©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Detection by Site-specific Disulfide Cross-linking of a Conformational Change in Binding of Escherichia coli Pyruvate Oxidase to Lipid Bilayers (*)

(Received for publication, January 10, 1995)

Ying-Ying Chang (1)(§) John E. Cronan Jr. (1) (2)

From the  (1)Departments of Microbiology and (2)Biochemistry, University of Illinois, Urbana, Illinois 61801

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

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.


INTRODUCTION

Escherichia coli pyruvate oxidase (PoxB) (^1)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.


MATERIALS AND METHODS

Strains, Media, and Chemicals

All bacteria strains are derivatives of E. coli K12. All mutant PoxB proteins were purified from strain CG3 carrying the respective mutant plasmids. Strain CG3 is DeltaaceEF pfl-1 pps-1 poxB1 recA. Strain EM588, kindly provided by S. Maloy, is thi supE Delta(lac-pro) mutS::Tn10/F`proAB lacI^qDelta(lacZ)M15. Strain JM109 is recA thi supE Delta(lac-proAB)/F`lacI^qDelta(lacZ)M15 proAB.

The media used were described previously(8) . CuSO(4) 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.

Construction of Cysteine Substitution Mutants

Plasmid pYYC170 encoding D560C was made by using the ung/dut site-directed mutagenesis method described previously(4) . Plasmid pYYC170 was derived from plasmid pYYC102, which encodes wild type PoxB (7) . Plasmids pYYC173, pYYC174, pYYC171, and pYYC180, which encode the mutant PoxB proteins V562C, E564C, D560C/E564P, and D560C/V562C, respectively, were obtained by using the unique site elimination method described by Deng and Nickoloff (9) and Diehl and McFadden(10) . Plasmids pYYC173 and pYYC174 were derived from the wild type PoxB plasmid pYYC77. Plasmid pYYC77 is similar to plasmid pYYC68(4) , except the vector is the Kan^r vector pK19(11) . Plasmid pYYC171 was derived from the mutant PoxB16 (E564P) plasmid pYYC69(4) , whereas plasmid pYYC180 was derived by mutagenesis of plasmid pYYC170.

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.

Enzyme Purification and Assay of Pyruvate Oxidase

The wild and mutant PoxB proteins were partially purified as described earlier (3) with some modifications. Cell pellets were suspended in buffer A (0.1 M potassium phosphate, pH 6.0, 20% glycerol, 1 mM EDTA) plus 0.1 mM phenylmethanesulfonyl fluoride. The cells were broken by sonication and centrifuged. The resulting supernatant was heated to 60 °C for 5 min, and denatured proteins were removed by centrifugation. Ammonium sulfate fractionation was done on the heat-treated supernatant, and the 40-70% ammonium sulfate cut was fractionated on a DEAE-cellulose column as described previously(13) . Due to the high level of enzyme production, the enzyme was found to be 90% pure by SDS-polyacrylamide gel electrophoresis following column fractionation. The wild type enzyme was stored at -10 °C in 0.1 M potassium phosphate (pH 6.0) containing 25% glycerol and was stable for 2 months. However, the cysteine-substituted PoxB proteins (especially V562C) tended to polymerize during storage and gradually lost activity.

Oxidase activity was assayed spectrophotometrically with K(3)Fe(CN)(6) as the electron acceptor(8) . Activity due to cross-linking was assayed by incubating the enzyme with pyruvate and TPP-MgCl(2) 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.

Cross-linking Conditions

Partially purified mutant PoxB (10 µg of protein) was incubated with 0.1 M sodium pyruvate, 0.2 mM TPP, and 10 mM MgCl(2) in a final volume of 10 µl for 5 min at room temperature. To this mixture was then added Tris-HCl (pH 6.8) to 25 mM and Cu-PHT (to 0.2 mM CuSO(4) and 0.6 mM 1,10-phenanthroline). The reaction mixture (25 µl) was incubated at 37 °C for various time intervals. When the high concentration of Cu-PHT was used, it was 0.4 mM CuSO(4) plus 1.2 mM 1,10-phenanthroline. Triton X-100 (when added) was at a final concentration of 1%. When DTT was used as an oxidizing reagent, the reaction mixture (10 µl) described above was supplemented with Tris-HCl (pH 7.4) to 50 mM and DTT to 0.1 M in a final volume of 25 µl followed by incubation for 1 h at 37 °C.

SDS-Polyacrylamide Gel Electrophoresis

After cross-linking, samples were withdrawn and an equal volume of NEM sample buffer (see below) was added. The samples were then heated to 100 °C for 2 min and loaded on a 10% SDS-polyacrylamide gel. The electrophoresis system was that of Laemmli(14) . The gel was stained with Coomassie Blue and dried, and the intensities of the cross-linked dimeric and the monomeric protein bands were measured by densitometry. The percentage of cross-linking was the integrated area of cross-linked protein band divided by the sum of the integrated area of cross-linked protein band plus that of monomeric protein band.

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.

Triton X-114 Partition

The enzyme was cross-linked by DTT as described above and was then partitioned between the aqueous and Triton X-114 phases as described previously(4) .

Western Blot Analysis

Extracts from strains carrying plasmid pYYC180 or plasmid pYYC184 were prepared as described above to the heat step. The supernatants from the heat step with or without cross-linking were loaded on a 10% SDS-polyacrylamide gel, and, following electrophoresis, the proteins were transferred to an Immobilon-P membrane (polyvinylidene difluoride; pore size, 0.45 µm) (Millipore Corp.). The membrane was treated first with anti-PoxB antibody and then with goat anti-rabbit IgG (H+L) antibody conjugated with horseradish peroxidase (Zymed Laboratories, Inc.). The protein bands were detected using 1-Step 4-CN (Pierce).


RESULTS

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(2) (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(2) 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.



Cross-linking of Introduced C-terminal Cysteines

We first converted Asp-560 of PoxB to cysteine because previous work showed that D560P PoxB was a fully functional enzyme(4) . As expected, the D560C oxidase was fully active both in vivo and in vitro, and the lipid binding properties of the protein were unchanged. Upon addition of Cu-PHT, virtually no cross-linking of the D560C oxidase was observed in the absence of pyruvate and TPP-Mg, but, in the presence of pyruvate plus TPP-Mg, about half of the enzyme subunits were converted to disulfide-linked dimers (Fig. 1, lanes 6-9). Cross-linking was very rapid and essentially complete within 2 min of adding the oxidation catalyst (Fig. 1). Some cross-linking was observed in the absence of Cu-PHT, presumably due to oxidation catalyzed by metal ion impurities in the buffer. Qualitatively similar results were observed with two other cysteine-substituted proteins, V562C (Fig. 2) and E564C (data not shown), although these mutant proteins gave differing extents of subunit cross-linking. The extent of cross-linking of the V562C oxidase was 80% (Fig. 2), whereas E564C cross-linking was only 15%. Cross-linking was also catalyzed by a very mild oxidizing agent, the oxidized form of DTT (Fig. 3). The addition of DTT gave similiar extents of cross-linking, although the rate of cross-linking was considerably slower than that seen with Cu-PHT (Fig. 3). Cross-links formed by either catalyst were readily reversed by addition of a reducing reagent (lanes 10 of Fig. 1and Fig. 2). Moreover, the D560C oxidase was efficiently cross-linked either when in aqueous solution or when bound to Triton X-100 micelles (Fig. 1, lanes 6 and 7 and Fig. 2, lanes 5, 6, 8, and 9). These results indicated that the paired C termini structure was compatible with both the aqueous and lipid bilayer environments.


Figure 2: Cross-linking of V562C oxidase. Partially purified V562C oxidase was incubated in the presence or absence of pyruvate (Pyr) and TPP-MgCl(2) 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(2) 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.



Enzyme Activation by Cross-linking

Lipid bilayer binding of E. coli pyruvate oxidase results in a significant increase in enzyme activity as assayed with artificial electron acceptors(2) . Therefore, if disulfide cross-linking stabilizes a structure involved in lipid binding, cross-linking might then result in enzyme activation. Indeed, cross-linking led to activation of PoxB. Cross-linking the subunits of the D560C oxidase resulted in an increase in activity from the basal level to half of that given by chymotrypsin treatment in the absence of lipid activators (Table 1). The extent of enzymatic activation due to cross-linking was proportional to the degree of cross-linking. Enzymatic activation associated with cross-linking was also observed with a mutant protein defective in lipid binding/activation (D560C/E564P based on E564P) (4) (Table 1).



Lipid Binding of the Cross-linked Proteins

The cross-linked D560C species retained the ability to associate with lipid micelles. PoxB is known to partition into Triton X-114 in the presence of pyruvate plus TPP-Mg(4) , and both the uncross-linked and cross-linked D560C oxidase species retained this property (Fig. 4). Moreover, cross-linked D560C was protected by Triton X-100 from chymotrypsin digestion (data not shown). These results indicate that the cross-linked D560C structure was compatible with the formation of a stable protein-lipid complex. However, cross-linking did not correct the lipid binding defect of a mutant protein lacking this property. The D560C/E564P mutant protein cross-linked normally, but both the cross-linked and uncross-linked tetramers failed to partition into Triton X-114 (Fig. 4).


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.



Cross-linking of More than Two C Termini

Recent data that argue strongly that the PoxB monomers are arranged in tetrahedral symmetry within the tetramer (see ``Discussion'') suggested that more than two subunits might be able to associate. To test this hypothesis we constructed a mutant PoxB containing cysteine substitutions at two positions within the PoxB C terminus. The cysteine residues were separated by only 1 residue, such that the geometric restraints of the peptide and disulfide bonds prohibit formation of intramonomer cross-links(17) . In this protein, PoxB D560C/V562C, a cross-linked dimer, could potentially cross-link to another monomer or a dimer. Western blot analysis of Cu-PHT-cross-linked D560C/V562C PoxB showed a significant new protein band that migrated more slowly than the dimer band in SDS-polyacrylamide gel electrophoresis (Fig. 5). This new protein band migrated faster than a 200-kDa standard protein, suggesting that it may be a cross-linked trimeric species. However, it was also possible that the new band was a dimer cross-linked at 2 different cysteine residues (e.g. cross-linking of D560C of one subunit to V562C of another subunit). Such heterologous disulfide cross-linking can give anomalous mobility on SDS-polyacrylamide gel electrophoresis as has been observed in disulfide cross-linkings of the Tar chemotaxis receptor protein(18) . In order to test this possibility, we constructed plasmid pYYC184, which carried two different poxB genes, one that encoded D560C PoxB and another that encoded V562C PoxB. Previous work (7) showed that when two PoxB gene alleles are co-expressed, heterotetrameric species are formed in addition to homotetramers, and thus heterologous disulfide cross-linking would occur in preparations from these cells. Western blot analysis of Cu-PHT-cross-linked crude extracts of a strain carrying plasmid pYYC184 showed only a single dimer protein band (Fig. 5, lane 4), indicating that the heterologous disulfide D560C-V560C dimers had the same mobility on SDS-polyacrylamide gel electrophoresis as the individual homologous disulfide dimers. We believe the new band of Fig. 5is the cross-linked trimer. Although other bands were present that migrated more slowly than the putative trimeric species, the trimer band was the most distinct and prominent band. Moreover, the putative trimer band was formed when the protein concentration was decreased during cross-linking (data not shown), indicating that it was due to cross-linking within tetramers rather between tetramers.


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.




DISCUSSION

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 alpha-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 H(2)O(2) 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(2) 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(2) 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(2) 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(2) 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.


FOOTNOTES

*
This work was supported by National Institutes of Health Grant GM 26156. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Dept. of Microbiology, University of Illinois, 407 S. Goodwin, Urbana, IL 61801. Tel.: 217-244-3466; Fax: 217-244-6697; Ying-Ying_Chang{at}qms1.life.uiuc.edu.

(^1)
The abbreviations used are: PoxB, pyruvate oxidase; TPP, thiamine pyrophosphate; Cu-PHT, CuSO(4)/1,10-phenanthroline; DTT, oxidized dithiothreitol; NEM, N-ethylmaleimide.


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