(Received for publication, January 10, 1995)
From the
Escherichia coli pyruvate oxidase, a peripheral
membrane homotetrameric flavoprotein, exposes its C-terminal lipid
binding site in the presence of substrate pyruvate and co-factor
thiamine pyrophosphate Mg and binds tightly to
phospholipid bilayers during catalysis. Using site-specific disulfide
cross-linking, we demonstrate that disulfide cross-links are formed
between C termini of D560C pyruvate oxidase and that the degree of
cross-linking is greatly increased by the presence of substrate and
co-factors indicating a conformational change that results in
juxtaposition of two subunit C termini. The cross-linked oxidase is
enzymatically active and remains able to associate with lipid micelles.
These results argue strongly that lipid bilayer binding of pyruvate
oxidase involves pairing of the C termini of two subunits.
Escherichia coli pyruvate oxidase (PoxB) ()is a tetramer of the 62-kDa protein encoded by the poxB gene(1, 2) . PoxB is a soluble
flavoprotein that binds tightly to phospholipid bilayers in the
presence of its pyruvate substrate and the loosely bound co-factor,
thiamine pyrophosphate (TPP)(3, 4) . In the absence of
these compounds, the oxidase lacks affinity for lipid
bilayers(3, 4) . Lipid bilayer binding is essential
for pyruvate oxidase function in vivo, because ubiquinone, the
electron acceptor required to reoxidize the catalytic flavin group of
the enzyme, resides within the lipid bilayer (5) . Lipid
binding also greatly increases the activity of the enzyme
(10-20-fold) and gives a 10-fold decrease in the Michaelis
constant for pyruvate(3, 4) . Previous studies have
localized lipid binding to the C terminus of the protein (4, 6) and have shown that single conservative amino
acid substitutions can abolish lipid binding(4) . Recently, we
obtained tetrameric species composed of mixtures of wild type subunits
and subunits defective in lipid binding and found that lipid binding
requires tetramers having at least two wild type subunits, a result
suggesting that pairing of two C termini is required for lipid bilayer
binding(7) . We now report evidence for such pairing obtained
by site-specific disulfide cross-linking.
The media used were described previously(8) . CuSO 1,10-phenanthroline (Cu-PHT), trans-4,5-dihydroxy-1,2-dithiane (oxidized dithiothreitol,
DTT
), N-ethylmaleimide (NEM), and other chemicals
were obtained from Sigma. Oligonucleotide primers were synthesized by
the University of Illinois Genetic Engineering Facility.
For unique site elimination mutagenesis, two oligonucleotide primers were used. The first primer was the EcoRI/AatII 21-mer (5`-ACGGCCAGTGACGTCGAGCTC-3`), used in each mutagenesis to substitute a unique AatII site for the unique EcoRI site of the multiple cloning sites of the parental plasmids and allow selection of the mutant plasmids. The second primers (21- to 30-mers), which contained the desired mutation, were D560C (5`-GATCACTTCACAACCGCGTCC-3`), V562C (5`-CGCCAGTTCGATACATTCATCACCGCGTCC-3`), E564C (5`-TGTTTTCGCCAGGCAGATCACTTCATC-3`), and D560C/V562C (5`-GCCAGTTCGATACATTCACAACCGCG-3`). (The underlined bases are those giving the desired mutation.) The EcoRI/AatII primer and the mutation primer were annealed to the parental plasmid template, and DNA syntheses were carried out as described(9) . The plasmid DNA was subsequently transformed into mutS strain EM588, and the DNAs isolated from overnight cultures were digested with EcoRI and transformed into strain JM109. Plasmids isolated from individual colonies that failed to cut with EcoRI but that cut with AatII were candidates for DNA sequencing. Plasmid preparations and DNA sequencing were done by conventional methods(12) .
Plasmid pYYC184 carried two genes (one encoding D560C PoxB and the other encoding V562C PoxB) and was derived from plasmids pYYC170 and pYYC173. Plasmid pYYC170 was digested with PstI, resected to blunt ends with T4 DNA polymerase, and then digested with SacI. The PstI-SacI fragment containing the D560C PoxB gene was isolated from an agarose gel and cloned between the SmaI and SacI sites of plasmid pYYC173 giving pYYC184.
Oxidase
activity was assayed spectrophotometrically with
KFe(CN)
as the electron acceptor(8) .
Activity due to cross-linking was assayed by incubating the enzyme with
pyruvate and TPP-MgCl
in the absence of lipid activators.
When the enzyme was cross-linked in the presence of Cu-PHT, the enzyme
was first passed through a Speedy desalting column (bed volume, 5 ml;
Pierce), and the eluant was collected into fractions and assayed. When
the enzyme was cross-linked in the presence of DTT
, the
reaction mixture could be assayed directly without desalting.
NEM sample buffer contained 0.1 M Tris-HCl, pH 6.8, 20 mM EDTA, 4% SDS, 20% glycerol, 0.02% bromphenol blue, 20 mM NEM, and the running buffer of Laemmli (25 mM Tris base, 0.192 M glycine, 0.1% SDS, pH 8.3). The NEM was added just before use. For reduction of the disulfide cross-links, the sample buffer contained 0.1 M Tris-HCl, pH 6.8, 4% SDS, 20% glycerol, 0.02% bromphenol blue, and 10% 2-mercaptoethanol.
The site-specific disulfide cross-linking approach, based on
that of Falke and Koshland(15) , was to introduce cysteine
residues into the C-terminal region of PoxB and assay disulfide subunit
cross-linking by SDS-polyacrylamide gel electrophoresis. A potential
difficulty in this approach was the 10 native cysteine residues of
PoxB(1) . However, prior chemical modification experiments
indicated that 9 of these cysteine residues were in nonreactive
environments and that the 10th was reactive only in the absence of
TPP(16) . These data suggested that disulfide cross-linking via
the native cysteine residues should not complicate the analysis of
cross-linking due to introduced cysteine residues. Indeed, no
cross-linking of wild type PoxB subunits was observed upon oxidation
with either O (catalyzed by Cu-PHT) (Fig. 1, lane 2) or DTT
(data not shown).
Figure 1:
Cross-linking of D560C oxidase.
Purified wild type or mutant D560C (10 µg of protein) was
incubated in the presence or absence of pyruvate (Pyr) plus
TPP-MgCl
and subjected to oxidation catalyzed by Cu-PHT as
described under ``Materials and Methods.'' The incubation
mixture of lane 7 contained 1% Triton X-100. At the times
indicated an equal volume of NEM buffer was added to each sample
(except the sample in lane 10) and loaded on a 10%
SDS-polyacrylamide gel. The sample of lane 10 contained
2-mercaptoethanol, which diffused into lane 9. The gel was
stained with Coomassie Blue.
Figure 2:
Cross-linking of V562C oxidase. Partially
purified V562C oxidase was incubated in the presence or absence of
pyruvate (Pyr) and TPP-MgCl and subjected to
oxidation by Cu-PHT as described under ``Materials and
Methods.'' In lanes 6 and 9, 1% of Triton X-100
(final concentration) was included in the incubation mixture before
cross-linking. In lanes 1-9, the samples were treated
with the NEM loading buffer, whereas in lane 10, the
mercaptoethanol loading buffer was used.
Figure 3:
Cross-linking of the D560C and V562C
oxidases by DTT. Samples of partially purified D560C or
V562C preparations were first incubated in the presence of pyruvate
plus TPP-MgCl
and subjected to oxidation by DTT
as described under ``Materials and Methods.'' At the
time intervals indicated, samples were taken into an equal volume of
NEM loading buffer and loaded on a 10% SDS-polyacrylamide gel. The gel
was stained with Coomassie Blue and dried, and the densities of the
dimeric and monomeric protein bands in the cross-linked samples were
measured by densitometry for calculation of the percentage of
cross-linking.
Figure 4:
Partition behavior of the D560C (wild
type) or D560C/E564P (mutant) oxidases. Partition of the proteins into
aqueous (aq) and Triton X-114 (Tx) phases in the low
or high concentration of pyruvate (Pyr) plus
TPP-Mg was assayed by SDS-polyacrylamide gel
electrophoresis. Lanes 1 and 6 contained cross-linked
samples (2 µg) of the wild type and mutant oxidases, respectively,
in the absence of Triton X-114. DTT
-catalyzed
cross-linking was done as described under ``Materials and
Methods'' for 1 h at 37 °C. DTT
-treated samples
(20 µl) were supplemented with 2 µl of 20% Triton X-114 and
followed by separation into aqueous and detergent phases and
SDS-polyacrylamide gel electrophoresis as described
previously(4) . Lanes 2 and 7 show the
aqueous phases of enzyme samples partitioned in the low concentrations
of pyruvate (40 mM) plus TPP-Mg
(0.08
mM, 4 mM) carried over from the cross-linking
mixture, whereas lanes 3 and 8 are the corresponding
Triton X-114 phases. Lanes 4 and 9 show the aqueous
phases of enzyme samples partition in the presence of saturating levels
of pyruvate (0.14 M) plus TPP-Mg
(0.28
mM, 14 mM), whereas lanes 5 and 10 are the corresponding Triton X-114
phases.
Figure 5: Immunoblot analysis of PoxB D560C/V562C. Extracts from strains carrying plasmid pYYC180 (encoding D560C/V562C PoxB) or plasmid pYYC184 (encoding D560C and V562C) were prepared and analyzed by immunoblotting as described under ``Materials and Methods.'' Lanes 1, 2, and 5-7 were pYYC180 (D560C/V562C, 2 cysteine substitutions/monomer (2C/m)) extracts, whereas lanes3 and 4 were pYYC184 (D560C and V562C, one cysteine substitution/monomer (1C/m)) extracts. Lanes 2, 4, and 6 were samples cross-linked by the low concentration of Cu-PHT for 2 min at 37 °C as described under ``Materials and Methods,'' whereas lanes1, 3, and 5 lacked the cross-linking catalyst. The protein concentrations during cross-linking of the samples in lanes2, 4, and 6 were 0.87, 0.78, and 1.85 mg/ml, respectively. Lanes 1-6, samples received equal volumes of NEM loading buffer. Lane 7 was identical to lane2, except that the 2-mercaptoethanol loading buffer was used. The migration positions of PoxB, monomer (62K), dimer (Di), and putative trimer (Tri) are indicated. pyr, pyruvate.
Our results strongly argue that a required step in lipid
bilayer binding of pyruvate oxidase involves a pairing of the C termini
of two subunits. We and others have argued that a putative -helix
proposed to involve residues 558-572 is involved in lipid
binding(4, 19) . The putative helix is strongly
amphipathic in character(19) , and a straightforward model for
lipid bilayer binding would involve pairing of two helices at their
hydrophilic faces followed by partition of the resulting hydrophobic
paired structure into the lipid bilayer. This is consistent with the
lipid binding properties of the isolated C-terminal
segment(20) , the effects of introduction of a proline residue
into the putative helix(4) , and the finding that PoxB-lipid
interaction is driven by hydrophobic
interactions(3, 4) . We believe that the paired C
termini structure penetrates deeply into the lipid bilayer because the
strength of binding of PoxB to phosphatidylcholine bilayers depends on
the length of the phospholipid acyl chain(3, 4) .
The finding that more than two subunits can be cross-linked in
double cysteine substitution mutants strongly suggests that the
subunits of the PoxB tetramer are arranged in a tetrahedron. In the
other possible arrangement, a square planar array, it would seem
difficult for more than two C termini to become juxtaposed. Consistent
with the proposed PoxB tetrahedron, the crystal structure of a Lactobacillus enzyme (also called pyruvate oxidase) has been
solved at high (2.1 Å) resolution(21, 22) . This
enzyme is not an analog of the E. coli PoxB, although it does
share a number of its properties. (The identical names are a historical
accident; both enzymes are misnamed.) The Lactobacillus enzyme, like PoxB, is a homotetramer. Each of the 66-kDa monomers
binds FAD and TPP, the latter co-factor being bound via a magnesium
ion. Like PoxB, the Lactobacillus enzyme decarboxylates
pyruvate in an oxidative manner, but the products are different (acetyl
phosphate and HO
rather than the free acetate
and reduced quinone produced by PoxB). Most importantly, the Lactobacillus enzyme shows no lipid binding or activation.
This lack of lipid interaction is expected, because the flavin is
directly reoxidized by O
rather than by quinone dissolved
in the lipid bilayer (Lactobacilli lack the standard
respiratory chain).
The Lactobacillus amino acid sequence can be readily aligned with the PoxB sequence (Fig. 6). Excepting the extreme ends, the alignment proceeds over 500 residues with only six gaps that are all less than 3 residues. The sequences show 29-34% identical residues, depending on the gaps chosen. If we use the most significant set of residue substitutions given by Bardo and Argos (23) for exchanges seen among topographical families of proteins, the similarity then rises to 50%. The only long segments of the two proteins lacking similarity are the C termini, a result consistent with the special lipid binding properties of PoxB. The x-ray structure shows that each subunit of the Lactobacillus protein is composed of three domains (Fig. 6). The protein core involved in subunit interaction is followed by the FAD binding domain, then the TPP binding domain, and finally a 50-residue C-terminal extension. The alignment gives 48% identical residues in the core, whereas the two co-enzyme binding domains contain 30% identical residues. The increased sequence identity in the core is expected from the lack of space available to accommodate amino acid side chain changes within protein cores. Indeed, when the Bardo-Argos (23) set of core residue substitutions is used, the core similarity approaches 60%. Virtually all of the sequences postulated or shown to play important roles in the structure and function of the Lactobacillus enzyme are found in PoxB. The conservation of the FAD binding residues is of special interest because the Lactobacillus enzyme binds FAD by a novel use of the classic Rossmann dinucleotide fold structure(21, 22) , and thus this mode of FAD binding can be extended to PoxB and accounts for the lack of standard glycine dinucleotide fold fingerprint sequence in PoxB.
Figure 6: Alignment of the deduced amino acid sequences of the Lactobacillus POX (Lb POX) and E. coli PoxB (PoxB). Identical residues are boxed, and the domain structure of Lactobacillus POX is given.
Consideration of the Lactobacillus structure provides good explanations for many known properties of PoxB. First, the lack of reactivity of the PoxB cysteine residues can be attributed to their locations within the protein structure (Fig. 6). All 10 PoxB cysteines are in regions of strong alignment between the two proteins, but only 1 residue remains a cysteine in the Lactobacillus protein. By analogy with the Lactobacillus structure, five PoxB cysteines are buried within the core, whereas 2 residues are in the FAD binding region (known from spectral studies to be a hydrophobic environment)(24) , and 3 are in the TPP binding region. One of these latter cysteine residues is adjacent to the DGG sequence involved in binding the pyrophosphate moiety of TPP and seems almost certain to represent the sole PoxB residue accessible to sulfhydryl reagents only in the absence of TPP or pyrophosphate(16) . Second, the FAD domain of PoxB is the N-terminal half of the molecule consistent with our prior hybrid protein studies(25) . Third, the ``business ends'' of the two co-enzymes are in very close contact, consistent with previous spectral studies of PoxB(24, 26) . Finally, the alignment also suggests that known protease-sensitive regions of PoxB reside in surface loops.
Given the above data we expect that the overall structure of the Lactobacillus and PoxB enzymes are very similar. The Lactobacillus enzyme has D symmetry, and the
structure is termed a dimer of dimers. PoxB dimers have been reported (27) , and the presence of these half-molecules indicate that,
consistent with D
symmetry, there is more than one type of
bond between subunits. Moreover, the high degree of similarity in the
core residues of Lactobacillus with PoxB together with the
finding of unusually tight packing of the Lactobacillus core
residues strongly suggests tetrahedral D
symmetry for PoxB.
It should be noted that, from preliminary electron micrographs, planar
symmetry was suggested for PoxB(28) . However, these
micrographs also contain images consistent with tetrahedral symmetry.