Metabolite Channelling and Metabolic Complexity
Substrate channelling in 2-0x0 acid dehydrogenase multienzyme complexes
Richard N. Perharn', D.Dafydd Jones2,Hitesh J. Chauhan and Mark J. Howard3
Cambndge Centre for Molecular Recognition, Department of Biochemistry, University of Cambridge,
80 Tennis Court Road, Cambridge CB2 I GA, U.K.
Abstract
complex, the three component enzymes are pyruvate decarboxylase ( E l p ; EC 1.2.4.1), dihydrolipoyl acetyltransferase (E2p; EC 2.3.1.1 2) and
dihydrolipoyl dehydrogenase (E3 ; EC 1.8.1.4)
(reviewed in [1-4]). Corresponding enzymes make
up the O G D H and BCDH complexes; in each
case, the E l and E2 are specific for the particular
2-0x0 acid undergoing decarboxylation. E l catalyses the initial decarboxylation, utilizing thiamin
diphosphate as a cofactor, and the subsequent
reductive acetylation of a lipoyl group covalently
attached to a lysine residue in E2. E2 catalyses the
transfer of the acyl group to CoA and E3, which
carries out an identical reaction in each complex
and is normally the same enzyme in all three
instances, concludes the process by reoxidizing
the dihydrolipoyl group and regenerating the
dithiolane ring at the expense of NAD+.
In the P D H complex from Escherichia coli
and most Gram-negative bacteria, E2p forms a
cubic core consisting of 24 polypeptide chains
arranged with octahedral symmetry, whereas
in Bacillus stearothermophilus and most Grampositive bacteria, the E2p core is icosahedral
and comprises 60 E2p chains [1,2]. E l and E3 are
bound tightly but non-covalently in peripheral
positions around the E2 core. The O G D H and
BCDH complexes follow the same structural
pattern, with E2 cores [dihydrolipoyl succinyltransferase (E20) and dihydrolipoyl branched
chain acyltransferase (E2b)l of octahedral symmetry. T h e intact complexes are thus of giant size,
with molecular masses of (5-10) x lo6 Da and
diameters of up to 50 nm, significantly bigger than
a ribosome.
In this article we review some of the structural
features of the 2-0x0 acid dehydrogenase complexes that are of particular relevance to the
mechanisms of active-site coupling and substrate
channelling in such vast catalytic machines [4].
Heteronuclear NMR spectroscopy and other experiments indicate that the true substrate of the E l
component of 2-0x0 acid dehydrogenase complexes is not lipoic acid but the lipoyl domain of
the E2 component. E l can recognize the lipoyllysine residue as such, but reductive acylation
ensues only if the domain to which the lipoyl
group is attached is additionally recognized by
virtue of a mosaic of contacts distributed chiefly
over the half of the domain that contains the
lipoyl-lysine residue. The lipoyl-lysine residue
may not be freely swinging, as supposed hitherto,
but may adopt a preferred orientation pointing
towards a nearby loop on the surface of the lipoyl
domain. This in turn may facilitate the insertion of
the lipoyl group into the active site of E l , where
reductive acylation is to occur. The results throw
new light on the concept of substrate channelling
and active-site coupling in these giant multifunctional catalytic machines.
Introduction
T h e 2-0x0 acid dehydrogenase multienzyme
complexes constitute a family of related enzymes that includes the pyruvate dehydrogenase
(PDH), 2-oxoglutarate dehydrogenase (OGDH)
and branched-chain 2-0x0 acid dehydrogenase
(BCDH) complexes. They catalyse the oxidative
decarboxylation of the relevant 2-0x0 acid, transferring the resultant acyl group to CoA and
generating NADH in the process. In the P D H
Key words: lipoic acid, lipoyl domain, protein-protein interaction,
pyruvate dehydrogenase.
Abbreviations used: BCDH, branched-chain 2-0x0 acid dehydmgenase; OGDH. 2-oxoglutamte dehydrogenase; E lo, 2-0x0glutarate decarboxylase; E I p. pyruvate decarboxylase; E2p,
dihydrolipoyl acetyltransferase; E20, dihydrolipoyl succinyltransferase; E3, dihydrolipoyl dehydrogenase; PDH. pyruvate dehydrogenase; T,, transverse relaxation time.
'To whom correspondence should be addressed (e-mail
r.n.perham@,bioc.cam.ac.uk).
'Present address: Novozymes AIS, Protein Screening, 6E2.06,
Smtarmosevej I I , DK-2880 Bagsvcerd, Denmark
3Present address: Research School of Biosciences, University of
Kent, Canterbury, Kent CT2 7N], U.K.
The lipoyl domain and active-site
coupling
The lipoyl group is covalently attached in an
amide linkage to the p - a m i n o group of a specific
lysine residue of an independently folded domain
(about 80 residues) that forms the N-terminal part
47
0 2002 Biochemical Society
Biochemical Society Transactions (2002) Volume 30, part 2
side of the domain (Figure 1). Exact positioning of
the target lysine within the protruding /?-turn is
essential for correct post-translational modification by the lipoylating system(s) of the E. cob
cell [ 121.
of the E2 chain. T h e number of lipoyl domains per
E2 chain can vary, e.g from one in the E2p chain of
B. stearothermophilus and the E20 chain of E. coli
to three in the E2p chain of E. coli [1,2]. T h e lipoyl
domain plays a vital role in coupling the reactions
within the complex in an organized and specific
manner. Acting as a ‘swinging arm ’ [S], the lipoyllysine group visits each of the three active sites of
the complex, carried by a lipoyl domain that is
itself rendered mobile by virtue of the conformational flexibility in the segment of polypeptide
chain that links it to the inner E2 core of the
complex.
T h e NMR solution structures of the single E.
coli E20 [6] and the innermost of the three E. coli
E2p [7] lipoyl domains have been solved, as have
those of others from various other organisms
[%lo], including that of the B. stearothermophilus
E2p chain [ l l ] . Their overall backbone structures
are virtually identical. They consist of a P-barrel
formed from two four-stranded P-sheets, arranged
with a 2-fold axis of quasi-symmetry. T h e lipoyllysine is located at the tip of a tight, type I /?-turn
in one P-sheet, and the N- and C-termini are
close in space in the other /?-sheet on the opposite
Substrate channelling
As shown first with the PDH complex of E. coli,
free lipoate can act as the substrate for E2p and E3
but is a very poor substrate for E l p . However,
the E2p lipoyl domain is an excellent substrate
(kcat/&, raised by a factor of lo4); moreover, the
E2p and E20 lipoyl domains from E. coli function
as substrates only for their cognate E l s ([13,14]
and references therein). Similar results have been
reported for the lipoyl domains of Azotobacter
vinelandii E2p and E20 [15]. T h u s the true
substrate in these complexes is not lipoic acid or
lipoyl-lysine but the lipoyl domain; recognition of
the domain by its cognate E l provides an elegant
mechanism for substrate channelling whereby
reductive acylation is confined to a lipoyl group
covalently attached to a specific lysine residue of
Figure I
Structure of the innermost lipoyl domain of € coli E2p
Left-hand panel, schematic structure of the lipoyl domain drawn using MOLSCRIPT [23], with a freely rotating lipoyl-lysine residue at the
tip of the p-turn between p-strands 4 and 5. Middle and right-hand panels, two views of the lipoyl domain with the lipoyl-lysine residue in
a prefered orientation, close in space to the surface loop between B-strands I and 2. Reproduced from c/] with permission from
Biochemistry 2000, 39, 8448-8459. 0 2000 Am. Chem. SOC.
N-lcrm
\
N-lcrm
L.
C-lcnn
c-tcrm
0 2002 Biochemical Society
48
Metabolite Channelling and Metabolic Complexity
the intended E2 component [ 2 4 ] . How the E2p
and E20 lipoyl domains are recognized by their
correct partner E l s yet have the same structural
scaffold thus becomes an important question.
thiamin diphosphate in the active site is buried at
the bottom of a 2 nm-deep funnel-shaped hole
at the interface between the a-and /?-subunits. In
order for the lipoyl group to reach the cofactor,
and assuming that no major conformational
changes are involved, the protruding /?-turn housing the lipoyl-lysine residue would have to point
into the entrance of the funnel and the lipoyllysine side chain (1.4 nm) to become fully extended. T h e lipoyl domain, and the surface loop
region between /?-strands 1 and 2 in particular,
would by necessity come into close contact with
the surface of Elp, as indicated by the earlier N M R
experiments [171.
T h e quaternary structures of the E. coli E l p
and 2-oxoglutarate decarboxylase ( E l o ; a2)and B .
stearothermophilus E l p (a$,)differ, and therefore
so may their interactions with the lipoyl domain.
However, more detailed experiments with heteronuclear N M R spectroscopy, involving studies of
both chemical shifts and transverse relaxation
times (T2),
have indicated that the same general
principles apply [20,21]. I n the B. stearothermophilus E l p , it is clear that the incoming lipoyl
domain makes contact with both E l a and El/?
subunits, consistent with the active site lying
between them. Moreover, although the surface
loop region between /I-strands 1 and 2 is a major
site of contact, there are other sites distributed
Interaction of the lipoyl domain
with E l
T h e binding of the lipoyl domain to E . coli E l p is
both weak ( K ,not less than 1 mM, despite a K , of
x 20 pM) and transient [16]. Thus co-crystallization of the lipoyl domain with E l is likely to be
impossible. However, N M R has been used to
identify regions on the apo-form of the B. steavothermophilus E2p lipoyl domain that provide important contact sites in the interaction with E l p . A
prominent surface loop, linking /?-strands 1 and 2,
is present only in the /?-sheet that contains the
lipoyl-lysine residue, and lies close in space to
the lipoyl-lysine /?-turn (Figure 1). Alterations in
chemical shift in the presence of E l indicated that
this loop is likely to make contact with El during
catalysis; likewise, the results of directed mutagenesis indicated that residues flanking the lipoyllysine residue in its /?-turn are also of critical
importance [171.
T h e crystal structures of the heterotetrameric
(a$,) E l p s from Pseudomonas putida [18] and
humans [19] have recently been solved. T h e
Figure 2
Points of contact between the lipoylated 15coli lipoyl domain and its partner E I p
Left-hand and middle panels,two views ofthe lipoyl domain rotated by I 80°,indicating residues (numbered in single-letter code) that exhibit
significant changes in chemical shift in the presence of E I p. Right-hand panel, residues that exhibit significant changes in J2 values in the
presence of E I p. Reproduced from [2 I] with permission. 0 (200 I) Academic Press.
4 1K (lipoyl-lysine)
41K (lipoyl-lysine)
L
41K (lipoyl-lysine)
3
42A
IK
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0 2002 Biochemical Society
Biochemical Society Transactions (2002) Volume 30, part 2
Given that the true substrate for the 2-0x0
acid dehydrogenase complexes is actually the
lipoyl domain, much slower to diffuse than lipoic
acid itself, even if free, the tethering of the lipoyl
domain in the complex assumes a much clearer
significance. T h e local concentration of the domain is raised to millimolar concentrations, way
above the K,,, values it exhibits as a substrate for
the component enzymes. Recognition of the lipoyl
domain by E l provides the molecular basis of an
elegant and efficient form of substrate channelling;
and the flexibility in the tethering linker regions
and the three-dimensional organization of the
enzyme complex, together with a preferred orientation of the classical swinging arm on the surface
of the domain, may promote catalytically advantageous trajectories of the domain between the
contributing active sites.
over the surface of the lipoyl domain physically
remote from the lipoyl-lysine residue [20]. These
experiments were carried out with the apodomain, as the lipoylated domain appeared to bind
too tightly to the B. stearothermophilus E l p and
substantial line-broadening was observed. However, with the E. coli E l p , the lipoylated domain
could be studied [21], and again it was inferred
that a significant number of residues on the
domain, largely restricted to the lipoyl-lysinecontaining half and dominated by the lipoyl-lysine
p-turn, come into contact with E l p (Figure 2).
E l o did not interact detectably with the apodomain from E. coli E2p, but some interaction was
detected with the holo-domain. T h e conclusion
must be that the E l components can recognize the
lipoyl-lysine residue as such, but that reductive
acylation ensues only if the domain to which the
lipoyl group is attached is additionally recognized
P11.
We thank the Biotechnology and Biological Sciences Research
Council (BBSRC) forthe award of a research grant (to R. N.P.) and
research studentships to D.D.J. and H.J.C., and the BBSRC
and The Wellcome Trust for support of the core facilities of
the Cambridge Centre for Molecular Recognition. W e also thank
Mr C. Fuller for skilled technical assistance and numerous
colleagues, in particular Dr Katherine Stott, for help and discussion.
Implications for catalysis
In comparing the NMR spectra of "N-labelled
apo- and holo-forms of the lipoyl domain of E. coli
E2p, it became apparent that the lipoyl-lysine
p-turn becomes less flexible following posttranslational modification. From the chemical
shift data, it appears that, rather than being freely
swinging, as hitherto supposed, the lipoyl group
may have a preferred orientation (Figure l ) ,
pointing towards the surface loop linking Bstrands 1 and 2 [7]. This is the self-same loop that
comes into contact with E l as the lipoyl domain
approaches the E l active site. It has also been
observed that limited proteolysis of a surface loop
in the E l a chain at the entrance to the active site of
the E l component of the B. stearothermophilus
P D H complex has a pronounced inhibitory effect
on the catalytic activity of the intact complex. At
the same time, there was no loss of catalytic activity
of E l itself and no effect on the ability of the
proteolysed E l to bring about the reductive
acetylation of a free lipoyl domain [22]. This has
raised the interesting possibility that the lipoyl
domain may have a restricted trajectory in the
intact complex, causing it to approach the E l
active site in a particular way. A preferred orientation of the lipoyl-lysine residue on the lipoyl
domain, directed towards the nearby surface loop
between /?-strands 1 and 2, may contribute to the
chance of an efficacious encounter and facilitate
the insertion of the lipoyl-lysine arm into the E l
active site, as discussed above.
0 2002 Biochemical Society
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Conversion of pancreatic cells to hepatocytes
David Tosh', Chia-Ning Shen and Jonathan M. W. Slack
Developmental Biology Programme, Department of Biology and Biochemistry, University of Bath,
Bath BA2 7AY, U.K.
germ layer (mesoderm, endoderm or ectoderm)
[l-31. Secondly, transdifferentiation leads to a
predisposition towards certain neoplastic transformations and therefore elucidating the molecular basis of the conversion will also provide
information on the processes underlying the development of cancer [2].
In order to demonstrate that transdifferentiation has occurred in a system, Eguchi and
Kodama [4] suggested that two prerequisites be
fulfilled. T h e first involves demonstrating (preferably with molecular evidence) the differentiation
state of the two cells types before and after the
transdifferentiation event. T h e second prerequisite involves showing a direct ancestor-descendent
relationship between the cells prior to and following transdifferentiation. It is difficult to fulfil
these prerequisites under in vivo conditions. However, in vitro culture systems are more amenable
to testing these prerequisites. One of the beststudied in vitro models for transdifferentiation is
the conversion of pigmented epithelial cells of the
retina to lens cells, so-called Wolffian lens regeneration [4]. Developing in vitro models for the
transdifferentiation of one cell type to another is
crucial as it will allow us to define the molecular
and cellular mechanisms which distinguish the
two cell types involved in the switch.
Abstract
Transdifferentiation is the name used to describe
the conversion of one differentiated cell type to
another. During development, the liver and pancreas arise from the same region of the endoderm
and cells from the two organs can transdifferentiate in the adult under different experimental
procedures. We have produced two in vitro models
for the transdifferentiation of pancreatic cells to
hepatocytes. T h e first utilizes a pancreatic exocrine cell line AR42J-B13 and the second comprises cultures of mouse embryonic pancreas. We
have analysed the pancreatic hepatocytes and they
express a range of liver markers including albumin, transferrin and transthyretin. We also present evidence for (i) the molecular mechanism
which regulates the conversion between pancreas
and liver and (ii) the cellular basis of the switch in
phenotype.
Introduction
T h e ability of one differentiated cell type to
convert to another is termed transdifferentiation
or metaplasia [l-31. T h e process of transdifferentiation is important for two reasons. Firstly, for
understanding the molecular and cellular basis of
embryonic development, as the conversion of one
cell type to another generally occurs between cells
which arise from neighbouring regions of the same
K e y words: C/ESPB, exocrine, glucocorticoid. liver, transdiffer-
Models for the transdifferentiation of
pancreas to liver
entiation.
Abbreviations used: HGF, hepatocyte growth factor: Dex,
dexamethasone: LETF, liver-enriched transcription factor: GFP,
green fluorescent protein: C/EBP, CCAAT/enhancer-binding
protein.
'To whom correspondence should be addressed (e-mail
[email protected]).
Numerous examples of transdifferentiation exist
but perhaps one of the most interesting is the
appearance of hepatocytes in the pancreas. Foci of
hepatocytes have been shown to be induced in the
pancreas of rats, mice or hamsters following
various experimental procedures, e.g. copper
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0 2002 Biochemical Society