Purification, characterization and subunits identification of the diolNicolas Sauvageot1, Vianney Pichereau1, Loı¨c Louarme2, Axel Hartke1, Yanick Auffray1 and Jean-Marie Laplace1 1 USC
Trang 1Purification, characterization and subunits identification of the diol
Nicolas Sauvageot1, Vianney Pichereau1, Loı¨c Louarme2, Axel Hartke1, Yanick Auffray1
and Jean-Marie Laplace1
1
USC INRA de Microbiologie de l’Environnement, Universite´ de Caen, France;2Chaire 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 a2b2c2composition The Kmfor the three
main substrates were 1.6 mMfor 1,2-propanediol, 5.5 mM for 1,2-ethanediol and 8.3 mM for glycerol The enzyme required the adenosylcobalamin coenzyme for catalytic activity and the Kmfor the cofactor was 8 lM Inactivation
of the enzyme was observed by both glycerol and cyano-cobalamin The optimal reaction conditions of the enzyme were pH 8.75 and 37C 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
a2b2c2 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 piquˆre acrole´ique, 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)
Trang 2different 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
M A T E R I A L S A N D M E T H O D S
Bacteria and culture conditions
The lactic acid bacterium used in this study was L
collino-idesLMG 18850, isolated from a French cider [17] Cultures
were grown in MRS medium [18] supplemented with 2%
(w/v) glucose at 30C 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 mM 1,2-propanediol and 15 mM glucose After
inoculation with 2% (v/v) of a 48-h culture of L collinoides,
the conical flask was incubated for 20 h at 30C The
Escherichia coliM15[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)1ampicillin 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 K2HPO4 I (10 mM, pH 7.2, 1 mM
dithiothreitol and 1 mM phenylmethanesulfonyl fluoride)
and suspended in 10 mL of degassed K2HPO4II (10 mM,
pH 7.2 containing 5 mM of dithiothreitol) The lysis was
performed by one passage through the one shot 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, 10 min and
15 500 g, 20 min)
Ammonium sulfate precipitation The extract was
homo-genized with 1 volume of ammonium sulfate solution at
456 gÆL)1to 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 K2HPO4II buffer described above
Gel filtration chromatography The preparation was
loaded onto a Sephacryl S300H (Sigma) column
(100· 1.6 cm) equilibrated with K2HPO4 II
Chromato-graphy was conducted at a flow rate of 0.6 mLÆmin)1and
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), 0.05MKCl, 0.035M potas-sium phosphate (pH 7.2) and 20 lMAdoCbl The reaction was proceeded for 10 min at 37C and stopped by addition
of 0.5 mL of 0.1M 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 37C, 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 0.1M1,2-propanediol, 0.1 M Hepes buffer (pH 8.75), 0.1 mM NADH, excess alcohol dehydrogenase (18 U) and 20 lM AdoCbl The 1,2-propanediol utilization was monitored by following the conversion of NADH to NAD+at 340 nm For the Km 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 1Mcitrate 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% polyacrylpolyacryl-amide 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 lL of 50 mM NH4HCO3 containing 1 lg of trypsin for 1 h Twenty microliters of
50 mMammonium 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 lL of 20 mM ammonium bicar-bonate, 20 lL of 20 mMammonium bicarbonate/acetonit-rile (1 : 1, v/v), twice in 5% formic acid/acetonitbicarbonate/acetonit-rile (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 lL of 0.1% 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
C18 capillary column (ThermoHyPurity C18 150· 0.18)
A linear 18-min gradient (flowrate, 3 lLÆmin)1) from 5 to
Trang 380% 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 flowrate, 30; capillarity
temperature, 200C; 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 ThermoFinniganBIOWORKSsoftware
(version 2)
Cloning of thepduCDE 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 mM for 4 h
The His-tag removal was obtained from the previous
construction The newconstruction (pQE30DD) contained
the sequence of the a, b and c subunits genes and their Shine
Dalgarno sequences
R E S U L T S
High-level expression of theL 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 newattempt was performed with a newconstruction (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 pduCDEencode for active diol dehydratase, we attempted to purify the enzyme from L collinoides
Purification of the diol dehydratase ofL collinoides Unlike the enteric bacteria, L collinoides was unable to growon 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 mMglucose and 50 mM 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-mM potassium phosphate buffer without 1,2-propane-diol and potassium Under this condition, the enzymatic complex was very unstable at 4C 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 coli 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
1 m 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
Trang 4containing 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 pasteurianumcan 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 Scorea
MS/MS sequenced peptides
% Protein coverageb
2 Propionaldehyde
dehydrogenase
147 8 24.9
a
Represents the score given by the SEQUEST software andb 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.
Trang 5150 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 mM 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 25C
and 45C was tested and the optimum was observed at
37C So, further kinetic experiments were performed at
these pH and temperature values
The Km 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 Km of 1.6 mM followed by 1,2-ethanediol (Km:
5.5 mM) and glycerol (Km: 9.4 mM) Affinity for the AdoCbl
coenzyme was considerably higher with a Kmof 8.3 lM
It has been shown that glycerol is both a substrate and a
suicide-inactivator for diol and glycerol dehydratase [8] In
order to showwhether 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 (0.2M), and
conse-quently must have resulted from the inactivation of the
L collinoidesdiol 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 lM 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 Ki of 26.4 lM 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 mM) on the diol dehydratase activity was then studied (data not shown) A slightly inhibitory effect was observed with the divalent Mg2+ion whereas both sodium and calcium ions caused complete inhibition The potassium concentration estimated at pH 8.75 in the reaction mixture was 100 mM Therefore, the inhibitory effect observed with Mg2+, Ca2+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 NH4+, which did not affect activity All characteristics are summarized in Table 3
D I S C U S S I O N
In cider, L collinoides is involved in glycerol degradation leading potentially to an alteration of the beverage known
as piquˆre acrole´ique 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) or glycerol (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) and 50 l M (h) of CNCbl.
Trang 6metabolism 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
growon glycerol or 1,2-propanediol, L collinoides can not
growon 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 lowsolubility 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 As all the
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
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 Km obtained was similar to that obtained for the enzyme of L brevis (7 lM) [20] The study of the effect of different cations on the reaction showed that Na+ and Ca2+were incompatible with catalysis This was probably due to a dissociation of the complex as reported by Schneider et al [3] for the glycerol dehydratase apoenzyme with Na+
Contrary to the glycerol dehydratase of L reuteri, w hich 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, w e 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
a2b2c2composition 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
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 – – –
Subunit molecular masses (kDa) 63, 25, 19 – 52 –
Ion activity Na > Ca >Mg Na > Li > Mg > Mn – Na
Trang 7A C K N O W L E D G E M E N T S
The authors wish to thanks the professor Jacques Nicolas, director of
the Chaire de Biochimie Industrielle et Agro-Alimentaire of the
CNAM of PARIS for his welcome of Nicolas Sauvageot in his
laboratory This work was partly supported by a grant from the conseil
re´gional pour lAgrobiologie 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.
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