(Received for publication, May 7, 1997, and in revised form, May 21, 1997)
From the Departments of Microbiology and
§ Biochemistry, University of Illinois, Urbana, Illinois
61801
Lipoic acid is an essential enzyme cofactor that
requires covalent attachment to its cognate proteins to confer
biological activity. The major lipoylated proteins are highly conserved
enzymes of central metabolism, the pyruvate and -ketoglutarate
dehydrogenase complexes. The classical lipoate ligase uses ATP to
activate the lipoate carboxyl group followed by attachment of the
cofactor to a specific subunit of each dehydrogenase complex, and it
was assumed that all lipoate attachment proceeded by this mechanism. However, our previous work indicated that Escherichia coli
could form lipoylated proteins in the absence of detectable
ATP-dependent ligase activity raising the possibility of a
class of enzyme that attaches lipoate to the dehydrogenase complexes by
a different mechanism. We now report that E. coli and
mitochondria contain lipoate transferases that use lipoyl-acyl carrier
protein as the lipoate donor. This finding demonstrates a direct link
between fatty acid synthesis and lipoate attachment and also provides the first direct demonstration of a role for the enigmatic acyl carrier
proteins of mitochondria.
Lipoic acid is a cofactor required for function of the citric acid
cycle (1). Two key enzyme multienzyme complexes use covalently bound
lipoic acid to carry reaction intermediates between the active sites of
the complexes. These enzymes are the pyruvate dehydrogenase complex
(PDC)1 that is responsible for the
synthesis of the acetyl-CoA needed to prime the cycle and the
-ketoglutarate dehydrogenase complex (KGDC) that catalyzes the
C5 to C4 step of the cycle (2). Covalently attached lipoic acid not only provides substrate channeling between the
different active sites of these complexes but also maintains the
activated carboxyl group in thioester linkage for delivery to coenzyme
A. Covalent attachment of lipoic acid to the E2 subunits of
these enzyme complexes is required for activity in vivo. The cofactor is attached via an amide linkage formed between the lipoate carboxyl group and the
-amino group of a specific lysine residue of
a well conserved E2 subunit N-terminal domain (2). The
first enzyme shown to attach lipoic acid to the lipoyl domains of PDC catalyzes a two-step reaction in which ATP activates lipoic acid to
lipoyl-AMP followed by transfer of the lipoyl moiety to the appropriate
E2 lysine residue (3). Lipoate attachment is also required
for activity of the glycine cleavage enzyme (GCV, the plant enzyme is
often called glycine decarboxylase) (4, 5) and the branched chain
-keto acid dehydrogenase complex (2).
All lipoate attachment was thought to proceed by the classical ATP-dependent mechanism, and Escherichia coli contains such a lipoate ligase (LplA protein) that functions primarily in utilization of exogenously supplied lipoic acid (6, 7). E. coli strains having null mutations in the lplA gene lack the ability to attach exogenously added lipoic acid to protein and also lack detectable ATP-dependent lipoate ligase activity (6, 8). However, these strains retain the ability to attach endogenously synthesized lipoate to PDC and KGDC in vivo indicating the presence of a second enzyme (6, 7). We report that this enzyme represents a second class of lipoate attachment enzyme that utilizes the lipoyl thioester of the fatty acid synthetic protein, acyl carrier protein (ACP), as the source of lipoic acid.
ACPs are an extensive family of small (70-80 residues) acidic proteins
modified by covalent attachment of 4-phosphopantetheine to a centrally
located serine residue. ACPs carry acyl groups via thioester linkage to
the 4
-phosphopantetheine sulfhydryl group and were first demonstrated
to function in the fatty acid synthetic pathways of bacteria and plants
(reviewed in Refs. 9 and 10). Subsequently, the ACPs of fatty acid
synthesis have been shown to act as acyl donors in the synthesis of
phospholipids (9), lipid A (11), the acylated homoserine lactone
signaling molecules of bacteria (12), and in the activation of
bacterial toxins (13). Other ACPs function in the synthesis of
polyketide (14) and polypeptide antibiotics (15), cell wall polymers (16), and compounds regulating bacteria-plant interactions (17).
The reaction we report not only provides a new role in central
metabolism for ACP but is an unusual reaction in that an enzyme cofactor (lipoic acid) is first bound by thioester linkage to another
cofactor, 4-phosphopantetheine, the covalently attached prosthetic
group of acyl carrier protein. This lipoyl-ACP is then used to donate
lipoic acid to the enzyme subunits of PDC, KGDC, and GCV. An alternate
way of viewing the reaction is that lipoic acid is transferred from one
protein to another. We have also demonstrated the presence of this new
lipoate transferase (the formal name is
lipoyl-[acyl-carrier-protein]-protein N-lipoyltransferase) in mitochondria of both plant and fungi thus providing a physiological role for the ACPs reported in mitochondria of mammals (18), plants
(19-21), and fungi (19, 22, 23).
The E. coli K-12 strains used
were derived from JK1 (a spontaneous rpsL mutant of the wild
type strain, W3110) by P1vir transductions with selection
for internal or closely linked antibiotic resistance determinants. The
mutant alleles used are all null alleles (deletions or transposon
insertions) (6, 7). SWJ39 (lplA) is a tetracycline-sensitive (24) derivative of TM134 (lplA::tet) and was
transduced with a P1vir stock grown on TM122 to give strain
SWJ43 (lplA (aroP-aceF) zca::Tn10). Strain TM122
(
(aroP-aceEF) zca::Tn10)
and strain SWJ43 were transduced with a P1vir stock grown on
KER184 to give strain SWJ46 (lipB::kan
(aroP-aceF)
zca::Tn10) and strain SWJ58 (lplA
lipB
(aroP-aceF)
zca::Tn10). Strains TM134
(lplA::tet), KER184
(lipB::kan), and TM136 (lplA::tet
lipB::kan) were described previously (6, 7).
A mixture of the apo (unmodified) PDC and KGDC complexes was purified from E. coli strain TM136 (lplA::Tn10 lipB::Tn1000dkan) grown on glucose minimal medium supplemented with acetate and succinate (6). This strain lacks the ability to attach either lipoate or octanoate to protein, and thus the PDC and KGDC from this strain are not lipoylated and lack activity (6). The mixed enzyme complexes were purified by differential centrifugation (25). When fully lipoylated in vitro the complexes had specific activities of 1.5 and 8.1 units/mg in the PDC and KGDC assays, respectively, where 1 unit is defined as the activity required to reduce 1 µmol of 3-acetylpyridine adenine dinucleotide/min at 25 °C.
Activation and Assay of the Apoenzyme ComplexesThe ligation substrate was 0.2 unit of apoPDC in 10 mM sodium phosphate buffer (pH 7.0) containing 0.3 mM dithiothreitol. The reaction (50 µl, final volume) also contained 80 µg of lipoyl-ACP and 10-100 µg of extract protein (the extracts were from strains that lacked PDC activity due to deletion of the genes encoding the AceE and AceF subunits). The reactions were incubated for 10-30 min at 37 °C (E. coli) or 25 °C (mitochondrial lysates), then stopped by bringing the volume to 500 µl with 120 mM Tris-HCl (pH 8.5). The reactions were then assayed (25) for PDC or KGDC activity in a total volume of 1 ml using 3-acetylpyridine adenine dinucleotide as the electron acceptor and either 4 mM pyruvate or 4 mM 2-ketoglutarate as the substrate. The acyl-ACPs and lipoyl-ACP were synthesized with acyl-ACP synthetase (26) except that in the latter case 0.7 mM lipoic acid was substituted for the fatty acid substrate. E. coli cell extracts were made as described previously (7). Purified mitochondria from pea (Pisum sativum cotyledons) and Neurospora crassa were gifts from Wada et al. (21).
Gel Shift AssayThe N-terminal lipoyl domain (L1) of the dihydrolipoyltransacetylase subunit of E. coli PDC was produced in the unmodified form and purified as described previously (27). The gel shift assay was done as described previously (27). The E. coli biotin domain protein was the gift of Chapman-Smith et al. (28).
Assay of Transfer of Octanoate from ACP to Lipoyl DomainThe transfer of octanoate was tested by utilizing tritium-labeled octanoate. Octanoyl-ACP was synthesized using [8-3H]octanoate (20 Ci/mmol) and was then used to modify the lipoyl domain using conditions identical to those of the gel shift assay. The reaction products were then subjected to electrophoresis using 15% SDS-polyacrylamide gels, and the tritium-labeled bands were detected by fluorography.
Two assays
were used to detect attachment of lipoic acid (lipoylation) to the apo
forms of PDC and KGDC. In the first assay lipoic acid attachment was
measured by conversion of the inactive apo forms of PDC or KGDC (Fig.
1) to the active holo forms whereas the second assay
followed the acylation-dependent shift in the electrophoretic mobility of a purified 80-amino acid residue apolipoyl domain from the E. coli PDC (Fig.
2A). The second assay is much less sensitive
but has the advantage that it can be used with any acyl donor because
the mobility shift is due to loss of the positive charge of the lysine
residue. Both assays detected an enzyme activity in E. coli
cell extracts that catalyzed the transfer of lipoic acid from
lipoyl-ACP to lipoyl domains. Moreover, this activity was present in
extracts of E. coli lplA null mutants which lack detectable
ATP-dependent ligase activity (7, 8) (Figs. 1 and
2B), and unlike the ATP-dependent ligase (8) the activity was not inhibited by chelating agents. As anticipated from
prior in vivo studies (6) the enzyme was also active with octanoyl-ACP (Fig. 2C). [3H]Octanoyl-ACP was
used to demonstrate direct transfer of the acyl chain from ACP to the
lipoyl domain (Fig. 3), and the stoichiometry of the
reaction was appropriate (1 mol of octanoyl-ACP was consumed per 1.4 mol of lipoyl domain modified, the discrepancy is attributed to
hydrolysis of a portion of the octanoyl-ACP thioester linkages during
electrophoresis). Conversion of the apo form of E. coli PDC
to the enzymatically active holo forms requires attachment of lipoate
to a specific lysine residue of the lipoyl domains (1, 29). Therefore,
activation of apoPDC (Fig. 1) demonstrated accurate modification of the
lipoyl domains (activation of KGDC was also readily detected).
Specificity of the Lipoate Transferase
The gel mobility shift assay was used to determine the chain length specificity of the enzyme. The lipoate transferase was found to be much more active with the C8 acid than with acids of shorter or longer chain lengths (Fig. 2C). The C7 acid showed some activity, but since odd chain length acids are not present in vivo (9) the transferase has an appropriate chain length specificity. The enzyme had no detectable activity with octanoyl-CoA (data not shown). Moreover, octanoyl-CoA failed to inhibit domain modification by octanoyl-ACP, indicating that the detergent character of the acyl-CoA could not explain the lack of activity (data not shown). The specificity of the lipoate transferase for the protein acceptor was tested by use of the apo form of the 87-residue biotin-accepting domain of E. coli acetyl-CoA carboxylase (28). Although this protein has a structure remarkably similar to the lipoyl domain structure (30), upon substitution of the biotin-accepting domain for the lipoyl domain no modification of the biotin acceptor protein was detected by a gel shift assay (28) (data not shown; the biotin domain gave the expected mobility shift upon biotinylation in the presence of E. coli biotin protein ligase, biotin, and ATP).
E. coli lipB Mutants Lack Lipoate Transferase ActivityIn vivo studies from this laboratory had indicated that E. coli strains having null mutations in both lplA and a second gene (lipB) were completely defective in the modification of lipoylated proteins (6). Moreover, lipB mutants were specifically defective in the attachment of endogenously synthesized octanoate (6). These data indicated that lipB mutants should be deficient in the transferase activity, and this prediction was confirmed. We were unable to detect lipoate transferase activity in extracts of lipB strains (Figs. 1 and 2B). Introduction of multicopy plasmids in which lipB was appropriately oriented downstream of a variety of powerful promoters gave only small increases in transferase activity. Moreover, in agreement with prior results from this laboratory (31) we were unable to detect high level expression of a protein having the molecular weight of LipB. Attempted optimization of translational initiation also failed to markedly increase transferase activity. Thus, we attribute our failure to detect a protein matching the LipB sequence to the very low level of lipB expression such that we failed to detect the protein band in our purified preparations (i.e. the visible bands are contaminating proteins). This very low cellular abundance is consistent with the codon bias of lipB and may be characteristic of vitamin attachment enzymes. We expect that lipB encodes an essential component of the lipoate transferase, although it remains possible that LipB is a positive regulator required for expression of the enzyme. We think this latter possibility is remote since other organisms including diverse bacteria and yeast contain DNA sequences that potentially encode proteins closely similar to LipB (data not shown). The sequences of regulatory proteins are seldom conserved among diverse organisms whereas enzymes often show strong sequence conservation. It should be noted that LipB may be only one subunit of a multisubunit transferase. If so, this would account for only modest increases in transferase activity upon introduction of multicopy lipB plasmids.
Mitochondria Contain Both Lipoate Transferase Activity and a Lipoyl DonorSeveral laboratories have reported that mitochondria contain ACPs (18-23), and nuclear genes encoding ACP-like proteins having mitochondrial targeting sequences have been detected in plants (20) and fungi (32). The role of these proteins has been a puzzle since the major fatty acid synthetic enzymes reside in other cellular compartments.
Since mitochondria contain lipoylated proteins as well as ACP we tested
lysates of plant (from pea, P. sativum) and fungal (N. crassa) mitochondria for a lipoate transferase active with lipoyl-ACP. Activity was readily detected in these preparations using
either the gel shift assay (data not shown) or the E. coli apoPDC activation assay (Fig. 4). In the latter case we
inactivated the endogenous mitochondrial PDCs by addition of ATP. In
the presence of ATP an endogenous PDC kinase converts the plant and
fungal mitochondrial PDCs to their phosphorylated and inactive forms (33, 34) (the kinase has no effect on E. coli PDC). The
N. crassa transferase was also active with octanoyl-ACP. We
also used apoPDC activation to test for the presence of lipoyl-ACP in
the mitochondrial lysates and detected appreciable levels of endogenous
lipoyl donors in both the plant and fungal preparations (Fig. 4). To
demonstrate a stronger dependence of the N. crassa transferase activity on added lipoyl-ACP we serially diluted the lysate
and proportionally extended the incubation time of each dilution (to
compensate for dilution); the rationale being to dilute the endogenous
lipoyl-ACP to concentrations at which the transferase could not
efficiently bind the endogenous substrate. Under these conditions
addition of E. coli lipoyl-ACP gave a 6.6-fold increase in
apoPDC activation (data not shown). The endogenous lipoyl donor present
in N. crassa lysates may be a better transferase substrate
than E. coli lipoyl-ACP, since addition of a low
concentration of E. coli lipoyl-ACP (22 µM)
decreased the rate of apoPDC activation, presumably through occupation
of the transferase active site by the less active E. coli
lipoyl-ACP substrate. Moreover, E. coli octanoyl-ACP (but
not nonacylated ACP) was a potent inhibitor of the endogenous
donor-dependent transferase reaction (22 µM gave complete inhibition in the presence of excess apoPDC) consistent with identity of the endogenous donors as lipoyl thioesters of the
mitochondrial ACPs.
Although lipoic acid attachment is required for activity of several enzymes essential to central metabolism (1, 2) and this cofactor is synthesized by most organisms (including plants and probably mammals), the mechanism of lipoic acid synthesis is not understood. Octanoic acid has been shown to be the precursor of the carbon chain (35), but neither the origin nor the mechanism whereby the sulfur atoms are inserted into the hydrocarbon chain are known. Our finding that lipoyl-ACP donates lipoate to PDC suggests that lipoic acid synthesis may proceed through ACP-bound intermediates, the first of which (octanoyl-ACP) is produced by the fatty acid synthetic pathway. The amino acid sequences of PDC and KGDC and of their lipoyl domains are highly conserved throughout biology, and the structures of the lipoyl domains are also highly conserved. This conservation extends to function in that mammalian and plant lipoyl domains are appropriately lipoylated upon expression in E. coli (4, 5, 36). Therefore, it seems reasonable to propose that the lipoate synthetic pathway is also conserved.
We suspect that the low rates of de novo fatty acid
synthesis reported for isolated mitochondria and mitochondrial lysates (20-23) represents (at least in part) lipoic acid synthesis. Although the lipoylated enzymes, PDC, KGDC, GCV, and the branched chain -keto
acid dehydrogenase complex can comprise major fractions of the
mitochondrial proteins (e.g. more than one-third of the total plant mitochondrial soluble proteins; Ref. 3), these are very
large protein assemblies (often larger than ribosomes) with each
complex carrying only a few hundred lipoate molecules. Thus, what
appears to be a low rate of fatty acid synthesis could readily suffice
for synthesis of lipoic acid. Indeed, a major fatty acid chain
synthesized by isolated mitochondria is octanoic acid, some of which is
found in thioester linkage to the mitochondrial ACPs (21, 23).
Moreover, Wada et al. (21) have recently reported that a
portion of the octanoic acid synthesized by purified pea and N. crassa mitochondria is covalently attached to the H subunit of
GCV, the subunit that normally carries the lipoate cofactor. Since the
typical lipoylated enzymes of eucaryotes are located within the
mitochondria, we propose that lipoic acid synthesis and attachment
proceed within this cellular compartment and that mitochondrial ACP
functions as an essential cofactor in this pathway.
We thank Drs. H. Wada and J. Ohlrogge for mitochondrial preparations and free exchange of experimental results and Dr. A. Chapman-Smith for the gift of biotin domain.