(Received for publication, May 24, 1995; and in revised form, August 9, 1995)
From the
The CobU protein of Salmonella typhimurium was
overexpressed and purified to 94% homogeneity. N-terminal
sequencing of purified CobU confirmed the first 22 amino acids. In
vitro assays showed that CobU has kinase and guanylyltransferase
activities which catalyze the synthesis of adenosyl-cobinamide-GDP from
adenosyl-cobinamide, via an adenosyl-cobinamide-phosphate intermediate.
We present evidence that the transfer of the guanylyl moiety of GTP to
adenosylcobinamide-phosphate proceeds via an phosphoramidate-linked,
enzyme-guanylyl intermediate.
In the presence of oxygen, kinase and
guanylyltransferase activities of CobU were lost. Treatment of inactive
CobU with dithiothreitol restored 20% of the kinase and
guanylyltransferase activities, indicating the involvement of
sulfhydryl groups in enzyme activity. The sulfhydryl modifying agents
5,5`-dithiobis(2-nitrobenzoic acid) and N-ethylmaleimide
abolished both CobU activities.
Native CobU protein was a dimer
(40 kDa) that functioned optimally at pH 8.8-9.0 and 37
°C. Substrates and kinetic parameters for both activities were
determined. The preferred corrinoid substrate for this enzyme was
adenosyl-cobinamide. In vitro experiments are consistent with
previous genetic studies which had suggested that adenosyl-cobinamide
was the preferred substrate of CobU, and that CobU functioned more
efficiently in the absence of oxygen.
Assembly of the nucleotide loop of cobalamin in Salmonella
typhimurium is thought to require the involvement of four enzymes,
CobU, CobS, CobT, and CobC (Fig. 1). CobT and CobC act
sequentially in the generation of dimethylbenzimidazole--riboside
(also known as
-ribazole) from nicotinic acid mononucleotide and
dimethylbenzimidazole. CobT catalyzes the transfer of the
phosphoribosyl moiety of nicotinic acid mononucleotide to
dimethylbenzimidazole to yield
-ribazole-5`-phosphate(1) ,
which is then dephosphorylated by CobC to generate
-ribazole(2) . Homology to the CobP protein of Pseudomonas denitrificans(3, 4) and
nutritional studies performed on S. typhimurium cob mutants (5) suggested that CobU was responsible for the synthesis of
Ado
-CBI-GDP from Ado-CBI, via a Ado-CBI-P intermediate (Fig. 1). The CobS protein is thought to be the cobalamin
synthase that catalyzes the synthesis of Ado-cobalamin from Ado-CBI-GDP
and
-ribazole(2, 4, 5) .
Figure 1:
Ado-CBI and the nucleotide loop
assembly functions. The top of the figure shows the structure
of CBI. X = the 5`-deoxyadenosyl moiety, the structure
of which is shown. The pyrrole rings and peripheral ring decorations
are labeled. The boxed HO moiety represents the portion of the
Ado-CBI modified by CobU-catalyzed reactions. At the bottom of
the figure is shown a schematic of the reactions required for assembly
of the nucleotide loop of cabalamin. The corrin ring is shown in a
simplified form. The asterisks (*) indicates the phosphate donated by
[-
P]ATP to Ado-CBI. The enzymes catalyzing
each reaction are shown.
We report
the overexpression and purification of the cobU gene product,
document that the CobU protein has both kinase and guanylyltransferase
activities required for the synthesis of Ado-CBI-GDP from Ado-CBI, and
provide evidence that Ado-CBI-P is an intermediate in the synthesis of
Ado-CBI-GDP. We also present evidence that the transfer of the guanylyl
moiety of GTP to Ado-CBI-P proceeds via a phosphoramidate-linked,
CobUGMP intermediate. The characterization of homogeneous CobU
protein includes an analysis of substrate specificity, physical
characteristics, and kinetic parameters.
Results from previous genetic studies suggested that oxygen negatively affected CobU activity and that the corrinoid substrate utilized by CobU was also affected by molecular oxygen in the environment(6) . Specifically, it appeared that under aerobic conditions CobU could utilize Ado-CBI only as its corrinoids substrate. These results contrasted with the ability of this enzyme to utilize either Ado-CBI or HO-CBI (i.e. nonadenosylated CBI) under anaerobic conditions (6) .
The in vitro data presented support the hypothesis that the preferred corrinoid substrate for the CobU enzyme is Ado-CBI. HO-CBI can also serve as substrate, albeit poorly. We document oxygen lability of the kinase and guanylyltransferase activities of CobU, both of which can be restored by DTT. Chemical modification of sulfhydryl groups with DTNB or NEM strongly suggested that CobU activities require one or more of the 4 cysteinyl residues present in the protein.
Figure 2: Site-directed mutagenesis of cobU. At the top of the figure is the wild-type sequence of cobU near its start codon (shown in bold, pJO49) and the stop codon (TGA). The intervening cobU sequence is represented by the four dots. The second line is the mutagenized cobU sequence which results in a NdeI restriction site, shown in capital letters (CATATG, pJO51). The NdeI-to-HindIII (located in the multicloning site, MCS) fragment of pJO51 was cloned into pT7-7 digested with NdeI and HindIII to generate pJO52. In pJO52, cobU is expressed under control of the phage T7 promoter and ribosome binding site.
The UV-visible spectrum
of Ado-CBI showed no significant absorbance at 525 nM,
indicating quantitative conversion of (CN)CBI to Ado-CBI
(data not shown). The extinction coefficient of Ado-CBI in
H
O at 457 nm was calculated to be 4,526 M
cm
. Absorption maxima
in H
O were observed at 305 and 457 nm.
As shown below, ATP did not serve as an efficient substrate for the transferase activity of CobU. Therefore, in the absence of GTP, the conditions for the Ado-CBI kinase assay allowed us to document the synthesis of the intermediate Ado-CBI-P.
Figure 3: Overexpression and visualization of CobU in crude extracts and visualization of purified CobU protein. Panel A, lane 1, crude cell-free extract of a strain carrying only the overexpression vector without any insert (pT7-7, JE3420, negative control). Lane 2, crude cell-free extract of a strain carrying a CobU overexpression plasmid (pJO52, JE3207, CobU overexpressing strain). Panel B, lane 1, size markers in kilodaltons. Lane 2, purified CobU from pooled DEAE fractions (5 µg). CobU is the band indicated by the arrow. Proteins were resolved by SDS-PAGE (12%) and stained with Coomassie Blue.
Figure 4:
ATP:Ado-CBI kinase assay and GTP:Ado-CBI
kinase, Ado-CBI-P guanylyltransferase assay. Panel A, a
representative kinase assay. Panel B, a representative
guanylyltransferase assay. In both panels, lane 1 shows the
results of assays using an extract generated from the CobU
overexpressing strain JE3207. Lane 2 shows the results of
assays using an extract generated from strain JE3420, which carries
only the overexpression vector pT7-7. The R values for each indicated compound are reported under
``Experimental Procedures.''
The relative mobility of radiolabeled (CN)CBI-P
on the chromatograph was 0.77, a value consistent with that reported in
the literature(15) . This radioactive product comigrated with
authentic (CN)
CBI-P; unlabeled (CN)
CBI-P was
identified on the chromatogram by its characteristic red-purple color
(data not shown).
In this paper chromatography system,
(CN)CBI migrated with an R
=
0.92, ATP migrated with an R
= 0.38, and
the hydrolysis of ATP resulted in labeled inorganic phosphate which
remained near the origin. The synthesis of Ado-CBI-P was dependent on
overexpression of CobU and the presence of Ado-CBI and ATP in the
reaction mixture. Heat treatment of cell-free extracts resulted in loss
of ATP:Ado-CBI kinase activity.
The relative mobility of radiolabeled
(CN)CBI-GDP was 0.56, a value consistent with the
literature(15) . This radioactive product comigrated with
authentic (CN)
CBI-GDP and was identified on the paper
chromatogram by its characteristic red-purple color (data not shown).
The synthesis of Ado-CBI-GDP was dependent on overexpression of CobU and the presence of Ado-CBI and GTP in the reaction mixture. Heat treatment of cell-free extracts resulted in loss of GTP:Ado-CBI kinase, Ado-CBI-P guanylyltransferase activities.
The
molecular mass of homogeneous CobU, as judged by SDS-PAGE, was
estimated at 22 kDa, a value in agreement with a previous
report(5) . The predicted mass of CobU, based on the DNA
sequence of the cobU gene, is 19.7 kDa(4) . As
determined by nondenaturing gel electrophoresis(22) , the
native molecular mass was estimated to be 49 kDa. These data were
consistent with CobU functioning as a dimer. The experimentally
determined isoelectric point of the native CobU enzyme identified two
species at pI of 7.2 and 7.5, a sharp departure from the pI predicted
for the denatured protein (pI = 5.3). The molar extinction
coefficient of CobU at 280 nm in 0.1 M Tris-HCl, pH 8.0 at 4
°C, was 52,205 M cm
.
The first 22 amino acid residues of the purified CobU protein, as
determined by N-terminal sequencing, were identical to the amino acid
residues predicted from the DNA sequence of cobU.
An
examination of the CobU amino acid sequence revealed two partial
nucleotide triphosphate binding domains(5) . Near the N
terminus of CobU there is a portion of the ATP binding domain which
specifies phosphate binding, suggesting some flexibility in substrate
binding. Consistent with this observation, either ATP or GTP served as
phosphate donors for the synthesis of Ado-CBI-P. In fact, the specific
activity for the kinase reaction was approximately 10-fold higher when
GTP was used as substrate than when ATP was provided at the same
concentration. These results did not necessarily imply that GTP was the
phosphate donor in vivo, because these increased reaction
rates could be the result of an active guanylyltransferase function.
For example, the presence of 100 µM unlabeled GTP
stimulated the [-
P]ATP-dependent kinase
activity of CobU approximately 3-fold (data not shown). A similar
effect was reported by Blanche et al.(3) for the CobP
protein of P. denitrificans. The stimulation of the kinase
activity by GTP makes it difficult to determine the preferred substrate
for the kinase reaction. Blanche et al.(3) suggested
that ATP was the preferred substrate for the CobP protein of P.
denitrificans based on kinetic arguments. The amino acid and
functional similarity between the CobU and CobP proteins makes it
likely that CobU also preferentially utilizes ATP in the kinase
reaction.
Near the C terminus of CobU a portion of a GTP binding domain conferring guanine specificity can be identified. This partial GTP binding domain probably represents the portion of CobU required for binding the substrate of the transferase activity. Consistent with this observation, GTP (but not ATP, UTP, or CTP) served as an efficient substrate for the CobU transferase activity (Table 4).
To test whether
CobU forms an CobUGMP intermediate, we performed the experiment
shown in Fig. 5. As shown in lane 2, Panels A and B, the incubation of CobU with
[
-
P]GTP resulted in substantial
radioactivity associated with the CobU protein band. Incubation of CobU
with [
P]GTP labeled in the
-position
instead of the
-position resulted in no radiolabel associated with
CobU (lane 1, Panels A and B). Lanes
3-5, Panels A and B, demonstrated that
both CobU and [
-
P]GTP were required for the
formation of the radiolabeled protein band.
Figure 5:
Evidence for the formation of a
CobUGMP intermediate. Panel A, the PhosphorImager
analysis of the polyacrylamide gel shown in Panel B. The
experiment was performed as follows. The indicated additions were
incubated in Tris-HCl (0.1 M, pH 8.8, at 37 °C) plus 2.5
mM MgCl
in a final volume of 10 µl. After a
5-min incubation at 37 °C, 2 µl of 6
SDS-PAGE loading
buffer were added, and the sample was heated to 80 °C for 10 min
and immediately analyzed by SDS-PAGE (12%). The amount of each addition
was: CobU, 2.5 nmol; [
-
P]GTP, 25 pmol
(specific radioactivity = 800 Ci mmol
); and
[
-
P]GTP, 25 pmol (specific radioactivity
= 30 Ci mmol
). Panel B, the reaction
mixtures analyzed by SDS-PAGE (12%) and stained with Coomassie Blue.
Size markers are shown at the left and labeled with their
molecular mass (kDa).
Inclusion of a 100-fold molar excess of unlabeled GTP (Fig. 5, Panel A, lane 6) resulted in a 30-fold decrease in label incorporated into the CobU protein when compared to lane 2. The inclusion of a 100-fold excess of unlabeled ATP resulted in only a 0.4-fold decrease in label incorporated into the CobU protein. As described above, both ATP and GTP can serve as substrates for the kinase activity, but only GTP is an efficient substrate for the guanylyltransferase activity. The ability of GTP (but not ATP) to compete with the radiolabeled GTP suggested that the association of radiolabel with CobU occurred at a site required specifically for GTP binding and presumably for the guanylyl transfer reaction.
Based on
the data presented in this section, it is formally possible that CobU
forms an enzyme-GDP intermediate. Two lines of evidence argue against
such a conclusion. The cyano-derivative of product of the
CobU-catalyzed reaction has a molecular mass identical to
(CN)CBI-GDP. Further, the phosphate moiety of Ado-CBI-P was
retained in Ado-CBI-GDP, suggesting that a GMP moiety (not a GDP
moiety) was transferred to Ado-CBI-P to generate Ado-CBI-GDP. Taken
together, these data indicated that the GMP moiety of GTP, not the GDP
moiety, was transferred to the CobU enzyme.
The data presented above indicated that GMP was associated with the CobU protein. Further, the label associated with CobU was retained after treatment of the protein with SDS, heating to 80 °C, and analysis by SDS-PAGE. These observations indicated that GMP had formed a stable covalent bond with the CobU protein. This point is addressed in more detail in the next section.
Fig. 6, lanes 1 and 2, shows that a 10-min incubation at 55 °C (with no addition of acid or base) resulted in CobU protein which retained the radiolabeled GMP. Treatment with acid (pH of 1.5) at 55 °C for 10 min resulted in a 38-fold decrease in radiolabel associated with the CobU protein (lanes 3 and 4). The CobU protein, however, was still visible on Coomassie Blue-stained polyacrylamide gels at levels comparable to the control lanes (data not shown). Treatment with base (pH of 13.0) resulted in no loss of radiolabel (lanes 5 and 6).
Figure 6:
Stability of the CobUGMP intermediate
to acid and alkali treatment. Shown is the PhosphorImager analysis of
samples analyzed by SDS-PAGE (12%). Lanes 1 and 2 contain the H
O-treated control reaction mixture. Lanes 3 and 4 contain the acid-treated samples. Lanes 5 and 6 contain the alkali-treated samples. The
position of CobU is identified by the arrow. The reactions
were performed as follows. Radiolabeled CobU
GMP intermediate was
generated as described in Fig. 5, except after 5 min at 37
°C, 2.5 µL of H
O, 4 M NaOH, or 4 M citric acid were added to the indicated reactions. After
incubation for 10 min at 55 °C, 2.5 µl of H
O, 4 M HCl, or 4 M NaOH, respectively, were added to
neutralize the reactions. The samples were analyzed by SDS-PAGE (12%),
followed by analysis with the
PhosphorImager.
These data showed that the
CobUGMP intermediate was sensitive to treatment with acid, but not
to treatment with base. These data argue against a phosphoester bond,
which should be sensitive to both acidic and alkali conditions. This
behavior was consistent with a phosphoramidate linkage.
To address this point, we performed the
experiment described in Table 5. Briefly, the CobUGMP
intermediate was generated and dialyzed against buffer to remove
unreacted [
-
P]GTP. As shown in Table 5, the purified CobU
GMP intermediate was capable of
driving Ado-CBI-GDP synthesis only if provided with Ado-CBI-P, or
Ado-CBI and ATP. These results allowed two conclusions. First, the
CobU
GMP intermediate was functional for the synthesis of
Ado-CBI-GDP. Second, the CobU
GMP intermediate was competent for
the synthesis of Ado-CBI-P from Ado-CBI and ATP. From this second
observation we inferred that the two activities of the protein function
independently, a result consistent with genetic experiments. (
)
A ratio of 1.56 ± 0.27 mol of GMP per mol of CobU dimer was calculated based on results from four trials. These data suggested that each CobU monomer bound a GMP moiety and, based on the results in the preceding section, can subsequently catalyze a guanylyl transfer. Based on this result, we propose that both monomers of the CobU dimer are functionally equivalent and can likewise catalyze the kinase reaction.
Thiol-reducing agents have been reported to react and
form complexes with corrinoids(27) . Therefore, it was not
clear whether DTT affected the protein, the corrinoid substrate, or
both. To address this concern we performed the following experiment. A
sample of purified CobU was stored at 4 °C in air for approximately
2 weeks. The kinase and guanylyltransferase activities of this sample
decreased to <1% of their original activity (the specific activity
for the kinase reaction decreased from 83 to 0.7, and for the
guanylyltransferase reaction the specific activity decreased from 76 to
0.4). This form of CobU enzyme was referred to as ``oxidized
CobU.'' Enzyme stored at 4 °C under a N headspace
retained >98% of its activity for at least 3 weeks (data not shown).
DTT (10 mM) was added to one half of the oxidized CobU
sample and incubated on ice for 2 h; an equal volume of HO
was added to the other half of the sample. The DTT-treated sample (0.5
ml) was dialyzed against 2
1 liter of anoxic Tris-HCl, 0.1 M, pH 8.0 at 4 °C, to remove the DTT (this represents a 4
10
-fold dilution). The untreated sample was
dialyzed against the same type and volume of oxic buffer. DTT treatment
of oxidized CobU resulted in an 18-fold increase in kinase activity
(specific activity increased from 0.7 to 12.6), and a 42-fold increase
in guanylyltransferase activity (specific activity increased from 0.4
to 17.0). The activities of the DTT-treated CobU represent 15-20%
of the original specific activities. Untreated CobU showed no change in
either activity. Addition of DTT to a reaction mixture containing
reduced CobU enzyme did not further stimulate activity, suggesting that
the effect of DTT was due to the reduction of disulfide bonds in the
enzyme (data not shown).
We quantitated the total moles of sulfhydryl groups in reduced and oxidized CobU by the method of Habeeb(28) . DTT-treated CobU had 4 mol of reduced sulfhydryl groups per monomer of CobU, a result consistent with the 4 cysteinyl residues per CobU polypeptide. Untreated CobU showed <1 mol of reduced sulfhydryl group per monomer of CobU. Therefore, the presence of reduced sulfhydryl groups correlated with active kinase and guanylyltransferase functions of CobU.
An analysis of available sulfhydryl groups in the native CobU protein with DTNB was performed as reported elsewhere(28) . We observed a single class of sulfhydryl residues, which were fully modified with DTNB (at 0.33 and 1 mM) in less than 2 min (assays were monitored for a total of 30 min). We determined a value of 4 available sulfhydryl groups per dimer of CobU; each CobU dimer has 8 cysteinyl residues. The rapid modification of DTNB by CobU suggests that the sulfhydryl residues are readily accessible to DTNB. In fact, the rate of modification of CobU by DTNB is similar to the rate reported for small molecules such as cysteine(28) .
We tested whether the
sulfhydryl modifying agents DTNB and NEM inhibited CobU kinase and
guanylyltransferase activities. CobU was incubated at 37 °C in the
presence of 1 mM DTNB and NEM for 10 min, then assayed
immediately for its kinase and guanylyltransferase activities. The
kinase activity of CobU (original specific activity = 231) was
inhibited 95% by treatment with 1 mM DTNB (specific activity
= 11) and inhibited 94% by treatment with 1 mM NEM
(specific activity = 14). The guanylyltransferase activity
(original specific activity = 93) was inhibited 92% (specific
activity = 8) and 87% (specific activity = 13) by
treatment with DTNB and NEM, respectively. The buffer used to prepare
the DTNB and NEM solutions (0.1 M NaP, pH 8.0) had
no effect on either CobU activity (data not shown). Taken together with
the study above, these data suggested that one or more of the rapidly
modified sulfhydryl groups is required for CobU kinase and
guanylyltransferase activities.
Ado-CBI and ATP (for the kinase activity) and Ado-CBI-P (for the guanylyltransferase activity) afforded no protection from inhibition by DTNB. After incubation with substrate and inhibitor, unreacted compounds were separated from CobU on a desalting column of Sephadex G-25. CobU was assayed immediately for the appropriate activity. In all cases, the activity of CobU treated with DTNB in the presence of substrate was no higher than CobU treated with DTNB in the absence of substrate (data not shown). Untreated CobU served as a positive control and retained both activities after the 20 min of incubation at 37 °C and passage over the sizing column (data not shown).
GTP provided slight protection from inhibition of the
guanylyltransferase activity. CobU was incubated with 150 µM GTP for 10 min to allow formation of the CobUGMP
intermediate, then treated with DTNB as above. Dialysis against 2
1 ml of 0.1 M Tris-HCl, pH 8.0 at 4 °C, in a
Centricon-10 concentrator removed the unreacted GTP and DTNB, but the
CobU
GMP intermediate is stable under these conditions (see section
on CobU
GMP intermediate formation above). GTP-treated CobU
(specific activity = 1.25 nmol of Ado-CBI-GDP/min/mg) had 3-fold
more activity than the enzyme treated with DTNB in the absence of GTP
(0.34 nmol/min/mg). However, control enzyme not treated with DTNB had a
specific activity of 50.4 nmol/min/mg. Therefore, even in the presence
of GTP, DTNB inhibited the guanylyltransferase activity >95%. This
GTP-protected CobU had no detectable kinase activity (data not shown).
The activity of DTNB-modified CobU could be partially restored by
treatment with DTT. CobU was treated with DTNB and dialyzed as
described above. The enzyme was subsequently incubated with 10 mM DTT at 37 °C for 15 min, then immediately assayed for both
kinase and guanylyltransferase activity. The kinase activity was
stimulated 4-fold by treatment with DTT (specific activity
increased from 1.0 to 3.8 nmol of Ado-CBI-P/min/mg). The
guanylyltransferase activity was stimulated 6-fold by treatment with
DTT (specific activity increased from 1.1 to 6.4 nmol of
Ado-CBI-GDP/min/mg). However, this DTT-mediated increase results in a
specific activity
10-fold below that of untreated control enzyme
(specific activity = 50.4). There was no increase in either
activity when the time of DTT treatment was increased to 60 min (data
not shown).
At present, it is not clear whether cysteinyl residues are required for one or both CobU activities or, if so, how many cysteinyl residues are involved. It is possible that a modified cysteinyl residue is required for only one activity, and the loss of the second activity is simply the consequence of an indirect effect(s) of the modifying agents. Site-directed mutagenesis of each cysteinyl residue is in progress to evaluate the involvement of each of these residues in CobU activities.
Control assays with the ATP:corrinoid adenosyltransferase enzyme
(CobA), resulted in the synthesis of Ado-CBI from co(I)binamide and ATP (12) . The synthesis of Ado-CBI was monitored by two methods.
In a spectrophotometric assay, the synthesis of Ado-CBI was monitored
at 457 nm(12) . In a bioassay, Ado-CBI (but not nonadenosylated
CBI) restored growth of a cobA mutant on minimal
medium(29) . This bioassay is capable of detecting picomolar
quantities of corrinoids. ()
When homogeneous CobU (20 µg per assay) was substituted for CobA in the assay, we were unable to detect Ado-CBI synthesis. We concluded that CobU did not have ATP:CBI adenosyltransferase activity in vitro. Alternative models to explain the role of CobU in the assimilation of nonadenosylated corrinoids under anaerobic conditions should focus on the specificity of the enzyme for its corrinoid substrate as a function of oxygen in the environment.
The kinase activity of CobU catalyzes the phosphorylation
of Ado-CBI at the expense of ATP to yield Ado-CBI-P. The
-phosphate of ATP is donated to Ado-CBI in this reaction, and
remains a part of the product of the CobU-catalyzed reaction,
Ado-CBI-GDP. In S. typhimurium, the
-phosphate of ATP
probably remains as part of the end product of the pathway,
Ado-cobalamin(1, 2) .
The second CobU activity catalyzes the transfer of the guanylyl moiety of GTP to Ado-CBI-P. We presented evidence that the transfer of the guanylyl moiety occurs via an enzyme-guanylyl intermediate (Fig. 5). This intermediate is acid-labile and alkali-resistant (Fig. 6). The properties of the enzyme-guanylyl intermediate are reminiscent of the phosphoramidate linkages observed in galactose-1-phosphate uridylyltransferase (23, 24, 25) and histidine kinases such as FixL(26) .
Release of the GMP moiety from the CobUGMP
intermediate is driven by the presence of Ado-CBI-P (provided
exogenously or generated in situ), but not by Ado-CBI. This
finding is consistent with the identification of Ado-CBI-P as an
intermediate in the synthesis of Ado-CBI-GDP and supports the sequence
of reactions as phosphorylation followed by guanylyl transfer.
Two additional lines of evidence support the bifunctionality of CobU. Overexpression of the CobU polypeptide (Fig. 3, Panel A) results in the overexpression of both kinase and guanylyltransferase activities (Fig. 4) and both enzymatic activities copurified through three chromatography steps to >94% homogeneity (Table 2).
Comparison of the CobU amino acid sequence to its homolog CobP in P. denitrificans showed that cysteine at position 81 in CobU is conserved between these two proteins, suggesting an important role of this residue in enzyme activity(ies). This idea has been verified by genetic means. Site-directed mutagenesis of CobU at this position was performed to change the cysteine to alanine (C81A). The C81A allele of cobU resulted in loss of CobU function in vivo and in vitro (data to be presented elsewhere).
Although CobU has 4 cysteinyl residues, CobP has only the single conserved cysteinyl residue and the CobP enzyme does not appear to require reducing agents to maintain its activities(3) . Interestingly, Wong et al.(24) reported that galactose-1-phosphate uridylyltransferase also had an essential sulfhydryl group, probably located outside of the active site of the enzyme. Studies on the involvement of the remaining cysteinyl residues in CobU enzyme activity(ies) is in progress.
An alternative model proposed that CobU could utilize nonadenosylated corrinoids as substrates in the absence of oxygen. Our in vitro data show that CobU can utilize HO-CBI, although Ado-CBI is clearly the preferred substrate. Therefore, it appears that CobU can catalyze the synthesis of CBI-GDP from non-adenosylated CBI, albeit inefficiently. This low level of CBI-GDP synthesis, however, appears to be sufficient to satisfy the cell's requirement for cobalamin under anaerobic growth conditions(6) . Structural work on CobU (currently in progress) will prove valuable in understanding how corrinoid substrate specificity is established by the primary structure of this protein.