Downloaded from on May 16, 2020 by guest · 2010. 6. 11. · 2 24 25 Abstract 26 The reactive...

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Transcriptional activation of YqhD aldehyde reductase by YqhC protein and its implication in 3

glyoxal metabolism of Escherichia coli K-12 4

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Changhan Lee,1 Insook Kim,

1 Junghoon Lee,

1 Kang-Lok Lee,

1 Bumchan Min,

2 and Chankyu Park

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1 Department of Life Sciences, Korea Advanced Institute of Science and Technology, Yuseong-gu, 10

Daejeon 305-701, Republic of Korea. 11

2Analytical Science Center, Samyang Central R & D Institute, 63-2, Hwaam-dong, Yuseong-gu, 12

Daejeon 305-717, Republic of Korea. 13

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* Corresponding author 18

Mailing address: Department of Biological Sciences, Korea Advanced Institute of Science and 19

Technology, Yuseong-gu, Daejeon 305-701, Republic of Korea. 20

Phone : 82-42-350-2629. Fax: 82-42-350-2610. 21

E-mail: [email protected] 22

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Copyright © 2010, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.J. Bacteriol. doi:10.1128/JB.01127-09 JB Accepts, published online ahead of print on 11 June 2010

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Abstract 25

The reactive α-oxoaldehydes such as glyoxal (GO) and methylglyoxal (MG) are generated in 26

vivo from sugars through oxdative stress. GO and MG are believed to be removed from cells by 27

glutathione-dependent glyoxalases and other aldehyde reductases. We isolated a number of glyoxal-28

resistant mutants from the E. coli strain MG1655 on LB plates containing 10 mM glyoxal. By 29

tagging the mutations with the transposon TnphoA-132 and by determining their co-transductional 30

linkages, we were able to identify a locus to which most of the GO resistance (GOR) mutations were 31

mapped. DNA sequencing of the locus revealed that it contains the yqhC gene, which is predicted to 32

encode an AraC-type transcriptional regulator of unknown function. The GO resistance mutations we 33

identified result in missense changes in yqhC, and were concentrated in the predicted regulatory 34

domain of the protein, thereby constitutively activating the product of the adjacent gene yqhD. The 35

transcriptional activation of yqhD by wild-type YqhC and its mutant forms was established by an 36

assay with a β-galactosidase reporter fusion as well as with real time quantitative RT-PCR. We 37

demonstrated that YqhC binds to the promoter region of yqhD, and that this binding is abolished by a 38

mutation in the potential target site, which is similar to the consensus sequence of its homolog SoxS. 39

YqhD facilitates the removal of glyoxal through its NADPH-dependent enzymatic reduction activity 40

by converting it to ethadiol via glycolaldehyde, as detected by NMR as well as by spectroscopic 41

measurements. Therefore, we propose that YqhC is a transcriptional activator of YqhD, which acts as 42

an aldehyde reductase with specificity for certain aldehydes, including glyoxal. 43


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Introduction 46

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Glyoxal (GO) is a reactive α-oxoaldehyde formed by the oxidative degradation of glucose, 48

by lipid peroxidation, and by autoxidation of ascorbic acid [1, 2]. Due to its two reactive carbonyl 49

groups, GO is capable of inducing cellular damage through multiple mechanisms. For example, GO 50

can modify proteins and nucleic acids, causing protein malfunction and mutagenesis, respectively [3, 51

4]. It has been reported that the advanced glycation (non-enzymatic glycosylation) end products 52

(AGEs) formed by glyoxal are associated with pathophysiological changes found in aging and 53

neurodegenerative disease [5-7]. Like GO, methylglyoxal (MG) and 3-deoxyglucosone also cause 54

protein glycation and other types of cellular damage [3, 4]. 55

Glyoxal can be converted to glycolaldehyde, presumably by an uncharacterized aldo-keto 56

reductase (AKR) or aldehyde reductase (Fig 1). Glycolaldehyde can be further oxidized or reduced to 57

glycolic acid or 1,2-ethandiol, respectively. Glycolaldehyde oxidation is mediated by aldehyde 58

dehydrogenase and NAD+

[8, 9], while its reduction is accomplished by 1,2-propanediol 59

oxidoreductase using NADH as a co-factor [10, 11]. Glycolic acid is also believed to be generated 60

directly from glyoxal by glutathione (GSH)-dependent glyoxalases [12]. Unlike glyoxal, 61

detoxification of MG is quite well characterized, and is known to involve the glyoxalase system and 62

AKRs [13-17]. The glyoxalase system consists of glyoxalases I and II (gloA and B, respectively), 63

which require GSH as a cofactor [18, 19], and which produce lactate from MG. The aldo-keto 64

reductases, including YafB, YqhE and YqhZ, are also reported to detoxify MG by generating 65

hydroxyacetone using NADPH [15]. 66

Although the YqhD protein was previously predicted to be a group III (iron-activated) 67

NADP-dependent alcohol dehydrogenase based on sequence analysis, YqhD was recently proposed 68

to function as an aldehyde reductase, detoxifying aldehyde compounds including propanal, butanal, 69


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and acrolein, which are likely derived from lipid peroxidation [20]. The dimeric structure of YqhD 70

has been determined to contain NADPH and zinc at its active site [21]. YqhD is structurally 71

analogous to other enzymes, such as FucO and AdhE, that catalyze L-lactaldehyde and acetaldehyde, 72

respectively [22, 23]. YqhD has been characterized as an enzyme involved in GO metabolism, which 73

is enzymatically similar to but structurally different from other members of the AKR family. 74

In this study, we isolated a number of GO-resistant mutants which overproduce the aldehyde 75

reductase YqhD. Based on analysis of GO-resistant mutations, we were able to characterize YqhC as 76

a transcriptional activator of the yqhD gene that binds directly to the putative promoter region of 77

yqhD. Interestingly, the GOR mutations we characterized are located in the regulatory region of 78

YqhC, and not in the DNA-binding region. 79

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Materials and Methods 81

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Bacterial strains and growth conditions All strains used were derivatives of E. coli K-12. MG1655 83

was used as the wild-type strain for gene disruption. Disruption of the yqhD and yqhE genes was 84

achieved using a previously described method for chromosomal gene inactivation [24]. Other AKR 85

mutants were obtained from the E. coli genome project (University of Wisconsin, Madison) and 86

transferred to MG1655 by P1 transduction [25]. The BL21(DE3) strain (Novagen) was used for 87

overproduction and purification of protein. 88

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Isolation and localization of GOR mutations GO-resistant mutants, including yqhC-P63S, were 90

isolated from the E. coli MG1655 strain on LB plates containing 10 mM glyoxal. For mapping, a 91

λTnphoA-132 hopping phage carrying a tet gene encoding tetracycline resistance was used for 92

insertional mutagenesis [26]. After infection of GO-resistant mutant (yqhC-P63S) cells with the 93

phage, more than 10,000 independent clones with transposon insertions were obtained, thereby 94


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generating a yqhC-P63S::TnphoA-132 library. By infecting the library of cells with P1 phage, we 95

generated a P1 pool containing different genomic fragments of TnphoA-132. These phages were used 96

to identify insertions near yqhC-P63S after transfection into wild-type MG1655 cells and selection 97

for GO- as well as tetracycline- resistant strains. 98

Transposon insertion sites were identified by inverse PCR. Chromosomal DNA was 99

prepared and completely digested with TaqI restriction enzyme, which cuts near the 5’ end of 100

TnphoA-132, generating a fragment containing the 5’ end of the transposon with a flanking sequence. 101

The digested chromosomal DNA was then ligated to form circular DNA. PCR amplification was 102

performed with a pair of outwardly directed primers matched to the 5’ end of the transposon: the 103

complement of bases 31 to 55 (TNPO, directed to the 5’ end) and the sequence of bases 67 to 91 104

(TNPI, directed to the 3’ end). The amplified DNA was purified after agarose gel electrophoresis and 105

sequenced using TNPO as a primer. The sequences flanking the transposon insertion were searched 106

for similarity against the E. coli genome database using the BLASTN program [27]. After 107

determining the locations of tags through inverse PCR and sequencing, the sites of GOR mutations 108

were mapped using co-transduction frequencies obtained with the insertion tags. Finally, genes 109

responsible for GOR mutations were identified by sequencing genes in the candidate region. 110

111

Sample preparation and metabolite analysis by NMR A Bruker AVANCE-400 NMR 112

spectrometer equipped with a temperature controller was used for NMR experiments. Samples were 113

placed in a 5 mm NMR tube and kept at 28°C during measurement. Quantitative 1

H-NMR 114

measurements were made using a 300 pulse with a long relaxation delay. The duration of acquisition 115

was approximately 5 min for each NMR spectrum. All measurements were made in 600 µl of 116

solution with 10% D2O as the locking substance. To detect GO and MG reducing activity, purified 117

YqhD protein was dialyzed for 15 hours against three successive changes of 50 mM potassium 118

phosphate buffer (pH 7.0). The enzymatic reaction for GO or MG contained purified YqhD (40 µg), 119


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GO (10 mM) or MG (3 mM), and coenzyme (2 mM NADPH) in buffer (50 mM potassium 120

phosphate, pH 7.0) with D2O. NMR data were collected at 0 and 6 hours after mixing. 121

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Purification of proteins The yqhD gene was inserted into the pET15b vector (Novagen) and 123

transformed into the BL21(DE3) pLysS strain. The transformed cells were grown at 37℃ to the 124

mid-exponential phase (OD 0.5 at 600 nm) in LB broth with ampicillin (100 µg ml-1

). Proteins were 125

induced by adding isopropyl-β-D-thiogalactopyranoside (0.25 mM). Cells were further grown for 4 h, 126

harvested, and resuspended in binding buffer (20 mM Tris-Cl, pH 7.9, 5 mM imidazole, and 500 mM 127

NaCl). After disruption by sonication, cell debris was removed by centrifugation at 15,000 x g for 15 128

min, and protein was purified following standard procedures for Ni-NTA columns (Novagen). 129

Concentration and purity were determined using the Bradford reagent and also by sodium 130

dodecylsulfate polyacrylamide gel electrophoresis. 131

132

Electrophoretic mobility shift assay (EMSA) DNA probes containing the YqhC binding site were 133

generated by PCR, and were end-labelled using [γ-32

P]-ATP (GE Healthcare) and polynucleotide 134

kinase (Takara). Unincorporated [γ-32

P]-ATP was removed using a nucleotide removal column 135

(Intron). For EMSA, reactions were carried out using 20 µl DNA probe (100 fmol), 1 µg poly dI-dC, 136

and 0-200 fmol purified protein. The probes were synthesized by PCR using 10 pmol of the 137

following primers: 5’-gcaattttgtagcatttctc-3’ and 5’-gtttttcattgtgatcgcc-3’. To generate the mutant 138

probes, the following site-specific mutant oligonucleotides were used: 5’-139

ctcgggatccagatgtagggagaaatgctacaaaattgcgc-3’ and 5’-ccctacatctggatcccgaggcaagacattggcagaaatg-3’ 140

for mutant 1, and 5’-ttttactaaatgatacgggagagccagggcgctggcgatc-3’ and 5’-tcccgtatcatttagtaaaa 141

tgtcttgcctatttctccag-3’ for mutant 2. After synthesis of the mutant templates, the mutant probes were 142

synthesized by PCR using the same primers used for preparation of the wild-type probe. The binding 143

buffer contained 50 mM KCl, 20 mM Tris-Cl (pH 7.9), 1 mM dithiothreitol, 10% glycerol, and 125 144


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µg bovine serum albumin/ml. The mixture was kept on ice for 45 min, loaded onto an 8% low-ionic-145

strength polyacrylamide gel, and electrophoresed at 100 V in TBE buffer for 9 h at 4℃. 146

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Determination of the yqhD transcriptional start site Total RNA was prepared from yqhC-P63S 148

cells using Tri-reagent solution (MRC). For 5’-RACE, 5 µg aliquots of total RNA were reverse-149

transcribed using SuperScript II reverse transcriptase (Invitrogen) with 0.5 pmol of the primer 5’-150

GCTGCGGCGATAAATTTGG-3’ and 10 µl of 150 mM NaOH. The mixture was incubated at 65℃ 151

for 1 hr for removal of RNA. A PCR purification kit (Labopass, Korea) was used to purify the DNA. 152

After the homopolymeric A-tail was added to the 3’-end of the cDNA using terminal transferase 153

(Roche), the sample was purified again using a PCR purification kit (Labopass, Korea). The tailed 154

cDNA served as a template for PCR amplification using the following primers: 5’- 155

CCAGTGAGCAGAGTGACGAGGACTCGAGCTCAAGCTTTTTTTTTTTTTTTTT-3’ and 5’- 156

GCTGCGGCGATAAATTTGG-3’. The PCR product was sequenced using an ABI3100 sequencer. 157

158

Construction of lac reporter plasmid and ββββ-galactosidase assay The pRS415 plasmid was used 159

to construct a transcriptional fusion for reporter assay [28]. The DNA fragment containing the 160

putative yqhD operator sequences (425 bp including 5’ 60 bp of the open-reading frame) were 161

inserted into EcoRІ-BamHІ site of lacZ 5’-UTR region of pRS415, which was introduced into 162

recipient cells. The β-galactosidase assay was performed according to Miller [29]. Cells were grown 163

in LB to OD600=1 and treated with 0 or 1 mM glyoxal for 70 min. 0.2 ml each of cell cultures were 164

suspended in Z-buffer to a final 1 ml. 100 µl chloroform and 50 µl of 0.1% sodium dodecyl sulfate 165

(SDS) were added and vortexed for 10 seconds. 0.2 ml of O-nitrophenyl-β-D-galactoside (ONPG, 4 166

mg/ml) was added to mixture, and the sample was vortexed again. Enzyme reaction was stopped by 167

adding 0.5 ml of 1 M Na2CO3 when the yellow color was developed. After measuring OD420 and 168

OD550, LacZ activity was calculated according to Miller [29]. 169


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Quantitative Real Time (qRT)-PCR The MG1655 and yqhCstop

strains were cultured in LB 171

medium at 37℃ with agitation to OD600=1. The cultures were then treated with 0 and 1 mM of 172

glyoxal for 30 min, which were sedimented and used for RNA purification. One µg of total RNA 173

from different samples was used for reverse transcription. cDNA synthesis was carried out using 174

ImPromIITM

Reverse Transcriptase (Promega). Two µl each of cDNAs synthesized were used for 175

real time qRT-PCR. Reaction mixtures (20 µl) included SYBR Green I, 1 unit of HS Taq polymerase, 176

5 mM MgCl2, 2 mM dNTPs (Prime Q-Mastermix), and 0.5 pmol of specific primers for yqhD and 177

glyceraldehydes-3-phosphate dehydrogenase (GAPDH, a reference). The primers for yqhD are F: 5’-178

ACGCGAACAAATTCCTCACGATGC-3’ and R: 5’-GCTCAATACCGCCAAATTCCAGCA-3’, 179

for GAPDH F: 5’-ACTTACGAGCAGATCAAAGC-3’ and R: 5’-AGTTTCACGAAGTTGTCGTT-180

3’. Bio-rad CFX-96 was used for PCR reaction and for detecting fluorescence change. The ratio of 181

cycle threshold (ct) values, as determined from the Bio-rad CFX manager software, were compared 182

between samples [30]. 183

184

Enzymatic assay Enzymatic activity of purified YqhD was measured at 25℃ using a Beckman 185

Coulter DU800 spectrophotometer by monitoring the initial rate of oxidation of NADPH at 340 nm. 186

Substrate specificity was assessed using 1 ml of 50 mM potassium phosphate buffer (pH 7.0) with 2 187

mM NADPH as a coenzyme. In all reactions, nonenzymatic conversions were subtracted from the 188

observed initial reaction rates. The apparent Km and kcat values were determined by measuring the 189

initial rate over a range of substrate concentrations. 190

191

Cell viability assay for determining lethal concentrations of aldehyde compounds Cell viability 192

after exposure to aldehyde compounds was determined by growing cells on LB plates containing 193

different concentrations of aldehyde compounds. Fresh colonies of wild-type and mutant strains were 194


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grown overnight in LB broth, diluted 100-fold in the same medium, and incubated with shaking until 195

the OD600 reached 1.0. The cells were diluted from 10-1

to 10-6

and spotted (4 µl) onto LB plates 196

containing different concentrations of aldehyde compounds. Growth was assessed after 12-14 h of 197

incubation at 37 ℃. 198

199

Measuring inhibitory concentrations (IC50) of aldehyde compounds To compare IC50 among 200

strains, we measured optical densities of cells growing in media containing different concentrations 201

of GO. A 96-well culture plate containing 180 µl LB media per well was treated with different 202

concentrations of GO. Twenty microliters of inoculum containing cells at an OD600=1 were then 203

added to each well. The 96-well plate was sandwiched between two acrylic plates (preheated) for 204

efficient temperature equilibration and then incubated at 37℃ with shaking (220 rpm) for 6 hours. 205

OD595 was measured using a microplate reader (Model 680, Bio-Rad). IC50 was estimated using 206

Sigmaplot (SPSS Inc., Chicago, IL) by fitting to the sigmoidal dose-response equation. 207

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Results 209

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Isolation and localization of glyoxal-resistant E. coli mutants 211

We isolated glyoxal-resistant (GOR) mutants from the E. coli MG1655 strain on LB plates 212

containing 10 mM glyoxal. The mutations were mapped by random tagging with TnphoA-132 213

insertions [26], and were further analyzed by co-transduction with phage P1. The insertion tags were 214

localized to two different chromosomal loci, one of which was found to be in the region around 70 215

min (ca. 3152 Kb of the E. coli genome). The tags at 70 min were found to be in the hybB (3,141 Kb) 216

and ygiN (3,176 Kb) genes (Fig. 2A), which were linked to the GOR locus with co-transduction 217

frequencies of 70 and 43%, respectively, indicating that the candidate locus was somewhere between 218

the two tags (Fig. 2A). We included two more markers, yqhE::cat [15] and yqhZ::kan, for fine 219


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mapping and found that the GOR locus was very closely linked to yqhE::cat and yghZ::kan, with co-220

transduction frequencies of 98% and 95%, respectively. Mapping of the other GOR locus is currently 221

underway (Lee et al., unpublished). 222

Based on the fact that YqhD was proposed to function as an alcohol dehydrogenase/aldehyde 223

reductase [21] and the fact that it is flanked by the divergently transcribed yqhC gene, which is 224

predicted to be an AraC-type transcriptional regulator, we chose to sequence the yqhC gene first, and 225

found mutations associated with GOR. The YqhC protein shows 13.6% amino acid identity with 226

AraC (Fig. 2B). The GOR mutants identified had single changes in the yqhC gene as shown in Table 227

2 and Fig. 2B. All the mutations were in-frame, and were clustered in the putative regulatory domain 228

of YqhC (Fig. 2B). There was variation in the effectiveness of GO resistance in different mutants as 229

shown in Fig. 3A, which was quantitated by measuring inhibition of cell growth after treatment with 230

different concentrations of glyoxal in order to determine the 50% inhibitory concentration (IC50, 231

Materials and Methods). 232

233

YqhC mutations result in increased expression of YqhD protein 234

When we examined total protein expression in GOR mutants and compared it with that of 235

wild-type cells, we found overproduction of a protein shown on a 12% SDS-PAGE gel to be 43 kDa, 236

which was characterized as YqhD after MALDI-TOF MASS fingerprinting (Fig. 3B). The 237

overexpression of YqhD was also confirmed by 2-dimensional gel electrophoresis as a spot with a 238

predicted pI value of 6.0, which is close to the predicted pI for YqhD of 5.72 (data not shown). No 239

overproduction of the yqhE gene product was detected, suggesting that only expression of YqhD, and 240

not the downstream gene yqhE, is likely to be regulated by YqhC. This result implies that enhanced 241

expression of YqhD specifically results from missense mutations in yqhC, which is consistent with 242

the fact that expression of YqhD protein was relatively low in wild-type cells (Fig. 3B). Our results 243


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also showed that levels of YqhD expression in mutant cells are roughly proportional to their degree 244

of GO resistance (Fig. 3B), supporting the notion that YqhD is responsible for resistance to GO. 245

246

YqhC acts as a transcriptional activator for yqhD gene 247

In order to assess the role of yqhC in yqhD regulation, we obtained a mutant yqhCstop

with a 248

nonsense change (E112) in the yqhC gene that produced a peptide with a deletion of two thirds of its 249

coding region (Table 1). This peptide lacked the helix-turn-helix that was predicted to serve as the 250

DNA binding domain, and we observed that the mutant strain confers sensitivity to GO (Fig. 4A). 251

Thus, we concluded that YqhC functions as an activator for yqhD, supporting the notion that GO-252

resistance mutations in yqhC are specific changes activating yqhD. We further demonstrated that the 253

yqhD activation by YqhC indeed occurs at the transcription level by constructing a β-galactosidase 254

reporter and also by analyzing transcription with real-time qRT-PCR (Fig. 4BC). The promoter 255

region of yqhC was cloned into the transcriptional fusion plasmid pRS415 containing lacZ (Materials 256

and Methods for details). When cells were grown in LB medium, the wild type exhibited some level 257

of yqhD transcription (Fig. 4B) as measured by β-galactosidase assay, which was increased about 258

two folds in the presence of glyoxal at 1 mM. The removal of YqhC by introducing the yqhCstop

259

mutation essentially abolished the transcription of yqhD, which is unchanged upon an addition of 260

glyoxal. As expected, the yqhD transcription was dramatically enhanced by the presence of 261

constitutively activating mutation of yqhC-P63S. Levels of yqhD transcripts measured by real-time 262

qRT-PCR were consistant with the data of reporter assay, again confirming the transcriptional 263

activation of yqhD gene by YqhC. It is of interest to note that the yqhD transcription is enhanced by 264

an addition of glyoxal. 265

The yqhD-defective strain exhibits a glyoxal-sensitive phenotype, which can be 266

complemented by a yqhD clone (pACYC184 yqhD) that produces the protein under its own promoter 267

(Fig. 4A). Since YqhD protein was not highly expressed from this plasmid (data not shown), the 268


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resistance of the plasmid to glyoxal was only slightly enhanced relative to that of wild-type MG1655. 269

In order to assess the contributions of the glyoxalases to intracellular removal of methylglyoxal and 270

glyoxal, we tested yqhC and yqhD mutants on a gloA-negative background (Fig 4A). While the gloA 271

mutant was susceptible to methylglyoxal, its sensitivity to glyoxal was essentially the same as that of 272

wild type (MG1655). In contrast, yqhD mutants showed greater sensitivity to glyoxal, but no 273

difference in methylglyoxal sensitivity. The sensitivity of yqhCstop

mutants to these compounds was 274

similar to that of the yqhD mutants. However, the effects of yqhD and yqhC mutations on 275

methylglyoxal sensitivity became visible in the gloA-deficient background (data not shown) or in the 276

yqhC-P53S mutant overexpressing YqhD, indicating that YqhD plays a role in MG detoxification, 277

which is consistent with its catalytic activity as shown in Table 3. 278

279

YqhC binds to the operator region of yqhD gene 280

Phylogenetic analysis of YqhC revealed its close similarity to AraC, and showed that it is 281

somewhat distant from the other AraC cluster containing GadX, GadY, and AppY (data not shown). 282

The consensus sequence of the AraC binding site is not yet clearly defined; however, the consensus 283

binding sequence for SoxS, another member of AraC family, is well characterized [31]. Using 284

sequence analysis of the 5’ region of yqhD gene to search for a promoter with the SoxS-binding 285

sequence, several candidates were found, one of which is located in the ORF region of yqhC as 286

shown previously [20]. The predicted binding site (shown with an asterisk in Fig. 5A) in the yqhC 287

ORF appeared not to bind to YqhC (data not shown). Others are clustered approximately 50 bp 288

upstream of the yqhD ORF (Fig. 5A), and these sequences overlap with a 24-bp palindrome 289

consisting of two 10-bp repeating units. We carried out a gel mobility shift assay on DNA fragments 290

amplified by PCR containing the putative operator regions, which were tested for their ability to bind 291

to purified YqhC protein. As shown in Fig. 5B, YqhC binds to DNA containing the operator region 292

(the SoxS-binding consensus sequence with the palindrome) in concentration-dependent manner 293


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(lane 1-5). In order to verify that YqhC indeed binds to this region, a gel-mobility shift assay was 294

carried out with two mutated probes in which ten residues in the two SoxS consensus sequences were 295

altered (shown with an inverted triangle in Fig. 5A). As shown in Fig 5B, YqhC did not bind to the 296

mutated probes (lane 10-15), indicating that the mutated residues are indeed part of the YqhC-297

binding sequence. Furthermore, the mutated probes were unable to compete with the wild-type probe 298

(lane 6-9, Fig. 5B), confirming that the altered residues in the yqhD operator region are directly 299

involved in YqhC binding. The location of this putative promoter sequence for the yqhD gene is 300

consistent with the transcription initiation site revealed by 5’-RACE analysis as shown in Fig. 5A. 301

302

YqhD is an aldehyde reductase converting glyoxal to ethandiol using NADPH 303

YqhD was initially characterized as an alcohol dehydrogenase based on its 3-dimensional 304

structure [21], but a recent report has indicated that it is an aldehyde reductase showing specificity 305

for certain aldehydes, including butanaldehyde, malondialdehyde, and acrolein, which are derived 306

from lipid peroxidation [20]. Thus, we suspected that the YqhD enzyme is involved in the 307

metabolism of glyoxal and/or glycolaldehyde. When we tested the ability of purified YqhD to 308

metabolize various short-chain aldehydes, it exhibited fairly broad specificity with the highest 309

specific activity for glycolaldehyde (Table 3), although its Km value was estimated to be a little bit 310

high (28.28 mM). Furthermore, the enzyme showed activity against glyoxal, generating 311

glycolaldehyde using NADPH as a cofactor. Thus, we have shown that the YqhD enzyme mediates 312

catalysis of the two consecutive reactions involved in the conversion from glyoxal to ethandiol, and 313

thereby efficiently removes two toxic aldehydes. When we incubated glyoxal (10 mM) with YqhD 314

for 6 hours and analyzed the resulting products by NMR, increased amounts of glycolaldehyde and 315

ethandiol were observed (Fig. 6). YqhD also exhibited activity against methylglyoxal, generating 316

hydroxyacetone as the major reaction product (Table 3, Fig. 6), which accounted for more than 90% 317

of the substrate converted. This estimation was based on molar quantification of hydroxyacetone 318


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generation versus methylglyoxal disappearance. Commercially available MG contains only a small 319

amount of acetate and no other compounds such as hydroxyacetone as shown on the NMR spectrum, 320

even after a longer period of incubation. 321

The enzymatic specificity of YqhD for both glyoxal and glycolaldehyde is consistent with 322

the prevalence of glyoxal-resistant mutants (Fig. 2). In fact, when we compared the 323

glyoxal/glycolaldehyde reducing activities of crude extracts from the yqhC-P63S mutant, which 324

expresses yqhD constitutively, with those of △yqhD strains, a significant difference was noted (78±325 5/90±2 versus 42 ± 3/62±3 nmol min

-1 mg

-1, respectively, with 100/20 mM glyoxal/glycolaldehyde, 326

2 mM NADPH, and 100 µg of protein). Since YqhD has been previously reported to remove 327

aldehyde compounds derived from lipid peroxidation, we tested the YqhD-overproducing strain for 328

its ability to remove butanal and propanal. Surprisingly, YqhD activity increased the toxic effects of 329

these aldehydes by converting them to their corresponding alcohols, i.e. butanol and propanol (Fig. 330

7A). These alcohols are already known to be highly toxic to cells [32, 33]. When we examined the 331

toxic effects of butanol and propanol added externally, we found that the lethal and effective 332

concentrations were much higher than those of the corresponding aldehydes; the IC50 values of 333

butanol and propanol in wild-type E. coli (MG1655) were more than 100 and 200 mM, respectively. 334

335

DISCUSSION 336

337

Certain short-chain carbohydrates are known to be highly reactive because of their 338

unprotected aldehyde groups [34]. Glyoxal is a member of the 2-oxoaldehydes, a reactive subgroup, 339

and is believed to be a product of glucose oxidation [35]. However, in contrast to methylglyoxal, the 340

intracellular presence and degradation of glyoxal have not been clearly elucidated. Here, we have 341

presented evidence that E. coli cells are capable of detoxifying glyoxal with a reducing enzyme, 342

using NADPH as a cofactor. Our genetic study to isolate glyoxal-resistance mutations revealed the 343


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identity of an enzyme YqhD that is overexpressed in constitutively active yqhC mutants. The 344

prevalence of such mutants might be due to the rather simple mechanism of activation of yqhD by 345

YqhC. We demonstrated that YqhC protein directly binds to the promoter region of the yqhD gene, 346

which contains the SoxS-like binding sequence as well as a 24-bp palindrome (Fig. 5). More 347

importantly, the isolation of yqhC mutants allowed us to characterize the YqhD enzyme, which 348

catalyzes the reaction of various aldehydes. 349

YqhD has been classified as an alcohol dehydrogenase [21], and it is also highly similar to 350

the 1, 2-propanediol oxidoreductase FucO and to the C-terminal half of AdhE, a glycolytic enzyme 351

producing ethanol from acetaldehyde. YqhD is structurally different from the members of the alde-352

keto reductase (AKR) family, which share a characteristic motif for NADPH binding; however, 353

growing evidence has indicated that YqhD homologs are primarily involved in the reduction of 354

aldehyde compounds [20]. It has been demonstrated that the enzyme AdhE acquired alcohol 355

dehydrogenase activity through the introduction of a simple missense mutation, A267T, an example 356

of the evolutionary adaptation to an oxidative enzyme [36]. The aldehyde reductase activity of YqhD 357

was first identified by its ability to convert 3-hydroxypropionaldehyde to 1,3-propanediol [37]. Later, 358

YqhD was proposed to function as a detoxifying enzyme removing short-chain aldehydes, such as 359

butanal and propanal, generated from lipid peroxidation [20]. However, the proposed role of YqhD 360

in the removal of such compounds seems unlikely based on our in vivo evidence indicating that the 361

enzymatic conversion of butanal and propanal results in intracellular accumulation of even more 362

toxic compounds, butanol and propanol, respectively (Fig. 7). The unexpected intracellular toxicity 363

of butanol and propanol may suggest the presence of a more sensitive in vivo target for these alcohols 364

that is not easily accessible from an exogenously added compound. 365

While the intracellular detoxification of methylglyoxal is known to occur through multiple 366

pathways, including the glutathione-mediated glyoxalase I/II system, the metabolic pathways for 367

glyoxal have not been well established. We observed that the glyoxal sensitivity of a gloA-deficient 368


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strain was not much different from that of the wild-type strain, suggesting that the glyoxalase system 369

does not serve as an efficient pathway for glyoxal detoxification. Thus, there may be other pathways 370

for intracellular removal of glyoxal. E. coli cells might have an advantage in detoxifying glyoxal 371

through their expression of the YqhD enzyme, since it is capable of converting glyoxal to nontoxic 372

ethandiol with a single enzyme. However, it appears that cellular strategies for removal of short-373

chain aldehydes are not specific, as exemplified by the presence of aldo-keto reductases with rather 374

non-specific substrate specificities. We have previously demonstrated that several AKRs, including 375

YghZ and YqhE, are involved in the catalysis of MG and related compounds [15]. 376

Intracellular production of 2-oxoaldehyde is enhanced by oxidative stress [35, 38]. Thus, it is 377

possible that regulation of aldehyde removal is associated with regulation of the oxidative stress 378

response. An example would be that glycolaldehyde induces the SoxR/S regulon [39]. Although the 379

YqhC-YqhD regulatory system was discovered through a specific class of regulatory mutations in 380

YqhC that confer cellular resistance to glyoxal, the mechanism underlying its activation still needs to 381

be elucidated. Although we observed that the transcription of yqhD was activated by GO, the identity 382

of intracellular signal for YqhC is yet to be discovered. Finding such signal may provide an insight 383

into the underlying regulatory mechanism for responses to aldehyde as well as oxidative stresses. 384

During this study, we isolated other glyoxal-resistance mutations localized at 37 centisome region 385

that were unlinked from yqhC (Lee et al., unpublished data). Further characterization of these 386

mutants may reveal other mechanism associated with carbonyl stress and its detoxification. 387

388

Acknowledgements 389

This work was supported by the 21C Frontier Microbial Genomics and Application Center Program, 390

Ministry of Education, Science & Technology, Republic of Korea to C. Park. The authors thank 391

Dongkyu Kim for the isolation of mutant strains. 392


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Table 1. Strains and plasmids used 482

----------------------------------------------------------------------------------------------------------------------- 483

Strain/plasmid Genotype or description Source or reference 484

----------------------------------------------------------------------------------------------------------------------- 485

Strains 486

MG1655 F-, λ

- Lab collection 487

CH3 MG1655 yqhC-P63S This work 488

CH5 MG1655 yqhC-A109D This work 489

CH6 MG1655 yqhC-R61H This work 490

CH9 MG1655 yqhC-P63Q This work 491

CH13 MG1655 yqhC-V99E This work 492

CH20 MG1655 yqhCstop

(E112 nonsense) 1 This work 493

CH21 MG1655 ΔyqhD::kan This work 494

CH26 MG1655 ∆gloA::kan Lab collection 495

CH27 MG1655 ΔyqhD::tet ∆gloA::kan This work 496

CH28 MG1655 ΔyqhD::tet ∆gloA::kan This work 497

BL21(DE3) F-ompT hsdSB(rB-mB

-) gal dcm(DE3) Novagen 498

CH29 MG1655 ∆lacZ This work 499

CH30 CH3 ∆lacZ This work 500

CH31 CH20 ∆lacZ This work 501

502

Plasmids 503

pACYC184 ori(p15A) Tcr Cm

r Lab collection 504

pCH1 pACYC184 yqhD This work 505

pCH2 pET15b yqhD This work 506


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pET-YafB-His pET21b yafB 15 507

pET-YqhE-His pET21b yqhE 15 508

pET-YghZ-His pET21b yghZ 15 509

pON1 pET21a yqhC This work 510

pRS415 Apr, lacZ operon fusion vector 28 511

pON2 pRS415 yqhD operator This work 512

1represents a nonsense mutation (334G→T, counted from the first ATG). 513


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Table 2. Glyoxal-resistance mutations found in the yqhC gene 514

------------------------------------------------------------------------------ 515

Mutational change 516

-------------------------------------------------------------------- 517

Allele Nucleotide (bp)a Amino acid IC50

b for GO (mM) 518

Wild type none 2.87 519

yqhC- P63S 187C→T P63S 5.99 520 yqhC- A109D 326C→A A109D 5.79 521 yqhC- R61H 182G→A R61H 5.91 522 yqhC- V99E 296T→A V99E 6.65 523 yqhC- P63Q 188C→A P63Q 5.49 524 ------------------------------------------------------------------------------------------------ 525

a Designates position from the beginning of the yqhC open-reading frame. 526

b The value (IC50) for the yqhD insertion mutant is 1.88 mM. 527


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Table 3. Substrate specificities and kinetics of YqhD 528

--------------------------------------------------------------------------------------------------------------- 529

Substrate Product Specific activitya Km (mM) kcat (min-1) kcat / Km (min

-1M-1) 530

(nmol min-1 mg-1) 531

---------------------------------------------------------------------------------------------------------------------------------------------------- 532

Glyoxal Glycolaldehyde 36250 11.53 618 5.36×104 533 Glycolaldehyde 1,2-Ethandiol 61902 28.28 3258 1.09×105 534 Butanaldehyde Butanol 58587 0.40 1983 4.99×106 535 Methylglyoxal Hydroxyacetone 26528 2.60 284 1.09×105 536 Glyceraldehyde Glycerol 26363 1.36 204 1.49×105 537 Hydroxyacetone 1,2-Propanediol 3050 76.90 144 1.87×103 538 -------------------------------------------------------------------------------------------------------------------------------------- 539

a Enzyme activities were measured at room temperature with 2 mM NADPH, unless otherwise stated 540

(0.1 mM for hydroxyacetone). Concentrations of the substrates were 200 mM glyoxal and 541

glycolaldehyde, 10 mM butanal and glyceraldehyde, 20 mM MG, and 500 mM hydroxyacetone. Ten 542

micrograms of YqhD was used. 543 on April 2, 2021 by guest

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544

Figure legends 545

Fig. 1 The proposed intracellular conversion of glyoxal. GO can be reduced to glycolaldehyde, and 546

further to 1,2-ethandiol through involvement of the AKRs, including YafB, YqhE, and YghZ. This 547

study revealed that YqhD mediates two-step reduction of GO to 1,2-ethandiol using NADPH. FucO 548

can also mediate glycolaldehyde reduction [10, 11]. Glycolic acid can be inefficiently produced from 549

GO by the glyoxalase system [12], and it is also produced by the oxidation of glycolaldehyde 550

involving AldA [8, 9, 12]. 551

552

Fig. 2 Mapping glyoxal-resistant mutations to yqhC using transposon insertions. (A) Using nearby 553

TnphoA-132 insertions isolated as described in Materials and Methods, the locations of yqhC 554

mutations were determined based on their co-transductional linkages. The candidate locus was found 555

at approximately 70 min on the E. coli linkage map with physical coordinates shown underneath. 556

Using insertions located close to the mutations, i.e. hybB (70% co-transduction frequency), ygiN 557

(43%), yghZ (95%), and yqhE (98%), the candidate locus was further narrowed down to the region 558

containing the yghB, yqhC and yqhD genes. Eventually, the mutation was revealed by sequencing the 559

yqhC gene (asterisk), a member of the AraC transcriptional activator family. (B) YqhC is a homolog 560

of the AraC transcriptional activator with 13.6% amino acid identity based on alignment with AlignX 561

(program provided in Vector NTI). The point mutations in Table 2 are marked by asterisks, with α-562

helices (cylinders) and β-sheets (arrows) shown as predicted by Jpred3 563

(http://www.compbio.dundee.ac.uk/www-jpred/). The predicted regulatory and DNA-binding 564

domains are shown with solid and dashed lines, respectively (http://www.genome.jp/kegg/). 565

566

Fig. 3 YqhC mutations confer resistance to 2-oxoaldehydes and alter protein expression patterns. (A) 567

Point mutations of yqhC exhibit a spectrum of resistance to glyoxal and methylglyoxal. The most 568

resistant mutant, yqhC-V99E, shows normal growth in media containing up to 7 mM GO and 0.9 569


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mM MG, conditions that are lethal for wild-type MG1655. The yqhD-P63Q mutation had the lowest 570

level of resistance to both substrates. (B) YqhC mutations result in overexpression of a 40-kDa 571

protein as visualized on a 12% SDS-PAGE gel, which was identified as YqhD by MALDI-TOF 572

MASS analysis. Levels of YqhD expression are roughly proportional to degrees of oxoaldehyde 573

resistance among the yqhC mutants . 574

575

Fig. 4 Transcriptional regulation of yqhD by YqhC and their roles in glyoxal detoxification (A) 576

Phenotypes of yqhC, yqhD and gloA deletion mutants and complementation with a cloned yqhD 577

plasmid. The yqhD deletion mutant showed an enhanced susceptibility to GO and MG relative to 578

wild-type cells. The oxoaldehyde sensitivity of yqhD deletion was rescued by pACYC184-cloned 579

yqhD expressed under its own promoter. The yqhC and yqhD deletion mutants show apparent GO 580

sensitivity but not MG sensitivity. In contrast, the gloA deletion mutant exhibits a sensitivity to only 581

MG , but not to GO. The transcriptional activity of yqhD was measured by the fusion of β-582

glalactosidase reporter (B) and by real time quantitative qRT-PCR analysis (C). Compared to the 583

wild type, yqhD expression was dramatically enhanced in yqhC-P63S, while the yqhCstop

mutant 584

exhibited almost no expression of yqhD. In wild-type MG1655, yqhD expression was increased by 585

an addition of GO. The cells at OD600=1 were treated 1 mM of GO for 70 and 30 min in β-586

glalactosidase assay and real time qRT-PCR, respectively. 587

588

Fig. 5 Binding of YqhC protein to the yqhC-yqhD intergenic region. (A) Relative locations of the 589

yqhC and yqhD genes and the predicted promoter and regulatory sequences. The -10 and -35 590

sequences of the yqhD promoter are underlined with the translational start site shown in bold. The 591

transcriptional start site of yqhD is also shown and was determined by 5’-RACE (see Materials and 592

Methods). The predicted YqhC binding sites are shown with broken lines and bold letters (I and II) 593

designating the consensus sequences ((N3)GCA(N9)AAA(N2)) for SoxS binding that are conserved 594


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in the target sequences of AraC-XylS transcriptional regulators [40]. The palindromic sequence is 595

shown with arrows. The DNA probe used for the gel-shift assay (shown in B) contained the region 596

from -98 to +1 of the yqhD promoter. (B) Gel mobility shift assay using YqhC to identify the 597

promoter region. The cores (▼) of the predicted binding sites were mutated to generate Mutant I and 598

II probes, each having 10 base pairs. Molar ratios of DNA to protein, as well as probe DNA to 599

competitor DNA, are shown; ‘WT comp’ represents addition of the cold wild-type probe as a 600

competitor, and ‘MUI comp’/‘MUII comp’ indicate cold mutant probe. Probes were labeled with [γ-601

32P]-ATP as described in Materials and Methods. 602

603

Fig. 6 1H -NMR spectra showing conversion of oxoaldehydes by YqhD. (A) Glycolaldehyde (▼) 604

and 1,2-ethandiol (▽) were produced after 6 hours of incubation with purified YqhD protein (40 µg), 605

10 mM glyoxal, and 2 mM NADPH in 50 mM potassium phosphate buffer (pH 7.0), and were 606

detected by 1H -NMR spectroscopy. The commercial GO reagent is contaminated with some 1,2-607

ethandiol. The GO peak is hidden by water noise at 5.0 ppm (not shown). (B) Hydroxyacetone (▽) 608

was produced from MG (▼, 3 mM) by YqhD (40 µg). For this reaction, the same condition 609

described for glycoaldehyde was used. It should be noted that the MG reagent is contaminated with 610

acetate showing a peak around 2 ppm. 611

612

Fig. 7 Activity of YqhD on butanal and propanal. (A) Spot assays on LB plates containing 10 mM 613

butanal or 50 mM propanal indicate that the yqhC-P63S strain overexpressing YqhD became highly 614

sensitive to these compounds, which was not observed in the strain lacking YqhD. (B) Growth 615

inhibition with various concentrations of butanal and propanal was observed and plotted to obtain 616

IC50 values. IC50 values for MG1655, yqhC-P63S, and △yqhD for butanal/propanal were 16.4/15.5, 617

9.5/9.6, and 24.9/18.5 mM, respectively. 618


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