K of fromScrutton 2 mM et m rare NADH-dependent GR available as recombinant enzyme in was increased by cat m E.coli we provided a new tool for GR dependent diagnostic assays a factor of 72 (24.4 min-1 -1) compared to the wild type with or for GSH dependent biotransformation reactions. avGR has a to 0.086 -1mM.-1 The. Based catalytic on this efficiency information k the/K homology model as µM 0.34 min µM

Figure 7 Structure model of avGR with FAD and NAD+. Residues leucine 197 and glutamate 198 are shown in the stick mode and the distances to the

2’ and 3’-hydroxy groups of the ribose ring are specified. JSM Biotechnol Bioeng 2(1): 1028 (2014) 6/8 Sieber et al. (2014) Email: Central

14. Backos DS, Franklin CC, Reigan P. The role of glutathione in brain E. coli. The enzyme can be produced in soluble form in good yield. superior catalytic efficiency than previously engineered GR from 15. tumor drug resistance. Biochem Pharmacol. 2012; 83: 1005-1012. wasStorage still stabilityavailable is showing high. At thattemperature the enzyme above can 20be °Cused the at enzyme higher Krauth-Siegel RL, Leroux AE. Low-molecular-mass in 16. parasites. Antioxid Redox Signal. 2012; 17: 583-607. temperature.has reduced activity. Recently However, we demonstrated even at 70 the °C significantapplication activity of this enzyme in a redox-neutral synthetic enzymatic pathway for cadmiumMendoza-Cózatl stress D,in ,Loza-Tavera protists H, and Hernández-Navarro plants. FEMS Microbiol A, Moreno- Rev. Sánchez R. and glutathione metabolism under the degradation of lignin [9]. In future it could even be feasible to couple this NADH-dependent GR with important toxic or 17. 2005; 29: 653-671. environmentally problematic industrial waste degradation López-Mirabal HR, Winther JR. Redox characteristics of the eukaryotic approaches. 18. cytosol. Biochim Biophys Acta. 2008; 1783: 629-640. Glutathione Reductase of Human Erythrocytes. J Biol Chem. 1963; ACKNOWLEDGEMENT 238:Scott 3928-3933. EM, Duncan IW, Ekstrand V. Purification and Properties of We would like to thank Kerstin Stadler and Benjamin Kick for 19. Vanoni MA, Wong KK, Ballou DP, Blanchard JS. Glutathione reductase: experimental support. We also thank the Bavarian government comparison of steady-state and rapid reaction primary kinetic isotope for funding this project within the BayernFit program. effects exhibited by the yeast, spinach, and Escherichia coli enzymes. REFERENCES Biochemistry.Perham RN, Scrutton 1990; 29: NS, 5790-5796. Berry A. New enzymes for old: redesigning 1. Foyer CH, Noctor G. Ascorbate and glutathione: the heart of the redox 20. Bioessays. 1991; 13: 515-525. the coenzyme and substrate specificities of glutathione reductase. 2. Noctorhub. G, Physiol. Mhamdi 2011; A, Chaouch 155: 2-18. S, Han Y, Neukermans J, Marquez- 21. Garcia B, et al. Glutathione in plants: an integrated overview. Plant Cell Chung YC, Hurlbert RE. Purification and properties of the glutathione 22. reductase of Chromatium vinosum. J Bacteriol. 1975; 123: 203-211. 3. Environ.Meyer AJ, 2012; Hell 35: R. Glutathione454-484. homeostasis and redox-regulation by Horocker BL. [Alternate pathways of carbohydrate metabolism and 23. theirBradford physiological MM. A rapid significance]. and sensitive Seikagaku. method 1965; for the37: 313-324. quantitation of 4. sulfhydryl groups. Photosynth Res. 2005; 86: 435-457. microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976; 72: 248-254. highlightsParisy V, Poinssot the importance B, Owsianowski of glutathione L, Buchala in A, disease Glazebrook resistance J, Mauch of F. Identification of PAD2 as a gamma-glutamylcysteine synthetase 24.

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Cite this article Reiter J, Pick A, Wiemann LO, Schieder D, Sieber V (2014) A Novel Natural NADH and NADPH Dependent Glutathione Reductase as Tool in Biotechnological Applications. JSM Biotechnol Bioeng 2(1): 1028.

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