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J. Biol. Chem., Vol. 280, Issue 49, 40948-40956, December 9, 2005

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Computer-assisted Docking of Flavodoxin with the ATP:Co(I)rrinoid Adenosyltransferase (CobA) Enzyme Reveals Residues Critical for Protein-Protein Interactions but Not for Catalysis^*

From the Department of Bacteriology, University of Wisconsin, Madison, Wisconsin 53726

Received for publication, June 21, 2005 , and in revised form, October 3, 2005.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
The activity of the housekeeping ATP:co(I)rrinoid adenosyltransferase (CobA) enzyme of Salmonella enterica sv. Typhimurium is required to adenosylate de novo biosynthetic intermediates of adenosylcobalamin and to salvage incomplete and complete corrinoids from the environment of this bacterium. In vitro, reduced flavodoxin (FldA) provides an electron to generate the co(I)rrinoid substrate in the CobA active site. To understand how CobA and FldA interact, a computer model of a CobA·FldA complex was generated. This model was used to guide the introduction of mutations into CobA using site-directed mutagenesis and the synthesis of a peptide mimic of FldA. Residues Arg-9 and Arg-165 of CobA were critical for FldA-dependent adenosylation but were catalytically as competent as the wild-type protein when cob(I)alamin was provided as substrate. These results indicate that Arg-9 and Arg-165 are important for CobA·FldA docking but not to catalysis. A truncation of the 9-amino acid N-terminal helix of CobA reduced its FldA-dependent cobalamin adenosyltransferase activity by 97.4%. The same protein, however, had a 4-fold higher specific activity than the native enzyme when cob(I)alamin was generated chemically in situ.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
The conversion of vitamin B₁₂ (also known as cyanocobalamin, CNCbl) to its coenzymic form requires replacing the cyano Co() upper ligand with an adenosyl moiety. The resulting coenzyme B₁₂ (also known as adenosylcobalamin (AdoCbl))² is essential for the function of AdoCbl-dependent enzymes (1-3). De novo corrin ring biosynthesis occurs via adenosylated intermediates, and adenosylation is also required for salvaging complete and incomplete corrinoids from the environment (4-6). Corrinoid adenosylation has been studied in several organisms (7-12), but the best understood system is the one in Salmonella enterica (13-16). In this bacterium the last step of the corrinoid adenosylation pathway, i.e. the transfer of the adenosyl group from ATP to the corrin ring, is catalyzed by the ATP:co(I)rrinoid adenosyltransferase (CobA, EC 2.5.1.17 [EC] ) enzyme (16). In addition to CobA, the S. enterica genome encodes two other ATP:cob(I)alamin adenosyl-transferases encoded by the eutT and pduO genes (17-19). However, unlike the EutT and PudO enzymes, CobA has broad specificity for its corrinoid substrate, allowing it to adenosylate de novo corrin ring intermediates as well as complete (e.g. cobalamin (Cbl)) and incomplete corrinoids lacking the nucleotide loop (e.g. cobinamide (Cbi)) (4, 16).

The Co-C bond in coenzyme B₁₂ is the result of a nucleophilic attack by the Co(I) ion on the C5' carbon of ATP. In vitro the Co(I) ion is generated by the transfer of an electron from reduced flavodoxin A (FldA) to Co(II), an event that is currently thought to occur in the active site of CobA (14).

FldA is an electron transfer protein essential to cell survival (20). In addition to its involvement in the corrinoid adenosylation pathway (14), FldA provides reducing equivalents for the reactivation of MetH, the B₁₂-dependent methionine synthase (21). Elegant NMR spectroscopy studies identified the interacting surfaces of a fragment of MetH with FldA (22).

One unanswered question regarding the mechanism of catalysis by CobA is how the redox potential of the Co(II) to Co(I) transition (-610 mV) (23) is increased enough so the electron transfer from reduced FldA (-450 mV; semiquinonine/hydroquinone) (21, 24) can occur. As mentioned above, we previously hypothesized that FldA reduces Co(II) to Co(I) in the CobA active site, triggering the attack of the Co(I) nucleophile on the 5' carbon of the ribosyl moiety of ATP (14). Stich et al. (25) recently reported that binding of the corrinoid substrate to the CobA/MgATP complex increases the Co(II)/Co(I) redox potential to within the range for FldA reduction (25), leading to the reduction of Co(II) to Co(I) in the active site of CobA. At present, the interactions of FldA with CobA are not understood. Here we report results of studies aimed at advancing our understanding of CobA·FldA interactions highlighting the use of computer modeling to generate experimentally testable models.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Strains and Plasmids—TABLE ONE lists all strains and plasmids used in this work. TABLE TWO lists all the mutagenic PCR oligonucleotides used to generate the plasmids used in these studies.

PCR Methods—An Eppendorf Mastercycler® thermocycler was used to amplify templates listed in TABLE TWO using primers listed in TABLE TWO. Amplification products were cloned in vector pET15b (Novagen) to fuse an N-terminal hexahistidine tag to the gene product.

Site-directed Mutagenesis—The Stratagene QuikChange® site-directed mutagenesis kit was used to create point mutants in plasmids pCOBA17 and pFLDA4 (TABLE TWO). Nonradioactive BigDye® (Amersham Biosciences) protocols for DNA sequencing were used to verify the presence of mutations. Plasmids were reconstructed and then sequenced at the DNA sequencing facility at the University of Wisconsin-Madison Biotechnology Center.

Purification of His-tagged CobA Proteins—Plasmids were transformed into Escherichia coli strain BL21(DE3) for overexpression (JE3892). Fresh transformants were used to inoculate 2 ml of fresh Luria-Burtani broth (LB)/ampicillin medium. A starter culture (1 ml) was used to inoculate 100-ml cultures of LB/ampicillin medium. After overnight growth at 37 °C with shaking, cells were harvested by centrifugation at 15,000 x g at 4 °C in a Beckman/Coulter Avanti J-25I centrifuge equipped with a JLA-16.250 rotor. Cell paste was frozen at -80 °C until use. To purify proteins 5 ml of lysis buffer (1x Bug Buster reagent (Novagen) in 1x binding buffer, 0.1 mM phenylmethanesulfonyl fluoride) was used to resuspend the cell pellet. Cells were lysed at room temperature for 20 min and centrifuged for 30 min at 43,667 x g. Cell-free extract was passed through a 0.45-µm syringe filter before loading onto HisBind 900 cartridges (Novagen). Protein was purified as per the manufacturer's instructions. Protein was eluted into 1 mM EDTA. Fractions containing highly purified protein were pooled and dialyzed once against 50 mM Tris-Cl buffer, pH 8, (at 4 °C) containing 10 mM EDTA. Subsequent dialyses were performed against buffer without EDTA. In some cases protein was concentrated using a YM10 Centricon unit (molecular weight cutoff = 10,000; Amicon). Glycerol was added to 10% (v/v) and dithiothreitol to 1 mM; 100-µl drops of protein solution were flash-frozen using liquid N₂, and pellets were stored at -80 °C until used.

Native Wild Type (WT) and CobA^N2-26 Protein Purification—Native WT CobA protein was overexpressed on strain JE2886 and purified as described (15). Allele cobA1328 encoding the mutant CobA protein with the N-terminal 2-26 amino acids deleted (CobA^N2-26) was cloned into plasmid pT7-7 (27) and overproduced in E. coli strain BL21(DE3); all chromatographic steps were identical to those used to isolate native WT CobA protein.

FldA Overexpression and Purification—Plasmids encoding WT or mutant His-tagged FldA proteins were freshly transformed into strain JE4182. Four 2-liter flasks containing 750 ml of LB medium containing kanamycin (50 µl/ml) and ampicillin (100 µl/ml) were each inoculated with 10 ml of an overnight starter culture grown in the same medium. After the culture reached mid-log phase at 30 °C and 160 rpm shaking, riboflavin was added to a final concentration of 10 µM, and cultures were shifted to 42 °C for 1 h and incubated at 37 °C overnight. Cells were harvested at 4 °C and 15,000 x g for 10 min; cell paste was frozen at -80 °C until used.

Frozen cell paste was resuspended in 20 mM sodium phosphate buffer, pH 7.5, (at 4 °C) containing 0.5 M NaCl and 1 mM phenylmethanesulfonyl fluoride. The suspension was passed through a French press twice, 10 mg of DNase was added, and the lysate was incubated on ice for 10 min. The lysate was centrifuged at 4 °C at 43,667 x g for 30 min, and soluble protein was passed through a 0.45-µm syringe filter. Proteins were isolated from crude cell extracts using a ÄKTA fast protein liquid chromatograph equipped with a 5-ml HisTrap column (Amersham Biosciences). After column equilibration, cell-free extract was loaded onto the column, which was washed with 5 column volumes (CV) of 20 mM sodium phosphate buffer, pH 7.5, (at 4 °C) containing 0.5 M NaCl. Mutant FldA proteins were eluted with a 0-50% linear gradient of 20 mM sodium phosphate, pH 7.5, (at 4 °C), 0.5 M NaCl, 0.5 M imidazole over 15 column volumes. Fractions containing pure protein were pooled, concentrated in an YM10 Centricon unit, and dialyzed against 50 mM Tris-Cl, pH 7.5, (at 4 °C) containing 1 mM EDTA. Protein concentration was determined by A₄₆₆ to measure flavin mononucleotide (FMN) bound to FldA (₄₆₆ = 8250 M^-1) (28). Glycerol was added to 10% (v/v), and dithiothreitol was added to 1 mM before protein was flash-frozen and stored at -80 °C until used. Fpr protein (ferredoxin (flavodoxin):NADP⁺ reductase) was purified as described (14).

Fre Protein Overexpression and Purification—H₆Fre (flavin reductase; EC 1.5.1.29 [EC] ) protein was overexpressed using E. coli BL21(DE3)/pFRE3 (13). The latter was re-constructed before overexpression by transforming strain JE3892 with plasmid pFRE3. A single colony was used to inoculate 5 ml of fresh LB/ampicillin medium and incubated to stationary phase at 37 °C. 2.5 ml of the starter culture was used to inoculate four 1-liter flasks containing 250 ml of LB/ampicillin medium. All cultures were incubated at 37 °C to late log phase and induced with 0.5 mM isopropyl--D-thiogalactopyranoside with shaking for an additional 4 h. Cells were harvested at 15,000 x g for 10 min; the cell pellet was frozen at -80 °C until used. To purify H₆Fre (flavin reductase) protein, 20 ml of lysis buffer (1x Bug Buster reagent (Novagen) in 1x binding buffer, 1 mM phenylmethanesulfonyl fluoride) was used to resuspend the cell pellet. Cells were lysed at room temperature for 20 min and centrifuged for 30 min at 43,667 x g. Cell-free extract was filtered through a 0.45-µm and then a 0.2-µm syringe filter before loading onto HisBind 900® cartridges (Novagen). H₆-Fre Protein was purified as per the manufacturer's instructions. Protein was eluted directly into 5 mM EDTA. Fractions containing homogeneous protein were pooled, concentrated in an YM10 Centricon unit, and dialyzed once against 50 mM Tris-Cl buffer, pH 7.5, (at 4 °C) containing 10 mM EDTA. Subsequent dialyses were against buffer without EDTA. Glycerol was added to 10% (v/v) and dithiothreitol to 1 mM before aliquots were flash-frozen and stored at -80 °C.

Growth Behavior Analysis—Ten µl of overnight cultures of all strains was used to inoculate 190 µl of fresh medium in each well of a 96-well microtiter dish; all strains were cultured in triplicate. Growth media contained 1x no-carbon E medium (29), 1 mM Mg⁺², 0.5 mM methionine, 1x trace minerals (30), 50 mM NH₄Cl, 50 mM ethanolamine, pH 7.0. When added, ampicillin was used at 100 µg/ml, kanamycin was used at 50 µg/ml, and AdoCbl, hydroxycobalamin, or dicyano-cobinamide was used at 200 nM. For growth on glycerol as the sole carbon and energy source, ethanolamine and methionine were omitted, and glycerol was added to 30 mM. Cultures were incubated with shaking for up to 96 h at 37 °C in a SpectraMAX Plus automatic plate reader (Molecular Devices). Western blots were performed to determine whether complementation defects were due to protein expression or stability issues. However, because of the sensitivity of the growth assay, even undetectable levels of CobA^WT allowed full complementation.

Cobalamin Adenosyltransferase Assays—Fig. 1 shows three different ways to generate the co(I)rrinoid substrate for CobA. Reactions contained 4 mg of potassium borohydride (KBH₄), 50 µM hydroxycobalamin, 0.8 mM Mn²⁺, 0.4 mM ATP, 3 µM, 0.5 µM CobA. AdoCbl formation was determined by measuring the decrease in A₅₂₅ after 15 min at 37 °C and after 5 min of photolysis. When FldA was used as the reductant instead of KBH₄, assays contained 50 µM hydroxycobalamin, 0.8 mM Mn²⁺, 0.5 mM NADPH, 0.4 mM ATP, 3 µM Fpr, 2 µM FldA, 2 µM CobA and were incubated for 30 min at 37 °C. Peptide inhibition assays contained concentrations of FldA ranging from 0.25 to 2 µM. For mutant FldA proteins, Fpr protein was omitted and replaced by 0.5 µM Fre protein and 50 µM FMN. FldA peptides were synthesized by the Peptide Sequencing Facility at the University of Wisconsin-Madison Biotechnology Center.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Modeling CobA·FldA Interactions—We artificially docked CobA with FldA using ExPasy Deep View (www.expasy.org/spdbv) software (Fig. 2). Without making any structural modifications, three positively charged surface residues on CobA (Arg-9, Arg-98, Arg-165) were aligned with three negatively charged residues on FldA (Glu-61, Asp-68, Asp-93). These FldA residues were previously shown (28) to be involved in its interactions with the Cbl-dependent methionine synthase (MetH) enzyme. In this hypothetical CobA·FldA complex, the FMN cofactor of FldA was within 10 Å of the Cbl substrate in the CobA active site, and hydrophobic patches on both proteins were brought together (Fig. 2D).

View larger version (13K): [in this window] [in a new window]   FIGURE 1. Assays used to assess the production of AdoCbl by CobA. A, KBH4 is used to reduce Co(III)Cbl to Co(I)Cbl directly. B, the Fpr/FldA system converts cob(III)alamin to cob(II)alamin. CobA (represented by the shaded box) binds cob(II)alamin and Mg/ATP. Reduced FldA then provides the electron to reduce cob(II)alamin in the CobA active site. C, in these studies we attempted to bypass Fpr by providing reduced flavins using Fre and NADPH.

The effect of all three peptides on FldA-dependent AdoCbl synthesis was assayed in vitro. In these assays, AdoCbl formation relied on productive CobA·FldA interactions for the generation of the cob(I)alamin nucleophile. To optimize the assay conditions, various concentrations of FldA were used in assays where substrates and CobA were held constant. The K_m for FldA was 5.65 µM and V_max = 31.8 nmol min^-1 mg^-1 (Fig. 4). For Lineweaver-Burk analysis, subsaturating levels of FldA were used such that the reaction proceeded at a rate between 4 and 50% V_max when no peptides were added. Each of the three peptides was then varied from 2-6 µM for each concentration of FldA.

Peptide TWYYGEAQCDWDD⁶⁸ inhibited AdoCbl formation by 30% when present at a 1.5:1 peptide:FldA ratio. At 59% V_max, the specific activities were 19 and 13, respectively. This level of inhibition was significant considering the small size of the peptide. According to Lineweaver-Burk analysis, this inhibition was competitive (K_m peptide = 29 µM and K_i = 1.3 mM). A 13-amino acid peptide mimic of FldA had only a 5-fold decrease in CobA binding efficiency. This decreased affinity might be due to several reasons. The peptide could have greater conformational flexibility than the FldA protein or lack a secondary structure needed for interactions with CobA. In addition, the peptide may not span all the residues involved in the CobA·FldA interaction.

In contrast, the mutagenized and control (TWYYGAAQCDWDA⁶⁸ and WPTAGYHFEASKG¹³² peptides, respectively) failed to inhibit AdoCbl production even when present at 10-fold increase over FldA. These results suggested that the TWYYGEAQCDWDD⁶⁸ peptide did bind to CobA and interfered with the FldA docking. Results with the mutagenized peptide indicated that this inhibition depended on residues Glu-61 and Asp-68.

In Vivo Assessment of CobA Activity—To test the effect of mutations in residues Arg-9, Arg-98, and Arg-165, we site-directed mutagenized each one of these residues to Ala or Glu. Permutations of mutated residues were also constructed, and their effects were tested in vitro and in vivo.

Plasmids containing mutant cobA alleles were transformed into a metE cobA strain of S. enterica (JE7180) to assess their level of function in vivo. Strains were grown under conditions that demanded low or high levels of cobalamin synthesis. When grown on glycerol as sole carbon source, a metE cobA strain cannot convert dicyano-cobinamide into Cbl because enzymes that assemble the nucleotide loop of Cbl require AdoCbi (4, 31). Therefore, in the absence of Cbl, the Cbl-dependent methionine synthase (MetH) cannot methylate homocysteine to produce methionine. Hence, the metE cobA strain is an AdoCbi, Cbl, or methionine auxotroph. When grown on ethanolamine as the carbon and energy source, AdoCbl synthesized by CobA is needed to activate transcription of the ethanolamine utilization (eut) operon and to provide AdoCbl for ethanolamine ammonia-lyase function (18, 32).

Of the CobA mutant proteins tested, only CobA^R98A, CobA^R98E, and CobA^R165A supported growth on ethanolamine as carbon and energy source, albeit to a reduced level relative to the wild-type strain (Fig. 5A). It was surprising to learn, however, that protein CobA^R98A with <20% of the CobA^WT activity (TABLE THREE, column C) supported growth on ethanolamine at 80% that of the growth rate observed with a strain synthesizing CobA enzyme (Fig. 5). Surprisingly, all cobA^WT alleles tested complemented the Cbl auxotrophy of the cobA mutant strain during growth on glycerol (data not shown). We interpret these data to mean that all mutant CobA proteins sufficiently interacted with FldA to produce enough Cbl to satisfy the methionine requirement of the cell. These results indicated that the wild-type CobA enzyme synthesizes an excess of AdoCbl. The inability of some CobA variants to complement a cobA strain during growth on ethanolamine (demands more AdoCbl) reflected the negative effects of the mutations on CobA·FldA interactions rather than being the result of expression problems or unstable CobA mutant proteins.

View larger version (38K): [in this window] [in a new window]   FIGURE 2. Computer model of the CobA·FldA complex. A, crystal structures of CobA (monomers are colored salmon and pink) and FldA (light blue) are shown. Selected basic surface residues on FldA are colored blue, whereas elected acidic side chains on CobA are colored red. B, the structures in A are rotated 90° toward each other. C, CobA and FldA were aligned so that negatively charged residues on FldA (blue) lined up with positively charged residues on CobA (red) to create the docked model. D, close-up of the CobA·FldA complex. At the docking interface FldA residue Asp-68 contacts residue Arg-9 of CobA, and FldA residue Asp-93 contacts CobA residue Arg-165. Residue Asp-11 of FldA is close to, but not contacting residue Arg-98 of CobA. Additionally, the FMN cofactor of FldA (yellow) is within 10 Å of bound Cbl (maroon) and ATP (orange) in the CobA active site. Surface hydrophobic patches on both proteins overlap (not shown).

View larger version (33K): [in this window] [in a new window]   FIGURE 3. Peptide inhibition of the CobA·FldA interaction. A, the primary sequence of FldA is shown above. A subset of acidic residues known to interact with methionine synthase (MetH) is shown in boldface. Peptides designed to inhibit CobA·FldA interaction (TWYYGEAQCDWDD68) and a control peptide (WPTAGYHFEASKG132) are underlined. The crystal structure of FldA was used to model the structure of each peptide (B and C). FldA residues Glu-61 and Asp-68 are shaded gray. Peptide TWYYGAAQCDWDA68 is identical to TWYYGEAQCDWDD68, except that the shaded residues are mutated to Ala.

View larger version (12K): [in this window] [in a new window]   FIGURE 4. Kinetics of FldA-dependent AdoCbl synthesis by CobA. Substrates and CobA were held constant, whereas FldA concentrations were varied. AdoCbl formation was determined after 30 min by detecting a change in A525 after photolysis. The Km for FldA is 5.7 µM, and the Vmax is 31.8 nmol min-1 mg-1. Inset, Lineweaver-Burk plot.

View larger version (13K): [in this window] [in a new window]   FIGURE 5. In vivo and in vitro assessment of CobA function. Plasmids encoding CobAWT and variants of it were transformed into strain JE7180 (metE205 ara-9 cobA366::Tn10d(cat+) eut1141(eutT)). When grown on ethanolamine as sole carbon and energy source, only CobAWT, CobAR98A, CobAR98E, CobAR165A, and CobAA134L proteins produced enough AdoCbl to support growth. + and - signs indicate whether cultures grew to A650 >0.2 after 12 h on ethanolamine; the bar graph indicates the growth rate compared with strains carrying the WT cobA allele. VOC, vector-only control.

Effect of Compensatory Mutations in FldA—Based on the model of the CobA·FldA complex (Fig. 2), we hypothesized that changing FldA residues Asp-68 and Asp-93 to Arg would compensate for the negative effects of CobA mutations R9E and R165E on FldA-dependent synthesis of AdoCbl. A problem with these experiments arose when the Fpr enzyme failed to reduce FldA^D68R and FldA^D93R mutant proteins (data not shown). In an attempt to circumvent this problem, we used the NADH:FMN reductase (Fre) enzyme (13, 33) to reduce free FMN, which would indirectly reduce the flavin cofactor in mutant FldA enzymes (Fig. 1C). If the Fre system worked, CobA^R9E was predicted to preferentially interact with either FldA^D68R or FldA^D93R proteins. Or else CobA^R9E would interact equally well with the FldA^D68R and FldA^D93R proteins if the orientation for docking were not important. However, CobA^R9E was not expected to interact with wild-type FldA or the FldA^D68R/D93R proteins. The first observation we made was that when we used the Fre enzyme, the specific activity of CobA^R9E increased 12-fold over the level measured when Fpr was used to reduce FldA (4.5 versus 0.4). CobA^R9E was not as active when either FldA^D68R (specific activity = 1.7) or FldA^D93R (specific activity = 2) was used in the assay. When FldA^D68R/D93R protein was used, the specific activity of CobA^R9E was as high as when wild-type FldA was used (both 4.5). Results from assays using permutations of CobA and FldA mutant proteins did not reveal any additional insights into CobA·FldA interactions.

Effect of Changes in the Hydrophobic Topology of the CobA Docking Surface—The hydrophobic patch of CobA that is proposed to be involved in CobA·FldA docking has a concave surface. To investigate the contributions of hydrophobic packing to the interactions of CobA with FldA, residue Ala-134 was mutated to Leu. Ala-134 was chosen for mutagenesis because it is the only Ala in the hydrophobic patch. Hence, a change to Leu would be tolerated by the polypeptide. In contrast, mutating other residues in the patch (Phe or Trp) to a less bulky side chain like Ala or Gly would be expected to affect the structural integrity of the protein. The CobA^A134L protein supported growth on glycerol or on ethanolamine as well as did CobA^WT (Fig. 5), and its specific activity under all assay conditions (KBH₄ or Fpr/FldA) was as high as that of the CobA^WT protein (TABLE THREE columns B, C; lines 1 versus 12). Further mutagenesis of CobA is necessary to determine the contributions (if any) of the hydrophobic patch of CobA to the interactions of CobA with FldA.

Deletion of the CobA N-terminal Helix Increases the Specific Activity of the Enzyme but Decreases CobA·FldA Docking—A striking feature of the CobA crystal structure is an N-terminal helix of 26 amino acids that remains disordered unless Mg/ATP and the corrinoid substrate are bound to the active site (12). The N-terminal helix of CobA may have several functions. It may help secure the corrinoid substrate in the active site, it may exclude water from the active site, thereby helping to stabilize the co(I)rrinoid nucleophile, or it may be important for FldA recognition or a combination of the above. To gain insights into the role of the N-terminal helix, we deleted it and tested the ability of the resulting CobA^N2-26 variant to function in vivo and in vitro. Deletion of this helix did not affect the ability of the mutant CobA protein to support growth of a metE cobA strain on glycerol (data not shown). However, the CobA^N2-26 protein failed to support growth on ethanolamine as sole carbon source (Fig. 5).

Results from in vitro assays showed that the specific activity of the CobA^N2-26 enzyme was only 3% of the CobA^WT enzyme when FldA was used as the reductant. The same protein has 4-fold higher specific activity than the wild-type when cob(I)alamin was generated with KBH₄. Eliminating the N-terminal helix resulted in a more efficient enzyme as long as cob(I)alamin is in excess. When the concentration of cob(I)alamin in the active site depends on interaction with FldA, the absence of the N-terminal helix of CobA affects AdoCbl production. Deletion of the N-terminal helix of CobA may affect electron transfer, catalysis, or both.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Computer Modeling as a Tool to Investigate Protein-Protein Interactions—At present, there is a great deal of interest in improving our molecular understanding of how protein complexes form. Fortunately, the accumulation of data in protein databases is rapidly increasing as a result of structural genomics initiatives. Here we report one example of how this valuable information can be used to generate experimentally testable models of protein docking. Sophisticated, userfriendly software for the manipulation of protein structure data is available to researchers interested in studying enzyme complexes. As shown here, a computer-assisted model facilitates the design of peptide mimics predicted to strongly inhibit the interactions between partner proteins. Structure models and peptide mimics can be used to investigate the contribution of specific amino acid residues to the formation of biologically relevant protein-protein contacts.

Insights into CobA·FldA Interactions—We have identified part of the regions of the CobA and FldA proteins that allow electron transfer from the flavin in FldA to the co(II)rrinoid in the active site of CobA. Four pieces of evidence support the conclusion that FldA residues TWYYGEAQCDWDD⁶⁸ are part of the FldA structure that interacts with CobA. First, a 30% inhibition of CobA activity when the TWYYGEAQCDWDD⁶⁸ peptide is present in the assay is significant given the small size of the peptide (13 amino acids). Second, there was a complete lack of an inhibitory effect by another FldA-derived peptide of similar size and hydrophobicity. Third, mutagenized peptide TWYYGAAQCDWDA⁶⁸ failed to inhibit adenosylation, demonstrating that residues Glu-61 and Asp-68 are key components of the FldA surface interacting with CobA. Fourth, this peptide is part of the surface of FldA that interacts with MetH and Fpr (28).

Residues of CobA Critical for Docking—We suggest that residue Arg-9 is essential for docking of CobA with FldA (TABLE THREE). Regardless of the nature of the change at this position, CobA cannot dock with FldA if Arg-9 is altered. A strong case for docking can also be made for residue Arg-165. However, there is more tolerance for change at this position. A CobA^R165A variant still supported growth on ethanolamine, indicating that FldA-dependent reduction of the substrate was sufficient to meet the high demand of AdoCbl required to grow on ethanolamine. Only a drastic change in the charge of the side chain (e.g. Arg to Glu) prevented docking of CobA with FldA. The role of residues Arg-9 and Arg-165 appears to be limited to CobA·FldA interaction, since the catalytic competence of mutant enzymes unable to dock with FldA was equal to that of the wild-type protein (TABLE THREE, column B, lines 1, 2-3, 6-7).

It is not surprising that a truncation of the N-terminal helix results in poor interactions of CobA with FldA. CobA^N2-26 protein is missing residue Arg-9 as well as other residues that may be involved in docking. What is surprising, however, is the substantial increase in the specific activity of the enzyme (4-fold higher than CobA^WT enzyme). We speculate that this increase in specific activity may be due to an increase in the rate of substrate binding, product release, or both.

Results from recent EPR and MCD studies by Stich et al. (25) have provided evidence for the existence of a four-coordinate co(II)rrinoid in the active site of the wild-type CobA enzyme. The effect that FldA docking to CobA may have on electron transfer or stability of the co(II)rrinoid substrate is under investigation. Use of the CobA^N2-26 protein might be especially useful in probing the role of residues in the N-terminal helix in activation and stabilization of the corrinoid substrate in the CobA active site.

Differences in the Sensitivity of the in Vivo Assays for AdoCbl Reveal the Coenzyme B₁₂ Requirement of S. enterica—We typically use low Cbl levels (1 nM) in the culture medium to satisfy the methionine requirement of S. enterica. In contrast, optimal growth rates on ethanolamine as carbon and energy source require a >2 orders of magnitude higher concentration of Cbl in the medium (150 nM (34)). It was surprising to learn that a mutant CobA protein that is one-tenth as active as the wild-type enzyme can meet the demand for AdoCbl during growth on ethanolamine. This result explains why it has been difficult to isolate cobA alleles that do not contain nonsense or missense mutations that severely destabilize the protein. The low requirement for endogenous AdoCbl when growing on ethanolamine means that Salmonella strains carrying cobA alleles encoding defective CobA variants appear indistinguishable from cobA⁺ strains on solid media.

	FOOTNOTES

 
^* This work was supported in part by National Institutes of Health Grant GM40313 (to J. C. E.-S.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Supported in part by a Howard Hughes Predoctoral Fellowship. To whom correspondence should be addressed: Dept. of Bacteriology, 1710 University Ave., Madison, WI 53726-4087. Tel.: 608-262-7379; Fax: 608-265-7909; E-mail: escalante{at}bact.wisc.edu.

² The abbreviations used are: AdoCbl, adenosylcobalamin; Cbi, cobinamide; CobA, ATP: co(I)rrinoid adenosyltransferase; FldA, flavodoxin; FMN, flavin mononucleotide; Fpr, ferredoxin (flavodoxin):NADP⁺ reductase; Fre, flavin reductase; WT, wild type; Fre, flavin reductase.

	ACKNOWLEDGMENTS

 
We thank Ivan Rayment for help with PyMOL and Perry Frey for critical review of the manuscript.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 

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