Direct enzyme assay evidence confirms aldehyde reductase function of Ydr541cp and Ygl039wp from Saccharomyces cerevisiaeby Jaewoong Moon, Z. Lewis Liu

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Direct enzyme assay evidence concur aldehyde reductase function of Ydr541cp and

Ygl039wp from Saccharomyces cerevisiae

Jaewoong Moon+ and Z. Lewis Liu *

BioEnergy Research Unit, National Center for Agricultural Utilization Research, USDAARS, Peoria, IL 61604

Running title

Aldehyde reductase function of Ydr541cp and Ygl039wp *Correspondence author:

Dr. Z. Lewis Liu

U.S. Department of Agriculture

Agricultural Research Service

National Center for Agricultural Utilization Research 1815 N University St.

Peoria, IL 61604

USA

Tel: +1 309 681 6294

Fax: +1 309 681 6427

Email: ZLewis.Liu@ars.usda.gov +

Current address: Xyleco Inc., 271 Salem St Woburn, MA 01801

This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1002/yea.3067

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Abstract

Aldehyde reductase gene ARI1 is a recently characterized member of intermediate subfamily under SDR (short-chain dehydrogenase/reductase) superfamily that clarified mechanisms of in situ detoxification of 2-furaldehyde and 5-hydroxymethyl-2-furaldehyde by

Saccharomyces cerevisiae. Uncharacterized open reading frames (ORF) are common among tolerant candidate genes identified for lignocellulose-to-advanced biofuels conversion. This study presents partially purified proteins of two ORFs, YDR541C and YGL039W, and direct enzyme assay evidence against aldehyde inhibitory compounds commonly encountered during lignocellulosic biomass fermentation processes. Each of the partially purified proteins encoded by these ORFs showed a molecular mass of approximately 38 kDa, similar to Ari1p, a protein encoded by aldehyde reductase gene. Both proteins demonstrated strong aldehyde reduction activities toward 14 aldehyde substrates with high levels of reduction activity for

Ydr541cp toward both aromatic and aliphatic aldehydes. While Ydr541cp was observed to have a significantly higher specific enzyme activity at 20 U/mg using co-factor NADPH,

Ygl039wp displayed a NADH preference at 25 U/mg in reduction of butylaldehyde. Amino acid sequence analysis identified a characteristic catalytic triad, Ser, Tyr and Lys; a conserved catalytic motif of Tyr-X-X-X-Lys; and a cofactor-binding sequence motif Gly-XX-Gly-X-X-Ala near the N-terminus that are shared by Ydr541cp, Ygl039wp,

Yol151wp/GRE2, and Ari1p. Findings of aldehyde reductase genes contribute to the yeast gene annotation and aids development of the next-generation biocatalyst for advanced biofuels production.

Key words: aldehyde reductase family; direct enzyme assay; gene annotation; in situ detoxification; Saccharomyces cerevisiae

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Introduction

For advanced biofuels production from lignocellulosic biomass including agricultural and industrial processing residues, a pretreatment of the biomass is needed in order to release fermentable sugars for microbial use. Depolymerization of cellulose and hemicellulose materials typically produces toxic byproducts that inhibit microbial growth and fermentation.

Knowledge on inhibitory compounds liberated from lignocellulose pretreatment is well known (Klinke et al., 2004; Larson et al., 1999; Liu and Blaschek, 2010; Palmqvist e al., 2000). Remediation of inhibitory compounds by additional physical and chemical means appears too expensive and economically impractical (Liu and blaschek, 2010). Using tolerant ethanologenic strains of Saccharomyces cerevisiae to in situ detoxify lignocellulosic inhibitors, such as 2-furaldehyde (2-furancarbaldehyde; or furfural) and 5-(hydroxymethyl)2-furaldehyde (5-hydroxythyl-2-furancarbaldehyde; or HMF) while producing ethanol was successful (Liu and Moon, 2009; Liu et al., 2009). It was concluded that multiple genemediated NAD(P)H-dependent aldehyde reduction is one of the most significant mechanisms of the in situ detoxification for the tolerant yeast (Heer et al., 2009; Liu et al., 2008). New pathways and reprogrammed metabolic pathways were discovered for an improved tolerant industrial yeast strain to maintain cofactor regeneration balance in S. cerevisiae (Liu et al., 2009). Key regulatory elements and molecular mechanisms were identified at the genome level for the yeast tolerance and the in situ detoxification (Ma and Liu, 2010; Yang et al., 2012). However, with a large number of genes involved in yeast tolerance and detoxification, many genes and open reading frames, including tolerance candidate genes, in

S. cerevisiae genome remain uncharacterized.

We recently characterized a novel NADPH-dependent aldehyde reductase gene ARI1

This article is protected by copyright. All rights reserved. from S. cerevisiae, as a member of the subclass intermediate under the SDR superfamily (short-chain dehydrogenase/reductase) (Liu and Moon, 2009; http://www.yeastgenome.org/].

Enzyme kinetics of this reductase has been characterized and its stereochemistry in furan aldehyde reduction established (Bowman et al., 2010; Jordan et al., 2011). Several ORFs such as YDR541C, YGL039W, and YOL151W (GRE2) were observed to have similar enhanced gene expression response to furfural or HMF (Liu, 2006; Liu and Slininger, 2006;

Liu and Moon, 2009; Moon and Liu, 2012). These ORFs encode similar amino acid sequences; however, evidence of direct enzyme assay is not available to establish a valid gene annotation. Gene annotation by computation and high throughput data is commonly used to predict functions of unknown genes. However, such predicted functions are often inconsistent with low confidence, such as on annotation of previously unknown ORF

YGL157W/ARI1 (Baxter et al., 2004; Joshi et al., 2004; Liu and Moon, 2009; Pir et al., 2006).

Gene function inferred by direct assay is more reliable and preferred as a functional annotation method. In this study, we isolated proteins and evaluated aldehyde reduction activities for two previously identified target ORFs YDR541C and YGL039W toward 14 aldehyde compounds related to lignocellulose hydrolysates. Evidence of direct enzyme assay aids identification and characterization of functions of unknown genes.