Metabolic fate of L-lactaldehyde derived from an alternative L-rhamnose pathway Seiya Watanabe 1,2,3 , Sommani Piyanart 1 and Keisuke Makino 1,2,3,4 1 Institute of Advanced Energy, Kyoto University, Japan 2 New Energy and Industrial Technology Development Organization, Kyoto, Japan 3 CREST, JST (Japan Science and Technology Agency), Japan 4 Innovative Collaboration Center, Kyoto University, Japan l-Rhamnose (l-6-deoxymannose) is a constituent of glycolipids and glycosides, such as plant pigments, pectic polysaccharides, gums and biosurfactants, and can be utilized as the sole carbon and energy source by most bacteria, including Escherichia coli and Salmonella typhimurium. In this pathway, l-rhamnose is converted into dihydroxyacetone phosphate and l-lactaldehyde via l-rhamnulose and l-rhamnulose l-phosphate by the Keywords Azotobacter vinelandii; L-lactaldehyde dehydrogenase; L-rhamnose metabolism; molecular evolution; Pichia stipitis Correspondence S. Watanabe, Institute of Advanced Energy, Kyoto University, Gokasho, Uji, Kyoto 611-0011, Japan Fax: +81 774 38 3524 Tel: +81 774 38 3596 E-mail: irab@iae.kyoto-u.ac.jp (Received 8 July 2008, revised 9 August 2008, accepted 15 August 2008) doi:10.1111/j.1742-4658.2008.06645.x Fungal Pichia stipitis and bacterial Azotobacter vinelandii possess an alter- native pathway of l-rhamnose metabolism, which is different from the known bacterial pathway. In a previous study (Watanabe S, Saimura M & Makino K (2008) Eukaryotic and bacterial gene clusters related to an alternative pathway of non-phosphorylated l-rhamnose metabolism. J Biol Chem 283, 20372–20382), we identified and characterized the gene clusters encoding the four metabolic enzymes [l-rhamnose 1-dehydrogenase (LRA1), l-rhamnono-c-lactonase (LRA2), l-rhamnonate dehydratase (LRA3) and l-2-keto-3-deoxyrhamnonate aldolase (LRA4)]. In the known and alternative l-rhamnose pathways, l-lactaldehyde is commonly pro- duced from l-2-keto-3-deoxyrhamnonate and l-rhamnulose 1-phosphate by each specific aldolase, respectively. To estimate the metabolic fate of l-lact- aldehyde in fungi, we purified l-lactaldehyde dehydrogenase (LADH) from P. stipitis cells l-rhamnose-grown to homogeneity, and identified the gene encoding this enzyme (PsLADH) by matrix-assisted laser desorption ioniza- tion-quadruple ion trap-time of flight mass spectrometry. In contrast, LADH of A. vinelandii (AvLADH) was clustered with the LRA1–4 gene on the genome. Physiological characterization using recombinant enzymes revealed that, of the tested aldehyde substrates, l-lactaldehyde is the best substrate for both PsLADH and AvLADH, and that PsLADH shows broad substrate specificity and relaxed coenzyme specificity compared with AvLADH. In the phylogenetic tree of the aldehyde dehydrogenase super- family, PsLADH is poorly related to the known bacterial LADHs, includ- ing that of Escherichia coli (EcLADH). However, despite its involvement in different l-rhamnose metabolism, AvLADH belongs to the same subfamily as EcLADH. This suggests that the substrate specificities for l-lactaldehyde between fungal and bacterial LADHs have been acquired independently. Abbreviations ALDH, aldehyde dehydrogenase; AvLADH, Azotobacter vinelandii LADH; EcLADH, Escherichia coli LADH; GAPDH, glyceraldehyde 3- phosphate dehydrogenase; LADH, L-lactaldehyde dehydrogenase; LAR, L-lactaldehyde reductase; L-KDR, L-2-keto-3-deoxyrhamnonate; LRA1, L-rhamnose 1-dehydrogenase; LRA2, L-rhamnono-c-lactonase; LRA3, L-rhamnonate dehydratase; LRA4, L-2-keto-3-deoxyrhamnonate aldolase; MjLADH, Methanocaldococcus jannaschii LADH; PsLADH, Pichia stipitis LADH. FEBS Journal 275 (2008) 5139–5149 ª 2008 The Authors Journal compilation ª 2008 FEBS 5139 sequential action of l-rhamnose isomerase (RhaA, EC 5.3.1.14), rhamnulokinase (RhaB, EC 2.7.1.5) and l-rhamnulose l-phosphate aldolase (RhaD, EC 4.1.2.19) (Fig. 1A). Most fungi, including Saccharomyces cerevi- siae, cannot grow on d-xylose, l-arabinose and l-rhamnose as the sole carbon source [1]. However, Pichia stipitis possesses the ability to metabolize these sugars through alternative pathways different from L-Rhamnose L-Rhamnono-γ-lactone L-Rhamnonate L -2-Keto-3-deoxyrhamnonate ( L -KDR) L-Rhamnose 1-dehydrogenase ( LRA1, EC 1.1.1.173) L-Rhamnono-γ-lactonase ( LRA2, EC 3.1.1.65) L-Rhamnonate dehydratase ( LRA3, EC 4.2.1.90) NAD(P) + NAD(P)H H 2 O H2O Pyruvate L-Lactaldehyde L-KDR aldolase ( LRA4, EC 4.2.1 ) L -Rhamnose L-Rhamnulose L-Rhamnulose 1-P ATP ADP L-Rhamnose isomerase (RhaA, EC 5.3.1.14) L -Rhamnulokinase (RhaB, EC 2.7.1.5) L-Rhamnulose 1-P aldolase (RhaD, EC 4.1.2.19) Dihydroxyacetone-P RhaD RhaA RhaB RhaS RhaR RhaT AAC76884 AAC76885 AAC76886 AAC76887 AAC76888 AAC76889 E. coli P. stipitis (L-Rhamnose:H + symporter) EAM07803 EAM07804 EAM07805 EAM07806 EAM07807 EAM07808 EAM07809 EAM07810 (Sugar transporter)(Sugar channel) A. vinelandii ABN68602ABN68405ABN68404 ABN68603Chr 8 Chr 2 ABN64318 AAC74497 Methylglyoxal NADPH NADP + Glutathione L-Lactaldehyde S-Lactoyl glutathione NAD(P) + NAD(P)H Lactate Glutathione NAD + NADH Pyruvate Dihydroxyacetone-P NADH NAD + 1,2-Propanediol P NAD + NADH L-Lactaldehyde dehydrogenase ( LADH, EC 1.2.1.22) Lactaldehyde:propanediol oxidoreductase ( EC 1.1.1.77(55)) FucO FucA FucP FucI FucK FucU AAC75841 AAC75842 AAC75843 AAC75844 AAC75845 AAC75846 OH H H HO OH H H O H 3 C HO H H HO OH H H O H 3 C HO H O CH 3 H OH H OH OH H OH H HOOC CH 3 H OH H OH H H O HOOC 3 CH H OH OHC CH 3 O HOOC L -Rhamnose D -Xylose L -Arabinose OH H H HOH 2 C H OH HO H O OH H HOH 2 C H H OH HO H O HOH 2 C OH H H OH CH 2 OPO 3 2- O A C D B Fig. 1. (A) Known bacterial L-rhamnose pathway. (B) Novel non-phosphorylating L-rhamnose pathway. In addition to L-rhamnose, Pichia stipi- tis (but not Saccharomyces cerevisiae) can metabolize D-xylose and L-arabinose to yield a common phosphorylated end-product, xylulose 5-phosphate. (C) Schematic gene clusters related to L-rhamnose metabolism. Chr 8 and Chr 2 in P. stipitis indicates chromosome number. Homologous genes are indicated in the same colour. Fungal and bacterial LRA4 enzymes are not related evolutionally [3]. LADH enzymes of P. stipitis and Azotobacter vinelandii (orange) were characterized in this study. L-Fucose is converted to pyruvate and L-lactaldehyde through the analogous pathway to L-rhamnose, and metabolic genes, including FucO, are also clustered on the Escherichia coli genome. (D) Metabolic network around L-lactaldehyde. In this study, we focused on LADH (black line). L-Lactaldehyde dehydrogenase S. Watanabe et al. 5140 FEBS Journal 275 (2008) 5139–5149 ª 2008 The Authors Journal compilation ª 2008 FEBS the well-known bacterial pathways. Although both d-xylose and l-arabinose are converted into a com- mon end-product, xylulose 5-phosphate, as in the bacterial pathway, it is believed that l-rhamnose is metabolized via non-phosphorylated intermediates (Fig. 1B) [2]. In this pathway, l-rhamnose is oxidized to l-rhamnono-c-lactone by NAD(P) + -dependent dehydrogenase. The lactone is cleaved by a lactonase to l-rhamnonate, followed by a dehydration reaction forming l-2-keto-3-deoxyrhamnonate (l-KDR). The last step is the aldol cleavage of l-KDR to pyruvate and l-lactaldehyde. We are in the process of enzy- matically and genetically characterizing the alterna- tive l-rhamnose pathway of P. stipitis, and recently identified four metabolic enzymes: l-rhamnose 1-dehydrogenase (LRA1, EC 1.1.1.173), l-rhamnono- c-lactonase (LRA2, EC 3.1.1.65), l-rhamnonate dehydratase (LRA3, EC 4.2.1.90) and l-KDR aldol- ase (LRA4) [3]. The LRA1–4 genes were clustered on the P. stipitis genome (Fig. 1C), and the homologous gene cluster was found on the genomes of many fungi as well as several bacteria, including Azoto- bacter vinelandii. In the known and alternative l-rhamnose pathways, the final reaction step is catalysed by each specific aldolase to commonly yield l-lactaldehyde as one of the products. There are two known enzymes for l-lact- aldehyde in bacteria (Fig. 1D). The first is oxidation by NAD + -dependent l-lactaldehyde dehydrogenase (EC 1.2.1.22, LADH) to produce l-lactate [4–6]. In E. coli, the enzyme is commonly responsible for both l-rhamnose and l-fucose metabolism, and is also iden- tical to the glycolaldehyde dehydrogenase (EC 1.2.1.21) involved in ethylene glycol metabolism and glyoxylate biosynthesis [4,5]. Under anaerobic conditions, l-lactal- dehyde is reduced by NADH-dependent l-lactaldehyde reductase (LAR, EC 1.1.1.77) and the l-1,2-propane- diol obtained is excreted in the medium. In an E. coli mutant that can grow on l-1,2-propanediol as a sole carbon source, LAR also functions as l-1,2-propanediol dehydrogenase, so-called ‘lactaldehyde : propanediol oxidoreductase’ [7]. In contrast with bacteria, the correct physiological role of l-lactaldehyde and related enzymes in fungi has not yet been clarified. Chen et al. [8] reported that the Gre2 (YOL151W) gene from S. cerevisiae encodes a NADPH-dependent methyl- glyoxal reductase (EC 1.1.1.283) catalysing the reduc- tion of methylglyoxal to d- and ⁄ or l-lactaldehyde. Furthermore, Inoue et al. [9] identified an aldehyde dehydrogenase (ALDH) with specificity for l-lact- aldehyde enzymatically but not genetically. However, it is well known that a toxic methylglyoxal is neutralized to lactate via lactoylglutathione (but not l-lactaldehyde) by glyoxalase I (EC 4.4.1.5, YML004C) and gly- oxalase II (EC 3.1.2.5, YDR272W). In this regard, the alternative l-rhamnose pathway is the significant physiological origin of l-lactaldehyde in fungi. In this study, we first identified a fungal LADH from P. stipitis. Furthermore, phylogenetic comparison with the LADH of A. vinelandii revealed that the same alternative l-rhamnose pathways appeared by convergent evolution between fungi and bacteria. Results Metabolic fate of L-lactaldehyde in P. stipitis When compared with d-glucose medium, approxi- mately 30-fold higher NAD + -dependent dehydroge- nase activity for l-lactaldehyde was observed in the cell-free extract from P. stipitis cells grown on l-rham- nose as the sole carbon source (Fig. 2A). Similar results were observed when d-lactaldehyde was used as a substrate instead of l-lactaldehyde. In Zymogram staining analysis, active bands of NAD + -dependent dehydrogenases for l-lactaldehyde and d-lactaldehyde appeared in the same position (Fig. 2B), and no active GR GR GR GR LD LD P. stipitis A. vinelandii Band A Band B NAD AC B + NADP + 0.5 0.4 0.3 0.2 0.1 0 0.06 0.04 0.02 0 GRGR GRGR L D L D Specific activity (unit mg –1 protein) P. stipitis A. vinelandii PsLADH PsALDH* AvLADH Fig. 2. Translational and transcriptional regulation of LADH. Pichi- a stipitis and Azotobacter vinelandii cells were cultured in synthetic medium containing D-glucose (G) or L-rhamnose (R) (2%, w ⁄ v). (A) NAD + - and NADP + -dependent dehydrogenase activity for L-lact- aldehyde (L) or D-lactaldehyde (D) in the cell-free extract. Values are the means ± SD, n = 3. (B) Zymogram staining. Fifty micrograms of the cell-free extract were applied to a 6% (w ⁄ v) non-denaturing PAGE gel. After electrophoresis, the gel was soaked in staining solution in the presence of 10 m ML-orD-lactaldehyde and 10 mM NAD + . (C) Transcriptional effect of carbon source on PsLADH, PsALDH* and AvLADH genes. Total RNAs (4 lg per lane) were isolated from microorganism cells grown on the indicated carbon sources. S. Watanabe et al. L-Lactaldehyde dehydrogenase FEBS Journal 275 (2008) 5139–5149 ª 2008 The Authors Journal compilation ª 2008 FEBS 5141 band was observed in the presence of NADP + (data not shown), suggesting that the l-rhamnose-inducible NAD + -dependent (or preferring) dehydrogenase for l-lactaldehyde and d-lactaldehyde seems to derive from the same enzyme, and that NADP + -dependent activity may be derived from the concomitant activity of other constitutively expressed ALDH(s). Under anaerobic conditions, P. stipitis could metabolize l-rhamnose (data not shown). These results indicate that the metabolic fate of l-lactaldehyde derived from the alternative l-rhamnose pathway in P. stipitis is dehydrogenation by LADH. Purification of LADH from P. stipitis (PsLADH) PsLADH was purified from P. stipitis cells grown on l-rhamnose as a sole carbon source in four chromato- graphic steps (Fig. 3A). During the purification proce- dure, the ratio of NAD + - to NADP + -linked activity remained almost constant (2.2–3.0), suggesting the presence of only one protein as LADH. The purified enzyme exhibited a clear preference for NAD + over NADP + , with NAD + - and NADP + -dependent spe- cific activities of 6.85 and 2.26 unitsÆ(mg protein) )1 , respectively. SDS-PAGE revealed only one subunit with an apparent M r value of  55 kDa. As it was impossible to determine the N-terminal sequence because of blocking, the peptide mass fingerprinting of trypsin-digested fragments was alternatively performed by MALDI-TOF MS, and LADH was identified as a protein annotated as a putative ALDH of P. stipitis CBS 6054 (ABN64318): 63% sequence coverage (Table S1). This protein consisted of a polypeptide of 495 amino acids with a calculated M r of 53 488.85 Da, comparable with that of the purified LADH deter- mined by SDS-PAGE. For the known dehydrogenases for l-lactaldehyde, the reaction product of the enzymes from E. coli [4,5], Methanocaldococcus jannaschii [10] and S. cerevisiae [9] is l-lactate (EC 1.2.1.22), whereas that from rat liver is methylglyoxal (EC 1.1.1.78) [11]. In HPLC analysis, the retention time of the reaction product for PsLADH (13.32 min) was almost the same as that of l-lactate (13.35 min), but not methylglyoxal (12.36 min); therefore, the enzyme catalyses the NAD(P) + -linked oxidation of l-lactaldehyde into l-lactate. The amino acid sequence of PsLADH was most closely related to E. coli LADH (EcLADH) of the ALDH-like proteins on the P. stipitis genome (34.5% identity), whereas the protein annotated as a putative mitochondrial ALDH (ABN68636) also showed similar homology to EcLADH (32.2% identity), indicating the possibility that the latter is an LADH isozyme (referred to as PsALDH*); therefore, both enzymes were expressed in E. coli cells (see below). Candidate of LADH gene from A. vinelandii As described in the Introduction, we have previously identified the gene cluster related to the alternative l-rhamnose pathway of A. vinelandii [3]. The LRA1–4 genes are clustered together with putative sugar trans- porters and the ALDH gene (EAM07810) (Fig. 1C). This ALDH showed highest sequential similarity to EcLADH (61.7% identity) of all the putative ALDHs in the A. vinelandii genome, indicating that the protein may function as LADH (referred to as AvLADH). Two active bands corresponding to NAD + -dependent LADH were found in Zymogram staining analysis using the cell-free extract prepared from A. vinelandii cells grown on l-rhamnose: strict l-rhamnose-inducible enzyme with l-lactaldehyde specificity (band A); mod- erate l-rhamnose-inducible enzyme that utilizes both d- and l-lactaldehyde (band B) (Fig. 2B). Subsequent characterization revealed that ALDH with EAM07810 may correspond to band A, a major LADH in l-rham- nose-grown cells (see below). Functional expression of LADH in E. coli PsLADH, PsALDH* and AvLADH genes were overex- pressed in E. coli cells as a His6-tagged enzyme and purified homogeneously with a nickel-chelating affinity 1 2 345M AB M1234 19.5 kDa 119 kDa 91 kDa 65 kDa 48 kDa 37 kDa 28 kDa Fig. 3. (A) SDS-PAGE purification of native PsLADH in 10% (w ⁄ v) gel. Lane 1, cell-free extracts (50 lg); lane 2, HiPrep 16 ⁄ 10 Q FF (50 lg); lane 3, HiLoad 16 ⁄ 60 Superdex 200 pg (20 lg); lane 4, CHT Ceramic Hydroxyapatite (20 lg); lane 5, Blue Sepharose Fast Flow (10 lg). (B) SDS-PAGE of native and His6-tagged recombinant enzymes. Lane 1, native PsLADH; lane 2, His6-tagged PsLADH; lane 3, His6-tagged PsALDH*; lane 4, His6-tagged AvLADH. Ten micrograms of the purified enzyme were applied. Bottom panel: immunoblot analysis using anti-His6-tag IgG. One microgram of the purified enzyme was applied. L-Lactaldehyde dehydrogenase S. Watanabe et al. 5142 FEBS Journal 275 (2008) 5139–5149 ª 2008 The Authors Journal compilation ª 2008 FEBS column (Fig. 3B). Western blot analysis with anti- His6-tag IgG confirmed the His6 tag in the enzyme (bottom panel in Fig. 3B). Substrate specificity Generally, ALDHs show relatively broad substrate specificity in addition to the physiological substrate; therefore, various aldehydes, including l-lactaldehyde, were tested as substrates for dehydrogenation by purified proteins in the presence of NAD + , and the activity values for the tested aldehydes relative to l-lactaldehyde are summarized in Table 1. l-Lactalde- hyde was the best substrate for PsLADH, and the specific activity [6.95 unitsÆ(mg protein) )1 ] was compa- rable with that of native enzyme [6.85 unitsÆ(mg pro- tein) )1 ]. Only five other aldehydes showed more than 50% activity relative to l-lactaldehyde. The significant utilization of d-lactaldehyde conformed to the preli- minary Zymogram staining analysis using the cell-free extract (Fig. 2B). By contrast, PsALDH* utilized C2, C3 and C4 aldehydes more efficiently than l-lactalde- hyde, and most of the remaining aldehydes were also good substrates at varying rates up to about one-half the rate with l-lactaldehyde. Overall, the specificity for l-lactaldehyde of PsLADH was significantly higher than that of PsALDH*, conforming to the physiologi- cal role as a LADH involved in the alternative l-rham- nose pathway. Comparable dehydrogenase activity of AvLADH with PsLADH was found only for l-lactal- dehyde and glycolaldehyde, and activities with d-lactal- dehyde and C7 aldehyde were only 10% less than those with l-lactaldehyde: band A in Zymogram stain- ing may correspond to AvLADH (Fig. 2B). These results suggest that the enzyme should be assigned to LADH, as expected from the sequential similarity to EcLADH. Kinetic analysis EcLADH functions as a glycolaldehyde dehydro- genase involved in ethylene glycol metabolism and glyoxylate biosynthesis [4,5]. PsLADH, PsALDH* Table 1. Substrate specificity of PsLADH, PsALDH* and AvLADH. Substrate a Relative activity (%) b PsLADH PsALDH* AvLADH L-Lactaldehyde 100 100 100 D-Lactaldehyde 75 54 8.6 Formaldehyde (C1) 13 13 0 Acetaldehyde (C2) 67 309 0 Propionaldehyde (C3) 81 350 0 Butylaldehyde (C4) 39 175 0 Valeraldehyde (C5) 38 105 0 Hexylaldehyde (C6) 41 82 0 Heptylaldehyde (C7) 28 70 7.4 Octylaldehyde (C8) 22 57 0 Isobutylaldehyde 82 54 0 Glutaraldehyde 47 251 0 Glycolaldehyde 74 70 91 Benzaldehyde 27 23 0 Betaine aldehyde 13 15 0 Glyceraldehyde 30 27 0 Glyceraldehyde 3-phosphate 12 11 0 a The assay was performed with standard assay solution containing 10% (v ⁄ v) ethanol, 1 m M aldehyde and 1.5 mM NAD + using purified His6-tagged recombinant enzymes. b Relative values were expressed as a percentage of the values obtained in L-lactaldehyde. Table 2. Kinetic parameters of PsLADH, PsALDH*, AvLADH and EcLADH. Enzyme Substrate Coenzyme Specific activity [unitÆ(mg protein) )1 ] a K m (lM) k cat (min )1 ) k cat ⁄ K m (min )1 ÆlM )1 ) PsLADH L-Lactaldehyde b NAD + 6.95 ± 0.10 42.8 ± 4.2 1390 ± 127 32.4 ± 0.2 NADP + 1.65 ± 0.04 9.79 ± 0.74 195 ± 8 20.0 ± 0.3 D-Lactaldehyde b NAD + 4.43 ± 0.05 52.9 ± 3.4 1460 ± 79 27.5 ± 0.3 Glycolaldehyde c NAD + 8.94 ± 0.40 78.0 ± 1.6 469 ± 8 6.01 ± 0.03 PsALDH* L-Lactaldehyde b NAD + 3.64 ± 0.10 350 ± 62 651 ± 112 1.88 ± 0.01 NADP + 0.355 ± 0.007 131 ± 9 15.4 ± 0.7 0.119 ± 0.001 D-Lactaldehyde b NAD + 2.10 ± 0.04 32.6 ± 5.6 89.5 ± 11.9 2.76 ± 0.10 Glycolaldehyde c NAD + 4.28 ± 0.30 287 ± 12 137 ± 5 0.478 ± 0.003 AvLADH L-Lactaldehyde b NAD + 17.2 ± 0.9 35.5 ± 0.8 554 ± 19 15.6 ± 0.2 D-Lactaldehyde b NAD + 2.82 ± 0.04 167 ± 5 47.0 ± 1.5 0.281 ± 0.001 Glycolaldehyde c NAD + 11.5 ± 0.4 274 ± 41 307 ± 44 1.12 ± 0.01 EcLADH d L-Lactaldehyde NAD + 5.73 40 418 10.5 Glycolaldehyde NAD + 14.7 380 993 2.61 a Under standard assay conditions in Experimental procedures. b Eight different concentrations of aldehyde between 2 and 100 lM were used. c Eight different concentrations of glycolaldehyde between 10 and 100 lM were used. d Calculation from data in [5]. S. Watanabe et al. L-Lactaldehyde dehydrogenase FEBS Journal 275 (2008) 5139–5149 ª 2008 The Authors Journal compilation ª 2008 FEBS 5143 and AvLADH also utilize glycolaldehyde efficiently as a substrate (Table 1); therefore, these enzymes were sub- jected to further kinetic analysis with l-lactaldehyde, d-lactaldehyde and glycolaldehyde, and the parameters determined are listed in Table 2. The catalytic efficiency (k cat ⁄ K m ) with l-lactaldehyde of PsLADH in the pres- ence of NAD + (32.4 min )1 Ælm )1 ) was 17.2-fold higher than that of PsALDH* (1.88 min )1 Ælm )1 ), caused by both higher K m and lower k cat values. However, by contrast with l-lactaldehyde, the two fungal enzymes possessed similar k cat ⁄ K m values with d-lactaldehyde, but their values with glycolaldehyde were significantly lower, mainly caused by decreased k cat values. When NADP + was used as a coenzyme, the k cat ⁄ K m value with l-lactaldehyde of PsALDH* decreased 15.8-fold compared with that in the presence of NAD + because of a decreased k cat value, whereas that of PsLADH decreased only 1.6-fold. These results suggest that PsLADH possesses a stricter substrate specificity for l-lactaldehyde and a more relaxed coenzyme specificity than does PsALDH*. Furthermore, the PsLADH gene was significantly induced by l-rhamnose in P. stipitis cells, but the PsALDH* gene was not (Fig. 2C). These results strongly suggest the physiological function of PsLADH in the alternative l-rhamnose metabolism. The k cat ⁄ K m value with l-lactaldehyde of AvLADH (15.6 min )1 Ælm )1 ) in the presence of NAD + was 55.5-fold higher than that with d-lactaldehyde (0.281 min )1 Ælm )1 ), and no activity was observed in the presence of NADP + , in contrast with fungal enzymes. The kinetic parameters of l-lactaldehyde and glycolaldehyde were similar to those of EcLADH [5]. Furthermore, the AvLADH gene was up-regulated dur- ing growth on l-rhamnose (Fig. 2C). As the activities of LRA1–4 proteins were also significantly induced by l-rhamnose-grown A. vinelandii cells (data not shown), the gene cluster containing LRA1–4 and AvLADH genes may be strictly regulated by l -rhamnose as a single transcriptional unit (Fig. 1C). Amino acid sequence analysis of LADH In the phylogenetic tree of the ALDH superfamily, PsLADH and PsALDH* fall into the fungal ALDH subfamily, one of the 14 ALDH subfamilies compiled by Perozich et al. [12] (Fig. 4), confirming the micro- organism source. The fungal ALDH subfamily belongs to the Class 1 ⁄ 2 branch of ALDHs, which consists of tetrameric ALDH subfamilies with variable substrate specificity, as well as two P. stipitis enzymes (Table 1). In S. cerevisiae, there is biochemical evidence of two types of ALDH [13,14]. The mitochondrial ALDHs, ScALDH4 and ScALDH5, show dual coenzyme speci- ficity between NAD + and NADP + and are activated by K + . The cytosolic ALDHs, ScALDH2, ScALDH3 and ScALDH6, are specific to NADP + ; only ALDH6 is activated by Mg 2+ . Higher degrees of similarity to PsLADH (probably cytosolic enzyme because of no mitochondrial leader sequence) were found in the mitochondrial ALDHs of S. cerevisiae, confirming the enzyme properties, including coenzyme specificity. Indeed, the activity of PsLADH is also absolutely dependent on K + (data not shown). However, PsALDH* (cytosolic enzyme as well as PsLADH) is more closely related than PsLADH to cytosolic ALDHs, indicating that this enzyme may be assigned as an acetaldehyde dehydrogenase rather than LADH, based on substrate specificity (Table 1). A branch of AvLADH and EcLADH was located on the root of the non-phosphorylating glyceraldehyde 3-phosphate dehydrogenase (GAPDH, EC 1.2.1.9.) subfamily in the Class 3 branch, consisting of substrate-specific ALDH subfamilies (Fig. 4), confirming their enzyme proper- ties of high specificity with l-lactaldehyde (Tables 1 and 2). (dl-)Lactaldehyde dehydrogenase of archaeal M. jannaschii (MjLADH) is also a member of this subfamily, and is involved in the production of lactate for coenzyme F 420 biosynthesis [10]. Discussion In this study, we have identified the LADHs involved in the alternative l-rhamnose pathways of fungi and bacteria. In particular, although fungi possess multiple ALDH genes, only one physiological substrate, acetal- dehyde, has been identified in fermentation and ⁄ or growth on ethanol. To our knowledge, this is the second report of fungal ALDH as an aldehyde substrate in addition to acetaldehyde; the other stated that ScALDH2 and ScALDH3 play a role as 3-amino- propionaldehyde dehydrogenases in pantothenic acid (vitamin B 5 ) and coenzyme A biosynthesis [15]. Enzyme catalysis of LADH Hempel et al. [16] proposed several characteristic con- served regions containing almost all active amino acid residues in ALDHs. In particular, glutamate in the motif of LELGGKSP participates as a general base for the activation of catalytic cysteine and deacylation of the enzyme, and cysteine in the motif of FXNXGQXCIA (where X is any amino acid) acts as a nucleophile. These motifs are also conserved in PsLADH and AvLADH with a few modifications (Fig. 5), indicating that the overall structure and fundamental catalytic mechanism may be similar to L-Lactaldehyde dehydrogenase S. Watanabe et al. 5144 FEBS Journal 275 (2008) 5139–5149 ª 2008 The Authors Journal compilation ª 2008 FEBS those in known ALDHs. Based on structural studies of other ALDHs, amino acid residues at equivalent positions of 190 and 193 in PsLADH are involved in the distinction between NAD + and NADP + . The structur- ally equivalent lysine residue to Lys190 is conserved in all ALDHs, and is unlikely to influence directly coen- zyme specificity. A glutamate residue at an equivalent position to 193 interacts with 2¢- and 3¢-hydroxyl groups of the ribose of the adenine moiety in strict NAD + - preferring enzymes, such as EcLADH, AvLADH and PsALDH*. However, the structurally equivalent gluta- mate is found not only in PsLADH with significant NADP + -dependent activity, but also in NADP + - preferring ScALDH4 and ScALDH5. However, it is Fig. 5. Partial alignment of amino acid sequences around several active sites. Open and filled circles indicate NAD + - and NADP + -dependent enzymes. ScALDH4 (grey circle) utilizes both NAD + and NADP + as a coenzyme. Grey-shaded letters are highly conserved. In the crystal structure of EcLADH (PDB ID, 2IMP), open and filled stars indicate amino acid residues bound to L-lactate and 2¢- and 3¢-hydroxyl groups of NADH, respectively. The catalytic glutamate and cysteine residues are indicated by grey stars. Class 1 Class 2 Fungal ALDH FTDH HMSALDH Group X BALDH SSALDH GAPDH Aromatic ALDH MMSALDH Turgor ALDH GGSALDH Class 3 ALDH Class 1/2 branch Class 3 branch PsALDH* ScALDH3 ScALDH2 ScALDH6 PsLADH ScALDH5 ScALDH4 Pichia angusta Alternaria alternata Cladosporium herbarum Aspergillus nidulans Aspergillus niger Ustilago maydis EcLADH AvLADH MjLADH Fig. 4. The overall phylogenetic tree of known ALDHs, including LADHs. Sequence names and references of ALDHs are avail- able on the ALDH website described in Experimental procedures. The three enzymes in the boxes were characterized in this study. S. Watanabe et al. L-Lactaldehyde dehydrogenase FEBS Journal 275 (2008) 5139–5149 ª 2008 The Authors Journal compilation ª 2008 FEBS 5145 known that ALDH isozyme B of E. coli (AldB, 33% identity to EcLADH) is a strict NADP + -dependent enzyme and possesses the structural equivalent arginine residue at this position (Arg197) [17]. When compared with the wild-type enzyme, the R197E mutant shows 10% NADP + -dependent activity, together with no detection of NAD + -dependent activity. In PsLADH and PsALDH*, each E193R mutant expressed a simi- lar level to the wild-type enzyme in E. coli cells as an inclusion body; we did not perform further enzy- matic characterization (data not shown). This indicates that, although the glutamate residue in PsLADH (and fungal ALDHs) should play a role in coenzyme bind- ing and ⁄ or structural maintenance, other amino acid residues may also influence coenzyme specificity. Inoue et al. [9] purified a NAD + -dependent dehydro- genase for l-lactaldehyde from S. cerevisiae cells cul- tured in a nutrient medium. Although the enzyme has not yet been characterized genetically, the molecular structures (monomeric form consisting of the subunit with M r of 40 kDa) are clearly different from those of general ALDH enzymes, including fungal ALDHs (tetrameric or dimeric form consisting of the subunit with M r of 50–55 kDa, see Fig. 3). Furthermore, the activity with d-lactaldehyde is only 0.2% of that with l-lactaldehyde, and acetaldehyde, dl-glyceraldehyde and propionaldehyde are inactive substrates, in contrast with PsLADH (Table 1); acetaldehyde is a common active substrate for the known ScALDH2–6. Therefore, although the genetic background and physiological functions of LADH in S. cerevisiae have not been eluci- dated so far, it has been reported recently that the Gre2 (YOL151w) gene encodes methylglyoxal reductase, related to the detoxification of methylglyoxal [8], in which the LADH(-like) enzyme may also be involved. Convergent evolution of LADHs in fungi and bacteria In the phylogenetic tree, substrate-specific ALDHs have a tendency to belong to subfamilies in the Class 3 branch, whereas ALDH families with broad substrate specificity are more often found in the Class 1 ⁄ 2 branch (Fig. 4). PsLADH (and also PsALDH*) shows significant activity for several aldehydes in addition to l-lactaldehyde (Table 1), and the fungal ALDH subfamily containing this enzyme belongs to the Class 1 ⁄ 2 branch. However, AvLADH, which shows high specificity to l-lactaldehyde, is similar to the GAPDH subfamily in the Class 3 branch. It is note- worthy that, although l-lactaldehyde is produced by the same alternative pathway of l-rhamnose in P. stipitis and A. vinelandii, their LADHs are classified into different subfamilies, strongly suggesting that their substrate specificities have been acquired by ‘conver- gent evolution’ rather than divergence from a common ancestor. Indeed, four ligands for the substrate (l-lac- tate) are not conserved between PsLADH and EcLADH (Fig. 5). PsLADH seems to have evolved from an ancestor with broader substrate specificity, such as PsALDH*, because PsALDH* is located at the root of the fungal ALDH subfamily (Fig. 4). It is certain that AvLADH and EcLADH, which are involved in different pathways of the same l-rhamnose metabolism, evolved from a common ancestor. EcLADH is responsible for not only l-rhamnose but also l-fucose metabolism [5], whereas the LRA1–4 proteins, components of the gene cluster containing the AvLADH gene (Fig. 1C), show no significant activ- ity with l-fucose-related intermediates [3]. Therefore, it is probable that the gene cluster is involved in l-rham- nose metabolism only, but not l-fucose. MjLADH is involved in different metabolism from l-rhamnose (coenzyme F 420 biosynthesis) [10] and is similar to EcLADH and AvLADH phylogenetically (Fig. 5). Although glyceraldehyde 3-phosphate is commonly an inactive substrate for AvLADH (see Table 1), EcLADH and MjLADH, MjLADH is capable of utilizing several aldehydes, such as glycolaldehyde, dl-glyceraldehyde, formaldehyde, acetaldehyde and propionaldehyde (the last three are inactive substrates for AvLADH and EcLADH). Furthermore, substrate- binding sites of bacterial LADHs are not completely conserved in MjLADH (Fig. 5). These results suggest that substrate specificity for l-lactaldehyde has also been acquired for bacteria and Archaea independently, similar to fungi. Of the 14 subfamilies in the ALDH superfamily, some subfamilies, such as c-glutamyl semialdehyde dehydro- genase, methylmalonyl semialdehyde dehydrogenase and succinic semialdehyde dehydrogenase, include sequences from organisms ranging from bacteria to mammals (Fig. 4) [12]. In contrast, the fungal ALDH subfamily (consisting of only fungal sequences) appears to have diverged much later in evolution, indicating that the acquisition of substrate specificity for l-lactaldehyde might have occurred after divergence between bacteria and eukaryotes (fungi). Four metabolic enzymes (genes) that convert l-rhamnose into pyruvate and l-lactalde- hyde are found on the genomes of several fungi, includ- ing P. stipitis, Debaryomyces hansenii, Candida species and Aspergillus species [3], but not S. cerevisiae, which is not capable of growth on l-rhamnose [1]. Therefore, the acquisition of these l-rhamnose metabolic genes might have led to the appearance of LADH from a common ancestor of fungal ALDHs under evolutionary pressure. L-Lactaldehyde dehydrogenase S. Watanabe et al. 5146 FEBS Journal 275 (2008) 5139–5149 ª 2008 The Authors Journal compilation ª 2008 FEBS Experimental procedures Microorganism strains, culture conditions and preparation of cell-free extracts Pichia stipitis CBS 6054 was kindly provided by T. W. Jef- fries (University of Wisconsin, Milwaukee, WI, USA). A. vinelandii NBRC 102612 was purchased from the National Institute of Technology and Evaluation (Chiba, Japan). P. stipitis and A. vinelandii were grown at 30 °Cin yeast nitrogen broth and Burk’s nitrogen-free medium supplemented with 20 gÆL )1 d-glucose or l-rhamnose as carbon source, respectively. Usually, l-rhamnose was sterili- zed separately by filtration and added to each medium. The grown cells were harvested by centrifugation at 30 000 g for 20 min, washed with 20 mm potassium phosphate (pH 7.5) containing 1 mm EDTA and 10 mm 2-mercapto- ethanol (referred to as Buffer A), and stored at )35 °C until use. Fungal cells were suspended in Buffer A, homogenized with an equal volume of glass beads (0.5 mm diameter, Sigma, St Louis, MO, USA) for 30 min with appropriate intervals on ice using a TORNADO Ò Laboratory Power Mixer (AS ONE Co., Ltd., Osaka, Japan) and then centrifuged at 108 000 g for 1 h at 4 °C to obtain cell-free extracts. Bacterial cells were suspended in Buffer A, disrupted by sonication for 20 min with appropriate intervals on ice using an ASTRASON Ò Ultrasonic Liquid Processor XL2020 (Misonix Incorporated, New York, NY, USA) and then centrifuged. Enzyme activity assay l- and d-lactaldehyde were chemically synthesized from l- and d-threonine, respectively, according to the method of Huff and Rudney [18] with a few modifications. LADH activity was assayed routinely in the direction of aldehyde oxidation by measuring the reduction of NAD(P) + at 340 nm and 30 °C. The standard assay mixture contained 1mml-lactaldehyde, 1 mm EDTA and 10 mm 2-mercapto- ethanol in 66.7 mm potassium phosphate (pH 7.5) buffer. The reaction was started by the addition of 15 mm NAD(P) + solution (100 lL) with a final reaction volume of 1 mL. In the case of water-insoluble aldehyde, 10 mm substrates in ethanol (0.1 volume) were added to the standard assay solution. Protein concentrations were determined by the method of Lowry et al. [19], with bovine serum albumin as a standard. Zymogram staining analysis for LADH Cell-free extracts were separated by non-denaturing PAGE with a 12% gel at 4 °C. The gels were then soaked in 10 mL of staining solution [20] consisting of 100 mm Tris ⁄ HCl (pH 9.0), 10 mmd-orl-lactaldehyde, 0.25 mm nitroblue tetrazolium, 0.06 mm phenazine methosulfate and 15 mm NAD(P) + at 30 °C for 15 min. Dehydrogenase activity appeared as a dark band. Purification of native LADH from P. stipitis All purification steps were performed below 4 °C. All chromatography was carried out using an A ¨ KTA purifier system (Amersham Pharmacia Biotech, Little Chalfont, UK) and ⁄ or BioAssist eZ system (TOSOH, Tokyo, Japan). Cell-free extracts prepared from l-rhamnose-grown P. stipitis cells were loaded onto a HiPrep 16 ⁄ 10 Q FF column (1.6 · 10 cm, Amersham Biosciences, Uppsala, Sweden) equilibrated with Buffer A, and washed thoroughly with the same buffer. The column was developed with 300 mL of a linear gradient of 0–0.5 m NaCl in Buffer A. Active fractions containing LADH were combined and concen- trated by ultrafiltration with a Centriplus YM-30 (Millipore, Bedford, MA, USA) at 18 000 g for approxi- mately 2 h. The enzyme solution was loaded onto a column of HiLoad 26 ⁄ 60 Superdex 200 pg (2.6 · 60 cm, Amersham Biosciences) equilibrated with Buffer A. The active fractions were pooled, concentrated and applied to a column of Ceramic Hydroxyapatite Type I (1.6 · 5 cm, Bio-Rad Laboratories, Hercules, CA, USA) equilibrated with Buffer A. The column was washed thoroughly with the same buffer and developed with 150 mL of a linear gradient of 0–0.3 m potassium phosphate in Buffer A. The fractions with high enzymatic activity were combined, con- centrated and loaded onto a column of Blue-SepharoseÔ 6 Fast Flow (1.6 · 5 cm, Amersham Biosciences) equili- brated with Buffer A. The column was washed with Buffer A containing 50 mm NaCl, and then the enzyme was eluted with Buffer A containing 1 m NaCl. The elutant was concentrated, dialysed against 50 mm potassium phosphate, pH 7.5, containing 1 mm EDTA, 1 mm dithiothreitol and 50% (v ⁄ v) glycerol, and stored at )35 °C until use. Determination of internal amino acid sequences Purified PsLADH ( 50 lg) was separated by SDS-PAGE with a 10% (w ⁄ v) gel. In-gel digestion by trypsin was performed according to a standard protocol [21] with a few modifications. The peptide masses were analysed using a matrix-assisted laser desorption ionization- quadruple ion trap-mass spectrometer (AXIMA QIT, Shimadzu, Kyoto, Japan) with 2,5-dihydroxybenzoic acid (Shimadzu GLC Ltd, Tokyo, Japan) as a matrix in positive ion mode. Identification of enzyme reaction product HPLC analysis was performed using a Multi-Station LC-8020 model II system (TOSOH). Purified native S. Watanabe et al. L-Lactaldehyde dehydrogenase FEBS Journal 275 (2008) 5139–5149 ª 2008 The Authors Journal compilation ª 2008 FEBS 5147 PsLADH ( 1 mg) was added to a reaction mixture (500 lL) consisting of 20 mm potassium phosphate, pH 7.5, 10 mml-lactaldehyde and 10 mm NAD + . After incubation at 30 °C for 1 h, 12% (w ⁄ v) trichloroacetic acid was added to the samples (0.1 volume) to remove proteins. The filtrate (100 lL) was applied at 35 °Ctoan Aminex HPX-87H Organic Analysis column (300 · 7.8 mm, Bio-Rad) linked to an RID-8020 refractive index detector (TOSOH), and eluted with 5 mm H 2 SO 4 at a flow rate of 0.6 mLÆmin )1 . Functional expression and purification of His6-tagged proteins Genomic DNA of P. stipitis and A. vinelandii was prepared using a DNeasy Ò Boold & Tissue Kit (Qiagen, Tokyo, Japan). To introduce the restriction sites for BamHI and PstI at the 5¢- and 3¢-termini of PsLADH, PsALDH* and AvLADH genes, respectively, genomic PCR was carried out using Ex Taq Ò DNA polymerase (TaKaRa, Otsu, Shiga, Japan) and appropriate primers (Table S2). Each amplified DNA fragment was introduced into BamHI-PstI sites in pQE-80L (Qiagen), a plasmid vector for conferring the N-terminal His6 tag on expressed proteins. E. coli DH5a harbouring the expression plasmid was grown at 37 °Ctoa turbidity of 0.6 at 600 nm in Super broth medium contain- ing 50 mgÆL )1 ampicillin. After the addition of 1 mm iso- propyl thio-b -d-galactopyranoside, the culture was grown for a further 6 h to induce the expression of His6-tagged protein. Cells were harvested and resuspended in Buffer B (pH 8.0, 50 mm sodium phosphate containing 300 mm NaCl, 10 mm 2-mercaptoethanol and 10 mm imidazole). The cells were then disrupted by sonication, and the solution was centrifuged. The supernatant was loaded onto a column of Ni-NTA Super Flow (Qiagen) equilibrated with Buffer B, using an A ¨ KTA purifier system and ⁄ or Bio- Assist eZ system. The column was washed with Buffer C (pH 8.0, Buffer D containing 10% (v ⁄ v) glycerol and 50 mm imidazole instead of 10 mm imidazole). The enzymes were then eluted with Buffer C containing 250 mm imid- azole instead of 50 mm imidazole. All His6-tagged enzymes were used within 1 week in further experiments. Amino acid sequence alignment and phylogenetic analysis For phylogenetic analysis, 145 ALDH sequences were obtained from a website devoted to ALDHs: http://www. psc.edu/biomed/pages/research/Col_HBN_ALDH.html [12]. The sequences were aligned using the program clustalw , distributed by GenomeNet (Bioinformatics Center, Kyoto University, Kyoto, Japan) (http://www.genome.jp). The phylogenetic tree was produced using the treeview 1.6.1 program. Northern blot analysis Pichia stipitis and A. vinelandii cells were cultured at 30 ° C to the mid-logarithmic phase (A 600 = 0.6–0.8) and har- vested by centrifugation. Total RNAs were prepared using an RNeasy Ò Mini Kit (Qiagen). Northern hybridization was carried out using a standard method. The PCR prod- ucts of PsLADH, PsALDH* and AvLADH genes, amplified by PCR using appropriate DNA primers, were labelled with [a- 32 P]dCTP using the Random Primer Labelling Kit (TaKaRa) and used as probes for hybridization. Acknowledgements This work was supported by a Grant-in-Aid for Young Scientists (B) (No. 18760592) from the Ministry of Education, Culture, Sports, Science and Technology of Japan (to S.W.), by the Fermentation and Meta- bolism Research Foundation, by the Japan Bioindustry Association (to S.W.), by the Research Foundation from the Association for the Progress of New Chemis- try (to S.W.), by the New Energy and Industrial Tech- nology Development Organization (to S.W.) and by CREST, JST (to K.M.). We thank Dr T. W. Jeffries (University of Wisconsin, Milwaukee, WI, USA) for the gift of P. stipitis CBS 6054. We are especially grateful to Dr M. Yamada (Shimadzu Corporation, Kyoto, Japan) for his help with MALDI-TOF MS analysis. References 1 van Maris AJ, Abbott DA, Bellissimi E, van den Brink J, Kuyper M, Luttik MA, Wisselink HW, Scheffers WA, van Dijken JP & Pronk JT (2006) Alcoholic fermentation of carbon sources in biomass hydrolysates by Saccharomyces cerevisiae: current status. 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Metabolic fate of L-lactaldehyde derived from an alternative L-rhamnose pathway Seiya Watanabe 1,2,3 , Sommani Piyanart 1 and Keisuke Makino 1,2,3,4 1 Institute of Advanced Energy,. the metabolic fate of l-lactaldehyde derived from the alternative l-rhamnose pathway in P. stipitis is dehydrogenation by LADH. Purification of LADH from P. stipitis (PsLADH) PsLADH was purified from. enzymes of P. stipitis and Azotobacter vinelandii (orange) were characterized in this study. L-Fucose is converted to pyruvate and L-lactaldehyde through the analogous pathway to L-rhamnose, and metabolic