YKL107W from Saccharomyces Cerevisiae Encodes a Novel Aldehyde Reductase for Detoxification of Acetaldehyde, Glycolaldehyde, and Furfural

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YKL107W from Saccharomyces cerevisiae encodes a novel aldehyde reductase for detoxification of acetaldehyde, glycolaldehyde, and furfural

Hanyu Wang, Qian Li, Zhengyue Zhang, Chang Zhou, Ellen Ayepa, Getachew Tafere Abrha, Xuebing Han, Xiangdong Hu, Xiumei Yu, et al.

Applied Microbiology and Biotechnology

ISSN 0175-7598 Volume 103 Number 14

Appl Microbiol Biotechnol (2019) 103:5699-5713 DOI 10.1007/s00253-019-09885-x

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Applied Microbiology and Biotechnology (2019) 103:5699–5713 https://doi.org/10.1007/s00253-019-09885-x

BIOTECHNOLOGICALLY RELEVANT ENZYMES AND PROTEINS

YKL107W from Saccharomyces cerevisiae encodes a novel aldehyde reductase for detoxification of acetaldehyde, glycolaldehyde, and furfural

Hanyu Wang1 & Qian Li1 & Zhengyue Zhang1 & Chang Zhou1 & Ellen Ayepa1 & Getachew Tafere Abrha1 & Xuebing Han1 & Xiangdong Hu1 & Xiumei Yu2 & Quanju Xiang2 & Xi Li3 & Yunfu Gu2 & Ke Zhao2 & Chengcheng Xie3 & Qiang Chen2 & Menggen Ma1,2

Received: 10 March 2019 /Revised: 24 April 2019 /Accepted: 29 April 2019 /Published online: 21 May 2019 # Springer-Verlag GmbH Germany, part of Springer Nature 2019

Abstract The aldehyde reductases from the short-chain dehydrogenase/reductase (SDR) family were identified as a series of critical enzymes for the improved tolerance of Saccharomyces cerevisiae to the aldehydes by catalyzing the detoxification reactions of aldehydes. Herein, we report that a novel aldehyde reductase Ykl107wp deduced from YKL107W from S. cerevisiae belongs to the classical SDR group and can catalyze the reduction reactions of acetaldehyde (AA), glycolaldehyde (GA), furfural (FF), formaldehyde (FA), and propionaldehyde (PA) but cannot reduce the six representative ketones. Ykl107wp displayed the best maximum velocity (Vmax), catalytic rate constant (Kcat), catalytic efficiency (Kcat/Km), and highest affinity (Km) to acetaldehyde. The optimum pH of Ykl107wp was 6.0 for the reduction of AA and 7.0 for the reduction of GA and FF, and the optimum temperatures were 40, 35, and 30 °C for the reduction of AA, GA, and FF, respectively. Ykl107wp for the reduction of AA was greatly affected by metal ions, chemical additives, and salts and showed poor thermal and pH stability, but its stability was slightly affected by a substrate. Ykl107wp was localized in endoplasmic reticulum and prevented the yeast cells from damage caused by furfural via the detoxification of furfural to furfural alcohol. This research provides guidelines for the study of uncharacterized classical SDR aldehyde reductases and exploration of their protective mechanisms on the corresponding organelles.

Keywords Aldehyde reductase . Open reading frame . Protein-GFP localization·Saccharomyces cerevisiae . Short-chain dehydrogenase/reductase (SDR) Introduction

Nicotinamide adenine dinucleotide (NADH)- and/or nicotin- Hanyu Wang, Qian Li and Zhengyue Zhang contributed equally to this work. amide adenine dinucleotide phosphate (NADPH)-dependent aldehyde reductases play critical roles for the improved toler- Electronic supplementary material The online version of this article ance of Saccharomyces cerevisiae to the aldehydes by in situ (https://doi.org/10.1007/s00253-019-09885-x) contains supplementary material, which is available to authorized users. detoxifying aldehydes to the less toxic corresponding alcohols (Gutiérrez et al. 2002;Liu2011; Liu et al. 2004). Recent studies * Menggen Ma found that aldehyde reductases not only catalyzed the reduction [email protected] of external aldehydes but also had abilities to degrade the endogenous aldehydes derived from the metabolism of amino 1 Institute of Resources and Geographic Information Technology, acids, carbohydrates, lipids, biogenic amines, vitamins, and ste- College of Resources, Sichuan Agricultural University, No. 211 Huimin Road, Wenjiang 611130, Sichuan, People’sRepublicof roids for sustaining the homeostasis of yeast cells (Hazelwood China et al. 2008; Vasiliou et al. 2000). To date, 24 aldehyde reduc- 2 Department of Applied Microbiology, College of Resources, Sichuan tases have been characterized in S. cerevisiae (Wang et al. Agricultural University, Wenjiang 611130, Sichuan, People’s 2018). Given the chain length, conserved functional motifs, Republic of China and structural features, these aldehyde reductases could be clas- 3 College of Landscape Architecture, Sichuan Agricultural University, sified into the medium-chain dehydrogenase/reductase (MDR) Wenjiang 611130, Sichuan, People’s Republic of China family, aldo-keto reductase (AKR) family, and short-chain Author's personal copy

5700 Appl Microbiol Biotechnol (2019) 103:5699–5713 dehydrogenase/reductase (SDR) family, but part of these reduc- Although Gre2p shows preference for NADH in the reduction tases, such as Adh4p, Ald4p, and Ald6p, could not be classified reactions of most of aldehydes, Gre2p has preference for (Kavanagh et al. 2008; Nordling et al. 2002; Persson et al. 1999, NADPH rather than NADH for the reduction of methylglyoxal 2008;Wangetal.2018). Protein sequence alignment analysis and isovaleraldehyde (Chen et al. 2003;Hauseretal.2007), found that most of the aldehyde reductases from the SDR fam- which is attributed to the hydrogen bonds between Asn9, ily are shorter than those from the MDR and AKR family in Arg32, and Lys36 and the phosphate moiety of adenosine (Guo length (Kavanagh et al. 2008). Significantly, Ykl071wp (256 et al. 2014). Additionally, substitutionofVal285byAspinGre2p amino acids, aa) as the classical SDR was identified as the can improve cofactor preference for NADPH than NADH in the shortest one among the 24 aldehyde reductases characterized reduction of FF and HMF (Moon and Liu 2012). However, the in S. cerevisiae (Wang et al. 2017a). The SDR aldehyde reduc- mechanisms of cofactor preference of other SDR aldehyde re- tases show similarity to the structure of the aldehyde reductases ductases were still unknown. Probably, the discoveries of novel in MDR family which contain cofactor-binding domain aldehyde reductases and the development of experiments on ami- (Rossmann fold) and substrate-binding sites (Kavanagh et al. no acid substitution will accelerate the elucidation of the molec- 2008; Persson et al. 2009;Wangetal.2018). Based on the ular mechanism of cofactor preference. Until now, the subcellular Rossmann fold, cofactor-binding domain, and C-terminal re- localization of the SDR aldehyde reductases was identified. gion, over 140,000 SDR members can be classified into 6 Ygl039wp, Yll056cp, and Ykl071wp were localized in the cyto- groups, namely Bclassical,^ Bextended,^ Bintermediate,^ plasm, Gre2p and Ari1p were localized in the cytoplasm/nucleus, Bdivergent,^ Bcomplex,^ and Batypical^ groups (Jornvall while Ydr541cp was localized in endoplasmic reticulum (ER) et al. 2015; Marchler-Bauer et al. 2013, 2015). Until now, the (Huh et al. 2003;Wangetal.2017b;Yofeetal.2016). Since six characterized SDR aldehyde reductases (Ari1p, Ygl039wp, exogenous aldehydes can pass through subcellular membranes Gre2p, Ydr541cp, Ykl071wp, and Yll056cp) were included in (such as mitochondrial membrane, vacuole membrane, and nu- the Bintermediate,^ Bclassical,^ and Batypical^ subfamilies in clear membrane) and enter the organelles and induce damages of the SDR family (Wang et al. 2018). Even though these alde- organelles (Allen et al. 2010; Voulgaridou et al. 2011), the aldehyde reductases belong to SDR family, their catalytic abilities to hyde reductases localized in different organelles may play a pro- aldehydes, the types of catalytic substrates, cofactor prefer- tective role to them by detoxification of endogenous aldehydes. ences, and subcellular localizations were distinct. According to sequence analysis and alignment of cofactor- Based on the enzyme activity analysis of aldehyde reduc- binding sites and active sites, we found that the structure of an tases, it was found that Ari1p, Ydr541cp, and Gre2p not only uncharacterized protein Ykl107wp displayed similar features could catalyze the reduction of furfural (2-furaldehyde, FF) but to Yll056cp and Ykl071wp, and this protein was close to also the reduction of 5-hydroxymethylfurfural (HMF) (Liu and Ykl071wp and Yll056cp in phylogenetic distance (Wang Moon 2009; Moon and Liu 2012, 2015), but Ygl039wp, et al. 2018). Basic Local Alignment Search Tool (BLAST) Yll056cp, and Ykl071wp could only catalyze the reduction re- analysis (https://blast.ncbi.nlm.nih.gov/Blast.cgi)of action of furfural (Moon and Liu 2015;Wangetal.2017a, b). Ykl107wp demonstrates that it is a classical SDR protein Although Ygl039wp, Ari1p, and Ydr541cp catalyzed the reduc- and shares the glycine-rich NAD-binding motif of the classi- tion reactions of propionaldehyde (PA) and isobutyraldehyde cal SDRs. The evidences mentioned above infer that (IBA) (Liu and Moon 2009;MoonandLiu2015), their enzyme Ykl107wp can mostly probably catalyze the reduction of al- activities were significantly higher than that of Yll056cp for dehydes and/or ketones. Up to now, the function of Ykl107wp reduction of these substrates (Wang et al. 2017b). These results still remains unknown and needs to be explored. To identify implied that different SDR aldehyde reductases may play a crit- the function of Ykl107wp, we cloned YKL107W from S. ical role in the detoxification of a certain aldehyde in cerevisiae, determined the catalytic abilities for reduction of S. cerevisiae. Additionally, most of SDR aldehyde reductases the selected aldehydes and ketones, studied the enzymatic (Ygl039wp, Yll056cp, Ari1p, Ydr541cp, and Gre2p) exhibit characteristics of Ykl107wp on the reductive reactions of al- broad-substrate activities for the reduction of aldehydes, except dehydes, analyzed the structure feature of Ykl107wp, con- for Ykl071wp. These catalytic characteristics would contribute firmed the subcellular localization of Ykl107wp, and detected to the improved tolerance of yeast strains to multiple aldehydes. the protective effects of Ykl107wp to organelles. The SDR aldehyde reductases were non-metallo-reductases, but NADH and/or NADPH as a cofactor was essential for the reduction reactions of aldehydes catalyzed by these enzymes. It Materials and methods was vital that these enzymes exhibited a different cofactor preference in the catalytic reactions. For example, Gre2p, Yll056cp, Strains, plasmids, media, and reagents and Ykl071wp have preference for NADH (Jayakody et al. 2013;MoonandLiu2012;Wangetal.2017a, b), but Ari1p The S. cerevisiae yeast strain BY4742 (MATα his3Δ1leu2Δ0 has strict preference for NADPH (Liu and Moon 2009). lys2Δ0ura3Δ0) from Open Biosystems (Huntsville, AL, Author's personal copy

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USA) was used to amplify the target gene YKL107W. The were screened by LB medium supplemented with 100 mg/l S. cerevisiae yeast strain INVSc1 (his3Δ1/his3Δ1 leu2/leu2 ampicillin and identified by the diagnostic PCR method with trp1-289/trp1-289 ura3-52/ura3-52)fromInvitrogen the primer pairs iYKL107W_FandiYKL107W_R (Carlsbad, CA, USA) was used for the overexpression of the (Supplementary Material Table S1) and restriction endonucle- target gene YKL107W. The amplified fragment of gene ase analysis. YKL107W and the shuttle plasmid pYES2/NT B (Invitrogen) were adopted to construct the recombinant plasmid. The shut- Protein expression and purification tle plasmid pDDGFP-2 was applied to construct the recombinant plasmid for subcellular localization study by fused green The recombinant plasmid pYES2/NT B-YKL107W was iso- fluorescent protein (GFP) labeling (Newstead et al. 2007). The lated from the above positive clones and introduced into the Escherichia coli strain DH5α purchased from Sangon Biotech S. cerevisiae strain INVSc1 by using the lithium acetate meth- (Shanghai, China) was used as the host strain to screen the od (Gietz et al. 1995). The yeast strain INVSc1 containing the recombinant plasmid. Yeast peptone dextrose (YPD) medium recombinant plasmid was selected on SC-U medium supple- (w/v, 1% yeast extract, 2% peptone, and 2% glucose) was mented with 2% glucose and identified by diagnostic PCR. A prepared to harvest the cells of the S. cerevisiae strain transformant containing pYES2/NT B was used as a control. BY4742. A synthetic complete medium lacking uracil (SC- According to the method mentioned above, the transformants U) supplemented with 2% w/v glucose was used to select the were pre-incubated and transferred into induction medium for yeast strain INVSc1 containing the recombinant plasmid, and 72-h incubation. Using a One Step Yeast Active Protein SC-U supplemented with 2% w/v galactose and 1% w/v raffi- Extraction Kit (Sangon Biotech), raw protein was extracted nose was employed to induce the overexpression of from the induced yeast cells. A Ni-NTA Sefinose™ Kit YKL107W (Ma et al. 2013). Luria-Bertani (LB) medium (w/ (Sangon Biotech) was adopted to purify the Ykl107wp with v 0.5% yeast extract, 1% tryptone, 1% NaCl, pH 7.0) contain- a His-tag from the raw protein. Given the methods described ing 100 mg/l ampicillin was prepared to incubate the trans- previously (Zhao et al. 2015), we measured the concentration formed E. coli DH5α for isolation of the recombinant plas- of the purified protein. In order to ensure the reliable data, the mid. Solid medium was prepared by adding 2% (w/v)agar. purified protein samples were immediately used for the deter- NADH, NADPH, and media ingredients were purchased from mination of the enzyme activity within 48 h. Sigma-Aldrich (St. Louis, MO, USA) or Sangon Biotech. ER- Tracker™ Red purchased from Thermo Fisher Scientific Enzyme activity assay (Waltham, MA, USA) as ER membrane dye was adopted to stain the yeast cells for monitoring the morphological struc- The enzyme activity assay was determined using a Lambda 35 ture of ER. The aldehydes and ketones were purchased from UV/VIS Spectrophotometer (PerkinElmer Inc., Fremont, CA, Best-Reagent (Chengdu, China). USA), based on the methods described previously (Liu et al. 2008). The reaction mixtures, 10 mM final substrate (alde- Gene cloning and expression plasmid construction hydes and ketones), 10 μl(~1.0μg) purified protein sample, 100 μM NADH/NADPH, and 100 mM reaction buffer, were Genomic DNA extracted from the S. cerevisiae BY4742 by prepared for enzyme activity assays according to the descrip- using a Yeast DNA Kit (Omega Bio-Tek, Norcross, GA, tions by Wang et al. (2017a). For the reduction of acetalde- USA) and the primer pair YKL107W_F and YKL107W_R hyde (AA), the final concentration of NADH was 500 μMin were applied for amplification of the DNA fragment of the reaction mixture. Via the Michaelis-Menten equation in a YKL107W, (Supplementary Material Table S1). By means of double-reciprocal Lineweaver-Burk transformation the PrimerSelect program 7.1 (DNAStar Inc., Madison, WI, (Lineweaver and Burk 1934), the kinetic parameters for the USA), we designed these primer pairs which contained the reduction of AA, glycolaldehyde (GA), and furfural (FF) were restriction endonuclease sites of NotΙ and XhoΙ. The procedure obtained. All enzyme assays were run in technical triplicate. of PCR reactions was as follows: 1 cycle of 5 min at 95 °C; 35 cycles of 1 min at 95 °C, 30 s at 55 °C, and 1 min at 72 °C; and Effect of pH and temperature and pH and thermal a final extension cycle at 72 °C for 10 min. After the above stability mentioned PCR reactions and purification of the PCR products, the purified products were verified by DNA sequencing Based on the method mentioned above (Wang et al. 2017a), and digested with NotΙ and XhoΙ (Takara, Dalian, Liaoning, the enzyme activity at pH levels of 4.5–9.0 and at tempera- China) and purified via a Cycle-Pure Kit (Omega Bio-Tek). tures of 20–60 °C was determined for defining the effects of The digested DNA fragment was ligated into the pYES2/NT pH and temperature on Ykl107wp and the optimum pH and B vector digested with the same restriction endonuclease and temperature for reduction of aldehydes. For the pH and ther- then transformed into the E. coli DH5α. The positive clones mal stability test, the purified protein samples kept in pH Author's personal copy

5702 Appl Microbiol Biotechnol (2019) 103:5699–5713 conditions from 4.5 to 9.0 (with 0.5 as an interval) in 100 mM Protein localization analysis potassium phosphate buffer (PBS) at 30 °C and at temperature ranged from 20 to 60 °C (with 5 °C as an interval) in 100 mM The genomic DNA extracted from the cells of S. cerevisiae PBSatpH7.0,respectively.At0.25,0.5,1,2,3,4,5,and6h, BY4742 was used as a template to amplify the YKL107W the protein samples stored in PBS were used to determine the sequences with primers YKL107W_GFP_F and retained enzyme activities for substrate reduction at pH 7.0 YKL107W_GFP_R (Supplementary Material Table S1). The and 30 °C. PCR products were purified by a Cycle-Pure Kit (Omega Bio- Tek) and verified by sequence analysis (Sangon Biotech). The Effects of metal ions, chemical additives, salts, purified PCR product containing homologous sequences and and substrates the shuttle plasmid pDDGFP-2 digested with SmaIwerein- troduced into S. cerevisiae INVSc1 by the lithium acetate To evaluate the effect of metal ions on the enzyme activity of method (Gietz et al. 1995) to realize the homologous recom- Ykl107wp for the reduction of aldehydes, the reaction mixture bination in vivo and construct the recombinant plasmid was complemented by different concentrations of the pDDGFP-2 containing YKL107W (Drew et al. 2008). The selected metal ions (0.25, 0.5, 1.0, and 2.0 mM), including positive transformants were selected on SC-U medium sup- Mg2+,Ca2+,Ni2+,Cu2+,Mn2+,Zn2+,Co+,Fe3+, and Ag+. plemented with 2% glucose and verified by diagnostic PCR. For the effect analysis of chemical additives and salts on the After pre-incubation in SC-U medium supplemented with 2% enzyme activity of Ykl107wp, the final concentration of glucose at 30 °C overnight, the positive transformants were chemical additives and salts in the reaction system ranged transferred into SC-U medium supplemented with 2% galac- from 1 to 10 mM and 100 to 1000 mM, respectively. In this tose and 1% raffinose and incubated at 30 °C for inductive study, six chemical additives, glycerol, β-mercaptoethanol, expression of Ykl107w-GFP protein. After incubation for 24 ethylenediaminetetraacetic acid (EDTA), dithiothreitol and 48 h, the yeast cells were harvested and delivered for (DTT), sodium dodecyl sulfate (SDS), and Tween-20, and green fluorescence analysis by using the GFP filter lens of two salts Na+ (NaCl) or K+ (KCl) were included. For effective Axio Imager A2 (Carl Zeiss AG, Oberkochen, Germany). analysis of the substrate on the enzyme activities of During the subcellular localization analysis, the observation Ykl107wp, the protein samples were kept in 100 mM PBS of the cell structure was conducted with DIC filter lens. To supplemented with 5, 10, and 20 mM substrates (AA, GA, and observe the ER structure of cells containing Ykl107w-GFP FF) at pH 7.0. After incubation for 0.25, 0.5, 0.75, 1, and 2 h, protein, the above cells were harvested and washed twice with the cofactor was added to the mixture, and the retained en- sterile deionized water and resuspended in 100 mM PBS at pH zyme activity was detected. The retained enzyme activities of 7.0 and then added with 1 μlofa1mMERdye(ER- untreated samples in the same conditions were designated as Tracker™ Red, Thermo Fisher Scientific E34250). After in- background values for calculating relative activities. cubation at 30 °C for 30 min, the stained cells were washed with sterile deionized water and resuspended in 100 mM PBS Sequence analysis at pH 7.0. The observation of ER structure was conducted with the Rhod filter lens of Axio Imager A2. Based on the DNA sequence of YKL107W in the S. cerevisiae S288c, the aa sequence of Ykl107wp (GenBank accession no. ER membrane damage NP_012815.1) was deduced and used for Blastp (protein- protein BLAST) analysis in the National Center for The S. cerevisiae strain INVSc1 containing recombinant Biotechnology Information (NCBI) website (https://www. plasmid pYES2/NT B-YKL107W (INVSc1+)wasincubat- ncbi.nlm.nih.gov/). Via the ClustalW method in the ed in SC-U medium supplemented with 2% galactose and MegAlign program 7.1 (DNAStar Inc.), the aa sequence of 1% raffinose at 30 °C for the inductive expression of Ykl107wp and the similar aa sequences from other species Ykl107wp. The S. cerevisiae strain INVSc1 containing (coverage > 90%, identity > 50% from NCBI) were aligned pYES2/NT B (INVSc1−) was used as a control. After in- and analyzed. Based on the Conserved Domain Database cubation for 24 h, the yeast cells were transferred into SC- (CCD) (Marchler-Bauer et al. 2015, 2017)inNCBI,theprob- U medium supplemented with 2% galactose, 1% raffinose, able cofactor and catalytic active binding sites of Ykl107wp and 30 mM FF at a final cell concentration of 0.4 (optical and the similar proteins were predicted. For the phylogenetic density at 600 nm, OD600). Based on the methods men- analysis, Ykl107wp, the other aldehyde reductases from S. tioned above, the morphological changes of the ER in- cerevisiae, and the similar proteins mentioned above were duced by FF stress were monitored with the Rhod filter used to construct a phylogenetic tree using the neighbor- lens of Axio Imager A2. To ensure the accuracy of the joining method in the MEGA 6.0 program (Tamura et al. results, at least 100 cells were examined on each bright- 2013;Wangetal.2018). field image. Author's personal copy

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Results Table 1 Specific activities of Ykl107wp protein from S. cerevisiae for the reduction of aldehydes and ketones with 100 μMNADHorNADPH as the cofactor at 30 °C in 100 mM potassium phosphate buffer (pH 7.0) Gene cloning, enzyme activity, and kinetic parameters Substrate Specific activity (U/mg protein)

NADH NADPH The DNA fragment of YKL107W amplified from the S. cerevisiae BY4742 and plasmid pYES2/NT B digested Aldehydes Ι Ι with Not and Xho were employed to construct the recombi- Formaldehyde 0.62 ± 0.10 n.d. nant expression plasmid pYES2/NT B-YKL107W. The results Acetaldehyde 60.91 ± 1.02 n.d. from the diagnostic PCR test and DNA sequencing indicated Propionaldehyde 0.58 ± 0.01 n.d. that the recombinant expression plasmid pYES2/NT B- Butyraldehyde n.d. n.d. YKL107W was successfully constructed (Supplementary Isobutyraldehyde n.d. n.d. Material Fig. S1). The recombinant plasmid pYES2/NT B- Glycolaldehyde 5.29 ± 006 n.d. YKL107W was introduced into the S. cerevisiae strain Glutaraldehyde n.d. n.d. INVSc1 for overexpressing YKL107W. The recombinant pro- Hexaldehyde n.d. n.d. tein was extracted and purified for enzyme activity assays. Cinnamaldehyde n.d. n.d. Previous studies have shown that aldehyde reductases Isovaleraldehyde n.d. n.d. could catalyze the reduction of linear and branch-chain ali- Benzaldehyde n.d. n.d. phatic aldehydes and phenolic aldehydes (Larroy et al. Phenylacetaldehyde n.d. n.d. 2002a, b). Additionally, some of them have good enzymatic Syringaldehyde n.d. n.d. activity for the reduction of cinnamaldehyde, vanillin, and Vanillin n.d. n.d. furan aldehydes (furfural and HMF) which are derived from Furfural 2.56 ± 0.15 n.d. lignocellulosic hydrolysate (Larroy et al. 2002a, b; Liu et al. HMF n.d. n.d. 2008; Petersson et al. 2006;Wangetal.2017a). Therefore, the Ketones representatives from aliphatic, phenolic, and furan aldehydes Acetone n.d. n.d. were adopted as substrates for enzyme activity assays and Pyruvic acid n.d. n.d. shown in Table 1. The results illustrated that Ykl107wp could catalyze the reductive reactions of AA, GA, FF, formaldehyde 2-Pentanone n.d. n.d. (FA), and PAwith NADH as the cofactor. The highest enzyme Cyclopentanone n.d. n.d. activity (60.9 U/mg) was found in the reductive reaction of Acetylacetone n.d. n.d. AA catalyzed by this enzyme, followed by GA (5.29 U/mg), 2,6-Dimethyl-4-heptanone n.d. n.d. FF (2.56 U/mg), FA (0.62 U/mg), and PA (0.56 U/mg). There Values are presented by mean ± standard deviation (SD) (n =3).When was no enzyme activity for the reduction of the other 11 alde- acetaldehyde was used as a substrate, the concentration of NADH was hyde compounds with NADH as the cofactor (Table 1). In increased by fivefold addition, no NADPH-dependent enzyme activity was detected n.d. not detected for the reduction of all the selected aldehyde compounds (Table 1). For the reduction of ketones, Ykl107w did not dis- (Fig. 1a, b) was tested. The highest enzyme activity of play the catalytic activities using either NADH or NADPH as Ykl107wp for the reduction of AA was detected at pH the cofactor (Table 1). 6.0 (Fig. 1a). However, the optimum pH was pH 7.0 for Due to the low catalytic activities of Ykl107wp for the the reductive reaction of GA and FF catalyzed by reduction of FA and PA, we only analyzed its kinetic param- Ykl107wp. The enzyme activity of Ykl107wp for the re- eters for the reduction of AA, GA, and FF with NADH as the duction of AA remained 90% of maximal activities in the cofactor. With AA as the substrate, Ykl107wp displayed the range of pH conditions from 4.5 to 6.0 but dropped quickly best maximum velocity (Vmax), catalytic rate constant (Kcat), under pH > 6.0 conditions and decreased to less than 35% catalytic efficiency (Kcat/Km), and highest affinity (Km). When of the maximal activity at pH 9.0 (Fig. 1a). The catalytic GAwas used as the substrate, the lowest Km, Kcat,andKcat/Km activity of this enzyme for the reduction of GA and FF of Ykl107wp were characterized (Table 2). retained more than 70% of the maximal activities under acidic conditions (pH ranged from 4.5 to 7.0) but dropped Effects of pH and temperature, pH, and thermal quickly as the pH increased in alkaline condition (pH stability ranged from 7.0 to 9.0) and decreased to less than 35% of the maximal activities at pH 9.0 (Fig. 1a). These results The effect analysis of pH and temperature on the enzyme indicated that this enzyme was sensitive to alkaline condi- activity of Ykl107wp for the reduction of AA, GA, and FF tions when AA, GA, and FF were used as substrates. Author's personal copy

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Table 2 Kinetic parameters of Ykl107wp from S. cerevisiae for the reduction of multiple aldehydes with NADH as the cofactor

−1 −1 −1 Substrate Vmax (μmol/min mg) Km (mM) Kcat (s ) Kcat/Km (mM s )

Acetaldehyde 357.06 ± 0.00 2.09 ± 0.01 286.03 ± 0.00 139.43 ± 1.23 Glycolaldehyde 144.33 ± 21.56 131.82 ± 24.87 28.88 ± 4.26 0.22 ± 0.01 Furfural 101.68 ± 2.22 23.23 ± 0.39 40.67 ± 0.87 1.76 ± 0.13

Values are presented by mean ± standard deviation (SD) (n =3)

The highest enzyme activity of Ykl107wp was determined enzyme was determined at 25, 35, and 25 °C, respectively, and at 30, 35, and 40 °C for the reduction of AA, GA, and FF, the relative activity of Ykl107wp remained more than 50% in respectively (Fig. 1b). When temperature decreased from the these conditions (Fig. 2d–f). In view of the determination re- optimum temperature to 20 °C, its catalytic activities for the sults, it demonstrated that the relative activity of Ykl107wp reduction of AA and GA dropped quickly (Fig. 1b). In con- remained more than 50% in the above conditions for the re- trast, the enzyme activity of this protein for the reduction of FF duction of all the selected aldehydes after 6 h (Fig. 2d–f). This dropped slowly as the temperature decreased from 30 to 20 °C enzyme lost its catalytic activities for all the selected alde- and retained more than 84% of maximal activity at 20 °C hydes at 60 °C after 15 min and 50 °C after 5 h (Fig. 2d–f). (Fig. 1b). Meanwhile, the enzyme activities of this protein At 55 °C, Ykl107wp lost its catalytic activities for the reduc- for the reduction of all the selected aldehyde compounds tion of FF after 3 h and for the reduction of AA and GA after showed a significant decrease when the optimum temperature 2 h (Fig. 2d–f). increased to 60 °C and remained less than 20% of maximal activity at 60 °C (Fig. 1b). At the pH range of 4.5 to 9.0 for 6 h, stabilities of the Effects of metal ions, chemical additives, salts, enzyme activities of Ykl107wp for the reduction of AA, and substrates on enzyme activity GA, and FF were detected (Fig. 2a–c). Ykl107wp exhibited the best pH stability at pH 7.0 for the reduction of all the In the reduction reactions of FF, Mg2+,Ca2+, and Co+ en- selected aldehydes (Fig. 2a–c). At pH 5.5–7.0after6h,the hanced the enzyme activities of Ykl107wp. Mg2+,Ca2+, relative activity of Ykl107wp for the reduction of AA retained Mn2+,andFe3+ also could promote the enzyme activities of more than 50% (Fig. 2a). For the reduction of GA, relative Ykl107wp for the reduction of GA (Table 3). Except for the activities of this enzyme retained more than 58% at pH 5.0– above promotional effects on the enzyme activities of 8.0 after 6 h (Fig. 2b). At pH 5.0–7.0 after 6 h, relative activ- Ykl107wp, all the selected metal ions have varying degrees ities of Ykl107wp retained more than 55% in the reactions for of impact of enzyme activities for the reduction of AA, GA, the reduction of FF (Fig. 2c). The results showed that acidic and FF (Table 3). For the reduction of AA, GA, and FF, the conditions were more suitable for maintaining the stability of inhibitory degrees caused by Ni2+,Cu2+,andAg+ were more its enzyme activities for the reduction of AA, GA, and FF severe than those caused by the other seven metal ions. (Fig. 2a–c). Especially, Ykl107wp completely lost its catalytic activities Between the temperature range of 20 to 60 °C for the re- for the reduction of AA, GA, and FF in the presence of duction of AA, GA, and FF, the best thermal stability of this Cu2+and Ag+ (Table 3).

a b 120.0 120.0 AA AA ) 100.0 GA 100.0 GA %(ytivitcaevi FF FF 80.0 80.0

60.0 60.0

40.0 40.0 t aleR 20.0 20.0 Relative activity (%) Relative activity 0.0 0.0 45678910 0 10203040506070 pH Temperature (°C)

Fig. 1 Effect of pH and temperature on the enzyme activity of Ykl107wp. with NADH as the cofactor were measured in 100 mM potassium phos- Effects of pH (a) and temperature (b) on the activity of Ykl071wp for the phate buffer. Mean values of relative activity are presented with vertical reduction of acetaldehyde (AA), glycolaldehyde (GA), and furfural (FF) error bars representing standard deviation (n =3) Author's personal copy

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Fig. 2 pH and thermal stability of a b Ykl107wp. pH stability of 120.0 pH 4.5 120.0 pH 4.5 pH 5.0 pH 5.0

Ykl107wp for the reduction of ) 100.0 pH 5.5 100.0

% pH 5.5 acetaldehyde (a), glycolaldehyde (ytivitcaevitaleR pH 6.0 pH 6.0 80.0 (b), and furfural (c) at pH 4.5, 5.0, pH 6.5 80.0 pH 6.5 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, and pH 7.0 60.0 60.0 pH 7.0 9.0, respectively, at 30 °C for 6 h. pH 7.5 pH 7.5 Thermal stability of Ykl107wp 40.0 pH 8.0 40.0 pH 8.0 for the reduction of acetaldehyde pH 8.5 pH 8.5 20.0 pH 9.0 20.0 pH 9.0

(d), glycolaldehyde (e), and activity Relative (%) furfural (f) was determined at 20, 0.0 0.0 25, 30 35, 40, 45, 50, 55, and 01234567 01234567 60 °C, respectively, at pH 7.0 for Time (h) Time (h) 6 h. Mean values of relative activity are presented with c d vertical error bars representing 120.0 pH 4.5 120.0 20 °C pH 5.0 25 °C standard deviation (n =3) )%(yti 100.0 100.0 pH 5.5 30 °C pH 6.0 80.0 35 °C 80.0 pH 6.5 vitcaevitaleR 40 °C 60.0 pH 7.0 60.0 pH 7.5 45 °C 40.0 pH 8.0 40.0 50 °C pH 8.5 55 °C 20.0 20.0 pH 9.0 activity (%) Relative 60 °C 0.0 0.0 01234567 01234567 Time (h) Time (h)

e f 120.0 120.0 20 °C 20 °C 25 °C 25 °C )%(y 100.0 100.0 30 °C 30 °C 80.0 35 °C ti 80.0 35 °C vitc 40 °C 40 °C 60.0 60.0 45 °C a 45 °C

e 50 °C vitaleR 40.0 50 °C 40.0 55 °C 55 °C 20.0 20.0 60 °C activity Relative (%) 60 °C 0.0 0.0 01234567 01234567 Time (h) Time (h)

Tween-20 severely hampered the enzyme activity of inhibition effects on enzyme activity for the reduction of AA Ykl107wp for the reduction of AA at either concentration caused by these two salts were more serious than that of GA (from 1 to 10 mM) (relative activity < 10%), followed by β- (Fig. 4a, b). In the reactions for the reduction of FF, NaCl mercaptoethanol and glycerol (Fig. 3a). In contrast, DTT could not lead to the inhibition of enzyme activity of slightly increased the enzyme activity of Ykl107wp for the Ykl107wp at any concentration (from 100 to 1000 mM), but reduction of AA only at higher concentrations (5 and KCl caused severe inhibition effect on its enzyme activity at a 10 mM) but not at low concentration (1 mM) (Fig. 3a). lower concentration (100 mM) (Fig. 4c). When GA was used as the substrate, only β- Previous studies showed that FA, malonaldehyde (MA), mercaptoethanol showed inhibitory effect on the enzyme ac- and other products of oxidative lipid degradation (alde- tivity of Ykl107wp at high concentrations (5 and 10 mM) hydes and ketones) could lead to the denaturation, texture, (Fig. 3b). For the reduction of FF, enzyme activities of and decreased enzymatic activities of myofibrillar proteins Ykl107wp were slightly inhibited by glycerol and EDTA (rel- (Kikugawa 1986; Rehbein and Orlick 1990). We speculat- ative activity > 75%) but greatly inhibited by β- ed that when the purified Ykl107wp was exposed to alde- mercaptoethanol and Tween-20 from concentrations of 1 to hydes, its enzyme activity was probably affected by these 10 mM (Fig. 3c). aldehydes. Therefore, we conducted several experiments to For the reduction of AA and GA, the activity of this en- evaluate the effects of AA, GA, and FF on the enzyme zyme showed a declined trend when the concentrations of activity of Ykl107wp (Fig. 5a–c). As shown in Fig. 5,after NaCl/KCl were increasing (Fig. 4a, b). In contrast, the treatment by various concentrations of AA, GA, and FF Author's personal copy

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Table 3 Effect of metal ions on enzyme activity of Ykl107wp from Protein sequence analysis S. cerevisiae for the reduction of multiple aldehydes

Metal ion Conc. (mM) Acetaldehyde Glycolaldehyde Furfural Based on the DNA sequence of YKL107W from S. cerevisiae S288c, we deduced the amino acid sequence of Ykl107wp Control 0 100.0 ± 4.8 100.0 ± 4.2 100.0 ± 0.1 with 309 aa residues (Fig. 6). The protein BLAST analysis 2+ Mg 0.5 83.1 ± 0.5 119.8 ± 0.2 107.8 ± 0.2 from NCBI showed that several proteins like Ykl107wp from 2+ Mg 1.0 81.5 ± 0.3 111.4 ± 2.2 103.9 ± 0.1 S. cerevisiae were discovered from other species, including Mg2+ 2.0 80.1 ± 2.3 103.0 ± 0.4 89.5 ± 1.0 Saccharomyces kudriavzevii × S. cerevisiae, S. kudriavzevii, Ca2+ 0.5 95.4 ± 0.6 115.2 ± 0.9 94.3 ± 0.2 Saccharomyces eubayanus,andNaumovozyma dairenensis. Ca2+ 1.0 95.4 ± 0.7 92.9 ± 0.7 110.6 ± 0.9 Given the alignment analysis of amino acid sequences, these Ca2+ 2.0 94.8 ± 0.6 70.3 ± 0.7 103.8 ± 0.2 proteins were found to share a cofactor-binding domain Ni2+ 0.5 28.1 ± 3.1 44.2 ± 2.2 89.0 ± 0.5 (GGxxGxG) and several conserved amino acid residues but Ni2+ 1.0 22.2 ± 0.7 48.2 ± 2.4 80.8 ± 0.1 no typical catalytic domain (Fig. 6). A comparison of the Ni2+ 2.0 18.2 ± 2.6 61.5 ± 0.2 73.2 ± 0.3 cofactor-binding domain with the classification standards in Cu2+ 0.25 9.6 ± 1.0 4.0 ± 0.2 3.8 ± 0.1 SDRs showed that Ykl107wp could be roughly classified into Cu2+ 0.5 n.d. n.d. n.d. the classical SDR group (Supplementary Material Table S2). Cu2+ 1.0 n.d. n.d. n.d. It was found that Ykl107wp from S. cerevisiae together with Mn2+ 0.5 80.1 ± 0.5 107.6 ± 1.2 99.4 ± 0.2 Ykl107wp-like proteins from other species and Ykl071wp Mn2+ 1.0 80.5 ± 0.6 110.4 ± 0.4 99.9 ± 0.2 were clustered in the classical SDR group (B), which has a Mn2+ 2.0 81.8 ± 0.5 122.2 ± 2.4 96.2 ± .0.2 greater distance from the AKRs (D), the non-zinc-containing Zn2+ 0.5 53.9 ± 2.6 70.8 ± 4.2 65.1 ± 0.4 MDRs (E), and the zinc-containing MDRs (F) but is closer to Zn2+ 1.0 59.9 ± 3.5 60.1 ± 0.6 60.4 ± 1.0 the atypical (A) and intermediate SDR (C) groups (Fig. 6). Zn2+ 2.0 60.8 ± 0.3 49.6 ± 0.4 58.3 ± 1.2 Co+ 0.5 58.7 ± 1.2 99.6 ± 0.4 105.8 ± 0.2 Co+ 1.0 57.7 ± 1.3 97.2 ± 0.9 116.7 ± 1.1 Protein localization and ER membrane damage Co+ 2.0 44.5 ± 1.7 87.6 ± 0.6 119.6 ± 0.3 Fe3+ 0.5 65.8 ± 0.5 88.3 ± 1.1 99.1 ± 1.0 After induction for 24 h, the Ykl107w-GFP proteins were Fe3+ 1.0 63.2 ± 0.8 104.0 ± 0.2 99.8 ± 0.2 localized in the structure-like network which was probably Fe3+ 2.0 64.8 ± 0.8 106.3 ± 2.1 98.8 ± 1.1 ER in our conjecture (Fig. 7a). Therefore, ER dye, ER- ™ Ag+ 0.25 5.3 ± 0.2 n.d. n.d. Tracker Red, was adopted to stain these cells for detecting Ag+ 0.5 4.5 ± 0.3 n.d. n.d. the morphological structure of ER (Fig. 7b). As illustrated in Ag+ 1.0 n.d. n.d. n.d. Fig. 7a, b, it was found that the subcellular localization of the Ykl107w-GFP proteins overlapped the structure stained by Values are presented by mean ± standard deviation (SD) of relative spe- ER dye. Given the above results, we confirmed that the sub- cific activity to control (n =3) cellular localization of Ykl107w-GFP proteins was ER. Conc. concentration, n.d. not detected However, after the overexpression of Ykl107w-GFP proteins for 48 h, it was discovered that the Ykl107w-GFP proteins in (except for 5 mM AA), the results demonstrated that the few cells localized to cytoplasm and ER (Fig. 7d–f). enzyme activities of Ykl071wp for the reduction of sub- Additionally, cells in the S. cerevisiae strain INVSc1 con- strates (FF and GA) were significantly lost and displayed a taining recombinant plasmid pYES2/NT B-YKL107W decreasing trend at the extension of time (Fig. 5a–c). (INVSc1+) after incubation in inductive medium for 24 h were Especially, after treatment by 20 mM FF and GA for 2 h, transferred into the SC-U medium with 30 mM FF. its relative activities for reduction of substrates were less Meanwhile, the S. cerevisiae strain INVSc1 containing than 25% (Fig. 5a–c). High concentrations of AA (10 and pYES2/NT B (INVSc1−) was used as a control. After treat- 20 mM) exhibited more impacts on the enzyme activity of ment by FF for 3 h, the ER structures of the treated cells were Ykl107wp for the reduction of AA than those in low con- observed. The ER structures were grouped into two types, centration (5 mM) condition (Fig. 5a–c). Under varied con- including normal (distinct) and abnormal (diffuse, unsharp) centrations, the relative activity of Ykl107wp treated by (Fig. 7g). As illustrated in Fig. 7h, the percentages of cells in GA was always significantly lower than that of FF and INVSc1+ and INVSc1− containing abnormal ER were 5.5% AA after treatment for 2 h, which implied that GA could and 5.0%, respectively, in condition without furfural. In con- lead to the most severe inhibition of enzyme activity of trast, after treatment for 3 h, the percentage of cells in Ykl107wp for the reduction of substrate, followed by FF INVSc1+ and INVSc1− containing abnormal ER soared to and AA (Fig. 5a–c). 85.7% and 93.3%, respectively (Fig. 7h), which implied that Author's personal copy

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Glycerol -Mercaptoethanol Glycerol -Mercaptoethanol a EDTA DTT b EDTA DTT 120.0 SDS Tween-20 120.0 SDS Tween-20

100.0 100.0

80.0 80.0

60.0 60.0

40.0 40.0 Relative activity (%) Relative activity (%) 20.0 20.0

0.0 0.0 0246810 0246810 Concentration (mM) Concentration (mM)

c Glycerol β-Mercaptoethanol EDTA DTT 140.0 SDS Tween-20 120.0

100.0

80.0

60.0

40.0 Relative activity (%) 20.0

0.0 0246810 Concentration (mM) Fig. 3 Effects of chemical additives on the enzyme activity of Ykl107wp. determined under different additive concentrations. Mean values of Effects of chemical additives on the enzyme activity of Ykl107wp for the relative activity are presented with vertical error bars representing reduction of acetaldehyde (a), glycolaldehyde (b), and furfural (c)were standard deviation (n =3)

Fig. 4 Effects of salts on the KCl KCl a b 120.0 enzyme activity of Ykl107wp. 120.0 NaCl NaCl Effects of salts on the enzyme 100.0 ） 100.0

activity of Ykl071wp for the % 80.0 reduction of acetaldehyde (a), （ 80.0 ytividaevitaleR glycolaldehyde (b), and furfural 60.0 (c)weredeterminedunder 60.0 40.0 different salt concentrations. 40.0 Mean values of relative activity 20.0 are presented with vertical error 20.0 activity Relative (%) 0.0 bars representing standard 0.0 0 200 400 600 800 1000 deviation (n =3) 0 200 400 600 800 1000 Concentration (mM) Concentration (mM)

KCl c 120.0 NaCl )%(ytivitcaevitaleR 100.0

80.0

60.0

40.0 20.0 0.0 0 200 400 600 800 1000 Concentration (mM) Author's personal copy

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Fig. 5 Effects of substrates on the a AA GA FF b AA GA FF enzyme activity of Ykl107wp. 120.0 120.0 Effects of acetaldehyde, 100.0 100.0 glycolaldehyde, and furfural on )%(ytivitcaevitaleR the enzyme activity of Ykl071wp 80.0 80.0 for their reductions were determined under different 60.0 60.0 substrate concentration, such as 5mM(a), 10 mM (b), and 20 mM 40.0 40.0 (c), for 2 h. Mean values of relative activity are presented with 20.0 activity (%) Relative 20.0 vertical error bars representing 0.0 0.0 standard deviation (n =3) 0306090120 0306090120 Time (min) Time (min)

AA GA FF c 120.0

100.0 )%(ytivitcaevitaleR

80.0

60.0

40.0

20.0

0.0 0306090120 Time (min) the damage of cell in INVSc1+ induced by furfural is signifi- AA, which implied that the overexpression of YKL107W cantly lower than that in INVSc1−. could accelerate the conversion of acetaldehyde to ethanol and probably improve the ethanol yield in the process of fermentation (Liu 2011;Wangetal.2018). Although Ykl107wp exhibited high catalytic activity on the reduction Discussion of AA but was greatly affected by metal ions, chemical additives, and salts, it showed poor thermal and pH stability for A previous study proposed that Ykl107wp was probably a the reduction of AA, except for the effect of substrate. palmitoylated membrane protein, but the molecular function According to the effect analysis of AA, GA, and FF on the of this protein remained unknown (Lopez-Villar et al. 2006). enzyme activity of Ykl107wp, we found that the enzyme Since Ykl107wp displayed the similarity in sequences and activity of Ykl107wp for the reduction of substrate was se- functional motifs of aldehyde reductases belonging to the riously affected by GA, followed by FF and AA (Fig. 5a–c), SDR family, such as Ykl071wp and Yll056cp, Ykl107wp which showed the same trend with the affinity of Ykl107wp was deduced to have catalytic activities on reduction of al- for aldehydes. As illustrated in Table 2, Ykl107wp showed dehydes. Therefore, we cloned YKL107W and harvested the the lowest affinity for GA, followed by FF and AA. purified protein via its overexpression for enzyme activity assays. The results showed that Ykl107wp could catalyze the reductive reactions of at least five aldehydes, including Fig. 6 Alignment analysis of amino acid sequences of Ykl107wp and phylogenetic analysis. (a) Amino acid sequences of Ykl107wp from aliphatic aldehydes of AA, GA, FA, and PA, and furan alde- S. cerevisiae were aligned with similar proteins from other four species. hyde of FF. According to the results from the enzyme activity Identical amino acid residues are marked with a star on top. The assays, Ykl107wp, like Ykl071wp and Yll056cp, exhibited conserved cofactor-binding region is boxed with bold black lines.(b) strict preference for NADH (Wang et al. 2017a, b). In con- The phylogenetic tree was constructed using Ykl107wp and other characterized aldehyde reductases and Ykl107w-like proteins. A, atypical trast, as the similarity in protein sequences of these three SDR group; B, Ykl107wp and Ykl107w-like proteins; C, classical SDR aldehyde reductases is slightly low, the molecular mecha- group; D, intermediate SDR group; E, AKR family; F, non-zinc- nism of their NADH preference could not be concluded. containing MDR cluster; G, zinc-containing MDRs cluster. Sc, Moreover, among the 25 aldehyde reductases identified in Saccharomyces cerevisiae; Sk, Saccharomyces kudriavzevii; Se, Saccharomyces eubayanus; and Nd, Naumovozyma dairenensis. The S. cerevisiae (Wang et al. 2018), Ykl107wp was found to GenBank accession number for each protein is provided after the species display the highest catalytic activity in the reduction of name Author's personal copy

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a ** *** * ** * * * * *** * ***** ** * * * *** * Ykl107wp (Sc)(NP_012815.1) MFW- -KKDPTVSWERKNINDIDFSRFNVAI IGGTGGLGRAI SRELAQRNARVTVVGQTFR 58 Ykl107wp (Sk)(EJT44183.1) MFW- -KKDPSVTWERKNINNIDFSHLNVAI IGGTGGIGRAISRELAQRDAKVTVVGQTFR 58 Ykl107wp (ScxSk)(EHN01530.1) MFW- -KKDPSVTWERKNINNIDFSHLNVAI IGGTSGIGRAISRELAQRDAKVTVVGQTFR 58 Di49_3269 (Se)(XP_018220937.1) MFW--KKDPSVTWERKNINNIDFSYLNVAI IGGTGGIGRAISRELAQRDAKVTVVGQTFK 58 Ndai_0c03930 (Nd)(XP_003669296.1) M F F G T K K D N S V K W E H K D I S Q L D L C K V N A V V F G G T S G I G R A I S H Q L A D R G A N V L V V G R H F K 60

*** ** * ***** * ** *** ** ****** * * * ** * * Ykl107wp (Sc)(NP_012815.1) DEDLKDKINFVKADLSLVSECKR ISHSDEIPYEELTHLIFTTGIFASRQRQATSEGLEKD 118 Ykl107wp (Sk)(EJT44183.1) DEDLKDKIKFVKADLSLASECKR ISHSDEIPYEELTHLIFTTGIFASRQKQITSEGLEKD 118 Ykl107wp (ScxSk)(EHN01530.1) DEDLKDKIKFVKADLSLASECKR ISHSDEIPYEELTHLIFTTGIFASRQKQITSEGLEKD 118 Di49_3269 (Se)(XP_018220937.1) D E D L A D K I K F V K A D L S L V S E C K R I S H S D E I P Y E K L T H L I F T T G I I A S R Q R Q A T S E G L E K D 118 Ndai_0c03930 (Nd)(XP_003669296.1) D E D M K - K I K F L H A D L S L I T E A D R I A N - - E I P A K D I T H M I F T T G I V A A P Q R E Q T P E G I E R D 117

* * * ***** * ** ****************** Ykl107wp (Sc)(NP_012815.1) MAVSYLSRY I IFHDVAKRLGISRTKKDDLPKVFIAGFPGNGQVGDPDDLNSDEKKYSAYA 178 Ykl107wp (Sk)(EJT44183.1) MAVSYLSRY I IFHDVANRLGINRMKKDDLPKVFVAGFPGNGQMGDPDDLNSDGKNYSAYA 178 Ykl107wp (ScxSk)(EHN01530.1) MAVSYLSRY I IFHDVANRLGINRMKKDDLPKVFVAGFPGNGQMGDPDDLNSDGKNYSAYA 178 Di49_3269 (Se)(XP_018220937.1) M A V S Y L S R Y I I F H D V A K R L G T N R T K K D D L P K V F I A G F P G N G Q L G D P D D L N S D E K N Y S A Y A 178 Ndai_0c03930 (Nd)(XP_003669296.1) M A V S Y L S R Y M L I R E L A G K M G K G L P H N A S K P R I F I M G F P G S G Q L G D P E N L N S D K K K Y N A L S 177

************** * ************** ** ** Ykl107wp (Sc)(NP_012815.1) THMNTVAANESLV IDAKDRYTNI DTFGLNPGLIKTNIRNNLLGSDTYLSRITEWI ISWTC 238 Ykl107wp (Sk)(EJT44183.1) THMNTVAANESLVLDAKYRFTNI DTFGLNPGLIKTNIRSNLLGSDSYLSRITEWI ISWTC 238 Ykl107wp (ScxSk)(EHN01530.1) THMNTVAANESLVLDAKYRFTNI DTFGLNPGLIKTNIRSNLLGSDSYLSRITEWI ISWTC 238 Di49_3269 (Se)(XP_018220937.1) T H T N T V A A N E C L V L D A K D R Y T N I D T F G L N P G I I K T N I R S N L L G S N T Y I G R I A E W I I S W T F 238 Ndai_0c03930 (Nd)(XP_003669296.1) T H S N T V A A N E A L V L D S K D R Y K N V E V F G L N P G I I K T E I R N N Y L G K D S I F S K A V E W V V G W T C 237

********** ********************* Ykl107wp (Sc)(NP_012815.1) QSAETYAKT ICTL IASPAIESRSGTMFSNKGDAILPSPGLTKDVVEKFMENSELLVEKAL 298 Ykl107wp (Sk)(EJT44183.1) QSAETYAKMICTL IVSPAIESRSGTMFSNKGDAILPTPGLTKDVVGKFMKNSELLVEKAL 298 Ykl107wp (ScxSk)(EHN01530.1) QSAETYAKMICTL IVSPAIESRGGTMFSNKGDAILPTPGLTKDVVGKFMKNSELLVEKAL 298 Di49_3269 (Se)(XP_018220937.1) Q S A E T Y A K M I C T L I V S P A I E S R S G T M F S N K G D A I L P T P G L T K N V V E K F M E N S E L L V E K A L 298 Ndai_0c03930 (Nd)(XP_003669296.1) Q T P E D Y A K N I C P L L V S P D L D N K S G T M F D N K G N A I L Q S A G L A P E V V H N F I H N S A V L V T K V L 297

** Ykl107wp (Sc)(NP_012815.1) RNQSPFTSSNE-- 309 Ykl107wp (Sk)(EJT44183.1) QSQSPSTSSNE-- 309 Ykl107wp (ScxSk)(EHN01530.1) QSRSPSTSSNE-- 309 Di49_3269 (Se)(XP_018220937.1) Q N Q S P P T S S N E - - 309 Ndai_0c03930 (Nd)(XP_003669296.1) A M K P N E T T S K L P V 311

b Yll056cp (Sc)(NP_013044.1) A

100 Ykl107wp (Sk)(EJT44183.1) 83 Ykl107wp (ScxSk)(EHN01530.1) 24 100 Ykl107wp (Sc)(NP_012815.1) B 100 Di49_3269 (Se)(XP_018220937.1) 54 Ndai_0c03930 (Nd)(XP_003669296.1) Ykl071wp (Sc)(NP_012352.1) C Sc 35 100 Ari1p ( )(NP_011358.3) Ygl039wp (Sc)(NP_011476.1) D 100 Gre2p (Sc)(NP_014490.1) 74 Ydr541cp (Sc)(NP_010830.4) Adh4p (Sc)(NP_011258.2) Sc 31 Gre3p ( )(NP_011972.1) E 100 Yjr096wp (Sc)(NP_012630.1) Ald4p (Sc)(NP_015019.1)

40 Yml131wp (Sc)(NP_013575.1) 22 F Ynl134cp (Sc)(NP_014265.3) Sfa1p (Sc)(NP_010113.1) 61 100 Adh6p (Sc)(NP_014051.3) 91 Adh7p (Sc)(NP_010030.1) G 82 Adh5p (Sc)(NP_009703.3) Sc 100 Adh3p ( )(NP_013800.1) 56 Adh1p (Sc)(NP_014555.1)

0.2

However, the molecular mechanism is still unknown and Based on protein sequence alignment analysis and phylo- maybe associated with the structure and substrate-binding genetic relationship, Ykl107wp was identified as the second motifs of this enzyme. aldehyde reductase in the classical SDR group (Fig. 6). Author's personal copy

5710 Appl Microbiol Biotechnol (2019) 103:5699–5713

Usually, proteins from the classical group (~ 250 aa residues) classical SDR group, retinol dehydrogenase (retinol-DH) were smaller than those from the intermediate (250–350 aa and light-dependent protochlorophyllide (Pchlide) oxidore- residues), extended (~ 350 aa residues), and atypical group ductase (LPOR) (Marchler-Bauer et al. 2015, 2017). To fur- in the SDR family (Kallberg et al. 2002;Wangetal.2017a, ther explore the conserved domains, we conducted the se- b). However, Ykl107wp, as a member of classical SDRs, is quence alignment analysis of Ykl107wp and these two pro- composed of 309 aa residues. The sequence length of this teins (Perrault et al. 2004; Yamazaki et al. 2006) (data not protein is significantly longer than that of typical classical shown). We found that the retinol-DH and LPOR owned SDRs, which proved that Ykl107wp is distinct from these NADH/NADPH-binding motif (GxxxGxxG) and active sites proteins. Based on the alignment analysis of aa sequence (YxxxK). In contrast, since the comparability coefficient be- and the exploration of conserved motifs, the results demon- tween the Ykl107wp and these two proteins was low, no typ- strated that Ykl107wp only had typical cofactor-binding sites ical active sites (YxxxK or YxxxN or YxxMxxxK) were dis- (GGxxGxG) (Fig. 6). Additionally, BLAST analysis showed covered in the structure of Ykl107wp (Kavanagh et al. 2008). that Ykl107wp was similar to the identified proteins in the Additionally, SWISS-MODEL analysis (https://www.

a b c GFP ER-Tracker DIC Red dye

5 μm 5 μm 5 μm

d e f GFP ER-Tracker DIC Red dye

5 μm 5 μm 5 μm

ER-Tracker g Red dye DIC h

INVSc1+ INVSc1- 120.0

Normal 100.0

80.0 5 μm 5 μm 60.0

40.0

20.0 Cells with abnormal ER ER (%) withabnormal Cells Abnormal 0.0 0 h 3 h 5 μm 5 μm

Fig. 7 Localization of Ykl107wp in S. cerevisiae and the damages of DIC lens and used as a control. The cells in the S. cerevisiae strain endoplasmic reticulum in different strains induced by furfural. INVSc1 containing recombinant plasmid pYES2/NT B–YKL107W Localization of Ykl107wp was analyzed by measuring the green (INVSc1+) and the S. cerevisiae strain INVSc1 containing pYES2/NT fluorescence of Ykl107wp-GFP protein using confocal microscopy. B (INVSc1−) were treated by 30 mM furfural for 3 h. Images of the Images of Ykl107wp-GFP protein after incubation in inductive medium treated cells (g) stained with ER-specific dye were shown in the left for 24 (a)and48h(d) were respectively taken by using a GFP lens, column, and images taken by using a DIC lens were shown in the right images of the same cells (b and e) stained with the endoplasmic reticulum column. Percentage of abnormal cells from strain INVSc1+ and INVSc1− (ER)-specific dye, ER-Tracker™ Red, were respectively taken by using a (h) under 30 mM furfural at 0 and 3 h. Data represent averages of three Rhod lens, and images of the same cells (c and f) were taken by using a experiments. At least 100 cells were examined on each bright-field image Author's personal copy

Appl Microbiol Biotechnol (2019) 103:5699–5713 5711 swissmodel.expasy.org/interactive)showedthatnotemplate Compliance with ethical standards is suitable for building the model of Ykl107wp and its conserved active sites could not be characterized. To sum Ethical approval This article does not contain any studies with human up, Ykl107wp is identified as an atypical aldehyde reductase participants or animals performed by any of the authors. containing unusual active sites in the classical group of SDR Competing interests The authors declare that they have no competing family. This research provides guidelines for the study of more interests. uncharacterized atypical aldehyde reductases in the classical SDR group from S. cerevisiae and other organisms. Subcellular localization analysis showed that Ykl107wp was References localized in ER. Given the previous studies about aldehyde reductases, we concluded that Ykl107wp was the first aldehyde Allen SA, Clark W, McCaffery JM, Cai Z, Lanctot A, Slininger PJ, Liu reductase completely localized in ER (Huh et al. 2003;van ZL, Gorsich SW (2010) Furfural induces reactive oxygen species Loon and Young 1986;Wangetal.2017a, 2018;Yofeetal. accumulation and cellular damage in Saccharomyces cerevisiae. Biotechnol Biofuels 3:2. https://doi.org/10.1186/1754-6834-3-2 2016) (Supplementary Material Table S3). Given the previous Chen CN, Porubleva L, Shearer G, Svrakic M, Holden LG, Dover JL, studies, we speculated that the aldehyde reductases localized in Johnston M, Chitnis PR, Kohl DH (2003) Associating protein ac- different organelles might play a protective role by detoxifica- tivities with their genes: rapid identification of a gene encoding a tion of endogenous aldehydes in these organelles (Allen et al. methylglyoxal reductase in the yeast Saccharomyces cerevisiae. Yeast 20(6):545–554. https://doi.org/10.1002/yea.979 2010; Voulgaridou et al. 2011). Thus, we conducted several Drew D, Newstead S, Sonoda Y, Kim H, von Heijne G, Iwata S (2008) experiments to evaluate the protective mechanism of GFP-based optimization scheme for the overexpression and purifi- Ykl107wp on the ER when the yeast cells were exposed to cation of eukaryotic membrane proteins in Saccharomyces – furfural. The results demonstrated that the damage of ER in cerevisiae. Nat Protoc 3(5):784 798. https://doi.org/10.1038/nprot. 2008.44 INVSc1+ induced by FF was significantly lower than that in − Gietz RD, Schiestl RH, Willems AR, Woods RA (1995) Studies on the INVSc1 (Fig. 7h), which proved that the overexpression of transformation of intact yeast cells by the LiAc/SS-DNA/PEG pro- Ykl107wp could reduce the damages of ER induced by alde- cedure. Yeast 11(4):355–360. https://doi.org/10.1002/yea. hydes. 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The overexpression of 98-100:1-9:327 Ykl107wp will effectively retain the ER homeostasis in lignocel- Hauser M, Horn P, Tournu H, Hauser NC, Hoheisel JD, Brown AJ, lulosic hydrolysate containing a series of aldehydes and then can Dickinson JR (2007) A transcriptome analysis of isoamyl alcohol- induced filamentation in yeast reveals a novel role for Gre2p as improve the tolerance of S. cerevisiae to the aldehyde inhibitors isovaleraldehyde reductase. FEMS Yeast Res 7(1):84–92. https:// derived from lignocellulose by detoxification of aldehydes and doi.org/10.1111/j.1567-1364.2006.00151.x shorten the lag phase of cell growth caused by furfural and other Hazelwood LA, Daran JM, van Maris AJ, Pronk JT, Dickinson JR (2008) aldehydes (Gutiérrez et al. 2002;Liuetal.2004). When Ykl107- The Ehrlich pathway for fusel alcohol production: a century of research on Saccharomyces cerevisiae metabolism. 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Appl Microbiol Biotechnol 97(14):6589–6600. https://doi.org/10. the corresponding organelles. 1007/s00253-013-4997-4 Jornvall H, Landreh M, Östberg LJ (2015) Alcohol dehydrogenase, SDR and MDR structural stages, present update and altered era. Chem Funding This work was supported by the National Natural Science Biol Interact 234:75–79. https://doi.org/10.1016/j.cbi.2014.10.017 Foundation of China (No. 31570086), the 2011 Collaborative Kallberg Y, Oppermann U, Jornvall H, Persson B (2002) Short-chain Innovation Center for Farmland Protection and Agricultural Product dehydrogenases/reductases (SDRs): coenzyme-based functional as- Safety in Sichuan Province, and the Talent Introduction Fund of signments in completed genomes. Eur J Biochem 269(18):4409– Sichuan Agricultural University (No. 01426100). 4417. https://doi.org/10.1046/j.1432-1033.2002.03130.x Author's personal copy

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