Download Biochemistry of coenzyme B12‐dependent glycerol and diol

FEMS Microbiology Reviews 22 (1999) 553^566
Biochemistry of coenzyme B12-dependent glycerol and diol
dehydratases and organization of the encoding genes
Rolf Daniel a; *, Thomas A. Bobik b , Gerhard Gottschalk
b
a
a
Institut fuër Mikrobiologie und Genetik der Georg-August-Universitaët, Grisebachstr. 8, D-37077 Goëttingen, Germany
Department of Microbiology and Cell, Science Building 981, Room 1220, University of Florida, Gainesville, FL 32611, USA
Received 26 June 1998 ; received in revised form 19 August 1998 ; accepted 19 August 1998
Abstract
Glycerol and diol dehydratases exhibit a subunit composition of K2 L2 Q2 and contain coenzyme B12 in the base-on form. The
dehydratase reaction proceeds via a radical mechanism. The dehydratases are subject to reaction inactivation by the substrate
glycerol which is caused by a cessation of the catalytic cycle because coenzyme B12 is not regenerated, instead
5P-deoxyadenosine and a catalytically inactive cobalamin are formed. The genetic organization of the dehydratase genes is
quite similar in all organisms. Downstream of the dehydratase genes an open reading frame encoding a polypeptide of
approximately 600 amino acids was identified which is apparently involved in the reactivation of suicide-inactivated
enzyme. z 1999 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.
Keywords : Glycerol dehydratase ; Diol dehydratase; Coenzyme B12 ; Glycerol utilization ; 1,2-Propanediol degradation ; dha regulon;
pdu operon
Contents
1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2. Distribution of glycerol and diol dehydratases . . . . . . . . . . . . . . . .
3. Physiology and regulation of dehydratase production . . . . . . . . . . .
3.1. Glycerol fermentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.2. 1,2-Propanediol fermentation . . . . . . . . . . . . . . . . . . . . . . . . .
4. Reaction mechanism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5. Assay systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
6. Biochemistry and molecular genetics of B12 -dependent dehydratases
7. Dehydratase in biotechnology . . . . . . . . . . . . . . . . . . . . . . . . . . . .
8. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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* Corresponding author. Tel.: +49 (551) 393827; Fax: +49 (551) 393793; E-mail: [email protected]
0168-6445 / 99 / $19.00 ß 1999 Federation of European Microbiological Societies. Published by Elsevier Science B.V.
PII: S 0 1 6 8 - 6 4 4 5 ( 9 8 ) 0 0 0 2 1 - 7
FEMSRE 622 25-1-99
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R. Daniel et al. / FEMS Microbiology Reviews 22 (1999) 553^566
1. Introduction
Glycerol dehydratase (glycerol hydro-lyase, EC
4.2.1.30) and diol dehydratase (D,L-1,2-propanediol
hydro-lyase, EC 4.2.1.28) can each catalyze the conversion of glycerol, 1,2-propanediol and 1,2-ethanediol to the corresponding aldehydes [1,2]. These enzymic reactions are known to proceed by a radical
mechanism involving coenzyme B12 as an essential
cofactor. Coenzyme B12 contains a unique covalent
Co-C bond, which is stable in aqueous solution, but
easily undergoes homolytic cleavage in coenzyme
B12 -dependent enzymatic reactions. This homolytic
cleavage is the initial step in the catalytic cycle of
all coenzyme B12 -dependent enzymatic processes
[3,4].
The coenzyme B12 -dependent glycerol and diol dehydratases are involved in the anaerobic utilization
of small molecules, they catalyze molecular rearrangements that generate an aldehyde which can be
reduced (i.e. glycerol fermentation) or dismutated to
more oxidized and more reduced compounds (i.e.
1,2-propanediol fermentation). Coenzyme B12 -containing ethanolamine ammonia-lyase of enteric bacteria converts ethanolamine to acetaldehyde and ammonia, the aldehyde is proposed to dismutate to
acetate and ethanol, reactions allowing the generation of ATP in the acetate kinase reaction [5,6].
Analogous reactions take place following the conversion of 1,2-propanediol to propionaldehyde by diol
dehydratase [6^8]. The anaerobic degradation of
glycerol is initiated by two enzymes, glycerol dehydrogenase forms dihydroxyacetone and glycerol dehydratase yields 3-hydroxypropionaldehyde which is
converted further to 1,3-propanediol [1,9,10]. Glycerol dehydratases and the closely related diol dehydratases have been extensively studied in genera of
Enterobacteriaceae such as Klebsiella and Citrobacter. It has been shown that these enzymes are
similar in molecular masses and substrate spectra,
but are di¡erent in monovalent cation selectivity patterns, a¤nity for coenzyme B12 , and substrate speci¢city [1,11^16].
2. Distribution of glycerol and diol dehydratases
The presence of glycerol and/or diol dehydratases
was shown in several Gram-positive and Gram-negative microorganisms, including enteric and propionic acid bacteria, solventogenic clostridia and lactobacilli [7,15,17^27]. All characterized dehydratases
are coenzyme B12 -dependent, except the diol dehydratase of Clostridium glycolicum [28]. This enzyme
seems to be entirely di¡erent from the B12 -containing
dehydratases and will not be discussed here any further. In the case of enteric bacteria, B12 -dependent
glycerol or diol dehydratases were detected in some
strains of the genera Salmonella, Klebsiella, Enterobacter and Citrobacter [7,15,18,22,23,25,29]. Both dehydratases may occur individually or together in
these organisms. The 1,2-propanediol- and non-glycerol-fermenting Salmonella typhimurium contain diol
dehydratase exclusively. The glycerol-fermenting Enterobacter agglomerans, Klebsiella pneumoniae and
Citrobacter freundii possess glycerol dehydratase,
some strains of the latter two microorganisms also
possess diol dehydratase. Growth on 1,2-propanediol
induces only the diol dehydratase, whereas both dehydratases are present in anaerobically grown glycerol cells [15]. In one Klebsiella strain (ATCC 8724),
the isofunctional diol dehydratase substitutes for the
defective glycerol dehydratase [2,16,22].
Glycerol dehydratase was detected in Clostridium
pasteurianum, Cl. butyricum, Lactobacillus sp., Lactobacillus reuteri, Lactobacillus brevis and Lactobacillus buchneri [19,20,24,27,30] and diol dehydratase in
the propionic acid bacterium Propionibacterium freudenreichii [22].
3. Physiology and regulation of dehydratase
production
3.1. Glycerol fermentation
The pathway of glycerol breakdown and the key
enzymes and genes involved have been extensively
studied in C. freundii and K. pneumoniae. Glycerol
is converted by these bacteria to 1,3-propanediol
(major product), ethanol, 2,3-butanediol, acetic and
lactic acids [31]. In the absence of an external oxidant, glycerol is fermented by a dismutation process
involving two pathways, one serving for glycerol oxidation, the other for the consumption of reducing
equivalents generated (Fig. 1A). Oxidation of glycer-
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Fig. 1. Anaerobic utilization of glycerol. (A) Pathway used by enteric bacteria. (B) Apparent genetic organization of the C. freundii dha
regulon. (C) Genetic organization of the reductive branch of glycerol utilization in Cl. pasteurianum. 1, dhaD, glycerol dehydrogenase;
2, dhaK, dihydroxyacetone kinase ; 3, dhaB, dhaC and dhaE, subunits of glycerol dehydratase ; 4, dhaT, 1,3-propanediol dehydrogenase ;
dhaR, regulatory protein; orfW, orfX, orfY and orfZ, open reading frames with unknown function
ol is catalyzed by NAD‡ -linked glycerol dehydrogenase which converts the substrate to dihydroxyacetone. This product is then phosphorylated by dihydroxyacetone kinase and funneled into the
glycolytic pathway [10]. Generation of NAD‡ is
achieved by the NADH-linked 1,3-propanediol dehydrogenase [32]. By the action of coenzyme B12 -dependent glycerol dehydratase glycerol is ¢rst converted to 3-hydroxypropionaldehyde [1] which then
is reduced to 1,3-propanediol accounting for about
50^66% of the glycerol consumed. All four key enzymes and the corresponding genes have been identi¢ed and characterized in C. freundii and K. pneumoniae [1,10,18,21,32^34]. In C. freundii, the structural
genes of the glycerol dehydratase (dhaBCE) are part
of the dha regulon along with the genes encoding the
three other key enzymes (dhaD, dhaK, dhaT) of the
pathway (Fig. 1B). In addition, the dha regulon encodes a transcriptional activator protein (DhaR)
[10], a protein probably involved in reactivation of
glycerol dehydratase (OrfZ) [35], and three presumptive proteins with unknown function (OrfW, OrfX,
OrfY). The expression of the dha regulon is induced
under anaerobic conditions when dihydroxyacetone
or glycerol are present. In contrast to the 1,3-propanediol-forming enteric bacteria, very little information is available about the genes and enzymes responsible for glycerol utilization by clostridia and
lactobacilli.
The glycerol fermentation pattern of clostridia is
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R. Daniel et al. / FEMS Microbiology Reviews 22 (1999) 553^566
Fig. 2. Anaerobic utilization of 1,2-propanediol. (A) Proposed degradative pathway. (B) Genetic and physical maps of the pdu locus of S.
enterica LT2. The known promoter sites are indicated with the letter `P', and the direction of transcription is indicated by the arrow. The
pduGHJ genes have not yet been correlated to the physical map. ORFs 1^15 include homologs of the diol dehydratase reactivating protein (DdrA) of K. oxytoca, alcohol dehydrogenase, acetate kinase, as well as four homologs of carboxysome shell protein genes.
di¡erent, they form butyric acid and some strains of
Cl. pasteurianum butanol and ethanol as additional
products ([17,30,36], for the entire pathway of solvent formation see [17]). All four key enzymes
known from the study of enteric bacteria were detected in crude extracts of Cl. pasteurianum [19] as
well as in Cl. butyricum, except that the dihydroxyacetone kinase was not measured in the latter organ-
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ism [20]. These results indicate that clostridia ferment
glycerol like the 1,3-propanediol-forming enteric bacteria by a dismutation process. Recently, the genes
encoding glycerol dehydratase and 1,3-propanediol
dehydrogenase of Cl. pasteurianum were identi¢ed
[19,37]. The deduced amino acid sequences and the
properties of the gene products are very similar to
those of 1,3-propanediol-forming enteric bacteria,
but the genetic organization is di¡erent (Fig. 1B,C).
In contrast to C. freundii and K. pneumoniae (not
shown) the genes encoding glycerol dehydratase
and 1,3-propanediol dehydrogenase of Cl. pasteurianum showed the same orientation and all presumptive genes were located upstream of the dhaT
gene.
The participation of glycerol dehydratase and 1,3propanediol dehydrogenase in glycerol fermentation
has also been shown for some Lactobacillus species
such as L. reuteri, L. buchneri and L. brevis
[24,26,27]. In contrast to the mentioned enteric bacteria and clostridia, lactobacilli are not able to grow
on glycerol as sole carbon and energy source. These
organisms require a second substrate such as glucose
for glycerol utilization because of the absence of the
enzymes for the oxidative branch.
3.2. 1,2-Propanediol fermentation
Diol dehydratase is used by some enteric and propionic acid bacteria for the degradation of 1,2-propanediol [7,22]. Catabolism of this small molecule is
likely to be important in a variety of nutritionally
complex environments. 1,2-Propanediol is an endproduct of the fermentation of rhamnose and fucose.
These sugars are common in plant cell walls and are
also found in the glycoconjugates of mammalian intestinal epithelial cells. Diol dehydratase catalyzes
the ¢rst step in the pathway of propanediol degradation, which produces propionaldehyde [38]. Subsequently, propionaldehyde is converted to equal
amounts of propanol and propionic acid. Coenzyme
A-dependent aldehyde dehydrogenase, phosphotransacylase, propionate kinase, and alcohol dehydrogenase are proposed to catalyze this disproportionation (Fig. 2A) [7,8]. Fermentation of 1,2propanediol provides one ATP per molecule of propanediol, but no source of carbon. Aerobically, 1,2propanediol can provide both carbon and energy. It
557
is thought that the aerobic and anaerobic pathways
are similar, but that oxygen allows the conversion of
some propionyl-CoA to cell carbon.
In S. enterica (formerly called S. typhimurium) the
structural genes for diol dehydratase (pduCDE) are
located in the pdu operon along with at least 20 additional genes involved in propanediol degradation
(Fig. 2B) [39,40]. The pdu operon encodes a propanediol di¡usion facilitator (PduF), a transcriptional activator protein (PocR), and homologs
of acetate kinase and alcohol dehydrogenase [39^
41]. In addition, the pdu operon includes genes for
the reactivation of diol dehydratase, genes for the
conversion of cobalamins to coenzyme B12 , and
genes that are homologs of the shell proteins of carboxysomes (polyhedral bodies that encase Rubisco
and which are thought to function in concentrating
CO2 ) [40^42].
The apparent use of the carboxysome shell protein
homologs is to encase diol dehydratase within a polyhedral shell. Thus far, four homologs of carboxysome shell protein genes have been identi¢ed in the
pdu operon [40]. Electron microscopy showed that S.
enterica forms polyhedral bodies during growth on
1,2-propanediol, and immuno-electron microscopy
indicated that diol dehydratase was located within
these polyhedra. The physiological reason for encasement of diol dehydratase within these polyhedral
shells is currently unknown. S. enterica is not an
autotroph, it does not express Rubisco, and there
is no known role for CO2 in the degradation of
1,2-propanediol.
Diol dehydratase is also subject to inactivation/reactivation. Inactivation occurs during catalysis with
glycerol as substrate. Inactivation results from loss
of the adenosyl group from the B12 cofactor [43].
Reactivation is proposed to involve replacement of
the inactive cofactor with coenzyme B12 . Work with
K. oxytoca has shown that two genes ddrA and ddrB
are involved in reactivation [44]. A homolog of ddrA
(orf1) is found in the pdu operon.
In S. enterica, expression of diol dehydratase is
regulated via transcriptional control of the pdu operon. Both 1,2-propanediol and poor growth conditions are required for high expression. In addition,
induction of the pdu operon is coordinated with induction of adjacent cobalamin biosynthesis (cob) operon [45]. Co-expression of the pdu and cob operons
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Fig. 3. Mechanism of glycerol dehydratase reaction (adapted from [4]). The coenzyme B12 is depicted in the base-on form. Ade, adenine;
RCH2 , adenosyl; [Co], cobalamin; E, enzyme.
is achieved through the PocR, an activator protein
which mediates induction of both operons in response to propanediol. Two global regulatory systems also control expression of the pdu and cob operons [46]. Aerobic induction by propanediol
requires Crp/cAMP, and is inhibited by glucose
and glycerol. Anaerobic expression is controlled by
both Crp/cAMP and the ArcAB two-component system, which have additive e¡ects.
4. Reaction mechanism
Glycerol and diol dehydratases belong to the class
II of coenzyme B12 -containing enzymes. Class I compromises enzymes such as methylmalonyl-CoA mutase catalyzing carbon^carbon rearrangements [47].
Recent X-ray crystallographic studies of this protein
revealed that cobalamin is bound to the enzyme via
the imidazole of a histidine residue coordinating to
the cobalt atom in the lower axial position instead of
the 5,6-dimethyl-benzimidazole moiety of the coenzyme (base-o¡ form) [48]. The sequence D-x-H-x-xG, which contains the coordinating histidine residue,
is reported to be conserved in this enzyme and some
other cobalamin-dependent enzymes. However, the
deduced amino acid sequences of the dehydratases
do not show the B12 -binding motif of the class I
enzymes (see Section 6). This fact indicates that
coenzyme B12 is bound in a di¡erent manner. EPR
measurements with 15 N-labeled dehydratase apoenzyme and unlabeled coenzyme suggests that cobalamin is bound to diol dehydratase with the 5,6-dimethylbenzimidazole ligand coordinating to the
cobalt atom (base-on form) [4,49].
The reaction mechanism of glycerol and diol dehydratases can be formulated according to Abeles
and coworkers [3,4] as depicted in Fig. 3. Binding
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of the coenzyme to the apoenzyme activates the CoC bond of the coenzyme. The substrate-induced homolytic cleavage of the Co-C bond leads to the formation of cob(II)alamin and an adenosyl radical.
The adenosyl radical plays an essential part in the
catalysis by abstracting a hydrogen atom from the
substrate. This leads to the formation of a substratederived radical and 5P-deoxyadenosine. The substrate-derived radical then rearranges to a product
radical by a hydroxyl group transfer from C-2 to
C-1. The product radical abstracts a hydrogen
atom back from 5P-deoxyadenosine. This results in
the formation of the ¢nal product and regeneration
of the coenzyme.
The radical intermediates formed during the catalytic cycle must sustain their high reactivity at the
active site and must become extinct in the only
way destined for the reaction. Once a radical intermediate is quenched by undesirable side reactions or
escapes from the active site, regeneration of the
coenzyme is impossible. This leads not only to cessation of the catalytic cycle, but also to inactivation
of the enzyme, since the modi¢ed coenzyme remains
tightly bound to the enzyme and is not exchangeable
with free intact coenzyme B12 . Therefore, the radical
species must be strictly protected from side reactions
or from leaving the reaction center of the enzyme.
Undesirable side reactions occur in the case of glycerol and diol dehydratases and these enzymes are
subject to mechanism-based suicide inactivation by
glycerol and some other substrates [2,13,50,51]. Inactivation by glycerol involves irreversible cleavage
of the Co-C bond of coenzyme B12 , forming 5P-deoxyadenosine and an alkylcobalamin-like species. Irreversible inactivation is then brought about by tight
binding of the modi¢ed coenzyme [50].
Such suicide inactivation seems enigmatic, because
glycerol is a growth substrate and the dehydratase is
essential for glycerol breakdown (see Section 3.1).
Work of Toraya and co-workers [43,44] showed
that glycerol-inactivated dehydratase undergoes rapid reactivation in permeabilized cells (in situ) by exchange of the modi¢ed coenzyme for intact coenzyme B12 in the presence of ATP and Mg2‡ or
Mn2‡ . Recently, two proteins (DdrA and DdrB),
which are probably involved in the reactivation reaction, were identi¢ed in K. oxytoca [44] and one
protein (OrfZ) in C. freundii [35].
559
5. Assay systems
For determination of glycerol or diol dehydratase
activity, the 3-methyl-2-benzothialzolinone hydrazone (MBTH) method is widely applied [52]. This
method is based on the ability of the aldehydes
formed during the dehydratase reaction to react
with MBTH. The resulting azine derivatives are detected spectrophotometrically. The usual assay mixture contains an appropriate amount of dehydratase,
0.2 M substrate (i.e. 1,2-propanediol), 0.05 M KCl,
0.035 M potassium phosphate bu¡er (pH 8.0), and
15 WM coenzyme B12 , in a total volume of 1 ml.
After incubation at 37³C for 1^10 min, the enzyme
reaction is terminated by adding 1 ml 0.1 M potassium citrate bu¡er (pH 3.6) and 0.5 ml of MBTH
hydrochloride. After 15 min at 37³C, the amount of
aldehyde formed (i.e. propionaldehyde) is determined from the absorbance at 305 nm. The apparent
molar extinction coe¤cient at 305 nm for the colored
product from propionaldehyde is 13.3U103 M31
cm31 . The MBTH method is suitable for various
applications, such as kinetic and mechanistic studies,
determination of substrate and cofactor speci¢city.
Because of its higher brevity and sensitivity, the
MBTH method replaced the 2,4-dinitrophenylhydrazine assay [52], but the latter compound is still applied for the identi¢cation of glycerol or diol dehydratase in polyacrylamide gels after separation of
crude extracts or protein preparations by electrophoresis under non-denaturing conditions [1,11,14,37].
The dehydratase band can be localized by the colored precipitate of the 2,4-dinitrophenylhydrazone of
propionaldehyde [11].
Another assay is a coupled reaction in which the
propionaldehyde formed during the glycerol or diol
dehydratase reaction with 1,2-propanediol as substrate is reduced to 1-propanol in the presence of
added excess L-NADH and yeast alcohol dehydrogenase. The decrease in the absorbance of NADH at
340 nm is used for calculation of the dehydratase
activity [50]. This method is suitable when 1,2-propanediol is used as substrate and the appropriate
assay conditions allow alcohol dehydrogenase activity.
A major problem during dehydratase assays is the
rapid inactivation of the enzyme with glycerol as
substrate. This problem can be circumvented by us-
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Fig. 4. Alignment of the amino acid sequences corresponding to the K, L and Q subunits of glycerol and diol dehydratases from various
organisms. The dhaBCE genes of Cl. pasteurianum (C. p.) [37] and C. freundii (C. f.) [1,13] and the gldABC genes of K. pneumoniae (K.
p.) [14] encode the K, L and Q subunits of coenzyme B12 -dependent glycerol dehydratases. The pddABC genes of K. oxytoca (K. o.) [11]
and the pduCDE genes of S. typhimurium (S. t.) [23] encode the K, L and Q subunits of coenzyme B12 -dependent diol dehydratases.
Dashed lines indicate gaps which were introduced to optimize the alignment. The amino acids conserved in all ¢ve enzymes are shaded.
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Table 1
Properties of the genes and the corresponding gene products for the three structural subunits of glycerol and diol dehydratases
Organism
Clostridium pasteurianum
Citrobacter freundii
Klebsiella pneumoniae
Klebsiella oxytoca
Salmonella typhimurium
Gene name
Gene length
Protein molecular mass (Da)
K
L
Q
K
L
Q
K
L
Q
dhaB
dhaB
gldA
pddA
pduC
dhaC
dhaC
gldB
pddB
pduD
dhaE
dhaE
gldC
pddC
pduE
1665
1668
1668
1665
1665
540
585
585
675
675
441
429
426
522
522
60 813
60 433
60 621
60 348
60 307
19 549
21 487
21 310
24 113
24 157
16 722
16 121
16 094
19 173
19 131
The dhaBCE genes of Cl. pasteurianum [37] and C. freundii [1] and the gldABC genes of K. pneumoniae [14] encode coenzyme B12 -dependent
glycerol dehydratases. The pddABC genes of K. oxytoca [11] and the pduCDE genes of S. typhimurium [23] encode coenzyme B12 -dependent
diol dehydratases. K, large subunit; L, intermediate subunit; Q, small subunit.
ing 1,2-propanediol as substrate or a short reaction
time (1 min). Work of Toraya and Fukui [16]
showed that the use of 1,2-propanediol does not result in signi¢cant enzyme inactivation and that the
reaction with glycerol is linear for approximately
1 min.
6. Biochemistry and molecular genetics of
B12-dependent dehydratases
The glycerol dehydratases of K. pneumoniae and
C. freundii and the diol dehydratase of K. oxytoca
are biochemically well studied. Most of the data
summarized in this section are derived from the
work with these three enzymes. Glycerol and the
mutually related diol dehydratases convert glycerol,
1,2-propanediol and 1,2-ethanediol to the corresponding aldehydes, with glycerol being the preferred
substrate for glycerol dehydratase and 1,2-propanediol being the preferred substrate for diol dehydratase [2,37,38,53,54]. All characterized dehydratases
consist of three types of subunits and have the subunit composition K2 L2 Q2 [1,55]. The native enzyme
complex of glycerol dehydratase and diol dehydratase dissociates into components A and B or F and S
when subjected to ion-exchange chromatography
[11,56,57]. Recent studies with the diol dehydratase
of K. oxytoca identi¢ed the components F and S as L
subunit and K2 Q2 complex, respectively. The K and Q
subunits require each other for correct folding forming the soluble, active component S. Expression of
component F in a soluble, active form is promoted
by coexpression with both the K and Q subunits,
probably by coexistence with component S [55].
The reported native molecular mass of glycerol dehydratase (approximately 190 kDa) [1,37,53] is lower
than that of diol dehydratase (approximately 230
kDa) [12,55]. Both types of dehydratases are activated by certain monovalent cations; K‡ , NH4 ‡
and Rb‡ are most e¡ective as cofactor [13,58]. In
contrast to glycerol dehydratase, diol dehydratase
is also partially active with Cs‡ and Na‡ . Glycerol
and diol dehydratases of various Klebsiella and
Citrobacter strains are distinguishable by their di¡erent immunochemical properties [16] and Km values
for coenzyme B12 (13^50 nM and 750^1400 nM, respectively) [13,15,55,59].
The DNA sequence for diol dehydratase has been
determined in two organisms and that for glycerol
dehydratase in three [1,11,13,14,23,37]. Each enzyme
is encoded by three genes that are transcribed in the
following order: large gene (K subunit); intermediate
gene (L subunit); small gene (Q subunit). The deduced molecular masses of the subunits are approximately 60.5, 19.5^24 and 16^19 kDa, respectively.
The intermediate and the small subunits of diol dehydratases possess a higher molecular mass than the
corresponding subunits of glycerol dehydratases (Table 1). This is caused by additional amino acids in
the N-terminal region of both diol dehydratase subunits (Fig. 4). Each subunit has signi¢cant amino
acid sequence homology to the analogous subunit
from the other organisms (Fig. 4). No signi¢cant
similarities of the dehydratases to other cobalamindependent enzymes or cobalamin-binding proteins
are apparent, and no putative binding motif for
coenzyme B12 matching the conserved sequence described for other cobalamin-dependent proteins [60]
is evident.
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In some cases, the DNA sequences that £ank diol
and glycerol dehydratase have also been determined.
Where the sequence downstream of the dehydratase
genes is published, an open reading frame follows
which encodes a polypeptide of approximately 600
amino acids (OrfZ, DhaB4 (Orf4), DdrA or Orf1)
[1,13,14,37,44]. The sequence comparison of the
orfZ gene product of C. freundii revealed 84, 63
and 59% identity to the corresponding homologous
gene products of K. pneumoniae, Cl. pasteurianum
and K. oxytoca, respectively. Since deletion of orfZ
has no e¡ect on enzyme activity, it was concluded
that orfZ does not encode a subunit required for
glycerol dehydratase activity [1]. Similar results
were obtained for the dehydratase of K. pneumoniae,
K. oxytoca and Cl. pasteurianum [11,14,37]. However, Northern blot experiments performed with C.
freundii RNA revealed a common transcription of
the orfZ gene and the genes encoding the three structural subunits of glycerol dehydratase [35]. Work of
Mori et al. [44] showed that the OrfZ homolog DdrA
is involved in the reactivation of glycerol- or cyanocobalamin-inactivated diol dehydratase of K. oxytoca.
Other genes that £ank glycerol dehydratase are
involved in the pathway of glycerol fermentation
(dhaD, dhaK, dhaT), or have unknown functions
([1,10,32,37]; see Section 3.1). In both, C. freundii
and Cl. pasteurianum, there are three such putative
genes that are conserved between the two organisms,
orfW, orfX, orfY (Fig. 1B,C).
Genes adjacent to the diol dehydratase genes of K.
oxytoca and S. typhimurium are conserved. This is
upstream pduB in S. typhimurium and orf1 in K. oxytoca with unknown function [11,23] and downstream
the already mentioned genes involved in reactivation
of diol dehydratase. Conservation of the genetic organization for the remaining genes involved in propanediol degradation is uncertain since the DNA
sequences are incomplete.
7. Dehydratase in biotechnology
1,3-Propanediol is of industrial importance, because it can be used as a starting material for producing plastics, such as polyesters, polyethers and
polyurethanes. Therefore, industrial interest emerged
563
to develop improved routes for 1,3-propanediol production. A production process for 1,3-propanediol
from glycerol relies on the activity of glycerol or
diol dehydratases, which convert glycerol to 3-hydroxypropionaldehyde (see Section 3.1). It has been
shown that the dehydratase activity is the limiting
factor for the biotechnological production of 1,3propanediol [9,61]. This is most likely related to
the role of coenzyme B12 in the catalytic cycle and
the reaction inactivation with glycerol as substrate.
Thus, increasing the level of active dehydratase in
microorganisms could increase productivity. To
achieve this, the genes for the dehydratase could be
overexpressed in 1,3-propanediol-producing organisms or the reactivation reaction of glycerol-inactivated dehydratase could be improved. As mentioned
above, it has been shown that the genes downstream
of the three structural genes for dehydratase, e.g.
orfZ, are not necessary for enzyme activity, but are
somehow involved in reactivation of the enzyme.
Work of Skraly et al. [34] revealed that the 1,3-propanediol synthesis is more e¡ective in the presence of
these genes.
8. Conclusions
Signi¢cant progress has been made in recent years
to understand the dehydratases acting on glycerol or
diols. This came from the elucidation of the role of
coenzyme B12 in these reactions and from the identi¢cation of the genes coding for these enzymes.
There still are a number of questions to be solved
by future work. The binding motif for coenzyme B12
has to be identi¢ed. This and the elucidation of the
protein structure may aid in understanding why
these enzymes are inactivated by glycerol. Also, the
exact function of the reactivating enzymes, e.g. OrfZ,
has to be unravelled. Further understanding of the
dehydratase reactions might also contribute to the
development of an economically feasible process
for 1,3-propanediol production from glycerol.
Acknowledgments
The work of G. Gottschalk and R. Daniel was
supported by the Deutsche Forschungsgemeinschaft
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R. Daniel et al. / FEMS Microbiology Reviews 22 (1999) 553^566
within the Forschungsschwerpunkt `Neuartige Reaktionen und Katalysemechanismen bei anaeroben
Mikroorganismen', by the Fonds der Chemischen
Industrie and by the Akademie der Wissenschaften,
Goëttingen, Germany.
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