Crystal Structure of Wheat Glutaredoxin and Its Application in

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Functional Structure/Activity Relationships

Crystal structure of wheat glutaredoxin and its application in improving processing quality of flour Xiaomei Sun, Meirong Chen, Feng Jia, Yi Hou, and Song-Qing Hu J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b03590 • Publication Date (Web): 22 Oct 2018 Downloaded from http://pubs.acs.org on October 23, 2018

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TOC Graphic

Journal of Agricultural and Food Chemistry

wGrx

Oxidized form

Transit state

+ wTrx

Improving processing quality of flour ACS Paragon Plus Environment


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Crystal structure of wheat glutaredoxin and its application in improving processing quality

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of flour

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Xiaomei Suna,#, Meirong Chend,#, Feng Jiaa, Yi Houc, Song-Qing Hua,b,*

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# Xiaomei Sun and Meirong Chen contributed equally to this work

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a School of Food Science and Engineering, South China University of Technology,

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Guangzhou, Guangdong 510641, China

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b Overseas Expertise Introduction Center for Discipline Innovation of Food Nutrition and

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Human Health (111 Center), Guangzhou, China

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c State Key Laboratory of Pulp and Paper Engineering, South China University of

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Technology, Guangzhou, Guangdong 510640, China

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d Graduate School of Life Science, Hokkaido University, Sapporo 060-0810, Japan

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* Corresponding author at: School of Food Sciences and Engineering, South China

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University of Technology, Guangzhou 510641, China.

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E-mail address: [email protected] (S.-Q. Hu).

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ACS Paragon Plus Environment

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ABSTRACT

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Glutaredoxin (Grx) is a ubiquitous oxidoreductase that plays a vital role in

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maintaining cellular redox homeostasis. Compared with Grx from other organisms, plant

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Grx is unique that has many isoforms and thus suggests probably diverse functions and

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mechanisms. Therefore, structure-function characterization of plant Grx is necessary to

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have in-depth knowledge and explore its application in industry. In this study, wheat Grx

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(wGrx) was overexpressed and purified, and the crystal structure of wGrx was determined

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at 2.94 Å resolution. Interestingly, the structure for the first time captured both the oxidized

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form and the transient state of reduced-oxidized wGrx in a crystal. The mutagenesis of

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wGrx suggestes it adopts a mono-thiol catalytic mechanism. wGrx has ability to reduce

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wheat thioredoxin (wTrx), and this is the first example of the reduction of thioredoxin

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subgroup h class II by Grx. Flour farinograph and dynamic rheological analysis showed

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that wGrx together with wTrx has a positive effect on dough formation, which is probably

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attributed to the increased SDS-insoluble gluten macropolymer (GMP) through increasing

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intermolecular disulfide bond induced by wGrx-wTrx system. The results indicate a great

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potential of wGrx-wTrx as a novel synergetic enzymatic additive and may be employed to

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fine-tune processing performance of food related to redox reaction.

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Keyword: wheat glutaredoxin, wheat thioredoxin, crystal structure, flour processing

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quality



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INTRODUCTION

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Glutaredoxin (Grx), a member of oxidoreductase, is essentially involved in regulating

40

cellular redox environment by reducing the disulfide bond or protein-glutathine adducts,

41

and the oxidized Grx will be regenerated into reduced form by glutathione produced from

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NADPH by glutathione reductase. Increasing evidences demonstrated great importance of

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Grx with multifaceted roles in cellular processes, such as scavenging reactive oxygen

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species, Fe-S cluster assembly 1, transcription regulation 2, and pathogen responses 3.

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Grx belongs to the thioredoxin superfamily, together with other two members, 4

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thioredoxin (Trx) and protein disulfide-isomerase (PDI)

sharing a common Trx fold

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structure, which is made up of four stranded β-sheets flanked by three α-helices. There are

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two different catalytic mechanisms for Grx, monothiol or dithiol mechanism, depending

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on the number of cysteine of Grx involved in the reaction 5, 6.

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While most studies of Grx were focused on Escherichia coli, yeast and mammalian

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Grxs, little biochemical and structural information is available for plant Grx. Higher plants

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process plenty of Grx isoforms compared with other organisms. Generally, according to

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the sequence of the active site motif, Grx can be divided into three classes 7: class I with

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the active site motif CXXC/S, consisting of five subgroups, GrxC1, GrxC2, GrxC3, GrxC4

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and GrxC5/S12, each containing YCGYC, [Y/S]CP[Y/F]C, YCPYC, CSYC, CSYS active

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motif, respectively; class II with a conserved motif CGFS; Class III possessing CCXX, the


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motif exclusively found in plants. The diversity of plant Grx suggests that they may have

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different biochemical and structural features. Therefore, crystal structure analysis of

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different plant Grx is necessary for better understanding on the working mechanism at atom

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level, and therefore could be instructive for its modification and specific application as an

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additive oxidoreductase.

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Wheat is one of the three most popularly grown crops, and flour is extensively used

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to make various products such as bread, cake, and noodles. The quality of flour and its use

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for different kinds of products is affected by the disulfide bond formation in the gluten

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network. Wang et al 8 reported that the addition of 1Dx5, a superior high molecular weight

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glutenin subunit (HMW-GS), could improve the quality of dough, which is attributed to

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the formation of the massive protein networks through the disulfide bonds. Since the

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disulfide bonds mostly contribute to the quality of wheat flour, it is rational that

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oxidoreductases may produce an effect on flour processing by altering the texture of gluten

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network. Indeed, in recent years increasing evidences showed that redox enzymes and

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protein disulfide isomerase could affect the processing quality of flour. Kobrehel et al

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found that thiordedoxin system can improve the dough quality by reducing the

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intramolecular disulfide bonds. Liu et al

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oxidoreductin 1 (wEro1) could enhance mixing characteristics and viscoelastic properties

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of dough. However, not all oxidoreductases give positive effect on wheat flour, as wheat

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protein disulfide isomerase (wPDI) would weaken the processing quality of flour 11. These

10

9

showed that wheat endoplasmic reticulum



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results suggest not uniform, but probably a structure-function based, specific effect of the

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additive enzymes in flour quality. Strikingly, the modification of wPDI that abolishes its

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oxidoreductase activity, generated two positive modifiers (mPDI and aPDI) for

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strengthening the dough, indicating the feasibility to reverse the deleterious effect of

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oxidoreductase by protein engineering 11.

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Given the variety of the effects of oxidoreductases on processing quality of wheat

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flour, therefore, it would be meaningful to explore the potential of wheat Grx (wGrx) as an

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enzymatic additive in improving processing quality of flour, and to extend its application

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in food industry by structure-based protein engineering. Furthermore, the relation between

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wGrx and wTrx and their potential synergistic effect is another point that is worth exploring.

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Thioredoxin subgroup h (Trx h) (although a reducing agent), which is usually reduced by

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NADPH via the flavoenzyme NADP thioredoxin reductase (NTR, EC 1.8.1.9), seems to

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have a positive effect on the flour processing quality. The addition of Trx h, NADPH and

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NTR to weak flour results in stronger dough, increased loaf volume and improved crumb

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structure 9. Interestingly, Gelhaye et al reported 12 that Populus trichocarpa Trx h class III

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(Trx h III) is not reduced by NTR but glutathione (GSH)/Grx systems. Therefore, it would

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be meaningful to test whether the addition of wTrx together with wGrx could be beneficial

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to dough quality, so as to develop an efficient synergistic enzymatic additive system for

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food industry.


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In this study, wheat Grx from Triticum aestivum (wGrx) was overexpressed in E.coli

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and obtained with a high purity. The crystal structure of wGrx was determined at 2.94 Å

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resolution. There are five molecules of wGrx in an asymmetric unit with only one having

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C23-C26 disulfide bond, presenting both oxidized state and transient state of wGrx. Wheat

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thioredoxin (wTrx, Trx h II) was overexpressed to study the characteristics and application

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of wGrx. An enzymatic reaction system constructed with wGrx and wTrx was applied to

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improve the flour processing qualities, which were investigated by flour farinograph,

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dynamic rheological analysis, scanning electron microscopy (SEM), gluten composition

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analysis, and free sulfhydryl determination. All of the results consistently suggested that it

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is likely valuable and feasible to develop wGrx-wTrx system as an enzymatic additive for

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wheat flour.



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MATERIAL AND METHODS

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Materials. The restriction enzymes and T4 ligase were purchased from the

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ThermoFisher Scientific (USA). The HED and GR were purchased from Sigma-Aldrich

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(USA). The low-gluten flour was bought from the market.

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Expression and purification of wGrx. The total RNA was extracted from wheat

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‘Yannong 19’ according to the kit protocol. After reverse transcription PCR, the DNA

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sequence encoding wGrx was amplified by Taq DNA polymerase (EnzyValley, China)

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using forward primer: TATGGATCCTATGGCGCTCGCCAAG and the reverse primer:

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GGGCTCGAGAGGAGTGACGGTGGTCTTC, which was ligated into the pET-30b

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vector using the BamH I and Xho I as restriction enzymes sites. The obtained clone was

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sequenced. The verified plasmid wGrx-pET-30b was transformed into E.coli BL21(DE3)

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cells. The cells were grown in LB medium containing Kanamycin (50 mg/ mL) at 37 º C

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until OD600 reached 0.6. Then protein expression was induced by adding 0.05 mM

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Isopropyl β-D-1-thiogalactopyranoside (IPTG) and cells were grown for another 14 hours

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at 16 ℃.

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The cultured cells were centrifuged at 5000 g for 15 min at 4 º C. The cell pellets were

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suspended and then disrupted by sonication. The cell lysate was centrifuged at 11000 g for

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40 min and the supernatant was loaded onto a HisTrap HP column (GE healthcare,

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American) equilibrated with buffer containing 50 mM Tris-HCl (pH 8.0), 300 mM NaCl,

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and 10 mM imidazole. The bound wGrx was eluted using a linear increasing of imidazole


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to 500 mM. The collected fractions containing wGrx were further purified by size-

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exclusion chomatography (Hiload 26/60 Superdex 75pg; GE healthcare, American)

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equilibrated with 50 mM Tris-HCl (pH 8.0), 300 mM NaCl.

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The protein concentration was measured by the Bradford protein assay 13 with BSA

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used as the standard. The size of purified wGrx (5 mg/mL) was analyzed by size-exclusion

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chomatography (Superdex 200 Increase 10/300GL; GE healthcare, American) using BSA

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(MW 67 kDa), wEro1 (MW 48.57 kDa), 𝛽-lactoglobulin dimer (MW 36 kDa), and

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myoglobin (MW 17 kDa) as standards.

137 138

Biochemical characterization of the recombinant wGrx. The activities of wild type

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wGrx and its mutant were measured using 2-hydroxyethyl disulfide (HED) as substrate

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according to Holgmen’s method 14 with some modifications. Briefly, the reaction system

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consists of 50 mM Tris-HCl (pH 7.4), 12.7 nM GR, 1 mM HED, 0.125 mM NADPH, 0.5

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mM GSH and 2.62 µM wGrx wild type or mutant protein in a total volume of 200 μL. The

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reaction was monitored by a decrease in absorbance at 340 nm. In control group, wGrx was

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replaced by an equivalent volume of ultrapure water.

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The effects of temperature and pH on activity of wGrx were investigated. The optimal

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temperature was assessed in the range from 20 ℃ to 70 ℃ at pH 8.0. The optimal pH was

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evaluated in different buffer, CH3COONa-CH3COOH (pH 4.0-6.0), Tris-HCl (pH 6.0-9.0),

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and Glycine-NaOH (pH 9.0-11.0) at 40 ℃, respectively.



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Crystallization, data collection and structure determination of wGrx. The initial

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crystallization screening was performed by mixing 16 mg/mL wGrx containing 10 mM

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GSH with equivalent volume of reservoir and incubated at 20 º C. A total of 676 conditions

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from commercially available kits: Crystal Screen, Crystal Screen2 (Hampton Research,

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American), JCSGs, and PEGs (Qiagen, Germany) were used to set up crystallization drops.

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The crystals were obtained in condition containing 1.65 M ammonium sulfate, 0.08 M

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Sodium acetate, pH 4.6, 20% glycerol, and 10 mM GSH. Diffraction data were collected

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on the BL19U1 beamline at the Shanghai Synchrotron Radiation Facility (SSRF, China).

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Total 720 images were collected with each image at an oscillation angle of 0.5°. Diffraction

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data were indexed, integrated and scaled using HKL2000 15. The structure was solved by

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the molecular replacement using Populus trichocarpa GrxC1 (PDB entry: 2E7P) as the

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searching model. The initial model was built using Coot, and the model was refined by

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manual and automatic rebuilding using Phenix. After several rounds of refinement, the

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final model was obtained, giving a Rwork/Rfree = 25.79%/28.79%. Diffraction data and

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refinement statistics are summarized in Table 1.

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Preparation of wGrx mutant C26A. The wgrx gene with mutation that encoding

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wGrx_C26A mutant was prepared by overlap extension polymerase chain reaction (OE-

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PCR). The universal primers of T7 promoter and terminator, together with the primers

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designed for wgrx mutation were used in two separate reactions: T7 promoter primer and


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reverse primer of wgrx mutation were used to amplify wgrx sequence before the mutation

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site, while T7 terminator primer and forward primer of wgrx mutation were applied to

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obtain DNA region after mutation site. The two fragments amplified from reactions were

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identified and purified by agarose gel, followed by another PCR reaction using the two

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fragments as templates, T7 primers as primers, to obtain the full length DNA sequence of

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wgrx mutant. The product was confirmed by agarose gel as a band in accordance with the

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size of wgrx mutant was observed. The DNA fragment was digested by Xho I and Xba I

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and inserted into pET-30b, which was then transformed into E.coli strain DH5α. The

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colony was selected on the agar plate containing 50 mg/mL kanamycin, and the positive

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clone was validated by colony PCR and DNA sequencing (Figure S1). The pET-30b-

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wgrx(C26A) was transformed into BL21(DE3), and expression and purification of

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wGrx_C26A followed the same protocol as wild type wGrx.

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Reduction of wTrx by wGrx. The reduction of wTrx by wGrx was investigated to

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characterize the enzymatic potential of recombinant wGrx for exploring a highly effective

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redox system applied to food industry. wTrx used in this experiment and the following

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application was expressed and purified from E.coli as recombinant protein: the wtrx gene

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encoding wTrx was amplified by PCR and then ligated to pET-30b. Validated pET-30b-

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wtrx was transformed into BL21(DE3) for expression, and the recombinant wTrx was

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sequentially purified by affinity and size exclusion chromatography. The wTrx reduction



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assay was performed in a system containing 50 mM PBS (pH 7.0), 2 mM EDTA, 40 µM

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insulin, 0.2 µM glutathione reductase (GR), 0.325 mM NADPH, 1.3 mM GSH, 16 µM

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wTrx, and 5.2 µM wGrx. The reaction was incubated at 30 C for 30 min and then the

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activity was measured by the absorption at 650 nm. No addition of wTrx or/and wGrx were

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used as control groups.

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Flour farinograph analysis. The effect of wGrx-wTrx system on rheological

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properties of dough was analyzed to probe the effectiveness of the redox system as

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enzymatic additives in food. The dough rheological properties were characterized using

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DoughLAB (Sweden, Perten). Four groups of experiments were conducted, 10 μg wGrx,

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10 μg wTrx, 0.12 μg GR, 0.02 μmol GSH, and 0.005 μmol GSH were dissolved in water

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beforehand for wGrx+wTrx group; the above-mentioned components were added without

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wTrx for wGrx group and without wTrx and wGrx for Control group, and no above-

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mentioned components were added for Blank group. Prior to adding water, 4 g flour was

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stirred for 1 min by the instrument. The flour was mixed with water at a constant speed of

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60 rpm for 15 min. All procedures were carried out at 30 C. The rheological parameters

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of dough, including peak resistance, development time, stability time, and soften in

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resistance, were automatically recorded by the DoughLab software. The experiment was

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performed in triplicate, and the data were analyzed by SPSS software.


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As the Control group have no obvious effects on the Flour farinograph, the

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wGrx+wTrx group and the Blank group were used in the following experiments for

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investigating the processing quality of flour.

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Dynamic rheological determination. The dough used for dynamic rheological

213

determination was prepared using the farinograph and was let stand for 30 min before the

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experiment. Dynamic rheological determination was performed according to the method

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of Wang et al

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Thermo Haake, Germany) with a parallel plate geometry (40 mm diameter) and a smart

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swap Peltier Plate that maintains the temperature at 25 °C during the measurement. The

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dough was placed between the parallel plates with the gap adjusted to 1 mm and the excess

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dough was removed. The dough rested for 5 min to allow relaxation prior to the

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measurement. The linear viscoelastic zone was determined by frequency sweep test first.

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The frequency sweeps test was carried out in a frequency range of 0.1 to 10 Hz. The storage

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modulus (G′ ) and loss modulus (G˝), complex modulus (G*,

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as a function of frequency.

8

using a system containing a controlled stress rheometer (RheoStress 1,

G'2 + G˝2) were determined

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Scanning electron microscopy (SEM). The microstructure of dough was analyzed

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by scanning electron microscopy (SEM) (Evo 18, Carl Zeiss, Germany) as described

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previously 8. The freeze-dried dough was used to perform the SEM analysis. The dough



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sample was cut into the sizes of 1 cm × 1 cm × 0.5 cm by knife and then the sample was

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placed on the sample holder covered with a double scotch tape, and then coated with gold,

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which was transferred to the microscope and the images were taken at a magnification of

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1000× with the accelerating voltage of 10.0 kV and vacuum value of 9 ×10 -5 MPa.

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Analysis of free sulfhydryl. Free sulfhydryls were measured according to the method

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of Pescador-Piedra et al 16. Briefly, 15 mg gluten with or without wGrx-wTrx treatment

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was suspended in 0.5 mL buffer A (86 mM Tris, 90 mM glycine, 4 mM EDTA, pH 8.0,

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and 8 M urea) and then vortexed for 10 min followed by centrifugation for 5 min at 15000

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g. A 0.1 mL of supernatant was mixed with 0.5 mL of 4 mg/L 5, 5'-Dithiobis-(2-

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Nitrobenzoic Acid) solution and the reaction mix was incubated at 30 ℃ for 30 min. After

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that, the absorption at 412 nm was recorded. GSH was used to make the standard calibration

240

curve. The free thiol concentration was calculated by the calibration curve.

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Extraction and concentration determination of gluten compositions. The SDS-

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soluble proteins and SDS-insoluble proteins were extracted from 50 mg freeze-dried dough

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according to previous studies with some modifications

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firstly extracted by 2% NaCl. Then the residual pellet of dough was suspended in 1.5%

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SDS solution and the supernatant of SDS-soluble proteins was removed. SDS-insoluble

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proteins were extracted from residual pellets by resuspending in another aliquot of 1.5%

17, 18.


Salt-soluble proteins were

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(w/v) SDS solution with the assist of sonication at 30W for 15 s using a Branston sonic

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disrupter (Scientz, China). Each step was carried out with constant stirring (200 rpm, 15

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min) and followed by centrifugation at 8000 g for 15 min. Each protein fraction was

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analyzed by both reducing and non-reducing SDS-PAGE. The protein concentration of

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each extracted fraction was determined using BCA protein concentration assay kit

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(GK5013, Jerore, China). Bovine serum albumin (BSA) was used to make the standard

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curve.

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RESULTS AND DISCUSSION

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Primary sequence analysis of wGrx. The ORF of grx gene from wheat 'yannong 19'

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is 342 bp long, encoding wGrx with 113 amino acids. The sequence alignment showed that

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wGrx

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Arabidopsis.thalliana Grx, and 39% with chlorella sorokiniana Grx. wGrx has a CPFC

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motif at the active site, which to the C2 subgroup of class I 7. Primary sequence alignment

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showed that the catalytic important lysine, which interacts with the glutathione moiety of

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glutathionylated disulfide substrates 19, is also conserved in Triticum aestivum. The TVP

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motif and CDD motif are rather conserved motifs that are critical for GSH binding 20(Figure

266

S2).

shares

57%

identity

with

Populus

trichocarpa

Grx,

40.4

%

with

267 268

Purification and characteristics of wGrx. The DNA sequence encoding wGrx was

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cloned into pET-30b vector and transformed into E.coli BL21(DE3) strain for

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overexpression. After optimizing the expression condition, the highest yield of wGrx was

271

obtained when expression was induced with 0.05 mM IPTG at 16 ℃. The recombinant

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wGrx was expressed as His-tagged protein. After Ni affinity and size exclusion

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chromatography two-step purification, wGrx with high purity was obtained (Figure S3).

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Under the same size-exclusion chromatography condition, the elution volume of wGrx is

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almost the same as 𝛽-lactoglobulin dimer (MW 36 kDa) (Figure S4). As the molecule

276

weight of wGrx monomer is 18.3 kDa, therefore, the wGrx may exist as dimer in solution.


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To check if recombinant wGrx was active, activity assay was carried out using 2-

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hydroxyethyl disulfide (HED) as substrate. Compared with buffer control, addition of

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wGrx caused a decrease of 0.0475 min-1 in absorption at 340 nm, suggesting that

280

recombinant wGrx is enzymatic active. Enzymatic characteristics of wGrx, including

281

optimal temperature and pH, were determined, as the highest activity was observed at 40 ℃,

282

pH 8.0. (Figure 1). These results were similar with Grxs from Chlorella sorokiniana

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potato 22 and Pseudoalteromonas 23.

21,

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It was previously reported that Populus trichocarpa thioredoxin (Trx h4) could be the

285

substrate of E. coli Grxs 12. To test whether it is the case for wheat Grx and Trx, and to

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develop a synergetic system using food source Grx and Trx as enzymatic additives in food

287

processing, the reduction assay of wTrx by wGrx was initially carried out. The reduction

288

level of wTrx was indirectly measured using the absorption of reduced insulin as indicators,

289

as only wTrx in reduced state could reduce insulin. Compared with control groups that

290

without addition of wGrx or/and wTrx, the absorption at 650 nm increased over 4-fold

291

when both wGrx and wTrx were present (Figure 1c), suggesting that wGrx could efficiently

292

reduce wTrx to recycle active wTrx for insulin reduction. wTrx belongs to thioredoxin

293

group h class II because there is a conserved tryptophan residue but no cysteine residue in

294

the N-terminal region (Figure S5). To our knowledge, Grx was discovered for the first time

295

to reduce Trx h II.

296



297

Structure analysis of wGrx. To have a better understanding on the working

298

mechanism of wGrx, crystallization of wGrx was performed. The crystal diffracted X-ray

299

at 2.94 Å resolution, belonging to orthorhombic space group I222. There are five molecules

300

of wGrx in one asymmetric unit (Figure 2a), with a VM of 3.85 Å3Da-1 and a solvent fraction

301

of 68.07%. Generally, VM and solvent fraction ranged 1.6-3.2 Å3Da-1 and 23-62% for

302

normal protein crystal, respectively. The diffraction quality of wGrx crystal was attempted

303

to improve, and 2.94 Å resolution was the highest that can be achieved probably due to the

304

unusual high solvent fraction.

305

Five monomers are in a line in the asymmetric unit, with a rotation angle of about 72o

306

between two adjacent molecules. Most parts of wGrx are well modeled, except that the

307

final several residues of the C-terminus (aa:159-164) are invisible in the structure.

308

Superposition of the five wGrx molecules (assigned as A, B, C, D, and E, respectively)

309

gives an average root-mean-square deviation (RMSD) of 0.6 Å, indicating that the structure

310

of wGrx is rather rigid. The crystal structure of wGrx comprises five α helices and three β

311

strands in the order of α1, β1, α2, β2, α3, β3, α4 and α5, and reveals a compact globular. It

312

has the typical thioredoxin fold, consisting of a core three-stranded 𝛽-sheet (𝛽1 and 𝛽2

313

strands are parallel, and 𝛽1 and 𝛽3 are antiparallel) flanked on one side by two (α1 and α3)

314

and the other side by three α-helices (α2, α4 and α5) to form a stable hydrophobic core

315

(Figure 2b).


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Superposition of wGrx and other published Grx structures from E.coli (PDB entry:

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1FOV), Saccharomyces cerevisiae (PDB entry: 3D4M), Populus trichocarpa (PDB entry:

318

2LKU), and Homo sapiens (PDB entry: 2HT9)(Figure 2c), showed that Grxs share a very

319

conserved fold, with RMSD of 2.22 Å, 1.29 Å, 1.78 Å and 1.24 Å, respectively.

320

The two cysteines C23 and C26 in CPFC motif at the active site of wGrx, have close

321

access to each other in the structure, with the side chain pointing to each other. The catalytic

322

C23 locates in a loop connecting the 𝛽1 strand and α2 helix, and C26 in the N-terminal

323

region of α2.

324

For the molecular A, there is continuous electron density between the sulfur atoms of

325

the C23 and C26 (Figure 2d, left). The distance between the two S atoms is 2.0 Å, which

326

is close to the length of disulfide bond 20. Thus, the molecule A of wGrx is oxidized form.

327

Interestingly, for wGrx molecular B, C, D, and E, the protruding shape of electron density

328

of C23 and C26 indicates the absence of disulfide bond (Figure 2d, right). Furthermore, the

329

distance between the S atoms of C23 and C26 is about 3.3 Å, which is too long to form a

330

disulfide bond, but is enough for a hydrogen bond. The distance between the two cysteines

331

of human Grx in the fully reduced form is 4.0 Å 24 and in Ectromelia virus Grx is 5.69 Å

332

25.

333

3.5 Å

334

four molecules of wGrx, presenting a transient state of reduced and oxidized wGrx.

Thiol-thiolate hydrogen bonds have been observed with a sulfur-sulfur distance of 3.025, 24.

Probably, a thiol-thiolate hydrogen bond forms between C23 and C26 in the



335

In principle, it is believed that the classification of mono-thiol and dual-thiol catalytic

336

mechanism of Grx depends on whether the second cysteine in the CXXC motif is directly

337

involved in the reaction 6 . It was reported that dual-thiol catalytic mechanism is utilized in

338

Populus trichocarpa and chlorella sorokiniana Grx that having CGYC motif in the active

339

site, as the activity dramatically decreased when the second cysteine was mutated 21, 26. To

340

reveal the catalytic mechanism of wGrx, whether it belongs to dual-thiol or mono-thiol

341

catalytic mechanism, the second cysteine Cys26 in the CPFC was mutated to alanine,

342

which, however, didn’t reduce activity of wGrx dramatically (Figure 3). It indicates that

343

the second Cys in the CPFC motif is not essential for the activity of wGrx and mono-thiol

344

catalytic mechanism is probably utilized in wGrx, where only Cys23 is responsible for the

345

reaction.

346 347

Improvement of flour processing quality by addition of wGrx-wTrx. It was well

348

demonstrated that the processing of dough made by wheat flour is majorly influenced by

349

the types and amount of glutenin subunits, where redox of the key cysteines for glutenin

350

assembly contributes mostly. As an oxidoreductase, thioredoxin is known to improve

351

processing quality of flour 9. Since here we show that wGrx has ability to reduce wTrx, it

352

would be interesting to test whether wGrx-wTrx system could be applied to enhance the

353

processing quality of flour. The farinograph analysis was thus performed to characterize

354

the effect of wGrx-wTrx system on the rheological properties of dough. The results showed


Page 20 of 39

Page 21 of 39


355

that the stability time of dough, which is the strong index of dough strength, was markedly

356

extended from 1.5 to 2.53 min when both wGrx and wTrx were added (Table 2), suggesting

357

that wGrx-wTrx system could effectively improve the dough quality.

358

Although no obvious difference in peak resistance, development time, and softening

359

in resistance was observed, it may demonstrate the complexity of dough formation that is

360

affected by many factors. In particular, as development time is basically related to the

361

protein amount

362

effect of wGrx-wTrx could not be explained by the increase of total amount of protein

363

alone, instead, the enzymatic characteristics of wGrx and wTrx are more important for the

364

observed effects. These results provide a potential that the wGrx-wTrx integrated system

365

may be applied in food processing to greatly promote the reduction thus processing

366

efficiency.

27,

no change in development time is likely to support the idea that the

367

Compared with the Control group and the Blank group, the wGrx test group showed

368

no evident changes in the farinography parameters (Table 2). The results suggest that the

369

improvement ability of wGrx on the flour processing quality should be exhibited through

370

reducing wTrx. Trx h was reported to improve the bread-making quality via reducing

371

intramolecular bonds of the storage proteins of wheat with the coexistence of NADPH and

372

NTR 28. The reducing system containing wGrx in this study should play the similar role to

373

that of NADPH and NTR during the improvement of wTrx on the flour processing quality.



Page 22 of 39

374

The effect of the wGrx-wTrx on the dynamic rheological properties of dough was then

375

investigated, and it showed that wGrx-wTrx has a positive effect on the viscoelastic

376

properties of dough. With addition of wGrx-wTrx, both the storage modulus G′ (Figure 4a)

377

and the loss modulus G˝ (Figure 4b) of dough increased compared with the Blank group,

378

indicating that the addition led to higher elasticity and viscosity of dough. A gel-like

379

viscoelastic behavior was observed with the addition of wGrx-wTrx, as the storage

380

modulus G′ (elasticity) was higher than the loss modulus G˝ (viscosity)

381

complex modulus G* increased accordingly (Figure 4c), suggesting that wGrx-wTrx could

382

enhance dough against deformation 30. From the above, it is convinced that wGrx-wTrx

383

has a positive effect on rheological properties of dough, which is expected to improve

384

dough quality.

29.

Besides,

385 386

wGrx-wTrx

could

enhance

gluten

network

structure

by

promoting

387

intermolecular disulfide bond formation. The microstructure of dough was analyzed to

388

comprehend the improvement of dough quality at micro level. As shown in figure 5, the

389

dough in Blank group had a weak network structure with the exposed starch granules in

390

the gluten matrix both on dough surface (Figure 6a) and inside (Figure 6c). With addition

391

of wGrx-wTrx, more apparent and substantial network structure was found on the gluten

392

matrix within dough surface (Figure 6b) and inside (Figure 6d), and the starch granules

393

were not as obvious as that of the Blank group, as the starch granules were masked by the


Page 23 of 39


31.

394

gluten network, indicating that a stronger and more resistant gluten network formed

395

Since the gluten network is positively correlated with the quality of wheat dough, the

396

results showed here demonstrate that wGrx-wTrx could enhance dough quality by

397

influencing the gluten network structures in dough.

398

To further understand how wGrx-wTrx affects the gluten network structure, the

399

content of glutein proteins after wGrx-wTrx treatment was measured. BCA assay showed

400

that with the addition of wGrx-wTrx, the SDS-insoluble proteins significantly increased

401

compared with Blank group, while no obvious decrease was observed for the amount of

402

SDS-soluble proteins (Figure 6a). Consistently, the intensity of bands around 116 kDa in

403

the non-reducing SDS-PAGE profiles, which represents high molecular weight (HMW)

404

polymers, was weakened in SDS-soluble gluten, while was strengthened in SDS-insoluble

405

gluten, indicating that wGrx-wTrx treatment could markedly increase SDS-insoluble

406

gluten macropolymer (GMP) (Figure 6b). It has been reported that the elastic behavior of

407

dough and the bread baking performance, specifically the volume of dough, are positively

408

correlated with the amount of GMP

409

analysis demonstrated that the wGrx-wTrx improves the dough quality by boosting the

410

GMP content. Interestingly, further analysis of free thiol group showed that compared with

411

Blank group, addition of wGrx-wTrx could remarkably reduce the amount of free thiol

412

group in dough by 25.7% (Figure 6a), which means more thiol groups are involved in

413

network formation via disulfide bond, consistent with the rise of GMP. Previous research

32

. Therefore, both BCA assay and SDS-PAGE



414

has pointed out that thioredoxin system can improve the dough quality by reducing the

415

intramolecular disulfide bonds 9, thus the results showed here is reasonable since wGrx-

416

wTrx is an efficient redox system and is likely to interfere intramolecular disulfide bond

417

and thus promote the intermolecular disulfide bond formation in dough. The combination

418

of these results leads to a conclusion: wGrx-wTrx treatment could raise the amount of GMP

419

through promoting extensive crosslinking of the proteins via intermolecular disulfide bond,

420

which greatly contributes to the stronger gluten network.

421

Based on above results, it is convinced that wGrx-wTrx system could improve the

422

dough quality, probably in a way that wGrx reduces wTrx which subsequently reduces the

423

intramolecular disulfide bond, making place for the formation of intermolecular disulfide

424

bond between glutenin proteins and promoting the extensive gluten network. Therefore,

425

wGrx-wTrx is a promising synergetic enzymatic additive to enhance flour products and

426

regulate the food processing that is associated with redox reactions.

427 428 429


Page 24 of 39

Page 25 of 39


430

ASSOCIATED CONTENT

431

Supporting Information

432

C26A mutant sequence, sequence alignment of Grx, wGrx purification, elution curves, and

433

sequence alignment of Trx

434

AUTHOR INFORMATION

435

Corresponding Author

436

*(S.-Q.H.) School of Food Sciences and Engineering, South China University of

437

Technology,

438

[email protected].

439

ORCID

440

Song-Qing Hu: 0000-0003-3262-8911

441

Funding

442

This work was supported by the National Science Foundation of China (31471691 and

443

31771906) and 111 Project (B17018).

444

Notes

445

The authors declare no competing financial interest.

446

ACKNOWLEDGEMENTS

447

We would like to thank Prof. Min Yao, Hokkaido University, for helping to determine the

448

structure of wGrx.

Guangzhou

510641,

China.

Phone/fax:

449


86-20-87113848.

E-mail:


450 451 452 453 454 455 456 457 458 459 460 461 462 463 464 465 466 467 468 469 470 471 472 473 474 475 476 477 478 479 480 481 482 483 484 485 486 487

REFERENCES (1) Bandyopadhyay, S.; Gama, F.; Molina-Navarro, M. M.; Gualberto, J. M.; Claxton, R.; Naik, S. G.; Huynh, B. H.; Herrero, E.; Jacquot, J. P.; Johnson, M. K.; Rouhier, N. Chloroplast monothiol glutaredoxins as scaffold proteins for the assembly and delivery of [2Fe-2S] clusters. EMBO J. 2008, 27, 1122–1133. (2) Ojeda, L.; Keller, G.; Muhlenhoff, U.; Rutherford, J. C.; Lill, R.; Winge, D. R. Role of glutaredoxin-3 and glutaredoxin-4 in the iron regulation of the Aft1 transcriptional activator in Saccharomyces cerevisiae. J. Biol. Chem. 2006, 281, 17661–17669. (3) Ndamukong, I.; Abdallat, A. A.; Thurow, C.; Fode, B.; Zander, M.; Weigel, R.; Gatz, C. SA-inducible Arabidopsis glutaredoxin interacts with TGA factors and suppresses JA-responsive PDF1.2 transcription. Plant J. 2007, 50, 128–139. (4) Berndt, C.; Lillig, C. H.; Holmgren, A. Thioredoxins and glutaredoxins as facilitators of protein folding. Biochim. Biophys. Acta. 2008, 1783, 641–650. (5) Rouhier, N.; Lemaire, S. D.; Jacquot, J. P. The role of glutathione in photosynthetic organisms: emerging functions for glutaredoxins and glutathionylation. Annu Rev Plant Biol. 2008, 59, 143–166. (6) Stroher, E.; Millar, A. H. The biological roles of glutaredoxins. Biochem J. 2012, 446, 333–348. (7) Couturier, J.; Jacquot, J. P.; Rouhier, N. Evolution and diversity of glutaredoxins in photosynthetic organisms. Cell. Mol. Life Sci. 2009, 66, 2539–2357. (8) Wang, J. J.; Liu, G.; Huang, Y.-B.; Zeng, Q.-H.; Song, G.-S.; Hou, Y.; Li, L.; Hu, S.-Q. Role of N-terminal domain of HMW 1Dx5 in the functional and structural properties of wheat dough. Food chem. 2016, 213, 682–690. (9) Buchanan, B. B. Thioredoxin: a photosynthetic regulatory protein finds application in food improvement. J. Sci. Food Agric. 2002, 82, 45–52. (10) Liu, G.; Wang, J.; Hou, Y.; Huang, Y.-B.; Zhang, Y.-P.; Li, C.; Li, L.; Hu, S.-Q. Recombinant Wheat Endoplasmic Reticulum Oxidoreductin 1 Improved Wheat Dough Properties and Bread Quality. J. Agric. Food. Chem. 2017, 65, 2162–2171. (11) Liu, G.; Wang, J.; Hou, Y.; Huang, Y.-B.; Li, C.-Z.; Li, L.; Hu, S.-Q. Improvements of modified wheat protein disulfide isomerases with chaperone activity only on the processing quality of flour. Food Bioprocess Technol. 2017, 10, 568–581. (12) Gelhaye, E.; Rouhier, N.; Jacquot, J.-P. Evidence for a subgroup of thioredoxinhthat requires GSH/Grx for its reduction. FEBS Lett. 2003, 555, 443–448. (13) Bradford, M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 1976, 72, 248–254. (14) Holmgren, A.; Aslund, F. Glutaredoxin. Methods in Enzymology, Academic Press: New York, 1995; Vol. 252, pp 283–292.


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(15) Minor, W.; Otwinowski, Z. HKL2000 (Denzo-SMN) Software Package. Processing of X-ray Diffraction Data Collected in Oscillation Mode. Macromolecular Crystallography. Methods in Enzymology, Academic Press: New York, 1997; Vol.276, pp 307–326. (16) Pescador-Piedra, J. C.; Farrera-Rebollo, R. R.; Calderon-Dominguez, G. Effect of Glucose Oxidase and Mixing Time on Soluble and Insoluble Wheat Flour Protein Fractions: Changes on SH Groups and H2O2 Consumption. Food Sci. Biotechnol. 2010, 19, 1485–1491. (17) Steffolani, M. E.; Ribotta, P. D.; Pérez, G. T.; León, A. E. Effect of glucose oxidase, transglutaminase, and pentosanase on wheat proteins: Relationship with dough properties and bread-making quality. J. Cereal. Sci. 2010, 51, 366–373. (18) Gerrard, J.; Fayle, S.; Brown, P.; Sutton, K.; Simmons, L.; Rasiah, I. Effects of microbial transglutaminase on the wheat proteins of bread and croissant dough. J. Food Sci. 2001, 66, 782–786. (19) Begas, P.; Liedgens, L.; Moseler, A.; Meyer, A. J.; Deponte, M. Glutaredoxin catalysis requires two distinct glutathione interaction sites. Nat. Commun. 2017, 8, 14835. (20) Yogavel, M.; Tripathi, T.; Gupta, A.; Banday, M. M.; Rahlfs, S.; Becker, K.; Belrhali, H.; Sharma, A. Atomic resolution crystal structure of glutaredoxin 1 from Plasmodium falciparum and comparison with other glutaredoxins. Acta Crystallogr. D Biol. Crystallogr. 2014, 70, 91–100. (21) Chuang, H. H.; Cheng, C. Y.; Chen, Y. T.; Shaw, J. F. Novel highly active recombinant glutaredoxin from Chlorella sorokiniana T-89. J. Agric. Food. Chem. 2014, 62, 927–933. (22) Chi, X. W.; Lin, C. T.; Jiang, Y. C.; Wen, L.; Lin, C. T. A dithiol glutaredoxin cDNA from sweet potato (Ipomoea batatas [L.] Lam): enzyme properties and kinetic studies. Plant Biol. 2012, 14, 659–65. (23) Wang, Q.; Hou, Y.; Qu, J.; Hong, Y.; Lin, Y.; Han, X. Cloning, expression, purification and characterization of thioredoxin from Antarctic sea-ice bacteria Pseudoalteromonas sp. AN178. BioMed. Res. Int. 2013, 40, 6587–6591. (24) Sun, C.; Berardi, M. J.; Bushweller, J. H. The NMR solution structure of human glutaredoxin in the fully reduced form. J Mol Biol 1998, 280, 687–701. (25) Bacik, J. P.; Hazes, B. Crystal structures of a poxvirus glutaredoxin in the oxidized and reduced states show redox-correlated structural changes. J. Mol. Biol. 2007, 365, 1545–1558. (26) Rouhier, N.; Gelhaye, E.; Jacquot, J. P. Exploring the active site of plant glutaredoxin by site-directed mutagenesis. FEBS Lett. 2002, 511, 145–149. (27) Ning-Bo li, X.-X. W., Lei Yu, Yi Qu, Hong Lei Dough rheology properties and its applications in the food processing industry. Food Sci. Technol. 2008, 2008, 35–38.



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(28) Joye, I. J.; Lagrain, B.; Delcour, J. A. Endogenous redox agents and enzymes that affect protein network formation during breadmaking – A review. J. Cereal. Sci. 2009, 50, 1–10. (29) Larrosa, V.; Lorenzo, G.; Zaritzky, N.; Califano, A. Optimization of rheological properties of gluten-free pasta dough using mixture design. J. Cereal. Sci. 2013, 57, 520– 526. (30) Gujral, H. S.; Rosell, C. M. Functionality of rice flour modified with a microbial transglutaminase. J. Cereal. Sci. 2004, 39, 22–230. (31) Zhang, C.; Zhang, S.; Lu, Z.; Bie, X.; Zhao, H.; Wang, X.; Lu, F. Effects of recombinant lipoxygenase on wheat flour, dough and bread properties. Food Res. Int. 2013, 54, 26–32. (32) Jekle, M.; Becker, T. Wheat dough microstructure: the relation between visual structure and mechanical behavior. Crit .Rev. Food Sci. Nutr. 2015, 55, 369–382.

539 540 541


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543


Table 1 Data collection and refinement statistics Diffraction data statistics Wavelength (Å)

0.9785

Space group

I 222

Cell dimension a, b, c (Å)

92.577, 173.636, 175.708

α, β, γ(°)

90, 90, 90

Resolution (Å)

50-2.94(2.99-2.94)

Rmerge (%)

9.4(69.5)

Mean I/σI

27.5(3.71)

Redundancy

13.0(12.4)

Completeness (%)

100(100)

No. of unique reflections

29640(1447)

Refinement statistics Resolution (Å)

31.86-2.96

Rwork /Rfree (%)

25.38/ 27.89

Rfree test set size (%)

6.86

Protein residues

527

Number of atoms

3961

Protein

3882

Water

79

r.m.s. deviation Bond lengths (Å)

0.025

Bond angles (°)

2.136

Average B-factor (Å2)

45.59

Ramachandran plot (%)

544

Most favored regions

76.21

Additionally allowed regions

14.51

PDB entry

5ZVL

Value in parentheses are for the highest resolution shell.



546

Page 30 of 39

Table 2 The effect of wGrx-wTrx system on the rheological properties of dough Peak Resistance

Development

Stability Time

Softening in

(FU)

Time (min)

(min)

Resistance (FU)

Blank

508 ± 7.64 a

0.97 ± 0.06 a

1.50 ± 0.17 a

128 ±15.27 a

Control

496 ± 5.77 a

1.03 ±0.06 a

1.67 ± 0.05 a

125 ± 13.22 a

wGrx

506 ±2.89 a

1.00 ± 0.10 a

1.53 ± 0.28 a

135 ± 8.66 a

wGrx+wTrx

506 ± 5.77 a

1.06 ± 0.06 a

2.53 ± 0.06 b

136± 12.58 a

Additives

547

Data are expressed as the means ± standard deviation, n=3. Different letters within the same

548

column represent significant differences (p < 0.05).

549


Page 31 of 39

550


Figure legend

551 552

Figure 1 Biochemical properties of wGrx. (a) optimal temperature of wGrx. (b) optimal pH

553

of wGrx. (c) Reduction of wTrx by wGrx using absorption by insulin as indicator.

554 555

Figure 2 Crystal structure of wGrx. (a) Five wGrx molecules in the asymmetric unit. (b)

556

overall structure of wGrx in cartoon representation. (c) Superposition of wGrx (green) with

557

Grxs from E.coli (PDB entry: 1FOV, salmon), S. cerevisiae (PDB entry: 3D4M, magentas),

558

Populus trichocarpa (PDB entry: 2LKU, cyan), Homo sapiens (PDB entry: 2HT9, yellow).

559

(d) wGrx molecules in asymmetric unit show different redox state. 2Fo-Fc electron density

560

map of C23-C26 pair was created at σ =1.5. left, C23 and C26 don't form disulfide bond;

561

right, C23-C26 form disulfide bond.

562 563

Figure 3 Activity of wGrx and its C26A mutant.

564 565

Figure 4 Effects of wGrx-wTrx on dynamic rheological properties, including (a) elastic

566

modulus Gˊ; (b) viscous modulus G"; (c) complex modulus G*

567



568

Figure 5 Effects of wGrx-wTrx on the microstructure of dough. SEM photos of dough

569

surface without (a) and with (b) wGrx-wTrx treatment and dough inside without (c) and

570

with (d) wGrx-wTrx treatment.

571

Figure 6 (a) Analysis of the concentration of gluten proteins and free thiol group in

572

dough. (b) SDS-PAGE analysis of wheat proteins of dough.

573


Page 32 of 39

Page 33 of 39


574 575

Figure 1

576 110

100

Relative activity(%)

90

80

70

60

50

40 10

20

30

40

50

60

70

80

Tepreature/C

a

b

577

578 579

c



581

Page 34 of 39

Figure 2

582

583 584

a

585

b

c

586

587 ACS Paragon Plus Environment

Page 35 of 39


588 589

d

Figure 3

590

591 592



593

Figure 4

594 595

596


Page 36 of 39

Page 37 of 39


597 598



600

Figure 5

601 602


Page 38 of 39

Page 39 of 39

603


Figure 6

604 605

a

606 607

b


Crystal Structure of Wheat Glutaredoxin and Its Application in

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