Purification, characterization and subunits identification of the diol dehydratase of Lactobacillus collinoides Nicolas Sauvageot 1 , Vianney Pichereau 1 , Loı¨c Louarme 2 , Axel Hartke 1 , Yanick Auffray 1 and Jean-Marie Laplace 1 1 USC INRA de Microbiologie de l’Environnement, Universite ´ de Caen, France; 2 Chaire de Biochimie Industrielle et Agro-Alimentaire, CNAM, Paris, France The three genes pduCDE encoding the diol dehydratase of Lactobacillus collinoides, have been cloned for overexpres- sion in the pQE30 vector. Although the three subunits of the protein were highly induced, no activity was detected in cell extracts. The enzyme was therefore purified to near homo- geneity by ammonium sulfate precipitation and gel filtration chromatography. In fractions showing diol dehydratase activity, three main bands were present after SDS/PAGE with molecular masses of 63, 28 and 22 kDa, respectively. They were identified by mass spectrometry to correspond to the large, medium and small subunits of the dehydratase encoded by the pduC, pduD and pduE genes, respectively. The molecular mass of the native complex was estimated to 207 kDa in accordance with the calculated molecular masses deduced from the pduC, D, E genes (61, 24.7 and 19,1 kDa, respectively) and a a 2 b 2 c 2 composition. The K m for the three main substrates were 1.6 m M for 1,2-propanediol, 5.5 m M for 1,2-ethanediol and 8.3 m M for glycerol. The enzyme required the adenosylcobalamin coenzyme for catalytic activity and the K m for the cofactor was 8 l M . Inactivation of the enzyme was observed by both glycerol and cyano- cobalamin. The optimal reaction conditions of the enzyme were pH 8.75 and 37 °C. Activity was inhibited by sodium and calcium ions and to a lesser extent by magnesium. A fourth band at 59 kDa copurified with the diol dehydratase and was identified as the propionaldehyde dehydrogenase enzyme, another protein involved in the 1,2-propanediol metabolism pathway. Keywords: Lactobacillus collinoides; diol dehydratase; purification; adenosylcobalamin; 1,2-propanediol. Diol dehydratase (EC 4.2.1.28) and glycerol dehydratase (EC.4.2.1.30) are two iso-functional enzymes that catalyse the conversion of 1,2-propanediol, 1,2-ethanediol and glycerol to propionaldehyde, acetaldehyde and 3-hydroxy- propionaldehyde, respectively [1]. This dehydration reaction is the first step of an anaerobic metabolism pathway. The aldehyde produced by these dehydratases can then be dismuted, allowing regeneration of NADH by an alcohol dehydrogenase and/or the ATP synthesis involving CoA- dependent propionaldehyde dehydrogenase, phosphotrans- acylase and kinase [2]. These dehydratases have been widely studied in bacteria such as Klebsiella pneumoniae [2,3], K. oxytoca [4], Citrobacter freundii [5], Clostridium pasteu- rianum [6] and Salmonella enterica LT2 [7]. They use adenosylcobalamin (AdoCbl) as a cofactor and exhibit a a 2 b 2 c 2 structure, where a, b and c represent the large, medium and small subunits of the protein, respectively. However, these two enzymes differ in their substrate specificities since diol dehydratase has a higher affinity for 1,2-propanediol and glycerol dehydratase for glycerol [8]. Except for the microorganisms mentioned above, the function of these enzymes in other bacteria is not well understood. Despite the fact that the presence of diol and glycerol dehydratases has already been reported in the genera Lactobacillus [9,10], researchers have only recently started to study these enzymes [11,12]. In Lactobacillus reuteri, a bacterium resident of the gastrointestinal tract of humans, the AdoCbl-dependent glycerol dehydratase has been purified. This enzyme seemed to exhibit a particular structure of four identical subunits of 52 kDa each [13]. Up to now, this is the only communication of the composition of a dehydratase enzyme obtained by purification in this bacterial genera. L. collinoides is a lactic acid bacterium commonly encountered in cider [14], in which it may be responsible for the alteration known as Ôpiqu ˆ re acrole ´ iqueÕ, as a result of the formation of acrolein (2-propenal), a lachrymatory chemical generating a peppery flavour [15]. Acrolein is not issued from the bacterial metabolism but rather is in chemical equilibrium with 3-hydroxypropionaldehyde formed by the dehydratase from glycerol. This aldehyde can spontaneously form acrolein by thermal dehydration under acid or heat conditions thus spoiling the quality of cider. During the course of our investigations on the glycerol metabolic pathway in L. collinoides, we have sequenced a genomic DNA region exhibiting strong homologies with the diol dehydratase pdu operon of Salmonella enterica [16]. The structure of the protein deduced from this sequence was Correspondence to N. Sauvageot, USC INRA de Microbiologie de l’Environnement, Universite ´ de Caen, 14032 CAEN Cedex, France. Fax: + 33 2 31 56 53 11, Tel.: + 33 2 31 56 59 30, E-mail: phdlme@ibba.unicaen.fr Abbreviations: AdoCbl, adenosylcobalamin; CNCbl, cyanocobalamin. Enzymes: Diol dehydratase (EC 4.2.1.28); glycerol dehydratase (EC.4.2.1.30). (Received 21 June 2002, revised 25 September 2002, accepted 2 October 2002) Eur. J. Biochem. 269, 5731–5737 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.03288.x different than that reported in L. reuteri. In this study, we report the purification, enzymatic characterization and analysis of the composition of the diol dehydratase of L. collinoides. MATERIALS AND METHODS Bacteria and culture conditions The lactic acid bacterium used in this study was L. collino- ides LMG 18850, isolated from a French cider [17]. Cultures were grown in MRS medium [18] supplemented with 2% (w/v) glucose at 30 °C without shaking. For the purification of the diol dehydratase, L. collinoides was grown in 3 L conical flasks containing 2.5 L MRS medium supplemented with 50 m M 1,2-propanediol and 15 m M glucose. After inoculation with 2% (v/v) of a 48-h culture of L. collinoides, the conical flask was incubated for 20 h at 30 °C. The Escherichia coli M15[pREP4] strain (Qiagen, Santa Clara, CA, USA), used for the overexpression, was cultured under the manufacturer’s recommended conditions, in 2 · TY medium [19] with 100 lgÆmL )1 ampicillin and 25 lgÆmL )1 kanamycin. Purification procedures Cellular lysis. The protocol for the purification of the diol dehydratase of L. collinoides was adapted from that of Schu ¨ tz and Radler [20]. Cells were harvested by centrifuga- tion (3000 g, 10 min) and washed twice in potassium phosphate buffer K 2 HPO 4 I(10m M ,pH7.2,1m M dithiothreitol and 1 m M phenylmethanesulfonyl fluoride) and suspended in 10 mL of degassed K 2 HPO 4 II (10 m M , pH 7.2 containing 5 m M of dithiothreitol). The lysis was performed by one passage through the Ôone shotÕ cell disrupter (ConstantSystem, Northants, UK) at 2.15 kbar. 1 mg of deoxyribonuclease I (Sigma, St Louis, MO, USA) was added to the disrupted solution and cell debris were removed by two centrifugations (3000 g,10minand 15 500 g,20min). Ammonium sulfate precipitation. The extract was homo- genized with 1 volume of ammonium sulfate solution at 456 gÆL )1 to obtain a final concentration of 40% saturation. The homogenate was maintained on ice for 1 h and centrifuged for 20 min at 15 500 g. The pink sediment (C40) containing the diol dehydratase was resuspended in 1 mL of the K 2 HPO 4 II buffer described above. Gel filtration chromatography. The preparation was loaded onto a Sephacryl S300H (Sigma) column (100 · 1.6 cm) equilibrated with K 2 HPO 4 II. Chromato- graphy was conducted at a flow rate of 0.6 mLÆmin )1 and fractions containing the highest dehydratase activity were pooled and stored until use at )20 °C. Dehydratase assays Two methods were used for the determination of the activity. During the purification and characterization enzyme steps, the assay was carried out using the 3-methyl-2-benzothiazolinone hydrazone method [21]. The reaction mixture (0.5 mL) was composed of 0.2 M 1,2-propanediol (or glycerol), 0.05 M KCl, 0.035 M potas- sium phosphate (pH 7.2) and 20 l M AdoCbl. The reaction was proceeded for 10 min at 37 °C and stopped by addition of 0.5 mL of 0.1 M potassium citrate buffer (pH 3.6) and 250 lL of 0.1% 3-methyl-2-benzothiazolinone hydrazone solution. After a 15-min incubation period at 37 °C, 0.5 mL of water was added and the absorbance was measured at 305 nm. The second procedure, used for the determination of kinetic constants, was reported by Bobik et al.[7].The aldehyde formation was coupled with the alcohol dehy- drogenase (Roche Diagnostics, Mannheim, Germany). A 1-mL reaction mixture contained 0.1 M 1,2-propanediol, 0.1 M Hepes buffer (pH 8.75), 0.1 m M NADH, excess alcohol dehydrogenase (18 U) and 20 l M AdoCbl. The 1,2-propanediol utilization was monitored by following the conversion of NADH to NAD + at 340 nm. For the K m determination with glycerol, assays were performed as described above but without NADH and alcohol dehy- drogenase since the 3-hydroxypropionaldehyde was not converted to 1,3-propanediol by the alcohol dehydrogenase. The reaction was stopped after 0, 2, 4 and 6 min with 1 volume of 1 M citrate buffer (pH 3.6) and the 3-hydroxy- propionaldehyde was monitored using the 3-methyl- 2-benzothiazolinone hydrazone method. One unit of diol dehydratase activity was defined as 1 lmol of aldehyde formed per minute. For the assays, between 0.001 and 0.01 units of enzyme were used. Protein concentration was determined by the method of Lowry [22] with BSA as a standard. PAGE PAGE under denaturing conditions was performed as described by Laemmli [23] in the MiniProtean(R)3 appar- atus (Bio-Rad, Hercules, CA, USA) with a 12% polyacryl- amide gel. A 6% polyacrylamide gel without SDS was used for electrophoresis under nondenaturing conditions. Pro- teins were stained with Coomassie Brilliant Blue R250. Trypsin digestion and mass spectrometry Bands of interest were excised from the gel, rinsed twice with ultra pure water and dehydrated for 10 min by incubation in acetonitrile (Sigma). Samples were dried for 30 min under vacuum and reswelled with 2 lLof50m M NH 4 HCO 3 containing 1 lg of trypsin for 1 h. Twenty microliters of 50 m M ammonium bicarbonate were added and digestion was continued overnight. After centrifugation (1000 g, 5 min), the supernatant was collected and gel pieces were placed successively in 20 lLof20m M ammonium bicar- bonate, 20 lLof20m M ammonium bicarbonate/acetonit- rile (1 : 1, v/v), twice in 5% formic acid/acetonitrile (1 : 1, v/v) and finally 20 lL of acetonitrile. Samples were centrifuged between each step and supernatants were collected, pooled, dried and resuspended in 10 lLof0.1% formic acid in ultra pure water. An electrospray ion trap spectrometer (LCQ DecaXP, ThermoFinnigan, San Jose, CA, USA) coupled on line with HPLC (SurveyorLC) was used for peptides analysis. Peptides were separated by reversed-phase HPLC on a C 18 capillary column (ThermoHyPurity C18 150 · 0.18). A linear 18-min gradient (flow rate, 3 lLÆmin )1 )from5to 5732 N. Sauvageot et al. (Eur. J. Biochem. 269) Ó FEBS 2002 80% B was used, where solvent A was 0.1% aqueous formic acid and solvent B was 0.1% formic acid in acetonitrile. The electrospray ionization parameters were as follows: spray voltage, 3.5 kV; sheath gas flow rate, 30; capillarity temperature, 200 °C; capillarity voltage, 30 V; Tube lens offset, 35 V. Mass spectrometry were acquired in a mode that alternated a full MS scan (mass range: 400–1600) and a collision induced dissociation tandem mass spectrometry (MS/MS) of the most abundant ion. The collision energy for the MS/MS scan was preset at the value of 35%. Data were analysed using the SEQUEST algorithm (version 2) incorporated with the ThermoFinnigan BIOWORKS software (version 2). Cloning of the pduCDE genes and overexpression DNA manipulation techniques were performed according to Sambrook et al. [19]. The three diol dehydratase genes were ligated into the expression vector pQE30 (Qiagen) downstream the His-tag sequence (pQE30HisDD). E. coli M15[pREP4] was used as the host strain. The absence of undesired mutation was confirmed by sequencing using the dideoxy chain-termination method [19] with the ABI Prism sequencing system (PE Biosystem, Warrington, UK). Transcription was induced by addition of isopropyl b- D - thiogalactoside to a final concentration of 1 m M for 4 h. The His-tag removal was obtained from the previous construction. The new construction (pQE30DD) contained the sequence of the a, b and c subunits genes and their Shine Dalgarno sequences. RESULTS High-level expression of the L. collinoides diol dehydratase The three genes pduCDE encoding the three subunits of the diol dehydratase were cloned in the pQE30 expression vector system. The synthesis of the three subunits of the protein was controlled by SDS/PAGE (Fig. 1A). Despite the high level of synthesis of the three proteins at 61, 24.7 and 19.1 kDa, no activity was detected in the extracts even using the same extraction protocol that detects activity in L. collinoides.A new attempt was performed with a new construction (pQE30DD) in which the His-tag coding sequence was removed. However, no dehydratase activity was detectable even with addition of 1,2-propanediol, Brij35 (detergent compatible with activity [24]), or by reducing the induction period or induction temperature (data not shown). To determine if the three subunits of the enzyme were expressed in a soluble form or in inclusion bodies, an SDS/PAGE analysis of the soluble and insoluble fractions was performed (Fig. 1B). The subunits of the diol dehydratase, mainly the medium subunit, were present in the insoluble fraction suggesting that the recombinant protein cannot form an active complex. In order to demonstrate that the three genes pduCDE encode for active diol dehydratase, we attempted to purify the enzyme from L. collinoides. Purification of the diol dehydratase of L. collinoides Unliketheentericbacteria,L. collinoides was unable to grow on 1,2-propanediol as the sole carbon source. Recently, we have shown that a high level of activity was detected during the stationary phase when this microorgan- ism was grown in MRS medium containing 15 m M glucose and 50 m M 1,2-propanediol [16]. Therefore, these growth conditions were used in order to purify the diol dehydratase enzyme. As reported by Talarico et al. [13] for the glycerol dehydratase of L. reuteri, the enzyme of L. collinoides was stable in media containing high potassium and 1,2-pro- panediol concentrations. A first purification was attempted with a phosphate buffer containing these two compounds. However, under these conditions, the enzyme started to precipitate at very high ammonium sulfate concentration (70% saturation) and the yield of active enzyme was very low. For this reason, we have purified the enzyme in a 10-m M potassium phosphate buffer without 1,2-propane- diol and potassium. Under this condition, the enzymatic complex was very unstable at 4 °C. Ninety percent of the activity was lost during 5 days storage (data not shown). The different steps of the purification of the diol dehydra- tase of L. collinoides are summarized in Table 1. The first step was a precipitation with ammonium sulfate. The enzyme precipitated between 20 and 60% saturation with a maximum specific activity at 40% saturation. A first strategy based on stepwise ammonium sulfate precipitation (step 20% followed by 60% saturation) resulted in complete loss of activity. Thus, the dehydratase was precipitated with 40% ammonium sulfate. The sample was loaded onto a Sephacryl S300H chromatography. The activity eluted from the column 28 mL after the void volume. The six fractions Fig. 1. SDS/PAGE of cell-free extracts of E. c oli showing the over- expression (A) and insolubility (B) of the three genes pduCDE coding for the diol dehydratase of L. collinoides. (A) Protein content of E. coli M15[pREP4] carrying the plasmid pQE30 (control, lane 1), pQE30HisDD (lane 2) and pQE30DD (lane3) after induction with 1m M isopropyl b- D -thiogalactoside and 4 h incubation. (B) Protein content of E. coli M15[pREP4] carrying the plasmid pQE30 (control, lanes 4,5,6), pQE30HisDD (lanes 7–9), total expression (lanes 4 and 7), insoluble fraction (lanes 5 and 8) and soluble fraction (lanes 6 and 9). Table 1. Purification of diol dehydratase of L. collinoides. Purification step Protein (mg) Activity (U) Specific activity (UÆmg )1 ) Yield (%) Purification factor Crude extract 129.34 28.68 0.22 100 1 C40 16.78 5.69 0.34 19.8 1.54 Sephacryl S300H 4.15 3.2 0.77 11.1 3.5 Ó FEBS 2002 Diol dehydratase purification and characterization (Eur. J. Biochem. 269) 5733 containing the maximal diol dehydratase activity were combined and used for the characterization of the enzyme. After Sephacryl S300H chromatography, the pooled fractions containing the highest dehydratase activity were analysed by SDS/PAGE. The pattern showed four main bands with molecular masses of approximately 63, 59, 28 and 22 kDa (proteins 1, 2, 3 and 4, respectively) (Fig. 2). The molecular masses of three of these bands (proteins 1, 3 and 4) were in good agreement with those calculated for the polypeptides encoded by the pduCDE genes (60.1, 24.7 and 19.1 kDa for the a, b, c subunit, respectively). All bands were excised from the gel, digested by trypsin, and their identities were ascertained by mass spectrometry (Table 2) with the help of the pduCDE nucleotide sequence. The three bands at 63, 28 and 22 kDa were identified as PduC, PduD and PduE, respectively. Regarding the fourth band at 59 kDa (Protein 2), we first supposed that it could represent the large subunit of the reactivation factor of L. collinoides (PduG). Indeed, recent studies have shown that the diol dehydratase of K. oxytoca and the glycerol dehydratases of K. pneumoniae and C. pasteurianum can form a complex with the reactivation factor [25]. However, this hypothesis was disproved by mass spectrometry analysis. Among the various attempts to purify diol dehydratase, the presence of a fourth band at 51 kDa has been reported in K. oxytoca and microsequenced [26,27]. N-Terminal sequence analysis revealed that it showed high homology to CoA-dependent propionaldehyde dehydrogenase of S. ent- erica PduP. By mass spectrometry analysis, nine short polypeptides have been microsequenced; six of these exhibit weak homologies with some dehydrogenases and one fragment seemed to be related to the L. monocytogenes and L. innocua propionaldehyde dehydrogenase. With the help of the propionaldehyde dehydrogenase sequence of L. collinoides (O. Claisse, University of Bordeaux, France, personnal communication), we confirmed that the copuri- fied protein corresponded to this enzyme (Table 2). In order to determine if the fourth band belonged to the enzymatic complex, two-dimensional electrophoresis was performed (Fig. 3). The first dimension was carried out under nondenaturing conditions and revealed one main band and two weaker bands. Their dissociation by SDS in the second dimension showed that the main band was released into three subunits migrating at the same positions as the large, medium and small subunits of the diol dehydratase. However, the band of 59 kDa was not aligned with the three Pdu proteins. Therefore, it seemed that the fourth protein copurified with the diol dehydratase does not belong to the dehydratase complex. Characterization of the diol dehydratase The molecular mass of the native dehydratase was estimated by the Sephacryl S300H gel filtration using five standards of known molecular mass (thyroglobulin 669 kDa, apoferritin 443 kDa, b-amylase 200 kDa, alcohol dehydrogenase Fig. 3. Two-dimensional PAGE of purified diol dehydratase. After the two-step purification, an aliquot containing dehydratase activity was first separated on 6% nondenaturing polyacrylamide gels. The lane was cut from the first gel and put on horizontally onto a 12% SDS polyacrylamide gel. Proteins corresponding to PduCDE and to the propionaldehyde dehydrogenase copurified are indicated. The propionaldehyde dehydrogenase does not align with the three diol dehydratase subunits (dashed line). Table 2. Mass-spectrometric identification of the protein components purified from SDS/PAGE. Protein number Best hit Score a MS/MS sequenced peptides % Protein coverage b 1 PduC 274 17 37.3 2 Propionaldehyde dehydrogenase 147 8 24.9 3 PduD 157 7 48.7 4 PduE 181 7 58.4 a Represents the score given by the SEQUEST software and b the percentage of amino acids effectively sequenced by LC-MS/MS. Fig. 2. SDS/PAGE analysis of the purification steps of the diol dehy- dratase from L. collinoides. Lane 1: molecular mass markers, lane 2: crude extract, lane 3: ammonium sulfate precipitation C40, and lane 4: pooled fractions from the Sephacryl S300H. 5734 N. Sauvageot et al. (Eur. J. Biochem. 269) Ó FEBS 2002 150 kDa and BSA 66 kDa) (Fig. 4) and was found to be approximately 207 kDa. The determination of the optimum pH for enzyme activity was performed by using a range from pH 6 to pH 9.5 in 100 m M Hepes buffer adjusted with KOH. The highest activity was obtained between 8.5 and 9.25 with a maximum at pH 8.75. This was in accordance with the optimum pH of the dehydratase of K. pneumoniae (pH 8.6) [28] but not with the L. reuteri enzyme (pH 7.2) [13]. Temperature was also studied for its influence on the 1,2-propanediol conversion to propionaldehyde. A range of temperatures between 25 °C and 45 °C was tested and the optimum was observed at 37 °C. So, further kinetic experiments were performed at these pH and temperature values. The K m for the three preferential substrates of the dehydratase and the AdoCbl cofactor were determined. The highest substrate affinity was obtained for 1,2-propanediol with a K m of 1.6 m M followed by 1,2-ethanediol (K m : 5.5 m M ) and glycerol (K m :9.4 m M ). Affinity for the AdoCbl coenzyme was considerably higher with a K m of 8.3 l M . It has been shown that glycerol is both a substrate and a suicide-inactivator for diol and glycerol dehydratase [8]. In order to show whether the enzyme of L. collinoides posses- ses this characteristic, a dehydration reaction time course was performed with 1,2-propanediol or glycerol (Fig. 5A). When 1,2-propanediol was used as substrate, a linear increase in aldehyde formation was observed for 20 min. In the case of glycerol, the initial kinetic was similar to that found using 1,2-propanediol but the reaction ceased after 4 min. This could not be explained by exhaustion of the substrate, which was present in excess (0.2 M ), and conse- quently must have resulted from the inactivation of the L. collinoides diol dehydratase. Cyanocobalamin (CNCbl) is a competitive inhibitor of diol and glycerol dehydratases and its effect on the enzyme of L. collinoides was studied. Figure 5B illustrated the time course reaction of L. collinoides diol dehydratase with 1,2- propanediol as substrate and a AdoCbl/CNCbl mixture as cofactor. In all kinetic experiments, AdoCbl was appointed to 15 l M . No inhibition was observed when CNCbl was absent. When increased concentration of CNCbl was added, the formation rate of propionaldehyde decreased reflecting the increased fixation of the inactive analogue of the AdoCbl. A K i of 26.4 l M was calculated for the cyanocobalamin. For all the dehydratases characterized [2–7], monovalent cations seem to be required for the catalytic activity. The influence of various mono and divalent cations in low concentrations (10 m M ) on the diol dehydratase activity was then studied (data not shown). A slightly inhibitory effect was observed with the divalent Mg 2+ ion whereas both sodium and calcium ions caused complete inhibition. The potassium concentration estimated at pH 8.75 in the reaction mixture was 100 m M . Therefore, the inhibitory effect observed with Mg 2+ ,Ca 2+ and Na + ions was not a competitive inhibition but rather due to an alteration of the complex. This effect was not observed with Li + and NH 4 + , which did not affect activity. All characteristics are summarizedinTable3. DISCUSSION In cider, L. collinoides is involved in glycerol degradation leading potentially to an alteration of the beverage known as Ôpiqu ˆ re acrole ´ iqueÕ. The first reaction of the glycerol Fig. 4. Gel filtration chromatography and molecular mass determination of the diol dehydratase of L. collinoides. Proteins were separated on Sephacryl S300H column and fractions containing diol dehydratase were identified by activity determination. The molecular mass calib- ration was performed with five standard proteins (d) (thyroglobulin 669 kDa, apoferritin 443 kDa, b-amylase 200 kDa, alcohol dehy- drogenase 150 kDa and BSA 66 kDa). Fig. 5. Time course of reaction of diol dehydratase with 1,2-propanediol or glycerol (A) and the effect of CNCbl on the enzymatic activity with 0.2 M of 1,2-propanediol as substrate (B). (A) The amount of aldehyde formed was determined by the 3-methyl-2-benzothiazolinone hydra- zone method with 0.2 M of 1,2-propanediol (j)orglycerol(d)and 0.0015 U of enzyme. (B) Time course of reaction with 0.007 U of diol dehydratase and 15 l M of AdoCbl plus 0 l M (j), 5 l M (e), 10 l M (n), 20 l M (s)and50l M (h)ofCNCbl. Ó FEBS 2002 Diol dehydratase purification and characterization (Eur. J. Biochem. 269) 5735 metabolism is catalysed by a dehydratase that converts glycerol to 3-hydroxypropionaldehyde, a precursor of acrolein [12]. The dehydratase enzyme plays therefore a key role in the development of the alteration in cider. Unlike the enteric bacteria and C. pasteurianum, which are able to grow on glycerol or 1,2-propanediol, L. collinoides can not grow on media containing these compounds as the sole carbon source. This strongly suggests an essential role for the regeneration of NADH, which is necessary to reduce 3-hydroxypropionaldehyde to 1,3-propanediol by 1,3-pro- panediol dehydrogenase. As the three genes encoding the three subunits of the dehydratase have been sequenced [16], the first strategy attempted to purify the enzyme was the expression of recombinant protein in E. coli. Although the three subunits were expressed, no activity could be detected. In all heterologous expressions of others dehydratases, it has been shown that no additional subunit was required for activity [5–7]. A reasonable explanation for our result was that the protein possesses a low solubility. This feature seems to be common to dehydratases and the use of detergents like Brij35 (0.5–1%) has been shown to increase considerably the solubility [24]. However, this was not the case for the diol dehydratase of L. collinoides.Asallthe recombinant diol dehydratases, overexpressed and recov- ered in an active form in E. coli, belonged to enterobacteria (S. enterica, K. oxytoca and K. pneumoniae), it is possible that an important compound for the formation of the L. collinoides protein folding complex or enzyme activity was lacking. In the second attempt, we purified the enzyme from crude extracts of L. collinoides by a two-step procedure. This strategy allowed us to purify the enzyme to near homogeneity and to increase the specific activity of the preparation 8 . The decrease of the total activity was prob- ably due to considerable enzyme instability during the purification process. The diol dehydratase exhibited a native molecular mass of the complex of 207 kDa. This was in accordance with the masses obtained for other dehydratases (180 kDa for L. brevis, 190 kDa for C. pasteurianum, 230 kDa for K. oxytoca, 188 kDa for K. pneumoniae). The enzyme was able to degrade the three substrates tested and the specificities observed confirmed that the enzyme belongs to the diol dehydratase family. Although L. collinoides is known to be involved in an alteration beginning with glycerol degradation, this latter was not the preferred substrate. 9 The enzyme showed highest affinity for 1,2-propanediol and 1,2-ethanediol. Moreover, it was coenzyme AdoCbl-dependent and had a strong affinity for the cofactor. The K m obtained was similar to that obtained for the enzyme of L. brevis (7 l M ) [20]. The study of the effect of different cations on the reaction showed that Na + and Ca 2+ were incompatible with catalysis. This was probably due to a dissociation of the complex as reported by Schneider et al.[3]forthe glycerol dehydratase apoenzyme with Na + . Contrary to the glycerol dehydratase of L. reuteri,which has been reported to be a homotetramer of 52 kDa subunits, the dehydratase of L. collinoides showed a classi- cal heterotrimer conformation similar to that of other described enzymes (K. oxytoca [4], K. pneumoniae [2,3], S. enterica [7], C. freundii [5] and C. pasteurianum [6]). However, since the enzyme of L. reuteri was purified with two other proteins (70 and 40 kDa) and no genetic studies have been published, one cannot eliminate the possibility that the dehydratase of this organism is also composed of several nonidentical subunits. Moreover, the copurification of the propionaldehyde dehydrogenase together with diol dehydratase observed in this work, has also been reported by McGee in K. oxytoca [26,27]. Therefore in L. reuteri,we can assume that the 52 kDa protein was incorrectly assigned as a subunit of the diol dehydratase and is rather a propionaldehyde dehydrogenase. It is interesting to note that the protein copurified with the diol dehydratase belongs to the same operon. However, as we showed, the propion- aldehyde dehydrogenase does not seem to be a part of the dehydratase. Recently, parts of diol dehydratase operon have been sequenced in L. hilgardii and L. diolivorans [29]. This confirms the presence of a diol dehydratase exhibiting a a 2 b 2 c 2 composition in the Lactobacillus genera. In conclusion, we confirm here that the pdu operon encodes the functional dehydratase enzyme of L. collinoides. This suggests that the structure with three nonidentical subunits protein represents the main model in the diol or glycerol dehydratase in enterobacteria, C. pasteurianum as well as in L. collinoides. Table 3. Characteristics and comparison of the diol dehydratase of L. collinoides with the others Lactobacillus enzymes. Data for L. brevis, L. reuteri and L. sp. 208 A are taken from [20] [13], and [10], respectively. p-CMB, p-chloromercuribenzoate, –, not determined. Characteristics L. collinoides L. brevis L. reuteri L. sp. 208 A K m 1,2-propanediol (m M ) 1.6 0.6 7 – K m glycerol (m M ) 8.3 4.0 3.3 – K m 1,2-ethanediol (m M ) 5.5 5.3 – – K m AdoCb (l M ) 8 7 0.3 – V max 1,2-propanediol (UÆmg protein )1 ) 0.054 – – – V max glycerol (UÆmg protein )1 ) 0.018 – – – V max 1,2-ethanediol (UÆmg protein )1 ) 0.012 – – – Molecular mass (kDa) 207 180 200 188 Subunit composition a2b2c2 – Homotetamer – Subunit molecular masses (kDa) 63, 25, 19 – 52 – pH optimum 8.75 7.0–7.5 6.5–8 5.8–6.0 Temperature optimum (°C) 37 30–40 – – Ion activity Na > Ca >Mg Na > Li > Mg > Mn – Na Inhibitor CNCbl p-CMB CNCbl CNCbl 5736 N. Sauvageot et al. (Eur. J. Biochem. 269) Ó FEBS 2002 ACKNOWLEDGEMENTS The authors wish to thanks the professor Jacques Nicolas, director of the ÔChaire de Biochimie Industrielle et Agro-AlimentaireÕ of the CNAM of PARIS for his welcome of Nicolas Sauvageot in his laboratory. This work was partly supported by a grant from the Ôconseil re ´ gional pour lÕAgrobiologie et la Bioindustrie (CRAB) de Basse- Normandie’ and from the European Union. N. Sauvageot is the recipient of an award from the Ministe ` re de la Recherche et de l’Enseignement Supe ´ rieur of France. We thank Mrs Monika Dabrowski-Coton for correcting the manuscript and Mr Olivier Claisse for providing us with the propionaldehyde dehydrogenase amino acid sequence. REFERENCES 1. Lee, H.A.J.R. & Abeles, R.H. (1963) Purification and properties of dioldehydrase, an enzyme requiring a cobamide coenzyme. J. Biol. Chem. 238, 2367–2373. 2. Toraya, T., Honda, S. & Fukui, S. (1979) Fermentation of 1,2-propanediol and 1,2-ethanediol by some genera of Enterobacteriaceae, involving coenzyme B 12 -dependent diol dehydratase. J. Bacteriol. 139, 39–47. 3. Schneider, Z., Larsen, E.G., Jacobson, G., Johnson, B.C. & Pawelkiewicz, J. (1970) Purification and properties of glycerol dehydrase. J. Biol. Chem. 245, 3388–3396. 4. Poznanskja, A.A., Tanizawa, K., Soda, K., Toraya, T. & Fukui, S. (1979) Coenzyme B 12 -dependent diol dehydrase: purification, subunit, heterogeneity, and reversible association. Arch. Biochem. Biophys. 194, 379–386. 5. Seyfried, M., Daniel, R. & Gottschalk, G. (1996) Cloning, sequencing and overexpression of the genes encoding coenzyme B 12 -dependent glycerol dehydratase of Citrobacter freundii. J. Bacteriol. 178, 5793–5769. 6. Macis, L., Daniel, R. & Gottschalk, G. (1998) Properties and sequence of the coenzyme B 12 -dependent glycerol dehydratase of Clostridium pasteurianum. FEMS Microbiol. Lett. 164, 21–28. 7. Bobik, T.A., Xu, Y., Jeter, R.M., Otto, K.E. & Roth. J.R. (1997) Propanediol utilization genes (pdu)ofSalmonella typhimurium: three genes for the propanediol dehydratase. J. Bacteriol. 179, 6633–6639. 8. Toraya, T., Shirakashi, T., Kosuga, T. & Fukui, S. (1976) Sub- strate specificity of coenzyme B 12 -dependent diol dehydratase, glycerol as both a good substrate and a potent inactivator. Biochem. Biophys. Res. Commun. 69, 475–480. 9. Sobolov, M. & Smiley, K.L. (1959) Metabolism of glycerol by an acrolein-forming Lactobacillus. J. Bacteriol. 79, 261–266. 10. Smiley, K.L. & Sobolov, M. (1962) A cobamide-requiring glycerol dehydrase from by an acrolein-forming Lactobacillus. Arch. Biochem. Biophys. 97, 538–543. 11. Schu ¨ tz, H. & Radler, F. (1984) Anaerobic reduction of glycerol to 1,3-propanediol by Lactobacillus brevis and Lactobacillus buchneri. System. Appl. Microbiol. 5, 169–178. 12. Sauvageot, N., Gouffi, K., Laplace, J M. & Auffray, Y. (2000) Glycerol metabolism in Lactobacillus collinoides:productionof 3-hydroxypropionaldehyde, a precursor of acrolein. Int. J. Food Microbiol. 55, 167–170. 13. Talarico, T.L. & Dobrogosz, W.J. (1990) Purification and Char- acterization of glycerol dehydratase from Lactobacillus reuteri. Appl. Environ. Microbiol. 56, 1195–1197. 14. Carr, J.G. & Davies, P.A. (1972) The ecology and classification of strains of Lactobacillus collinoides nov. spec. a bacterium com- monly found in fermenting apple juice. J. Appl. Bacteriol. 35, 463– 471. 15. Butzke, V.C.E., Bobmeyer,M.,Scheide,K.&Misselhorn,K. (1990) Anmerkungen zur Acrolein-Problematik in der Alkoho- lindustrie. Branntweinwirtschaft. 117, 286–289. 16. Sauvageot, N., Muller, C., Hartke, A., Auffray, Y. & Laplace, J M. (2002) Characterisation of the diol dehydratase pdu operon of Lactobacillus collinoides. FEMS Microbiol. Lett. 209, 69–74. 17. Laplace, J M., Sauvageot, N., Hartke, A. & Auffray, Y. (1999) Characterization of Lactobacillus collinoides response to heat, acid and ethanol treatments. Appl. Microbiol. Biotechnol. 51, 659–663. 18. de Man, J.C., Rogosa, M. & Sharp, M.E. (1960) A medium for the cultivation of Lactobacilli. J. Appl. Bacteriol. 23, 130–135. 19. Sambrook, J., Fritsch, E.F. & Maniatis, T. (1989) Molecular Cloning: a Laboratory Manual, 2nd edn. Cold Spring. Harbor Laboratory Press, Cold Spring Harbor, New York. 20. Schu ¨ tz, H. & Radler, F. (1984) Propanediol-1,2-dehydratase and metabolism of glycerol of Lactobacillus brevis. Arch. Microbiol. 139, 366–370. 21. Toraya, T., Kazutoshi, U., Kukui, S. & Hogenkamp, H.P.C. (1977) Studies on the mechanism of the adenosyl-cobalamin- dependent diol dehydratase reaction by the use of analogs of the coenzyme. J. Biol. Chem. 252, 963–970. 22. Lowry, O.H., Rosenbrough, N.J., Farr, A.L. & Randall, R.J. (1951) Protein measurement with the folin phenol reagent. J. Biol. Chem. 193, 265–275. 23. Laemmli, U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–685. 24. Tobimatsu, T., Sakai, T., Hashida, Y., Mizoguchi, N., Miyoshi, S. & Toraya, T. (1997) Heterologous expression, purification, and properties of diol dehydratase, an adenosylcobalamin-dependent enzyme of Klebsiella oxytoca. Arch. Biochem. Biophys. 347, 132– 140. 25. Mori, K. & Toraya, T. (1999) Mechanism of reactivation of coenzyme B 12 -dependent diol dehydratase by a molecular cha- perone-like reactivating factor. Biochem. 38, 13170–13178. 26. McGee, D.E. & Richards, J.H. (1981) Purification and subunit characterization of propanediol dehydrase, a membrane-asso- ciated enzyme. Biochem. 20, 4293–4298. 27. McGee, D.E., Carroll, S.S., Bond, M.W. & Richards, J.H. (1982) Diol dehydratase: N-terminal amino acid sequences and subunit stoichiometry. Biochem. Biophys. Res. Commun. 108, 547–551. 28. Schneider, Z. & Pawelkiewicz, J. (1966) The properties of gly- cerol dehydratase isolated from aerobacter aerogenes,andthe properties of the apoenzyme subunits. Acta Biochim. Polon. 13, 311–328. 29. Gorga, A., Claisse, O. & Lonvaud-Funel, A. (2002) Organisation of the encoding glycerol dehydratase of Lactobacillus collinoides, Lactobacillus hilgardii and Lactobacillus diolivorans. Sci. Aliments. 22, 151–160. Ó FEBS 2002 Diol dehydratase purification and characterization (Eur. J. Biochem. 269) 5737 . study, we report the purification, enzymatic characterization and analysis of the composition of the diol dehydratase of L. collinoides. MATERIALS AND METHODS Bacteria. encoding the three subunits of the diol dehydratase were cloned in the pQE30 expression vector system. The synthesis of the three subunits of the protein