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FEMS Microbiology Letters, 362, 2015, 1–7 doi: 10.1093/femsle/fnu014 Advance Access Publication Date: 4 December 2014 Research Letter

R E S E A R C H L E T T E R – Physiology & Biochemistry

Deciphering the role of the type II glyoxalase isoenzyme YcbL (GlxII-2) in Escherichia coli Matthias Reiger1,2,3,# , Jurgen Lassak1,# and Kirsten Jung1,∗ ¨ 1

Munich Center for integrated Protein Science (CiPSM) at the Department of Biology I, Microbiology, ¨ Munchen, Ludwig-Maximilians-Universitat Großhaderner Straße 2-4, D-82152 Martinsried, Germany, ¨ 2 ¨ Institute of Environmental Medicine, UNIKA-T, Neusasser Straße 47, 86156 Augsburg, Germany ¨ Munchen, and 3 Faculty of Medicine, Technische Universitat Ismaningerstr. 22, 81675 Munich, Germany ¨ ∗ Corresponding author. Ludwig-Maximilians-Universitat ¨ Munchen, Department Biologie I, Bereich Mikrobiologie, Großhaderner Str. 2-4, D-82152 ¨ Martinsried, Germany. Tel: +49-89-2180-74500; Fax: +49-89-2180-74520; E-mail: [email protected] One Sentence Summary: GlxII-2 (YcbL) is an accesory type II glyoxalase. Editor: Prof. Dieter Jahn # These authors contributed equally to this work.

ABSTRACT In Escherichia coli, detoxification of methylglyoxal (MG) requires glyoxalases I and II. Glyoxalase I (gloA/GlxI) isomerizes the hemithioacetal, formed spontaneously from MG and glutathione (GSH) to S-lactoylglutathione (SLG), which is hydrolyzed by glyoxalase II (gloB/GlxII) to lactate and GSH. YcbL from Salmonella enterica serovar Typhimurium is an unusual type II glyoxalase whose role in MG detoxification has remained enigmatic. Here we show that YcbL (gloC/GlxII-2) acts as an accessory type II glyoxylase in E. coli. The two isoenzymes have additive effects and ensure maximal MG degradation. Key words: methylglyoxal; gloB; Vibrio campbellii; VIBHAR 02708; VIBHAR 03213

INTRODUCTION Methylglyoxal (MG) is a naturally occurring, ubiquitous ketoaldehyde which is cytotoxic (Carrington and Douglas 1986; Clugston et al., 1997) (Fig. 1). Under conditions leading to the accumulation of phosphorylated glycolytic intermediates and consecutive phosphate limitation, MG is formed via dephosphorylation of dihydroxyacetone phosphate by the methylglyoxal synthase (MGS) (Green and Lewis 1968; Totemeyer et al., 1998). Monoamine oxidase may also contribute to MG formation by oxidizing aminoacetone, an intermediate in threonine catabolism (Mathys et al., 2002; Kim et al., 2004). The toxicity of MG originates from its reactivity as an electrophile that damages DNA as well as proteins, leading eventually to cell death (Colanduoni and Villafranca 1985; Russell 1993; Kang 2003). Bacteria have evolved various strategies to cope with MG stress. For example, E. coli possesses an arse-

nal of detoxification enzymes that can convert MG into nontoxic products. Whereas MG reductase and α-ketoaldehyde dehydrogenase play a minor role in detoxification, the major route uses glyoxalases to convert MG into D-lactate (Inoue and Kimura 1995; Ferguson et al., 1998; Thornalley 2003) (Fig. 1). Metabolization can occur directly via the glyoxalase GlxIII, formerly known as heat shock protein Hsp31 (Subedi et al., 2011), but the more important route is provided by the GlxI–GlxII detoxification system. Upon spontaneous reaction of MG with glutathione (GSH), the resulting hemithioacetal (HTA) is isomerized by glyoxalase I (EC 4.4.1.5, Glx I, gloA) to S-lactoylglutathione (SLG), which is then hydrolyzed to D-lactate and GSH by glyoxalase II (EC 3.1.2.6, Glx II, gloB) (O’Young, Sukdeo and Honek 2007; Sukdeo and Honek 2008). SLG also serves as an activator of the K+ efflux system KefGB (Ozyamak et al., 2010). K+ efflux is accompanied by influx of H+ and Na+ (Bakker and Mangerich 1982), and the resulting decrease in intracellular pH might slow

Received: 11 September 2014; Accepted: 27 October 2014  C FEMS 2014. All rights reserved. For permissions, please e-mail: [email protected]

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FEMS Microbiology Letters, 2015, Vol. 362, No. 1

were grown in Lysogeny Broth (LB) (Bertani 1951) and/or K0.2 medium (Epstein and Kim 1971) at 30◦ C (V. campbellii) and 37◦ C (E. coli), respectively. Media were solidified by adding 1.5% (w/v) agar. If necessary, antibiotics and supplements were added at the following concentrations: ampicillin sodium salt, 100 μg ml−1 ; kanamycin sulfate, 50 μg ml−1 ; chloramphenicol, 10 μg ml−1 ; meso-diaminopimelic acid (300 μM) or MG (various concentrations up to 1 mM) as indicated elsewhere.

Plasmid and strain construction

Figure 1. MG synthesis and detoxification in E. coli. MG is synthesized either from dihydroxyacetone phosphate (DHAP) by MGS or is derived from catabolism of threonine. Various detoxification enzymes convert MG into non-toxic products. MG is detoxified to pyruvate via L-lactaldehyde by MG reductase, while αketoaldehyde dehydrogenase converts MG directly to pyruvate. Glyoxalases generate D-lactate (D-lac) from MG. Direct conversion of MG into D-lac is mediated by glyoxalase III (GlxIII or Hsp31). Upon spontaneous reaction of MG with GSH, the resulting HTA is first isomerized to SLG by glyoxalase I (GlxI, gloA), and SLG is subsequently hydrolyzed to D-lac and GSH by glyoxalase II (GlxII, gloB). This work focuses on the role of YcbL (gloC/GlxII-2).

down the reaction of MG with guanine in DNA and with other macromolecules (Krymkiewicz 1973; Ferguson and Booth 1998; MacLean et al., 1998; Ko et al., 2005; Xu et al., 2006; Sukdeo and Honek 2008). In 2010, Stamp et al. showed that YcbL from Salmonella enterica serovar Typhimurium—a distant homolog of GlxII now designated as GlxII-2—exhibits robust GlxII activity in vitro (Stamp et al., 2010). Like GlxII, GlxII-2 contains zinc as a cofactor, but only one ion was found in the crystal structure rather than the two normally observed in type II glyoxalases (Campos-Bermudez et al., 2007, 2010; Stamp et al., 2010). Moreover, GlxII-2 lacks the typical C-terminal domain, including some recognition determinants for GSH (Stamp et al., 2010). The in vivo role of this enzyme has not been investigated thus far. In this study, we used E. coli to explore the physiological roles and enzymatic properties of GlxII and GlxII-2 (97% identical to S. enterica GlxII-2) in vitro and in vivo. Both gloB and gloC (ycbL is referred to as gloC in this work) mutants displayed decreased tolerance to MG and the double deletion mutant (gloBC) exhibited almost no resistance to exogenously supplied MG. In vitro studies confirmed that GlxII and GlxII-2 are isoenzymes, but have different turnover rates. Only when both glyoxalases II are present can E. coli realize its maximal potential for detoxification of MG. Experiments with Vibrio and a phylogenetic analysis suggest that the enzymatic activity of GlxII-2 is conserved in other bacteria.

MATERIALS AND METHODS Strains and growth conditions R Escherichia coli and Vibrio campbellii ATCC BAA-1116 (formerly R known as V. harveyi ATCC BAA-1116) (Lin et al., 2010) strains

Molecular methods followed standard protocols (Sambrook and Russel 2001) or were carried out according to manufacturer’s instructions. Plasmid and genomic DNA was isolated with the HiYield Plasmid Mini-Kit (Sued-Laborbedarf) and the DNeasy Blood and Tissue Kit (Qiagen), respectively. DNA fragments were purified from agarose gels using the Hi-Yield PCR Clean-up and Gel Extraction Kit (Sued-Laborbedarf). Q5 High-Fidelity or Phusion DNA-polymerases (New England Biolabs) were used according to supplier’s instructions. Restriction enzymes and other DNA modification enzymes were purchased from New England Biolabs. Replicative plasmids were transferred into E. coli strains by transformation using chemically competent cells (Dagert and Ehrlich 1979; Inoue, Nojima and Okayama 1990). Markerless in-frame deletion mutants of V. campbellii were constructed according to the method described previously (Lassak et al., 2010). Escherichia coli deletion mutants were constructed with the R R help of the pRED /ET recombination technique. Subsequently, resistance cassettes were excised using Flp recombinase encoded on plasmid pCP20 (Cherepanov and Wackernagel 1995). Genotypes of strains and plasmids used in this study are listed in Table S1 (Supporting information). The corresponding primer sequences are available on request. For complementation analysis of in-frame deletions, we used E. coli gloABC, which lacks GlxI and both type II glyoxalases GlxII and GlxII-2 to ectopically express the appropriate gene sequences from the arabinose-inducible plasmids pBAD33 and pBAD24.

Overproduction and purification of proteins. C-terminally-tagged His6 -GlxI, His6 -GlxII and His6 -GlxII-2 proteins were overproduced in E. coli gloABC (EC007), harboring the corresponding expression plasmids (Table S1). Expression was induced by the addition of L-arabinose at a final concentration of 0.2% (w/v) to exponentially growing LB cultures (OD600 = 0.5) followed by incubation for 6 h at 37◦ C. Cells were harvested and resuspended in lysis buffer (50 mM NaH2 PO4 [pH 8.0], 300 mM NaCl, 0.5 mM phenylmethanesulfonyl fluoride, 2 mM β-mercaptoethanol, 0.5 mg ml−1 lysozyme) and lysed by three passages through a French press (Thermo Electron Corporation) at 18 000 lbf/in2 . The lysate was clarified by centrifugation at 100 000 × g for 1 h at 4◦ C. To isolate His6 -tagged proteins, supernatants containing the soluble cytosolic protein fraction were subjected to affinity chromatography using Ni2+ -nitrilotriacetic acid Superflow (Qiagen, Hilden, Germany) for binding and 300 mM imidazole for elution. Protein eluates were further purified and transferred to storage buffer [40 mM NaH2 PO4 (pH 7.5), 240 mM NaCl] via isocratic size exclusion chromatography using a ‘SuperdexTM 200 10/300 increase’ column (GE Healthcare, Uppsala, Sweden) at a flow rate of 0.5 ml min−1 . Protein fractions were pooled and analyzed by SDS-PAGE.

Reiger et al.

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Figure 2. In vivo response of E. coli glo mutant strains to exogenous MG stress. (a) MG degradation: exponentially growing E. coli WT and the glo mutant strains (OD600 = 0.4) were exposed to 0.7 mM MG in K0.2 medium. Supernatants were collected at the indicated time points, and MG concentrations were determined colorimetrically. Control: MG in K0.2 medium without cells. (b) MG susceptibility: overnight cultures of WT E. coli and the glo mutant strains were diluted into fresh K0.2 medium to an optical density at 600 nm of 0.005 and exposed to increasing concentrations of MG (≤0.5 mM). The OD600 was determined after 5 h of cultivation. All data were obtained from three independent experiments, and average values are presented.

Figure 3. In vitro enzymatic activities of E. coli type II glyoxalases GlxII and GlxII-2. (a and b) Dependence of GlxII (a) and GlxII-2 (b) on metal ions: specific activities of the respective enzymes were measured in reaction buffer in the presence or absence of 100 μM bivalent metal ions or EDTA. (c) Specific activities of purified GlxII and GlxII-2: enzymatic conversion of increasing SLG concentrations was measured at 30◦ C by colorimetric detection of accumulating GSH. (d) SLG turnover rates for E. coli type II glyoxalases: 10 nM GlxII and 10 nM GlxII-2 were incubated at 30◦ C with 0.4 mM SLG, and the concentration of GSH liberated was monitored over 90 min. All data were obtained from three independent experiments, and average values are presented.

MG survival test and MG degradation Exponentially growing E. coli and V. campbellii strains (OD at 600 nm ∼ 0.4) were diluted to an OD at 600 nm of 0.05 in K0.2 and LB medium, respectively, containing different concentrations of MG (