Novel coenzyme B12-dependent interconversion of isovaleryl-CoA and pivalyl-CoA

Article abstract of DOI:10.1074/jbc.M111.320051

5′-Deoxyadenosylcobalamin (AdoCbl)-dependent isomerases catalyze carbon skeleton rearrangements using radical chemistry. We have recently characterized a fusion protein that comprises the two subunits of the AdoCbl-dependent isobutyryl- CoA mutase flanking a G-protein chaperone and named it isobutyryl-CoA mutase fused (IcmF). IcmF catalyzes the interconversion of isobutyryl-CoA and n-butyryl-CoA, whereas GTPase activity is associated with its G-protein domain. In this study, we report a novel activity associated with IcmF, i.e. the interconversion of isovaleryl-CoA and pivalyl-CoA. Kinetic characterization of IcmF yielded the following values: a Km for isovaleryl-CoA of 62 ± 8 μM and V_max of 0.021 ± 0.004 μmol min^-1 mg^-1 at 37 ° C. Biochemical experiments show that an IcmF in which the base specificity loop motif NKXD is modified to NKXE catalyzes the hydrolysis of both GTP and ATP. IcmF is susceptible to rapid inactivation during turnover, and GTP conferred modest protection during utilization of isovaleryl-CoA as substrate. Interestingly, there was no protection from inactivation when either isobutyryl-CoA or n-butyryl-CoA was used as substrate. Detailed kinetic analysis indicated that inactivation is associated with loss of the 5′-deoxyadenosine moiety from the active site, precluding reformation of AdoCbl at the end of the turnover cycle. Under aerobic conditions, oxidation of the cob(II)alamin radical in the inactive enzyme results in accumulation of aquacobalamin. Because pivalic acid found in sludge can be used as a carbon source by some bacteria and isovaleryl- CoA is an intermediate in leucine catabolism, our discovery of a new isomerase activity associated with IcmF expands its metabolic potential.

Full text of DOI:10.1074/jbc.M111.320051

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 287, NO. 6, pp. 3723–3732, February 3, 2012
© 2012 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A.
Novel Coenzyme B12-dependent Interconversion of
Isovaleryl-CoA and Pivalyl-CoA*
Received for publication, November 2, 2011, and in revised form, December 9, 2011 Published, JBC Papers in Press, December 13, 2011, DOI 10.1074/jbc.M111.320051
Valentin Cracan and Ruma Banerjee1
From the Department of Biological Chemistry, University of Michigan, Ann Arbor, Michigan 48109-0600
Background: IcmF is a fusion between a coenzyme B12-dependent isobutyryl-CoA/n-butyryl-CoA isomerase and a G-pro-
tein chaperone.
Results: IcmF also isomerizes isovaleryl-CoA to pivalyl-CoA and is partially protected from inactivation in the presence of GTP.
Conclusion: The isovaleryl-CoA mutase activity of IcmF might be important in leucine catabolism where isovaleric acid is an
intermediate.
Significance: IcmF might be critical for microbial bioremediation of the anthropogenic compound pivalic acid.
5؅-Deoxyadenosylcobalamin (AdoCbl)-dependent isomer-
ases catalyze carbon skeleton rearrangements using radical
chemistry. We have recently characterized a fusion protein that
comprises the two subunits of the AdoCbl-dependent isobu-
tyryl-CoA mutase flanking a G-protein chaperone and named it
isobutyryl-CoA mutase fused (IcmF). IcmF catalyzes the inter-
conversion of isobutyryl-CoA and n-butyryl-CoA, whereas
GTPase activity is associated with its G-protein domain. In this
study, we report a novel activity associated with IcmF, i.e. the
interconversion of isovaleryl-CoA and pivalyl-CoA. Kinetic
characterization of IcmF yielded the following values: a Km for
isovaleryl-CoA of 62 ؎ 8 ␮M and Vmax of 0.021 ؎ 0.004 ␮mol
min (AdoCbl)-dependent isomerases that catalyze 1,2 rear-
rangements of methylmalonyl-CoA to succinyl-CoA and
isobutyryl-CoA to n-butyryl-CoA, respectively (Fig. 1) (1, 2).
MCMs are widely distributed in nature, ranging from bacteria
and Archaea to animals, including humans. ICMs were initially
believed to be restricted to the genus Streptomyces, which
belongs to the Actinobacteria phylum (1, 2). With our discovery
of IcmF, the fusion protein between ICM and its G-protein
chaperone, the known distribution of ICM has expanded to
include four more phyla: Proteobacteria, Bacteroidetes, Firmi-
cutes, and Spirochaetes (3). The only known function of ICM in
the genus Streptomyces is its participation in polyketide biosyn-
thesis (4). In contrast, the relatively wide distribution of IcmF in
diverse organisms points to a broader range of roles in bacterial
metabolism that remain to be elucidated.
؊1
؊1
min mg at 37 °C. Biochemical experiments show that an
IcmF in which the base specificity loop motif NKXD is modified
to NKXE catalyzes the hydrolysis of both GTP and ATP. IcmF is
susceptible to rapid inactivation during turnover, and GTP con-
ferred modest protection during utilization of isovaleryl-CoA as
substrate. Interestingly, there was no protection from inactiva-
tion when either isobutyryl-CoA or n-butyryl-CoA was used as
substrate. Detailed kinetic analysis indicated that inactivation is
associated with loss of the 5؅-deoxyadenosine moiety from the
active site, precluding reformation of AdoCbl at the end of
the turnover cycle. Under aerobic conditions, oxidation of the
cob(II)alamin radical in the inactive enzyme results in accumu-
lation of aquacobalamin. Because pivalic acid found in sludge
can be used as a carbon source by some bacteria and isovaleryl-
CoA is an intermediate in leucine catabolism, our discovery of a
new isomerase activity associated with IcmF expands its meta-
bolic potential.
A family of enzymes that are similar in their primary
sequence to MCM catalyzes AdoCbl-dependent carbon skele-
ton rearrangements and include, in addition to ICM, 2-hy-
droxyisobutyryl-CoA mutase (HCM) (5) and ethylmalonyl-
CoA mutase (ECM) (6) (Fig. 1). The three-dimensional
structure of these proteins is expected to resemble the overall
structure of MCM (7, 8). The B12-binding domain of MCM
exhibits a typical Rossman-like fold, and the AdoCbl cofactor is
bound in a “base-off/His-on” conformation (9). The substrate-
binding domain is a triose-phosphate isomerase barrel com-
prising a core of eight (␣␤)-repeats (8). A limited number of key
amino acid substitutions in the active site distinguish these
closely related enzymes and accommodate chemical differ-
ences in the substrate (6, 10). Previous studies from our labora-
tory had pointed out that the two key substitutions that accom-
modate the switch from a carboxylate to a methyl group in the
substrates of MCM versus ICM are Tyr 3 Phe and Arg 3 Gln,
respectively (Figs. 2 and 3) (3). The Phe and Arg substitutions
are conserved in all ICM/IcmFs (Figs. 2 and 3). In contrast, in
HCM, the corresponding amino acids that interact with sub-
strate are Ile and Gln, respectively. Thus, although the Gln res-
Methylmalonyl-CoA mutase (MCM)2 and isobutyryl-CoA
mutase (ICM) are two closely related 5Ј-deoxyadenosylcobala-
* Thisworkwassupported, inwholeorinpart, byNationalInstitutesofHealth
Grant DK45776.
1 To whom correspondence should be addressed: Dept. of Biological Chem-
istry, University of Michigan, 3220B MSRB III, 1150 W. Medical Center Dr.,
Ann Arbor, MI 48109-0600. Tel.: 734-615-5238; E-mail: rbanerje@umich.
edu.
isobutyryl-CoA mutase; AMPPNP, adenosine 5Ј-(␤,␥-imido)triphosphate;
LIC, ligase-independent cloning; OH2Cbl, aquacobalamin; HCM, 2-hy-
droxyisobutyryl-CoA mutase; ECM, ethylmalonyl-CoA mutase; Gk, G. kaus-
tophilus; Cm, C. metallidurans.
2 The abbreviations used are: MCM, methylmalonyl-CoA mutase; AdoCbl,
5Ј-deoxyadenosylcobalamin; IcmF, isobutyryl-CoA mutase fused; ICM,
FEBRUARY 3, 2012•VOLUME 287•NUMBER 6
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IcmF Is a Pivalyl-CoA Mutase
MCM are replaced by Gly and Pro, respectively (6) (Fig. 3).
Notably, the Tyr and Arg residues in the active site of MCM
that interact with the carboxylate moiety of the substrate are
also conserved in ECM (Fig. 3).
In B12-dependent isomerases, AdoCbl serves as a radical res-
ervoir, and a common first step that initiates the isomerization
reactions is homolytic cleavage of the cobalt-carbon bond (Fig.
4), leading to formation of the 5Ј-deoxyadenosyl radical and a
paramagnetic cob(II)alamin species (1, 11). Inadvertent side
reactions of the reactive radical intermediates render AdoCbl-
dependent enzymes susceptible to inactivation (12). Alterna-
tively, inactivation can result from escape of the 5Ј-deoxyade-
nosine intermediate during the catalytic cycle (13). In both
cases, inactivation results from the inability to reform AdoCbl
from the 5Ј-deoxyadenosyl and cob(II)alamin radicals at the
end of the turnover cycle. MeaB, the G-protein chaperone of
MCM, protects against inactivation in the presence of GTP (13,
14). A similar role for the homologous G-protein chaperone of
ICM, MeaI, has not been studied. In IcmF, the MeaI domain is
sandwiched between the AdoCbl- and substrate-binding
domains (3). In this study, we report a novel AdoCbl-dependent
1,2 rearrangement reaction catalyzed by IcmF and demonstrate
that in the presence of GTP the isovaleryl-CoA mutase activity
of IcmF is partially protected from the inactivation.
EXPERIMENTAL PROCEDURES
FIGURE 1. Reactions catalyzed by MCM, ICM, IcmF, HCM, and ECM.
Materials
AdoCbl, ADP, AMPPNP, ATP, GDP, GTP, isobutyryl-CoA,
n-butyryl-CoA, isovaleryl-CoA, DL-3-hydroxybutyryl-CoA,
and pivalic acid were purchased from Sigma. Tris(2-carboxy-
ethyl)phosphine hydrochloride was from GoldBio (St. Louis,
MO). Valeryl-CoA was purchased from Crystal Chem
(Downers Grove, IL). A pET28a vector expressing IcmF from
Cupriavidus metallidurans was a generous gift from the lab-
oratory of Dr. Catherine Drennan (Massachusetts Institute
of Technology).
DNA Manipulations
Cloning of IcmF—In a previous study, we used IcmF from
Geobacillus kaustophilus cloned into pET30 Ek/LIC expression
vector (3). The S-tag, which is located just downstream of the
N-terminal His tag in pET30 Ek/LIC, was removed by subclon-
ing the full-length IcmF DNA into the ligation-independent
cloning vector pMCSG7 (15). The insert was amplified with the
following primers: forward, 5Ј-TACTTCCAATCCAATGCC-
ATGGCGCACATTTACCGTCC-3Ј; and reverse, 5Ј-TTATC-
CACTTCCAATGCTATTACATATTCGCCGGTATTG-
TCC-3Ј. In pMCSG7, which is a derivative of the pET21 vector,
the N-terminal His tag can be cleaved using tobacco etch virus
protease. The His tag was not removed in this study because its
FIGURE 2. Comparison of active site residues in related AdoCbl-depen-
dentmutases. TheMCMstructurefromPropionibacteriumshermanii(Protein
Data Bank code 4REQ) was used as a template to show that Tyr and Arg in
MCM/ECM correspond to Phe and Gln in ICM/IcmF. In HCM, this pair of resi-
dues is Ile and Gln.
idue is conserved in both ICM/IcmF and HCM, the Phe is sub- presence does not affect IcmF activity.
stituted by Ile to accommodate the bulkier 2-hydroxy-
isobutyryl-CoA substrate (Figs. 2 and 3).
Construction of K213A Mutant of IcmF from G. kaustophilus
(Gk)—The K213A mutation was created using the QuikChange
In contrast to ICM, ECM, which catalyzes the interconver- XL site-directed mutagenesis kit (Agilent) and the following
sion of ethylmalonyl-CoA and methylsuccinyl-CoA, is very sense primer: 5Ј-CCGGCACAGGCGGAGCTGGGGCAAGC-
similar to MCM (6). It has been proposed that two key substi- TCGCTCACCGATG-3Ј. The sequence of the reverse primer
tutions in the active site of ECM dictate specificity for the bulk- was complementary to the sequence of the forward primer. The
ier ethylmalonyl-CoA substrate: a His and an Asn residue in Gk IcmF cloned in pMCSG7 was used as a template. All con-
3724 JOURNAL OF BIOLOGICAL CHEMISTRY
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IcmF Is a Pivalyl-CoA Mutase
FIGURE 3. Multiple sequence alignment of substrate-binding domains of different AdoCbl-dependent mutases. IcmF from G. kaustophilus (YP_149244),
ICM from S. cinnamonensis (AAC08713), MCM from Methylobacterium extorquens (YP_001642233), MeaA from M. extorquens (YP_002961419), ECM from
Rhodobacter sphaeroides (YP_354045), and HCM from M. petroleiphilum (YP_001023546) are shown. Four residues recognized to be important for substrate
binding are highlighted in gray and indicated with asterisks. Two residues, Phe and Gln, found in ICM and IcmF are substituted by Tyr and Arg in MCM and ECM
or Ile and Gln in HCM. ECM is different from other acyl-CoA mutases by the substitution of His and Asp to Gly and Pro, respectively. ECM was previously known
as MeaA in M. extorquens. All accession numbers are from NCBI Proteins database.
ATPase/GTPase Assays
NTPase activity of IcmF was measured by HPLC as described
previously (3) or by a modification of the malachite green assay
involving inclusion of citrate in the reaction mixture (16).
Briefly, the color reagent was prepared by mixing 4.2% ammo-
nium molybdate in 4 N HCl with 0.045% malachite green hydro-
chloride (1:3, v/v). The resulting solution was shaken for 30 min
at 250 rpm in a Falcon tube and filtered through a 0.2-␮m Mil-
lipore filter. After that, 10% Tween 20 solution (v/v) was added
(200 ␮l for every 100 ml of color reagent). This solution was
stable for a week at 4 °C with only a minor increase in the back-
FIGURE 4. Generalized scheme for 1,2 rearrangement reaction catalyzed
by AdoCbl-dependent mutases. The binding of substrate to the enzyme
ground absorbance at 650 nm. The reaction was performed in
induceshomolyticcleavageofthecobalt-carbonbondofAdoCbl, resultingin
Buffer A in a total volume of 0.6–1 ml containing 0.5–2.5 ␮M
formation of a pair of radicals, the 5Ј-deoxyadenosyl radical and cob(II)ala-
IcmF or 10–20 ␮M K213A IcmF, 10 mM MgCl2, and nucleotides
min. The 5Ј-deoxyadenosyl radical abstracts a hydrogen atom from the
substrate to form 5Ј-deoxyadenosine and a substrate radical, which subse-
quently undergoes isomerization to the product radical. The latter re-
abstracts a hydrogen atom from the methyl group of 5Ј-deoxyadenosine to
form the product and regenerates the 5Ј-deoxyadenosyl radical. Finally,
cob(II)alamin and the 5Ј-deoxyadenosyl radicals recombine to give AdoCbl.
(0.02–1.2 mM GTP or 0.02–6 mM ATP). At the desired time
points, 200-␮l aliquots were removed, and the reaction was
quenched with 20 ␮l of 2 M trichloroacetic acid (TCA). After
centrifugation, 150 ␮l of supernatant was added to 750 ␮l of
color reagent. After 3 min, 100 ␮l of 34% sodium citrate was
structs were confirmed by nucleotide sequence determination
added to the sample. The color was allowed to develop for 30
at The University of Michigan DNA Sequencing Core.
min at room temperature, and the absorbance was measured at
650 nm. The calibration curve for phosphate was prepared
Protein Expression and Purification
using a 5–200 ␮M solution of monobasic potassium phosphate,
Purification of Gk Wild Type and K213A IcmF—Escherichia
which was dried in an oven (to remove traces of moisture) for
coli BL21(DE3) cells containing plasmids expressing the IcmF
4 h at 120 °C.
constructs were grown at 37 °C in Luria-Bertani (LB) medium
supplemented with 100 ␮g/ml ampicillin to an absorbance at
IcmF Assay
600 nm of 0.5–0.6. Cells were grown for 12–14 h at 15 °C after
A GC-based assay was used to measure both ICM activity
and the new, isovaleryl-CoA/pivalyl-CoA mutase activity (3). In
all assays, apoenzyme was preincubated with AdoCbl Ϯ nucle-
otides, and the reaction was started by addition of substrate.
Pivalyl-CoA Mutase Activity—The reaction was performed
in Buffer A in a total volume of 0.8–1.4 ml containing 600–
2500 ␮g of Gk IcmF or Cm IcmF, 100 ␮M AdoCbl, 20–2000 ␮M
isovaleryl-CoA, and 15 mM MgCl2 Ϯ 5 mM GTP. For every
concentration of substrate, two to six aliquots (200 ␮l each)
were removed and treated as described below.
induction with 0.5 mM isopropyl 1-thio-␤-D-galactopyranoside.
Proteins were purified as described previously for wild-type
IcmF (3) with the following difference. Gel filtration chroma-
tography was performed with 50 mM HEPES, pH 7.5 containing
100 mM NaCl (Buffer A).
Purification of C. metallidurans (Cm) IcmF—E. coli BL21(DE3)
cells containing plasmid expressing IcmF were grown in LB
medium supplemented with 50 ␮g/ml kanamycin and induced
at 15 °C with 0.1 mM isopropyl 1-thio-␤-D-galactopyranoside.
Cells were harvested 12–14 h after induction. Cm IcmF was
purified as described previously for Gk IcmF (3). Gel filtration
Isobutyryl-CoA Mutase Activity—The reaction was per-
chromatography was also performed on this enzyme using formed in Buffer A in a total volume of 0.8–1.4 ml containing
Buffer A.
10–60 ␮g of Gk IcmF or Cm IcmF, 100 ␮M AdoCbl, a saturating
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IcmF Is a Pivalyl-CoA Mutase
concentration of substrates (600–2000 ␮M isobutyryl-CoA or enzyme-bound aquacobalamin (OH2Cbl) as indicated by the
n-butyryl-CoA), and 10 mM MgCl2 Ϯ 3–6 mM GTP or ATP. At
various time points (0.5–30 min), 200-␮l aliquots were
removed and quenched with 100 ␮l of 2 N KOH containing 0.18
mM valeric acid used as an internal standard. Following addi-
tion of 100 ␮l of H2SO4 (15%, v/v), the reaction mixture was
saturated with NaCl and extracted with ethyl acetate (250 ␮l).
The extract was analyzed directly by GC using a DB-FFAP (30-
m ϫ 0.25-mm-inner diameter, 0.25-␮m) capillary column
(Agilent). A 5-␮l sample was injected in the pulsed splitless
mode. The oven temperature was initially at 80 °C. Following
sample injection, the temperature was raised to 150 °C at a rate
of 10 °C/min and maintained at 150 °C for 2 min. Retention
times for the compounds of interest was as follows: isobutyric
acid, 5.85 min; pivalic acid, 5.99 min; n-butyric acid, 6.5 min;
isovaleric acid, 6.96 min; and valeric acid, 7.78 min.
appearance of a 350 nm absorption peak. The increase at 350
nm was fitted to a single exponential function, A ϭ A0 ϩ A1(1 Ϫ
e
Ϫbt) where A is the absorbance at 350 nm, b is the observed rate
constant for inactivation, A0 is the initial absorbance at 350 nm,
t is time in minutes, and A1 is the amplitude.
Enzyme-monitored Turnover of Gk IcmF under Anaerobic
Conditions
To assess the effect of oxygen on enzyme inactivation,
enzyme-monitored turnover experiments were performed
under anaerobic conditions. For this, Buffer A containing 10
mM MgCl2 was bubbled with N2 for 3 h before use and intro-
duced into an anaerobic chamber (containing Ͻ0.3 ppm O2).
Stock solutions of AdoCbl, n-butyryl-CoA, isobutyryl-CoA,
and isovaleryl-CoA were prepared in the chamber using anaer-
obic buffer. The enzyme solution was deoxygenated by blowing
a stream of N2 over its surface at 4 °C for 40 min. UV-visible
spectra were collected using a Mikropack DH-2000 UV-visible
light source connected with fiber optics to a cuvette holder
inside the glove box.
IcmF Assays with Alternative Substrates
We used an HPLC-based assay to evaluate whether 3-hy-
droxybutyryl-CoA can be converted by IcmF to 2-hydroxy-
isobutyryl-CoA. The assay mixture contained in a total volume
of 0.5 ml Buffer A, 10 mM MgCl2, 100 ␮M AdoCbl, 2 mM GTP,
0.4–1 mM DL-␤-hydroxybutyryl-CoA, and 0.2–0.4 mg Gk
IcmF. The reaction was initiated by addition of enzyme, and the
reaction mixture was incubated at 37 °C. At different time
points (0.5–10 min), 60-␮l aliquots were removed, quenched
with 2 M TCA (10%, v/v), centrifuged, and subjected to HPLC
analysis.
HPLC Characterization of Inactivation Products
A solution containing 64 ␮M Gk holo-IcmF (containing 2 eq
of AdoCbl) in Buffer A and 10 mM MgCl2 Ϯ 5 mM GTP was
incubated with 1 mM isobutyryl-CoA at 37 °C in the dark. At
various times (0–60 min), 15-␮l aliquots were removed and
immediately quenched with 60 ␮l of 0.5% trifluoroacetic acid
(TFA). The aerobic inactivation products of AdoCbl, 5Ј-deoxy-
adenosine and OH2Cbl, were monitored by HPLC using an All-
tima HP 5-␮m C18 (250 ϫ 4.6-mm) column (Grace). All steps of
sample preparation and HPLC analysis were performed in the
dark. Initial buffer conditions were 92% Solvent C (0.1% TFA in
water) and 8% Solvent D (0.1% TFA in acetonitrile) at a flow rate
of 1.0 ml/min. Between 10 and 35 min, Solvent D was increased
to 32%. Between 35 and 36 min, Buffer D was decreased to 8%
and held for 5 min at that composition to equilibrate the col-
umn between injections. 50 ␮l of the sample was injected, and
elution was monitored at 254 and 350 nm. Under these condi-
tions, the following retention times were obtained: 6.92 min for
5Ј-deoxyadenosine, 22.77 min for OH2Cbl, and 29.27 min for
AdoCbl. The control reaction was performed in the absence of
isobutyryl-CoA. Calibration curves were generated for all three
compounds prepared and treated similarly to the assay sam-
ples. An extinction coefficient of ⑀260 nm ϭ 15 mMϪ1 cmϪ1 was
used to estimate the concentration of 5Ј-deoxyadenosine (17).
The data were well fitted by a single exponential function for
the disappearance of AdoCbl and appearance of 5Ј-deoxyade-
nosine and OH2Cbl. To improve recovery of OH2Cbl, protein-
ase K (Roche Applied Science) was used to digest the protein
sample for 1–2 h at 37 °C before precipitation with acid.
The acyl-CoA esters were separated using an HPLC system
equipped with an Alltima HP 5-␮m C18 (250 ϫ 4.6-mm) col-
umn (Grace). The detector was set at 254 nm. Solvent A was 50
mM monobasic potassium phosphate, pH 5.4. Solvent B was
prepared as follows. 500 ml of MeOH was added to 500 ml of
100 mM monobasic potassium phosphate, pH 5.4 (to give a final
concentration of 50 mM potassium phosphate and 50% MeOH).
Initial conditions used for separation were 10% solvent B and a
flow rate of 1.0 ml/min. Between 5 and 30 min, solvent B was
increased to 100% and then held at 100% solvent B for 5 min. At
36 min, solvent B was decreased to 10% and held for 10 min at
that composition to equilibrate the column between injections.
Under these conditions, the retention time for 3-hydroxybu-
tyryl-CoA was 20.1 min. An authentic standard of 2-hydroxy-
isobutyryl-CoA was not available, but based on the known per-
formance of the C18 column, branched acyl-CoAs elute earlier
than linear isomers.
Enzyme-monitored Turnover of IcmF
Changes in the spectra of Gk IcmF-bound AdoCbl were
monitored by UV-visible spectroscopy at 24 °C in Buffer A con-
taining 5–15 mM MgCl2. Substrates (final concentration of
0.5–4 mM) were added to 20–65 ␮M apoIcmF loaded with 1 or
2 eq of AdoCbl. To check the influence of the MeaI domain on
catalytic turnover, the reaction was also supplemented with
1–5 mM GTP or ATP. The amount of cob(II)alamin formed
under steady-state turnover conditions was calculated from the
decrease in absorbance at 525 nm upon substrate addition
using a value of ⌬⑀525 nm of Ϫ4.8 mMϪ1 cmϪ1 (14). During the
Bioinformatics Analysis
Operon and regulon browsers on the Microbes Online web
site were used for the elucidation of functional predictions for
course of the reaction, AdoCbl was gradually converted to the genes of interest (18).
3726 JOURNAL OF BIOLOGICAL CHEMISTRY
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IcmF Is a Pivalyl-CoA Mutase
FIGURE 5. Organization of genes in mmgABC operon harboring icmF
gene. Top, A. flavithermus; middle, B. megaterium; bottom, B. brevis. The fol-
lowing genes are found in the same operon with icmF: tetR (TetR/Acr-like
family transcriptional regulator), acdA (acyl-CoA dehydrogenase), mmgA
(acetyl-CoA transferase), mmgB (3-OH-butyryl-CoA dehydrogenase), mmgC
(acetyl-CoA dehydrogenase), and fadF (medium-/long-chain fatty acyl-CoA
dehydrogenase). rpoE gene, which encodes the ␴ subunit of RNA polymer-
ase, is found just downstream of icmF. The numbers below the genes indicate
the distance in nucleotides between two adjacent genes. Negative numbers
indicate overlapping genes. For a complete list of bacteria showing similar
operonic organization, see the text.
FIGURE6. Pivalyl-CoAmutaseactivityofIcmF. ArepresentativeGCchromat-
ogram showing separation of isovaleric and pivalic acids following enzymatic
conversion of isovaleryl-CoA to pivalyl-CoA is shown. The reaction mixture con-
tained in Buffer A 15 mM MgCl2, 2000 ␮g of Gk IcmF, 100 ␮M AdoCbl, 5 mM GTP,
and 1.56 mM isovaleryl-CoA. At different time points, aliquots were removed, the
esters were hydrolyzed, and the corresponding acids were separated by GC as
described under “Experimental Procedures.” The traces represent 1 min (black
line), 5 min (light gray line), 25 min (dark gray line), and pivalic acid standard
(dashed line). Valeric acid was used as an internal standard.
RESULTS
Gene Neighborhood Analysis for icmF—In several bacteria,
the icmF gene is located in the same operon with or in close
proximity to genes encoding enzymes involved in fatty acid
metabolism (3). For example, enzymes found in the operon
with icmF in several bacteria (Bacillus selenitireducens, Lysini-
TABLE 1
Isobutyryl-CoA mutase and pivalyl-CoA mutase activities of recombi-
nant IcmFs
Values represent the average of at least three independent experiments. In all exper-
bacillus sphaericus, Anoxybacillus flavithermus, Bacillus mega- iments, a saturating concentration (2 mM) of substrate was used.
Specific activity
terium, Bacillus halodurans, Bacillus pseudofirmus, Bacillus
coagulans, Bacillus sp. NRRL B-14911, Geobacillus sp.
WCH70, and Brevibacillus brevis) are annotated as enzymes in
the mother cell metabolic gene (mmg) operon, which has been
described in Bacillus subtilis (19) (Fig. 5). Three genes in this
operon are annotated as mmgA (acetyl-CoA transferase),
mmgB (3-OH-butyryl-CoA dehydrogenase), and mmgC
Substrate
Gk IcmF
Cm IcmF
Ϫ1
␮mol minϪ1 mg
Isovaleryl-CoA
Isobutyryl-CoA
n-Butyryl-CoA
0.021 Ϯ 0.004
1.2 Ϯ 0.1
0.015 Ϯ 0.004
13.8 Ϯ 1.1
3.3 Ϯ 0.4
33.0 Ϯ 1.4
(acetyl-CoA dehydrogenase) (19). A similar set of enzymes in and analyzed by GC. A time-dependent decrease in the
Ralstonia eutropha encoded by the H16_A0459–H16_A0464 isovaleric acid peak at 6.96 min was accompanied by the
operon allows growth on long-chain fatty acids. The absence of appearance of a peak at 5.99 min, suggesting conversion of
this operon together with the H16_A1526–H16_A1532 operon isovaleryl-CoA to a new compound (Fig. 6). The expected prod-
renders R. eutropha unable to grow on plant oils or long-chain uct of AdoCbl-dependent 1,2 rearrangement of isovaleryl-CoA
fatty acids as a carbon source (20). The gene acdH from Strep- (or 3-methyl-butyryl-CoA) is pivalyl-CoA (or 2,2-dimethylpro-
tomyces coelicolor and Streptomyces avermitilis that is homol- pionyl-CoA) (Fig. 1) (1). The retention time of a standard
ogous to mmgC is an acyl-CoA dehydrogenase and plays a role pivalic acid sample (5.99 min) exactly coincided with that of the
in the catabolism of branched-chain amino acids (21). Finally, product formed from isovaleryl-CoA (Fig. 6). The isomeriza-
the presence of the rpoE gene (which encodes the ␴ subunit of tion of isovaleryl-CoA to pivalyl-CoA by both Gk IcmF and Cm
RNA polymerase) just downstream of icmF strongly suggests IcmF is slow in comparison with the isomerization of n-butyryl-
that IcmF is linked to fatty acid metabolism (22) (Fig. 5).
CoA to isobutyryl-CoA (Table 1). The Km value for isovaleryl-
Alternative Substrates for IcmF—Based on the above gene CoA for Gk IcmF is 62 Ϯ 8 ␮M, and the specific activity of the Gk
neighborhood analysis, we sought to assess whether IcmF plays IcmF with isovaleryl-CoA is 0.021 Ϯ 0.004 ␮mol minϪ1 mg
,
Ϫ1
a role in the metabolism of branched fatty acids and can cata- which is ϳ150-fold lower than the activity with n-butyryl-CoA
lyze the isomerization of substrates other than isobutyryl-CoA (3.25 Ϯ 0.35 ␮mol minϪ1 mgϪ1). With the Cm IcmF, an ϳ2200-
and n-butyryl-CoA. We decided to use isovaleryl-CoA, a fold difference in the specific activities was obtained with the
building block for iso odd branched fatty acids, and 2-methyl- two substrates (Table 1).
butyryl-CoA, a building block for anteiso branched fatty acids,
Isomerization of valeryl-CoA is expected to produce 2-meth-
as potential substrates for IcmF. Isovaleryl-CoA and 2-methyl- ylbutyryl-CoA. However, consumption of valeryl-CoA was not
butyryl-CoA are synthesized from the branched-chain amino observed in the presence of IcmF, AdoCbl, and GTP/MgCl2 in
acids leucine and isoleucine, respectively, in reactions catalyzed the GC-based assay (data not shown). Because HCM (5, 23)
by the branched-chain ketoacid dehydrogenase complex (21).
from Methylibium petroleiphilum is very similar to the stand-
Gk IcmF was mixed with isovaleryl-CoA in the presence of alone ICM from Streptomyces cinnamonensis and catalyzes the
AdoCbl and GTP/MgCl2, and the products were hydrolyzed interconversion of 3-hydroxybutyryl-CoA and 2-hydroxy-
FEBRUARY 3, 2012•VOLUME 287•NUMBER 6
JOURNAL OF BIOLOGICAL CHEMISTRY 3727

IcmF Is a Pivalyl-CoA Mutase
FIGURE7. SpectralchangesinGkholo-IcmFinpresenceofisobutyryl-CoAandisovaleryl-CoA. A, spectraofholo-IcmF(40␮M)inbufferAat24 °C(solidline)
and after addition of 4.1 mM isobutyryl-CoA, which resulted in formation of cob(II)alamin (dotted line). B, spectra of holo-IcmF (31 ␮M) in Buffer A at 24 °C (solid
line) and after addition of 1.5 mM isovaleryl-CoA (dotted line).
isobutyryl-CoA (5, 24), we decided to examine their substrate
specificity overlap. However, conversion of DL-3-hydroxybu-
tyryl-CoA to 2-hydroxyisobutyryl-CoA was not observed as
judged by an HPLC-based assay (data not shown).
Absorption Spectrum of Gk IcmF during Steady-state
Turnover—The binding sites for AdoCbl in the Gk IcmF dimer
are nonidentical and exhibit an ϳ25-fold difference in affinities
(KD1 ϭ 0.08 Ϯ 0.01 ␮M and KD2 ϭ 1.98 Ϯ 0.4 ␮M) (3). For
enzyme-monitored turnover experiments, Gk IcmF was recon-
stituted with 1 eq of AdoCbl to avoid the presence of free cofac-
tor. The absence of free AdoCbl was confirmed by analyzing the
spectrum of the flow-through obtained upon concentrating the
reaction mixture using a Microcon 30 kDa concentrator. Addi-
FIGURE 8. Inactivation of Gk IcmF during turnover with isobutyryl-CoA.
Rapid oxidation of cob(II)alamin to OH2Cbl was observed during reaction of
holo-IcmF (with 1 eq; 41 ␮M AdoCbl bound) with 1.4 mM isobutyryl-CoA in
Buffer A with 5 mM MgCl2 at 24 °C in the dark. The spectra were recorded
between 0 and 60 min. Inset, the time-dependent increase at 350 nm was
fitted to a single exponential function in the absence of nucleotides (filled
circles) (kobs ϭ 0.11 Ϯ 0.01 minϪ1) or in the presence of 5 mM GTP (open circles)
(kobs ϭ 0.063 Ϯ 0.002 minϪ1) or 5 mM ATP (triangles) (kobs ϭ 0.10 Ϯ 0.01
minϪ1).
tion of substrate (isobutyryl-CoA or n-butyryl-CoA) to holo-
IcmF resulted in cob(II)alamin formation as evidenced by the
decrease in absorbance at 525 nm and increase at 480 nm (Fig.
Ϫ1
Ϫ1
7A). Using a ⌬⑀525 nm of Ϫ4.8 mM cm (14), the ratio of
AdoCbl:cob(II)alamin under steady-state turnover conditions
was estimated to be ϳ2:1 when isobutyryl-CoA was used as a
substrate. With isovaleryl-CoA, the ratio of AdoCbl:cob(II)ala-
min under steady-state turnover conditions was 9:1 (Fig. 7B).
Inactivation of IcmF and Effect of Nucleotides—Cobalamin ϳ7% of substrate was consumed (Fig. 9A). Very similar behav-
spectra under turnover conditions were used for monitoring ior was also observed with Cm IcmF (data not shown). Protein
the effect of nucleotides on the IcmF-catalyzed reaction (Fig. 8). stability, the presence of metal ions (K and Ca2ϩ), reductants
ϩ
Addition of isobutyryl-CoA results in formation of cob(II)ala- (DTT and tris(2-carboxyethyl)phosphine hydrochloride), and
min, which is converted over time to OH2Cbl (Fig. 8). The different buffers (50 mM sodium phosphate, pH 7.5, 100 mM
Ϫ1
increase in absorption at 350 nm (kobs ϭ 0.11 Ϯ 0.01 min
)
NaCl; 50 mM potassium phosphate, pH 7.5, 100 mM KCl; and 50
(Fig. 8, inset) and the distinctive double peaks at 505 and 540 nm mM HEPES, pH 7.5, 100 mM NaCl) were assessed and ruled out
indicated formation of OH2Cbl, a hallmark of inactivation for as contributors to the observed inactivation (data not shown).
AdoCbl-dependent enzymes (12, 25, 26). The amplitude but Nucleotides (ATP and GTP) decreased the activity of Gk IcmF
not the rate of OH2Cbl formation was diminished in the pres- (Fig. 9A) as reported previously with GDP and GTP (3).
ence of GTP (kobs ϭ 0.063 Ϯ 0.002 minϪ1) and ATP (kobs
0.10 Ϯ 0.01 minϪ1) (Fig. 8, inset).
ϭ
With isovaleryl-CoA, the Gk IcmF reaction was almost com-
pletely inhibited after 25–30 min (Fig. 9B). However, in the
Under standard in vitro assay conditions, Gk IcmF exhibits a presence of GTP, the enzyme was inactivated to a lesser extent
linear reaction time course for only 40–60 s before the activity in comparison with the reaction without GTP (Fig. 9B). Similar
plateaus (Fig. 9A). The specific activity with isobutyryl-CoA behavior was seen for Cm IcmF (data not shown). Interestingly,
(1.1 Ϯ 0.1 ␮mol minϪ1mgϪ1) was calculated using the linear for the first 2 min, no difference was seen in the isovaleryl-CoA/
portion of the curve and was comparable with values obtained pivalyl-CoA mutase activity ϮGTP (Fig. 9B).
previously in the coupled enzyme assay (3). When n-butyryl-
Loss of 5Ј-Deoxyadenosine Leads to Inactivation of IcmF—
CoA was used as a substrate, a specific activity of 3.25 Ϯ 0.35 The steady formation of OH2Cbl during IcmF turnover under
Ϫ1
Ϫ1
␮mol min mg was obtained. With both isobutyryl-CoA aerobic conditions suggests that inactivation might result from
and n-butyryl-CoA, inactivation of Gk IcmF resulted in termi- oxidative interception of cob(II)alamin (27). An alternative
nation of the reaction in ϳ6–10 min during which time only possibility is that inactivation is associated with loss of 5Ј-de-
3728 JOURNAL OF BIOLOGICAL CHEMISTRY
VOLUME 287•NUMBER 6•FEBRUARY 3, 2012

IcmF Is a Pivalyl-CoA Mutase
FIGURE 9. Effect of nucleotides on time course of reactions catalyzed by IcmF. A, time course of the isobutyryl-CoA mutase reaction catalyzed by Gk IcmF
at 37 °C. The assay mixture in Buffer A with 10 mM MgCl2 contained 40 ␮g of holo-IcmF, 100 ␮M AdoCbl, 1.5 mM isobutyryl-CoA, and either no nucleotides (black
circles), 6 mM ATP (triangles), or 3 mM GTP (open circles). B, time course of the isovaleryl-CoA mutase reaction catalyzed by Gk IcmF. The reaction mixture in Buffer
A with 15 mM MgCl2 contained 2500 ␮g of IcmF, 100 ␮M AdoCbl, and 1.5 mM isovaleryl-CoA with (white circles) or without 5 mM GTP (black circles) at 37 °C.
Aliquots of the reactions were removed at different time points and analyzed by GC as described under “Experimental Procedures.” Data represent the average
of three independent experiments. The data represent the mean Ϯ S.D. of three independent experiments.
FIGURE 10. Inactivation of Gk IcmF under anaerobic conditions. A, spectral changes upon incubation of 33 ␮M holo-IcmF (containing 1 eq of bound AdoCbl)
with 4.8 mM n-butyryl-CoA in Buffer A with 5 mM MgCl2 under anaerobic conditions at 24 °C in the dark. Spectra were recorded at time 0 (black), immediately
after addition of substrate (light gray), and 60 min after addition of substrate (dark gray). Formation of cob(II)alamin (480 nm peak) without further conversion
toOH2Cblwasobserved. B, comparisonoftimecoursesforthereactioncatalyzedbyGkIcmFat37 °Cunderaerobic(circles)andanaerobic(triangles)conditions
in the presence of GTP. The aerobic and anaerobic assay mixtures in Buffer A with 15 mM MgCl2 contained 40 ␮g of IcmF, 2 mM n-butyryl-CoA, and either no
nucleotides (solid symbols) or 4.3 mM GTP (open symbols). The data represent the mean Ϯ S.D. of three independent experiments.
oxyadenosine from the active site, and the uncoupled
cob(II)alamin is subsequently oxidized to OH2Cbl. To distin-
guish between these possibilities, enzyme-monitored turnover
experiments were conducted under anaerobic conditions (Fig.
10A). Addition of n-butyryl-CoA to holo-IcmF resulted in for-
mation of cob(II)alamin, which was not further converted to
OH2Cbl even after 60 min (Fig. 10A).
Next, we compared the time courses of the reaction catalyzed
by Gk IcmF with n-butyryl-CoA under aerobic and anaerobic
conditions (Fig. 10B). Under anaerobic conditions, the reaction
exhibited a linear dependence for a longer time period (2 versus
1 min) but eventually ceased after 10 min. Because spectro-
scopic analysis of the reaction time course shows that OH2Cbl FIGURE 11. Formation of OH2Cbl and 5؅-deoxyadenosine during enzyme-
monitored turnover. Holo-IcmF (64 ␮M IcmF active site concentration con-
taining 2 eq of AdoCbl) was mixed with 1.5 mM isobutyryl-CoA in Buffer A at
37 °C. All manipulations with the samples and HPLC were performed in the
dark. The decay of AdoCbl (open triangles) and the appearance of OH2Cbl
(solid triangles) and of 5Ј-deoxyadenosine (open circles) were monitored over
50 min. As a control for AdoCbl stability during sample handling, the analysis
was repeated without addition of isobutyryl-CoA (open squares). The data
were fitted by a single exponential function for the disappearance of AdoCbl
is not formed under these conditions, it demonstrates that inac-
tivation is not directly correlated with OH2Cbl formation. As
seen under aerobic conditions, GTP also led to decreased
OH2Cbl formation under anaerobic conditions (Fig. 10B).
Characterization of Inactivation Products by HPLC—The
reaction of 64 ␮M Gk holo-IcmF (containing 2 eq of AdoCbl)
and appearance of 5Ј-deoxyadenosine and OH2Cbl.
with 4 mM isobutyryl-CoA was monitored as described under
“Experimental Procedures” (Fig. 11). Under these conditions, 0.01 minϪ1) corresponded to the rate of AdoCbl disappearance.
AdoCbl disappeared with a kobs of 0.40 Ϯ 0.04 minϪ1 (Table 2). The concentration of 5Ј-deoxyadenosine recovered (20.6 Ϯ 0.8
The rate of appearance of 5Ј-deoxyadenosine (kobs ϭ 0.32 Ϯ ␮M) was equal to the concentration of AdoCbl consumed (23 Ϯ
FEBRUARY 3, 2012•VOLUME 287•NUMBER 6
JOURNAL OF BIOLOGICAL CHEMISTRY 3729

IcmF Is a Pivalyl-CoA Mutase
TABLE 2
Kinetics of OH2Cbl and 5؅-deoxyadenosine formation
Values represent the average of at least two independent experiments.
AdoCbl
OH2Cbl
Concentration
5؅-Deoxyadenosine
kobs Concentration
Nucleotide
kobs
Concentration
kobs
min
0.23 Ϯ 0.02
0.25 Ϯ 0.04
Ϫ1
Ϫ1
Ϫ1
min
␮M
␮M
4.34 Ϯ 0.01
5.9 Ϯ 1.4
min
␮M
20.6 Ϯ 0.8
19.9 Ϯ 1.3
None
GTP
0.4 Ϯ 0.04
23 Ϯ 1
0.32 Ϯ 0.01
0.35 Ϯ 0.05
0.37 Ϯ 0.04
20.65 Ϯ 1.02
first we tested the effect of introducing the K213A mutation in
the GtgGaGKSS sequence of the P-loop motif, which is impor-
tant for phosphate binding (29). The K213A Gk IcmF mutant
was devoid of both GTPase and ATPase activities. Notably, the
mutase activity of the K213A mutant was similar to that of
wild-type protein (0.6 ␮mol minϪ1 mgϪ1 with isobutyryl-CoA).
Furthermore, in the presence of 3 mM AMPPNP, the kcat for the
Ϫ1
FIGURE 12. Multiple sequence alignment of IcmFs and MeaB from M.
extorquens showing base specificity loop NKX(D/E). Accession numbers
are as follows: G. kaustophilus, YP_149244; C. metallidurans CH34, YP_582365;
R. eutropha H16, YP_724799; Frankia alni, YP_716016; Nocardia farcinica,
YP_117245; B. coagulans, ZP_01696637; Thauera sp., ZP_02841697; Rubriv-
ivax gelatinosus, ZP_00242991; and MeaB, AAL86727. Asp (D) substitution by
Glu (E) is indicated by an asterisk. All accession numbers are from the NCBI
protein database.
GTPase activity was inhibited ϳ3.6-fold to 2.7 Ϯ 0.14 min
.
Taken together, the above results are consistent with the
ATPase and GTPase activities being intrinsic to Gk IcmF.
Although it has been suggested that replacement of Asp by
Glu in the NKXD motif has no effect on nucleotide specificity
(30), our results suggest the contrary. Thus, in Cm IcmF, the
base specificity loop sequence is NKFD, and although this pro-
tein does not exhibit ATPase activity, it is an active GTPase
(kcat ϭ 18 Ϯ 1.3 minϪ1 and KGTP ϭ 40 Ϯ 8 ␮M). These results
provide an interesting illustration of a G-protein losing its spec-
ificity due to a single substitution in the NKXD motif.
1 ␮M). In contrast, the rate of OH2Cbl formation (kobs ϭ 0.23 Ϯ
0.02 minϪ1) was slower, and only 4.34 Ϯ 0.01 ␮M OH2Cbl was
recovered (Table 2). Treatment of inactivated enzyme with pro-
teinase K increased OH2Cbl recovery only marginally (6.3 Ϯ 0.2
␮M). The reason for the low yield of OH2Cbl is presently not clear.
To ensure that AdoCbl is not cleaved non-enzymatically to 5Ј-de-
oxyadenosine during sample preparation and/or chromato-
DISCUSSION
Previously, it was believed that ICM-like activity was
graphic separation, a control reaction was performed in which restricted to the Streptomyces genus where it is involved in
substrate was omitted from the reaction mixture (Fig. 11).
monensin A and B production (4). Our discovery of ICM activity
IcmF appears to show half-of-sites activity because reconsti- in the IcmF fusion protein, which is widely distributed in bacteria,
tution with 1 versus 2 eq of AdoCbl resulted in the same steady- suggests its involvement in metabolic processes beyond polyketide
state concentration of cob(II)alamin (data not shown). Surpris- synthesis(3). InoureffortstoelucidatethepossiblerolesofIcmFin
ingly, in the inactivation experiments, not all the AdoCbl bound bacterial metabolism, we noticed that icmF genes co-localize with
to Gk IcmF was converted to OH2Cbl and 5Ј-deoxyadenosine. genes encoding enzymes involved in ␤-oxidation of fatty acids.
Instead, ϳ66% of the AdoCbl at one site (i.e. 32 ␮M) was con- Subsequently, we probed branched organic acids, which are syn-
verted to 5Ј-deoxyadenosine; this corresponded to the concen- thesized from the branched amino acids valine, leucine, and iso-
tration of cob(II)alamin (ϳ21 ␮M) under steady-state turnover leucine, as substrates for IcmF. This in turn led to the discovery of
conditions. This provides further evidence for half-of-sites a new IcmF activity, i.e. isomerization of isovaleryl-CoA/pivalyl-
activity in Gk IcmF. It is unclear why complete inactivation at CoA (Figs. 1 and 6).
one of the two active sites was not observed.
Although AdoCbl-dependent pivalyl-CoA mutase activity
ATPase Activity of IcmF—In the MeaI domains of many has been predicted to exist (10, 31), an enzyme with this cata-
IcmFs, the base specificity loop motif NKXD is modified to lytic activity has not been identified previously. In this study, we
NKXE (3). In the Gk IcmF, the NKXE sequence is present, report that IcmFs from G. kaustophilus and C. metallidurans
whereas in the Cm IcmF, the sequence is NKXD (Fig. 12). The can convert isovaleryl-CoA to pivalyl-CoA (Fig. 1). Depending
Lys residue in the NKXD motif interacts via hydrophobic inter- on the organism, the isovaleryl-CoA/pivalyl-CoA mutase activ-
actions with the plane of the guanine ring, whereas the Asp ity of IcmF was ϳ150–2,200-fold lower than the conversion of
coordinates two nitrogen atoms in the purine ring. Gk IcmF n-butyryl-CoA to isobutyryl-CoA (Table 1). Recently, it was
catalyzes the hydrolysis of both GTP and ATP (Table 3). The shown that pivalic acid can be incorporated as a starter unit in
kinetic parameters for IcmF measured in the presence of vari- fatty acids in several bacteria (32). Thus, the production of piva-
Ϫ1
ous nucleotides are comparable: kcat with ATP is 19 Ϯ 1 min
,
lyl-CoA catalyzed by IcmF might be important in bacteria that
and kcat with GTP is 10 Ϯ 1 minϪ1. Although the Km for ATP is use this starter unit for the biosynthesis of branched fatty acids
high (1290 Ϯ 300 ␮M) relative to that for GTP (51 Ϯ 3 ␮M), the containing a quaternary carbon. Our discovery of the
concentration of ATP (3–5 mM) is higher than of GTP (1 mM) in isovaleryl-CoA/pivalyl-CoA mutase activity adds to the grow-
bacterial cells (28).
ing list of carbon skeleton rearrangements catalyzed by
To rule out the possibility that the ATPase activity of Gk AdoCbl-dependent isomerases (Fig. 1). Thus, it appears that
IcmF is due to a contaminant that co-purifies with the mutase, the “mutase core” is quite versatile for two reasons. First, sub-
3730 JOURNAL OF BIOLOGICAL CHEMISTRY
VOLUME 287•NUMBER 6•FEBRUARY 3, 2012

IcmF Is a Pivalyl-CoA Mutase
TABLE 3
Comparison of GTPase and ATPase Activities of Gk IcmF
GTPa
ATPa
Enzyme
kcat
Km(GTP)
␮M
51 Ϯ 3
k
cat/KGTP
kcat
Km(ATP)
␮M
1290 Ϯ 300
k
cat/KATP
Ϫ1
Ϫ1
Ϫ1
Ϫ1
min
Ϫ1 min
min
Ϫ1 min
Wild type
K213Ab
10 Ϯ 2
(1.96MϮ 0.37) ϫ 105
19 Ϯ 1
(1.47MϮ 0.03) ϫ 104
ND
ND
a All experiments were performed in Buffer A with 20 mM MgCl2 at 37 °C as described under “Experimental Procedures.” Values represent the average of at least five inde-
pendent experiments. ND, not detected.
b When a 40-fold higher concentration of the K213A mutant was used in the assay, GTPase/ATPase activity above the detection limit of 5 ␮M inorganic phosphate released
in the malachite green assay was not observed.
stitutions of a limited number of key active site residues alter
The presence of nucleotides has virtually no effect on the inac-
substrate specificity, and second, relaxed substrate specificity tivation kinetics of IcmF with either isobutyryl-CoA or n-butyryl-
allows alternative reactions to be catalyzed by the same active CoA as substrate (Fig. 9A). In contrast, when isovaleryl-CoA is
site as exemplified by IcmF.
used as substrate, protection, albeit modest, was seen in the pres-
We also report relaxed substrate specificity in the MeaI ence of GTP (Fig. 9B). The activity of Gk IcmF with isobutyryl-
domain of Gk IcmF. It has been reported that some G-proteins CoA is reduced in the presence of nucleotides (3). This is unex-
have either lost or switched nucleotide specificity (33). For pected because MeaB increases the kcat of MCM 1.8-fold in
example, in centaurin ␥-1 GTPase where the sequence of the addition to protecting it from inactivation (14). The rate of IcmF
base specificity loop NKXD is modified to TQDR (34), nucleo- inactivation (0.11 Ϯ 0.01 minϪ1) in the presence of isobutyryl-
tide specificity is lacking, and it is described as a general NTPase CoA is ϳ14-fold higher than inactivation of MCM in the presence
(34). Proteins belonging to the YchF subfamily of the Obg fam- of methylmalonyl-CoA (0.0072 minϪ1) (14).
ily of G-proteins harbor the NXXE motif instead of the NKXD
(35). For example, human OLA1, which belongs to the Obg CoA was linear for a longer duration than in the presence of oxy-
family, hydrolyzes ATP more efficiently than GTP. gen (Fig. 10B). Because OH2Cbl formation is observed only under
Under anaerobic conditions, the IcmF reaction with n-butyryl-
In our study, we show that in a subset of the MeaI chaperone aerobic conditions, it argues against an internal electron transfer
domains of IcmF the NKXD motif is modified to NKXE. This from cob(II)alamin to the substrate being responsible for inactiva-
constitutes an interesting example where only some members of a tion as described for lysine 5,6-aminomutase (26). Because IcmF is
protein family have lost specificity for GTP. We note that although also inactivated under anaerobic conditions where OH2Cbl is not
the MeaI domain of Gk IcmF exhibits ATPase activity it is func- formed, we conclude that inactivation is primarily signaled by the
tionally distinct from the ATP-dependent chaperones for loss of 5Ј-deoxyadenosine from the active site.
AdoCbl-dependent eliminases, e.g. diol dehydratases (36, 37).
In conclusion, we report that IcmF catalyzes the formation
A remarkable feature of AdoCbl-dependent enzymes is that pivalyl-CoA from isovaleryl-CoA. There is virtually no infor-
they catalyze reactions involving radical intermediates under mation on bacterial metabolism of pivalic acid, which has a
aerobic conditions. However, this comes with a price, i.e. their mostly anthropogenic origin (10). We suggest that pivalyl-CoA
susceptibility to inactivation (12). Under standard in vitro assay mutase activity of IcmF might be important for biodegradation
conditions, IcmF is inactivated quite rapidly with either isobu- of branched compounds where pivalic acid is a central interme-
tyryl-CoA or isovaleryl-CoA as substrate (Fig. 9). In a subclass diate (10, 43). The activity of IcmF would reduce branching of
of AdoCbl-dependent enzymes, reactivating factors mediate an compounds with a quaternary carbon. It will be important to
ATP-dependent exchange of enzyme-bound OH2Cbl with free follow the fate of pivalyl-CoA in IcmF-containing bacteria and
AdoCbl (36–39). These reactivases have sequence similarity to test whether this compound is incorporated in fatty acids
DnaK and to other members of the Hsp70 family of molecular and/or other compounds.
chaperones and lower sequence similarity to the large subunits
Acknowledgment—We gratefully acknowledge Dr. Thore Rohwerder
(Helmholtz Center for Environmental Research) for helpful discus-
sions on the distribution of acyl-CoA mutases in nature and their
potential roles in microbial metabolism.
of corresponding mutases (37). In contrast, the G-protein chap-
erones associated with AdoCbl-dependent mutases belong to
the SIMIBI (signal recognition particle, MinD, and BioD) sub-
class of the G3E family of P-loop metallochaperones (3, 13, 29).
In MCM where the role of the G-protein chaperone MeaB is
best characterized (14), the chaperone uses GTP hydrolysis to
power the expulsion of cob(II)alamin when 5Ј-deoxyadenosine
is lost from the active site (13). Unlike the ATP-dependent reac-
tivases, MeaB is unable to release OH2Cbl bound to MCM (13,
14). Instead, MeaB exerts a protective effect on the MCM reaction
by reducing the inactivation rate in the presence of nucleotides. In
numerous bacterial genomes, MeaB-like proteins are found in the
same operon as the mutases (40). Mutations in the MeaB ortholog
in humans results in methylmalonic aciduria, an inborn error of
metabolism(41, 42), pointingtotheimportantroleofthisauxiliary
protein in maintaining MCM function.
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IcmF Is a Pivalyl-CoA Mutase
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3732 JOURNAL OF BIOLOGICAL CHEMISTRY
VOLUME 287•NUMBER 6•FEBRUARY 3, 2012

Enzymology:
Novel Coenzyme B12-dependent
Interconversion of Isovaleryl-CoA and
Pivalyl-CoA
Valentin Cracan and Ruma Banerjee
J. Biol. Chem. 2012, 287:3723-3732.
doi: 10.1074/jbc.M111.320051 originally published online December 13, 2011
Access the most updated version of this article at doi: 10.1074/jbc.M111.320051
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