IcmF is a fusion between the radical B12 enzyme isobutyryl-CoA mutase and its G-protein chaperone

Article abstract of DOI:10.1074/jbc.M109.062182

Coenzyme B₁₂ is used by two highly similar radical enzymes, which catalyze carbon skeleton rearrangements, methylmalonyl-CoA mutase and isobutyryl-CoA mutase (ICM). ICM catalyzes the reversible interconversion of isobutyryl-CoA and n-butyryl-CoA and exists as a heterotetramer. In this study, we have identified >70 bacterial proteins, which represent fusions between the subunits of ICM and a P-loop GTPase and are currently misannotated as methylmalonyl-CoA mutases.Wedesignate this fusion protein as IcmF (isobutyryl-CoA mutase fused). All IcmFs are composed of the following three domains: the N-terminal 5′-deoxyadenosylcobalamin binding region that is homologous to the small subunit of ICM (IcmB), a middle P-loop GTPase domain, and a C-terminal part that is homologous to the large subunit of ICM (IcmA). The P-loop GTPase domain has very high sequence similarity to the Methylobacterium extorquens MeaB, which is a chaperone for methylmalonyl-CoA mutase. We have demonstrated that IcmF is an active ICM by cloning, expressing, and purifying the IcmFs from Geobacillus kaustophilus, Nocardia farcinica, and Burkholderia xenovorans. This finding expands the known distribution of ICM activity well beyond the genus Streptomyces, where it is involved in polyketides biosynthesis, and suggests a role for this enzyme in novel bacterial pathways for amino acid degradation, myxalamid biosynthesis, and acetyl-CoA assimilation.

Full text of DOI:10.1074/jbc.M109.062182

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 285, NO. 1, pp. 655–666, January 1, 2010
© 2010 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.
IcmF Is a Fusion between the Radical B12 Enzyme
□
S
Isobutyryl-CoA Mutase and Its G-protein Chaperone*
Received for publication, September 3, 2009, and in revised form, October 25, 2009 Published, JBC Papers in Press, October 28, 2009, DOI 10.1074/jbc.M109.062182
Valentin Cracan, Dominique Padovani1, and Ruma Banerjee2
From the Department of Biological Chemistry, University of Michigan, Ann Arbor, Michigan 48109
Coenzyme B12 is used by two highly similar radical enzymes,
which catalyze carbon skeleton rearrangements, methylmalo-
nyl-CoA mutase and isobutyryl-CoA mutase (ICM). ICM cata-
lyzes the reversible interconversion of isobutyryl-CoA and
n-butyryl-CoA and exists as a heterotetramer. In this study, we
have identified >70 bacterial proteins, which represent fusions
between the subunits of ICM and a P-loop GTPase and are cur-
rently misannotated as methylmalonyl-CoA mutases. We desig-
nate this fusion protein as IcmF (isobutyryl-CoA mutase fused).
All IcmFs are composed of the following three domains: the
N-terminal 5ꢀ-deoxyadenosylcobalamin binding region that is
homologous to the small subunit of ICM (IcmB), a middle
P-loop GTPase domain, and a C-terminal part that is homolo-
gous to the large subunit of ICM (IcmA). The P-loop GTPase
domain has very high sequence similarity to the Methylobacte-
rium extorquens MeaB, which is a chaperone for methylmalo-
nyl-CoA mutase. We have demonstrated that IcmF is an active
ICM by cloning, expressing, and purifying the IcmFs from
Geobacillus kaustophilus, Nocardia farcinica, and Burkholderia
xenovorans. This finding expands the known distribution of
ICM activity well beyond the genus Streptomyces, where it is
involved in polyketides biosynthesis, and suggests a role for this
enzyme in novel bacterial pathways for amino acid degradation,
myxalamid biosynthesis, and acetyl-CoA assimilation.
Gram-positive, filamentous soil bacterium Streptomyces cinna-
monensis. ICM is an ␣2␤2 heterotetramer composed of two
large subunits (IcmA) of 62.5 kDa and two small subunits
(IcmB) of 14.3 kDa. The genes encoding the subunits of MCM,
an ␣␤ heterodimer in some bacteria, are usually located in a
single operon (Fig. 2). In contrast, the icmA and icmB genes are
distant from each other in the genome of S. cinnamonensis (2).
The sequence of IcmA is very similar to the sequences of the
large subunit of MCM, with the exception of the AdoCbl bind-
ing region, which is missing. Thus, IcmA lacks a C-terminal
AdoCbl binding domain containing the signature DXHXXG
motif that is present instead in the small IcmB subunit, which
binds the cofactor (5). In this respect, ICM resembles some
other AdoCbl-dependent mutases that exhibit a similar organi-
zation. For example, glutamate mutase is also composed of two
subunits of very different sizes as follows: the large subunit
MutE, which binds substrate, and the small subunit MutS,
which binds AdoCbl (6).
The ICM-catalyzed reaction plays an important role in
polyketide biosynthesis in Streptomyces. In studies with 13C-
labeled isobutyrate, it was shown that this compound effi-
ciently incorporates into monensin A tylosin and leucomy-
cin at positions derived from n-butyrate (1). Although ICM
was believed to have a rather limited distribution, its close
sequence relative, MCM, is present in organisms ranging
from bacteria to man (7).
Isobutyryl-CoA mutase (ICM)3 (EC 5.4.99.13) is a coenzyme
B12 (or 5Ј-deoxyadenosylcobalamin (AdoCbl))-dependent en-
zyme, which catalyzes the rearrangement of isobutyryl-CoA
to n-butyryl-CoA (1–3). This reaction is very similar to that
catalyzed by methylmalonyl-CoA mutase (MCM), which is bet-
ter studied and more widely distributed in nature (4). In both
reactions, carbon skeleton rearrangements take place where
the carbonyl-CoA substituent and a hydrogen atom on neigh-
boring carbon atoms exchange positions (Fig. 1) (2, 3). The
genes encoding ICM were first cloned and sequenced from the
A G-protein chaperone, MeaB, shows strong operonic asso-
ciation with MCM, and mutations in the human ortholog, the
product of the cblA locus, result in methylmalonic aciduria due
to dysfunctional MCM activity (8). MeaB from Methylobacte-
rium extorquens has been characterized most extensively and is
a P-loop GTPase (9, 10). Other members of this subfamily
include HypB, UreG, and CooC, which are important in the
assembly of the following metalloenzymes: nickel hydrogenases
(11), urease (12), and CO dehydrogenase (13), respectively.
MeaB has been proposed to function in the GTP-dependent
assembly of holo-MCM and shown to protect the radical inter-
mediates formed during MCM catalysis from oxidative inter-
ception (14). MCM, in turn, influences the GTPase activity of
MeaB increasing it by Ͼ100-fold. Hence, MCM exhibits
GTPase-activating protein activity for MeaB (14, 15). In addi-
tion, MCM modulates the affinity of MeaB for nucleotides. The
crystal structure of MeaB in the presence of GDP has been
solved and confirms that it is a member of the G3E family of
GTPases but differs from other family members in possessing
N- and C-terminal extensions of unknown function (9). Struc-
tural insights into the interaction between MeaB and MCM are
lacking.
* Thisworkwassupported, inwholeorinpart, byNationalInstitutesofHealth
Grant DK45776.
□S
The on-line version of this article (available at http://www.jbc.org) contains
supplemental Tables S1–S2 and Figs. S1–S3.
1 Present address: Laboratoire d’Enzymologie et Biochimie Structurales,
UPR3082 CNRS, 91198 Gif/Yvette, France.
2 To whom correspondence should be addressed: 3220B MSRB III, 1150 W.
Medical Center Dr., Ann Arbor, MI 48109-5606. Tel.: 734-615-5238; E-mail:
rbanerje@umich.edu.
3 The abbreviations used are: ICM, isobutyryl-CoA mutase; MCM, methyl-
malonyl-CoA mutase; GMPPNP, guanosine-5Ј-[(␤,␥)-imido]triphosphate;
TCEP, tris(2-carboxyethyl)phosphinehydrochloride;AdoCbl, 5Ј-deoxyade-
nosylcobalamin; BDH, butyryl-CoA dehydrogenase; LIC, ligase-indepen-
dent cloning.
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IcmF Is an Isobutyryl-CoA Fusion Protein
In this study, we show that in Ͼ70 bacteria ICM is fused to a
icmF from G. kaustophilus—The icmF gene from G. kausto-
P-loop GTPase, which is a paralog of MeaB. This fusion protein philus was amplified from genomic DNA using nested-primer
that we have named IcmF (for ICM-fused) is described as a PCR as attempts to amplify the full-length gene in a single PCR
putative MCM-like protein in the data bases. The misannota- were unsuccessful. The first round of PCR was performed with
tion has led to the ascription of this gene product as represent- the following primers: forward 5Ј-TCTACCGATCTGCTA-
ing a fusion between MCM and MeaB (16) and to its function in AAGTTCAACG-3Ј and reverse 5Ј-GGATTATGGAGAAA-
pathways that are unlikely to be correct (17). Using bioinfor- CAGCGAGTC-3Ј. The second round of amplification was per-
matics and biochemical approaches, we demonstrate that IcmF formed with the following primers containing NheI and BamHI
is an ICM with ICM and GTPase activities. IcmF represents an restriction sites (underlined): forward 5Ј-TAGGCTAGCATG-
important paradigm for elucidating the cross-talk between a GCGCACATTTACCGTCCG-3Ј and reverse 5Ј-TAGGGA-
mutase and its auxiliary protein during the catalytic cycle.
TCCTTACATATTCCGCCGGTATTGTCC-3Ј.
The resulting fragment was cloned into pGEM-T easy (Pro-
mega, WI) and subsequently used as a template for LIC. The
insert was amplified with the following primers for LIC cloning,
forward 5Ј-GACGACGACAAGATGGCGCACATTTACCG-
TCCGAAG-3Ј and reverse 5Ј-GAGGAGAAGCCCGGTTTA-
CATATTCCGCCGGTATTG-3Ј), and inserted in the pET30
Ek/LIC vector according to the manufacturer’s protocol.
icmF from B. xenovorans—The first round of nested-primer
PCR was performed on genomic DNA of B. xenovorans with the
following primers: forward 5Ј-TGTCGACTTCCTCGCTGA-
GCGGTT-3Ј and reverse 5Ј-CGCGACGCGTTGTGGTTGT-
GCGTT-3Ј. The second round of nested PCRs was performed
with primers for LIC: forward 5Ј-GACGACGACAAGATGA-
CCGATCTGTCCACGCCG-3Ј and reverse 5Ј-GAGGAGAA-
GCCCGGTTTACATATTGCGGCGGTACTG-3Ј.
EXPERIMENTAL PROCEDURES
Materials
AdoCbl, GTP, GMPPNP, GDP, isobutyryl-CoA, and n-bu-
tyryl-CoA were purchased from Sigma. Tris(2-carboxyeth-
yl)phosphine hydrochloride (TCEP) was purchased from
Pierce. Butyric, isobutyric, and valeric acids were purchased
from Fluka. [14C]CH3-malonyl-CoA (56 Ci/mol) was pur-
chased from PerkinElmer Life Sciences. A construct harboring
butyryl-CoA dehydrogenase from Megasphaera elsdenii was a
generous gift from Donald Becker (University of Nebraska,
Lincoln).
Cloning and Expression of IcmF
The icmF gene from three organisms was cloned into the
pET-30 Ek/LIC expression vector (Novagen, CA). The genomic
DNA of Geobacillus kaustophilus and Burkholderia xenovorans
(formerly known as Burkholderia fungorum) were generous
gifts from Hideto Takami (Japan Agency for Marine-Earth Sci-
ence and Technology, Kanagawa, Japan) (18). The genomic
DNA clone KNL023_G20 in the pTS1 plasmid containing the
Nocardia farcinica icmF gene was obtained from Jun Ishikawa
(National Institute of Infectious Diseases, Tokyo, Japan) (19).
icmF from N. farcinica—The icmF gene was amplified from
the pTS1 plasmid (genomic DNA clone KNL023_G20) with the
following primers containing NdeI and HindIII restriction sites
underlined: forward 5Ј-ATATATCATATGGCCGACAGTA-
CGCTCCACCAA-3Ј and reverse 5Ј-ATATCTAAGCTTTCA-
CACGTTGCGCCGGTACTG-3Ј. The resulting PCR product
was subcloned into the pGEM-T vector (Promega, WI) and
used for LIC cloning with the following primers: forward
5Ј-GACGACGACAAGATGGCCGACAGTACGCTCCAC-3Ј
and reverse 5Ј-GAGGAGAAGCCCGGTTCACACGTTGCG-
CCGGTA-3Ј. The sequences of all the resulting constructs
were verified by nucleotide sequence determination at the
Genomics Facility, University of Nebraska.
Protein Expression and Purification
Recombinant M. elsdenii butyryl-CoA dehydrogenase ex-
pressed in Escherichia coli BL21 (DE3) was purified as
described previously (20). The pET-30 Ek/LIC vector with the
G. kaustophilus icmF gene was transformed into E. coli BL21
(DE3) cells, which were grown at
FIGURE 1. Reactions catalyzed by MCM and ICM.
37 °C in Luria Bertani (LB) medium
containing 50 ␮g/ml kanamycin to
an A600 of 0.6. Cells were grown for
14–16 h after induction with 0.5
mM isopropyl 1-thio-␤-D-galacto-
pyranoside at 15 °C. The cells were
resuspended in ϳ150 ml of lysis
buffer (50 mM sodium phosphate
buffer (NaPi), pH 8.0, 500 mM NaCl,
5 mM dithiothreitol, and 8 mM imid-
azole) containing three protease
FIGURE 2. Comparison of domain and gene organizations of bacterial IcmF, MCM, and ICM. MeaB and
MeaI represent the P-loop GTPase chaperones for MCM and ICM, respectively.
inhibitormixturetablets(RocheAp-
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IcmF Is an Isobutyryl-CoA Fusion Protein
plied Science) and disrupted by sonication on ice (output set- fixed-time GC/MS-based assay was employed by a modifica-
ting of 5.5 for 10 min with 30-s bursts and 3-min breaks). Fol- tion of a method described previously (1, 22). In this assay,
lowing centrifugation, the cell lysate was subjected to dilution
to a final concentration of 3–5 mg/ml and loaded onto a 50-ml
nickel-nitrilotriacetic acid-Sepharose column. After washing
with 10–20 column volumes of lysis buffer, the protein was
eluted with a gradient of 8–250 mM imidazole in lysis buffer.
Fractions containing IcmF were pooled, concentrated, and dia-
lyzed against 50 mM NaPi, pH 7.5, and applied to a 2.5 ϫ 7.5-cm
Source 15Q column equilibrated at a flow rate of 10 ml/min
with Buffer A (50 mM NaPi, pH 7.5, 50 mM NaCl). The column
was then washed at the same flow rate with 100 ml of Buffer A
and eluted with a 500-ml gradient from 50 to 250 mM NaCl in 50
mM NaPi, pH 7.5, over 50 min at the same flow rate. The puri-
fied IcmF was concentrated and loaded on a 160-ml Superdex-
200 column (GE Healthcare) equilibrated at a flow rate of 0.75
ml/min with 50 mM NaPi, pH 7.5, 250 mM NaCl. Under these
conditions, IcmF eluted with a retention volume of ϳ77.5 ml.
Fractions containing active IcmF were pooled, concentrated,
flash-frozen in liquid nitrogen, and stored at Ϫ80 °C until fur-
ther use. Approximately 25–35 mg of recombinant G. kausto-
philus IcmF was obtained from a 6-liter culture.
normal and isobutyryl-CoA thioesters were saponified, and the
resulting free acids were extracted into ethyl acetate. Product
formation was followed in a 200-␮l assay mixture containing 50
mM KPi, pH 7.5, 100 mM KCl, normal butyryl-CoA or isobu-
tyryl-CoA (0.1 to 1 mM), 50 ␮M AdoCbl, and 0.5–5 ␮g of IcmF.
The reaction was stopped by the addition of 100 ␮l of 2 N KOH
containing 0.18 mM valeric acid as an internal standard fol-
lowed by addition of 100 ␮l of H2SO4 (15%, v/v). In the last step
of sample preparation, the reaction mixture was saturated with
NaCl and extracted with ethyl acetate (250 ␮l). An aliquot of the
extract (5 ␮l) was subjected to analysis by GC/MS using a DB-
FFAP 30-m ϫ 0.25-mm inner diameter, 0.25-␮m capillary col-
umn (Agilent, CA). This column is especially designed for the
separation of organic acids without derivatization.
A continuous assay was developed to determine the kinetic
parameters for IcmF. In this assay, n-butyryl-CoA, which is
produced from isobutyryl-CoA by IcmF, is converted to croto-
nyl-CoA by butyryl-CoA dehydrogenase (BDH). BDH activity
was followed by the decrease in absorbance at 300 nm over 1–2
min upon reduction of ferricenium hexafluorophosphate
ϩ
Ϫ
(Fc PF6 ) (⌬⑀ ϭ 4.3 mMϪ1 cmϪ1) (23). BDH is able to use both
isobutyryl-CoA and n-butyryl-CoA as substrates but with a
preference for the latter. We found that with isobutyryl-CoA,
E. coli BL21 (DE3) cells transformed with recombinant N.
farcinica or B. fungorum IcmF were grown, and the cell extracts
were prepared as described above for the G. kaustophilus
enzyme using potassium phosphate (KPi) buffer. The cell
extracts were loaded onto a 4-ml nickel-nitrilotriacetic acid col-
umn, washed with 50 mM KPi, pH 8, containing 50 mM imid-
azole, and eluted with the same buffer containing 250 mM imid-
azole. IcmF-containing fractions were pooled, and the protein
was obtained in ϳ40% purity from this one-step purification
procedure.
K
m ϭ 311 Ϯ 26 ␮M and Vmax ϭ 1.66 Ϯ 0.06 units (␮mol/min)/
mg, and with n-butyryl-CoA, Km ϭ 68 Ϯ 4 ␮M and Vmax ϭ 33 Ϯ
1 units/mg. The reaction mixture for the coupled assay con-
tained the following in a final volume of 200 ␮l: 2–4 ␮g of IcmF,
50 ␮M AdoCbl, 0.2 ␮g of BDH, varying concentrations of isobu-
ϩ
Ϫ
tyryl-CoA (10–1000 ␮M), 250 ␮M (Fc PF6 ) Ϯ 1–2 mM GDP,
GTP, or GMPPNP in 50 mM NaPi, pH 7.5, 250 mM NaCl. Under
these conditions, the consumption of isobutyryl-CoA by BDH
is negligible and similar to the background rate observed in the
absence of BDH. The dye was preincubated for 3 min at 37 °C
before adding the substrate. After 1 min of incubation, BDH
was added, and the reaction was started 1 min later by the addi-
tion of holo-IcmF.
GTPase Activity of IcmF
The GTPase activity of IcmF (5 ␮M) was determined in the
presence of varying concentrations of GTP (50–5000 ␮M) at
37 °C in 0.4 ml of 50 mM KPi buffer, pH 7.5, 100 mM KCl, and 5
mM MgCl2. For each GTP concentration, aliquots (50 ␮l) were
removed at varying time points (2–60 min), quenched with 2 M
trichloroacetic acid (10% v/v), centrifuged, and filtered through
a 0.1-␮m Ultrafree-MC filter (Millipore) to remove the precip-
itated protein.
UV-visible Spectroscopy
UV-visible spectra were recorded on a Cary 100 spectropho-
tometer (Varian, Inc., Walnut Creek, CA). Holo-IcmF (10–12
␮M) in 50 mM NaPi, pH 7.5, 0.25 M NaCl Ϯ 5 mM MgCl2, and
1–2 mM GDP, GTP, or GMPPNP was incubated in the presence
of 3–5 mM isobutyryl-CoA at 20 °C. The spectra were acquired
after 2–5 min of incubation.
The nucleotides were analyzed by ion exchange chromatog-
raphy on a ␮Bondapak NH2 300 ϫ 3.9-mm high pressure liquid
chromatography column (Waters). Initial conditions were
100% Buffer B (50 mM monobasic KPi, pH 4.5) and 0% Buffer C
(800 mM monobasic KPi, pH 4.5) and a flow rate of 1.0 ml/min.
Between 5 and 20 min, Buffer C was increased to 80% and held
at that concentration for 5 min. Between 25 and 26 min, Buffer
C was decreased to 0% and held for 10 min at that composition
to equilibrate the column between injections. Under these con-
ditions, the retention time for GDP was 9.5 min and for GTP
was 13.1 min.
Isothermal Titration Calorimetry
The isothermal titration calorimetric experiments were per-
formed as described previously (14, 15). Each experiment was
performed in triplicate. IcmF was dialyzed for 10–12 h against
50 mM NaPi, pH 7.5, 0.25 M NaCl containing 1–2 mM TCEP
(Buffer D) before use. The protein (8–24 ␮M) Ϯ 1–2 mM GDP
or GMPPNP in Buffer D was titrated with 30–42 7–9.7-␮l ali-
Enzyme Assays
Initially, recombinant IcmF was assayed for MCM activity as quots of a 15–20 M excess solution of AdoCbl at 20 °C. The
described previously using the radioactive assay (21). To mon- calorimetric signals were integrated, and the data were ana-
itor IcmF activity, one of two assay methods was used. First, a lyzed with Microcal ORIGIN software using a two-sites binding
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IcmF Is an Isobutyryl-CoA Fusion Protein
model to determine the thermodynamic parameters associated
with AdoCbl binding to IcmF.
EPR Spectroscopy
EPR spectra were recorded on a Bruker EMX spectrometer
(Bruker Biospin Corp., Billerica, MA), equipped with an Oxford
ITC4 temperature controller, a Hewlett-Packard model 5340
automatic frequency counter, and a Bruker gaussmeter. Unless
otherwise noted, the following parameters were used: temper-
ature, 100 K; microwave power, 25 milliwatts; microwave fre-
quency, 9.38 GHz; receiver gain, 2 ϫ 105; modulation ampli-
tude, 10 G; modulation frequency, 100 kHz. Cob(II)alamin was
generated by treating a solution of hydroxocobalamin with 4–7
molar excess of TCEP. Formation of cob(II)alamin was fol-
FIGURE3.ComparisonoftheactivesiteresiduesinP.shermaniiMCMwith
those predicted for S. cinnamonensis ICM. The MCM structure was
obtained from the Protein Data Bank code 4REQ. The two striking differences
in the active site residues are the substitutions of Tyr-89 and Arg-207 in MCM
by Phe-80 and Gln-198 in ICM.
lowed by UV-visible spectroscopy, and the concentration of the
solution was estimated using ⑀473 nm ϭ 9.2 mMϪ1 cm
.
Ϫ1
Bioinformatics Analysis
MCM from Propionibacterium shermanii to identify residues
that might be involved in specific substrate binding in ICM
from S. cinnamonensis. They identified two key substitutions in
the large subunit of MCM, Tyr-89 and Arg-207, which are
replaced by Phe-80 and Gln-198 in the large subunit of ICM
(Fig. 3). These differences in active site residues can be rational-
ized based on the structural difference between the respective
substrates despite the very similar reactions catalyzed by the
two enzymes (Fig. 1). In MCM, the carboxylate group of meth-
ylmalonyl-CoA is engaged in electrostatic interactions with the
guanidinium group of Arg-207 and the phenolic group of
Tyr-89 (Fig. 3). The presence of a methyl group in the ICM
substrates instead of the carboxylate is reflected in the loss of
the hydrogen bond donating arginine and tyrosine residues.
Instead, a glutamine and phenylalanine in ICM substitute for
the arginine and tyrosine residues, respectively in MCM (Fig.
STRING was used to find functional linkages for proteins of
interest, as well as gene fusions and gene neighborhoods (24). A
protein-protein blast search (ncbi.nlm.nih.gov) was used to
perform distant searching of homologs. A multiple sequence
alignment and phylogenetic tree were constructed using a
stand-alone version of ClustalX version 1.8. Figures with mul-
tiple sequence alignments were generated using BOXSHADE
3.21. Phylogenetic analysis was carried out using default param-
eters in ClustalX. The trees were visualized using TreeView
1.6.6. Operon and regulon browsers on the Microbes on-line
web site were used for the elucidation of functional predictions
for the genes of interest (25).
RESULTS AND DISCUSSION
Bioinformatics Analysis of IcmF
Analysis of Mutase Domains—Based on bioinformatics anal- 3). Apart from these two differences, the remaining residues in
ysis, it was previously concluded that MCM either colocalizes the active sites of both mutases are highly conserved.
in the same operon with its chaperone, MeaB, or that MeaB is
Multiple sequence alignment of the predicted substrate-
fused to the large subunit of MCM in some bacteria (16). binding site in the C termini of all the identified fusion proteins
Indeed, the putative fusion protein between MCM and MeaB in clearly reveals conservation of the Phe and Gln residues (Fig. 4
B. xenovorans was reported to possess MCM activity (16). Our and supplemental Fig. S1). This analysis strongly suggests that
laboratory has been elucidating the influence of MeaB and the substrate for the fusion protein is n-butyryl-CoA/isobu-
MCM on the substrate binding and catalytic activities of each tyryl-CoA, and hence the fusion protein is predicted to be an
other (14). Because the kinetics of a fusion protein are easier to ICM and not an MCM. We thus designate this fusion protein as
characterize than the stand-alone versions of the component IcmF, for isobutyryl-CoA mutase fused. The IcmF designation
proteins, which interact with varying affinities depending on for this group of fusion proteins also distinguishes it from the
the ligand, we chose to focus on the putative MCM-MeaB “stand-alone” ICM described for the genus Streptomyces.
fusion protein. A BLAST search using the fusion protein from
All IcmFs are predicted to be composed of three domains as
B. xenovorans (NCBI code YP_556774) as a query sequence follows: the N-terminal AdoCbl binding region that is homol-
resulted in the identification of Ͼ70 proteins in bacteria, ogous to the small subunit of ICM, a middle P-loop GTPase
including the seven proteins that were previously identified as domain, and a C-terminal region that is homologous to the
examples of fusions between MCM and MeaB (supplemental large substrate-binding subunit of ICM (Fig. 2). Clear sequence
Table S1) (16). In the data bases, homologs of this fused protein similarities are seen between the AdoCbl binding regions of
are annotated as putative MCM-like proteins. However, a care- the large subunit of MCM, the small subunit of ICM (IcmB), and
ful examination of the domain organization and sequence anal- the N-terminal portion of IcmF (Fig. 5). The signature
ysis of the substrate-binding site in the B12-dependent isomer- DXHXXG…SXL…GG motif (where X is any amino acid) used
ase component (Figs. 2–4) suggested that this group of fusion for binding B12 in the “base-off/His-on” conformation is
proteins might in fact be misannotated.
observed in all three proteins (5). However, in IcmF, this motif
Based on the high sequence similarity between MCM and is similar but not identical to that seen in ICM and MCM. First,
ICM, Robinson and co-workers (2) used the crystal structure of a Gly 3 Ala/Ser change is found in IcmFs in the following
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IcmF Is an Isobutyryl-CoA Fusion Protein
FIGURE 4. Multiple sequence alignment of the C-terminal sequences of IcmFs, the large subunit of ICM (IcmA) from S. cinnamonensis
(AAC08713), MCM from M. extorquens (YP_001642233), and MCM from P. shermanii (CAA33090). The C-terminal region of the IcmFs is homolo-
gous to IcmA and MCM. The two conserved residues in IcmF (Phe and Gln-1 in IcmA) that are important for substrate binding are highlighted in gray and
indicated with asterisks. In MCM, these residues are substituted by Tyr and Arg, respectively. The sequence alignment for a more extensive list of putative
IcmFs is shown in supplemental Fig. S1.
conserved sequence: DXHXX(A/S)…SXY..GGGG. This substi- Expression and Initial Activity Analysis of IcmF
tution is not surprising because glycine is often replaced by
alanine or serine and vice versa in sequences of orthologous
proteins from different organisms. Second, an SXL3SXY sub-
stitution is seen in IcmF. The rationale for the leucine to tyro-
sine substitution and the insertion of two glycines at the end of
the motif are not clear. Thus, it appears that the sequence of the
AdoCbl binding domain of IcmF has diverged from the corre-
sponding sequences in IcmB and MCM.
To test the prediction from bioinformatics analysis that IcmF
harbors ICM rather than MCM activity, the icmF gene from
three organisms, G. kaustophilus, B. xenovorans, and N. far-
cinica, were cloned into the expression vector pET30 Ek/LIC.
Multiple IcmF-encoding genes were subcloned and purified so
that the enzymatic activity of the fusion protein from more than
one organism could be assessed.
Because the fusion protein from B. xenovorans was reported
to have MCM activity (16), we initially tested the activity of all
three IcmF proteins in the standard radiolabeled assay for
MCM (21). However, none of the three IcmFs exhibited detect-
able MCM activity. On the other hand, all three IcmFs exhib-
ited ICM activity. Two assays have been described for monitor-
ing ICM activity and are based on either gas chromatography
(GC) or NMR-based detection of the reactant and product (27).
Because the NMR-based method is not amenable for routine
enzymatic assays, we used a modification of the previously
described GC assay (1, 22) using mass spectrometry (MS) for
detection of the reaction components. A specific activity of
0.6 Ϯ 0.04 ␮mol minϪ1 mgϪ1 protein at 37 °C was obtained for
the G. kaustophilus IcmF.
Analysis of G-protein Domains—The P-loop GTPase domain
has very high sequence similarity to MeaB from M. extorquens
that was shown to be a chaperone for MCM (14, 15). We des-
ignated it as MeaI to distinguish it from the MeaB-like chaper-
one. The sequence encoding the MeaI domain of IcmF includes
ϳ250–300 amino acids in the middle of the protein (Fig. 6).
Sequence analysis strongly suggests that this domain belongs to
the G3E family of P-loop GTPases (10). All four GTPase
sequence fingerprints, the so-called G domains, which define
this family, are present in the MeaI domain of IcmF. These
include the following: (i) the Walker A motif (G1), which binds
the triphosphate moiety of GTP (as is typical for the G3E sub-
family, a slight modification of the GXXGXGK(ST) sequence to
GXXGXGK(SS) is seen); (ii) the Mg2ϩ-binding motif (G2)
(LXXD in all IcmF sequences); (iii) the DXXXXEXXG Walker B
motif (G3); and (iv) the nucleotide specificity NKXD motif
(G4). The replacement of Asp by Glu in NKXD motif in some
proteins (Fig. 6) might not affect the specificity for the gua-
nine nucleotide and is also seen in other G-proteins (26).
Based on this analysis, we conclude that the MeaI domain of
IcmF, like MeaB, belongs to the SIMIBI subclass of G-pro-
teins because two key aspartate residues at the N terminus of
the Walker B motif and in the Mg2ϩ-binding motif are pres-
ent (Fig. 6). Within the SIMIBI subclass, the MeaI domain of
IcmF belongs to the G3E family (the conserved glutamate
residue in the Walker B motif is a signature of this family) as
well as the intact nucleotide specificity motif (10). We pre-
dicted that the MeaI domain of IcmF functions like its para-
log MeaB, i.e. as a chaperone for ICM in the fusion protein
(14).
As an alternative to the gas chromatography-mass spectrom-
etry assay that depends on access to specialized instrumenta-
tion, a coupled spectrophotometric assay was developed to
measure IcmF activity as described under “Experimental Pro-
cedures.” The specific activity determined in the coupled assay
for the N. farcinica IcmF was 1.1 Ϯ 0.1 ␮mol minϪ1 mgϪ1 and
Ϫ1
for the B. xenovorans IcmF was 0.34 Ϯ 0.04 ␮mol minϪ1 mg
Ϫ1
Ϫ1
at 37 °C. A specific activity of 0.75 Ϯ 0.01 ␮mol min mg
protein at 37 °C was measured for G. kaustophilus IcmF, which
is comparable with the value from the gas chromatography-
mass spectrometry assay. In comparison, a Vmax of 38 Ϯ 3 ␮mol
minϪ1 mgϪ1 at 37 °C has been reported for purified stand-alone
ICM from S. cinnamonensis (2).
The recombinant G. kaustophilus IcmF was the most stable and
soluble of the three proteins and was further purified to ϳ95%
purity as described under “Experimental Procedures” to perform
biochemical and biophysical characterizations. Based on its elu-
tion from a calibrated gel filtration column, the G. kaustophilus
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IcmF Is an Isobutyryl-CoA Fusion Protein
IcmF appears to be a dimer with a
native molecular mass of ϳ286 kDa.
Binding of AdoCbl to IcmF ؎
Nucleotides
We investigated the energetics of
AdoCbl binding to G. kaustophilus
IcmF Ϯ nucleotides by Fig. 7 and
Table 1. These experiments re-
vealed the presence of two non-
equivalent binding sites with an
ϳ9–25-fold difference in affinity
for AdoCbl that was influenced by
the presence and identity of the gua-
nine nucleotide (Table 1). Binding
of AdoCbl to the high affinity site in
the absence of nucleotides (KD
ϭ
81 Ϯ 14 nM) is accompanied by a
⌬G10 of Ϫ9.5 Ϯ 0.1 kcal/mol that is
enthalpically favored, whereas bind-
ing to the low affinity site (KD
ϭ
2.0 Ϯ 0.4 ␮M) is entropically driven
(Table 1). These data suggest a pos-
sible difference in the flexibility of
the two AdoCbl-binding sites in
IcmF. A Kact of 12 Ϯ 2 ␮M for
AdoCbl for ICM from S. cinna-
monensis has been reported (2).
We next analyzed the influence of
nucleotides on cofactor binding.
The affinity for AdoCbl for the high
affinity site was slightly increased in
the presence of GDP (132 Ϯ 9 nM),
which resulted from changes in
both enthalpic and entropic contri-
butions (⌬⌬H1 ϳ2.5 kcal/mol and
⌬T⌬S1 ϳ2.8 kcal/mol). GDP did not
substantially influence binding of
AdoCbl to the second site. Binding
of AdoCbl in the presence of
GMPPNP, a nonhydrolyzable ana-
log of GTP (Fig. 7 and Table 1), indi-
cates that GTP hydrolysis is not
required for binding of AdoCbl to
IcmFꢁGTP. GMPPNP decreased by
ϳ2-fold the affinity for AdoCbl to site
1 (KD1 ϭ 154 Ϯ 71 nM) and slightly
increased the affinity at site 2 (KD2 ϭ
1.3 Ϯ 0.5 ␮M). Changes in both the
enthalpic and entropic terms contrib-
uted to this change. Although cofac-
tor binding to site 1 is enthalpically
FIGURE 5. Multiple sequence alignment of the N-terminal AdoCbl binding domain of IcmFs, the small
subunit of ICM (IcmB) from S. cinnamonensis (CAB59633), and MCM from M. extorquens (YP_
001642233). In B12 proteins that bind the cofactor in a base-off/His-on conformation, a signature …SXL…GG
motif is found (highlighted in blue). In IcmF, this motif is similar but not identical as follows: DXHXX(A/S)…(S/
T)XY…GGGG (highlighted in red). In Leptospira borgpetersenii, the histidine that is predicted to coordinate to
AdoCbl appears to be substituted by arginine. The Salinibacter ruber sequence is not included in the alignment
because the AdoCbl region is truncated at the N terminus (DXHXXG motif is missing). IcmFs, which were
previously annotated as MCM-like enzymes, are indicated in boldface. For accession numbers see supplemen-
tal Table S1.
␮M and the kcat to be 3.1 Ϯ 0.1 sϪ1 in the absence of nucleotides.
driven, it is almost entirely entropically driven at site 2.
In comparison, for isobutyryl-CoA a Km of 57 Ϯ 13 ␮M has been
reported for ICM from S. cinnamonensis (2). Thus, the kcat/Km
values for the stand-alone S. cinnamonensis ICM and G. kaus-
IcmF Is an Active Isobutyryl-CoA Mutase
Using the coupled assay, we further characterized the kinetic
tophilus IcmF are 6.8 Ϯ 2.7 ϫ 105 Ϫ1 sϪ1 (2) and 1.5 Ϯ 0.1 ϫ
M
parameters for IcmF from G. kaustophilus (Fig. 8A and Table 2).
The Km value for isobutyryl-CoA was determined to be 20 Ϯ 1 105
M
s
respectively. We note that the activity of the
Ϫ1 Ϫ1
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IcmF Is an Isobutyryl-CoA Fusion Protein
Absorption Spectroscopy of IcmF
under Steady-state Turnover
Conditions
As IcmF like ICM is expected to
deploy radical chemistry with AdoCbl
(Fig. 1), we analyzed whether the
presence of nucleotides affected the
cob(II)alamin levels under steady-
state turnover conditions (Fig. 8B). In
the presence of isobutyryl-CoA, the
spectrum of holo-IcmF was a ϳ1:2
mixture of cob(II)alamin:AdoCbl.
In the presence of nucleotides,
accumulation of cob(II)alamin was
diminished to ϳ1:4 (GDP) and ϳ1:9
(GMPPNP) (Fig. 8B). These results
indicate that the nucleotides influ-
ence the steady-state distribution of
intermediates, which might be
related to their effects on kcat
.
EPR Spectroscopy
The existence of a biradical inter-
mediate has been demonstrated by
EPR spectroscopy for MCM from P.
shermanii with cob(II)alamin cou-
pled to the product radical (29).
However, an EPR spectrum was not
observed when 40 ␮M holo-IcmF
was mixed with 7 mM isobutyryl-
CoA and frozen rapidly. Because
the cob(II)alamin intermediate is
observed by UV-visible spectros-
copy (Fig. 8B), the lack of a para-
magnetic signal suggests strong
coupling between it and the organic
radical species in the IcmF active
site. This has also been observed
with MCM from M. extorquens.4
The EPR spectrum of cob(II)alamin
bound to IcmF was recorded (Fig. 8C).
Binding of cob(II)alamin by IcmF
yields an EPR spectrum that is diag-
FIGURE 5—continued
nostic for the presence of an axial
G. kaustophilus was measured at 37 °C in the coupled
enzyme assay, which is significantly lower than the optimal
growth temperature (60 °C) for the organism (28). Because
purified recombinant IcmF was found to be unstable at high-
er temperatures, its activity at 60 °C could not be measured.
Based on a coefficient of 2 for every 10 °C rise in tempera-
ture, we estimate that the kcat for this enzyme might be
ϳ4-fold higher at 60 °C.
nitrogen ligand. Hyperfine coupling between the unpaired elec-
tron and the S ϭ 7/8 cobalt nucleus results in an eight-line spec-
trum, which is further split into triplets due to superhyperfine cou-
pling to the I ϭ 1 axial nitrogen ligand (Fig. 8C, spectrum 1). The
spectrum of cob(II)alamin bound to IcmF differs from that of free
cob(II)alamin (Fig. 8C, spectrum 4) particularly in the S-shaped
absorption feature at g Ϸ2.3 and probably results from immobili-
zation of the cofactor in the active site. When IcmF was reconsti-
tuted with cob(II)alamin and 5Ј-deoxyadenosine in the presence
or absence of isobutyryl-CoA (Fig. 8C, spectra 2 and 3), the spectra
showed sharpening and resolution of additional hyperfine struc-
Surprisingly, the presence of GDP or GTP affected both the
kcat and Km values (Fig. 8A and Table 2). Thus, the presence of
nucleotides decreased kcat ϳ1.6–1.7-fold while increasing the
Km ϳ2-fold. Consequently, the catalytic efficiency kcat/Km
value of IcmF decreased 3.5- and 4-fold, respectively, in the
presence of GDP and GTP.
4 D. Padovani and R. Banerjee, unpublished results.
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IcmF Is an Isobutyryl-CoA Fusion Protein
ture in the S-shaped feature was
observed. These spectral differences
suggest conformational changes that
influence the electronic properties of
the cob(II)alamin radical.
GTPase Activity of IcmF
Because IcmF possesses an MeaI-
like domain, it was expected that this
protein, like MeaB, can hydrolyze
GTP. Hence, the kinetics of GTP hy-
drolysis catalyzed by apo-IcmF was
characterized. A Michaelis-Menten
analysis of the data yielded the follow-
ing parameters: Km(GTP) ϭ 51 Ϯ 3 ␮M
and kcat ϭ 1.8 Ϯ 0.05 minϪ1 (Fig. 8D).
In comparison, MeaB alone exhibits a
lowerintrinsicGTPaseactivity(kcat ϭ
0.039 Ϯ 0.003 minϪ1), which is
increased ϳ100-fold in the presence
of MCM (15).
MeaI Domain of IcmF Is Distinct
from MeaB
The phylogenetic relationship be-
tween MeaB, the chaperone for
MCM, and the MeaI domain of IcmF
was evaluated. A dendrogram con-
structed from the analysis of MeaB
and MeaI sequences found in the
same organisms reveal that the two
gene groups cluster separately (Fig. 9).
MeaB and MeaI are thus paralogs that
have evolved to serve specific partner
proteins, i.e. MCM and ICM.
The observation of a MeaI do-
main in IcmF raises the obvious
question of whether MeaI chaper-
ones also exists for stand-alone
ICMs. Indeed, as discussed below,
analysis of genomic sequence re-
veals that two MeaB-like proteins
are found in bacterial genomes,
one associated with MCM (MeaB)
and the other with ICM (MeaI). The
diversification of the G domain
sequences within each subgroup
strongly suggests that the MeaI-like
FIGURE 6. Multiple sequence alignment of the MeaI domain in IcmF sequences and MeaB from M.
domain of IcmF is evolutionarily dis-
tinct from MeaB related to MCM.
extorquens (YP_001637793). The MeaI domain of IcmF includes ϳ250–300 amino acids in the middle of the
protein. SequenceanalysisstronglysuggeststhatthisdomainbelongstotheG3EfamilyofP-loopGTPases. The
G domains, which define this family, are present in the MeaI domain of IcmF and are highlighted in red. These
include the following: (i) the Walker A GXXGXGK(SS) motif; (ii) the Mg2ϩ-binding motif, which is typically
(V/I)XXD in proteins in the G3E family and is LXXD in all IcmF sequences; (iii) the DXXXXEXG Walker B motif; and
(iv) the nucleotide specificity NKX(D/E) motif. IcmFs, which were previously annotated as MCM-like enzymes,
are indicated in boldface. For accession numbers see supplemental Table S1.
Identification of Stand-alone ICMs
That Do Not Belong to the Genus
Streptomyces
To investigate the relationships
between the chaperones for ICM versus IcmF, we analyzed
and icmB genes are not necessarily located close to each other,
other bacterial genomes for the presence of stand-alone ICMs and (ii) the amino acid substitutions corresponding to Phe-
and MeaIs. In our search we assumed the following: (i) the icmA 80 and Gln-198 in the S. cinnamonensis sequence are always found
662 JOURNAL OF BIOLOGICAL CHEMISTRY
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IcmF Is an Isobutyryl-CoA Fusion Protein
mophilum. However, in other orga-
nisms, the two subunits are not
close to each other in the genome,
e.g. in Haloarcula marismortui,
Halobacterium sp., and in Natrono-
monas pharaonis (supplemental
Table S2). In some organisms, the
gene order in an ICM-encoding
operon is the following: MeaI, small
subunit of ICM, large subunit of
ICM, and in others the small sub-
unit and MeaI colocalize in an
operon whereas the large subunit is
independently transcribed.
A phylogenetic tree based on
the alignment of stand-alone MeaIs,
the MeaI domain of IcmF and the
MeaBs associated with MCM,
reveals significant overall similarity
between these proteins (supple-
mental Fig. S3). However, a careful
examination of all available MeaI
sequences reveals that this group is
evolutionarily distinct from MeaB
because they form two separate
clusters in the dendrogram. In con-
trast, MeaIs associated with stand-
alone ICMs are closely related to the
corresponding domain in IcmF.
Hence, the MeaI and MeaB are
paralogs that probably evolved from
a common ancestor and have di-
verged to support specific B12-
dependent isomerases.
Implications of the Presence of
IcmF
Gene fusion events occur during
evolution resulting in the physical
coupling of functionally coupled
proteins. It is speculated that gene
fusions that facilitate functional
interactions
between
and/or
coregulation of proteins might be
maintained by selective pressure
and are more common than gene
FIGURE 6—continued
in the large subunit of ICM. A BLAST search using both sub- fissions (30, 31). In this study, we have characterized IcmF, a
units of the stand-alone ICM from S. cinnamonensis as the protein that likely arose by fusion of three genes encoding the
query sequence identified several stand-alone ICM sequences, large and small subunits of ICM and the chaperone MeaI.
primarily in thermophilic archaea but also in halophilic archaea Bioinformatics analysis has allowed identification of Ͼ70
and in a limited number of bacteria (supplemental Table S2 and IcmFs in bacterial and archaeal genomes. However, as noted
supplemental Fig. S2). Furthermore, using the MeaI domain of earlier, all these proteins are incorrectly assigned as represent-
IcmF as a query sequence revealed that genes encoding stand- ing fusions between MCM and MeaB. There are several reasons
alone MeaIs can be associated with either the large or the small that could have led to misannotation of IcmF in the data bases.
subunit of ICM (supplemental Table S2). Interestingly, in sev- First, ICM activity was believed to be restricted to the genus
eral organisms, both ICM subunits are localized in the same Streptomyces, whereas the MCM-catalyzed reaction is impor-
operon or are in close proximity, e.g. in Desulfitobacterium tant in secondary metabolism and is widely distributed in bac-
hafniense, Archaeoglobus fulgidus, and Symbiobacterium ther- teria. Second, the two signature active site substitutions in the S.
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IcmF Is an Isobutyryl-CoA Fusion Protein
cinnamonensis ICM was missed in the IcmF sequence (16). The
importance of these residues in substrate selectivity was previ-
ously demonstrated by mutagenesis studies in which the MCM
double mutant, Y89F/R207Q, was designed to mimic the active
site residues in ICM (32). In contrast to wild-type MCM, the
double mutant bound the ICM substrates, n-butyryl-CoA or
isobutyryl-CoA, but instead of catalyzing an isomerization it
led to inactivation via an internal electron transfer.
In certain bacteria (e.g. Butyrivibrio fibrisolvens and Streptomy-
ces collinus), acetyl-CoA is converted to butyryl-CoA via four
reactions, involving acetyl-CoA acetyltransferase (thiolase), 3-hy-
droxybutyryl-CoA dehydrogenase, 3-hydroxybutyryl-CoA dehy-
dratase (crotonase), and butyryl-CoA dehydrogenase (33–35).
Subjecting the IcmF sequences to operon analysis reveals
that in eight bacterial genomes (Lysinibacillus sphaericus,
Bacillus sp., Bacillus halodurans, Bacillus coagulans, Bacillus
selenitireducens, Bdellovibrio bacteriovorus, Geobacillus sp.,
and Anoxybacillus flavithermus) IcmF is located in the same
operon with enzymes involved in formation of butyryl-CoA
from acetyl-CoA. Based on this analysis, we posit that IcmF is
involved in butyryl-CoA, rather than methylmalonyl-CoA
metabolism in these bacteria.
Third, the role for a MeaB-like chaperone protein has only
been described so far for MCM and could have contributed to
the erroneous assignment. IcmF (previously described as
McmC) was reported to have very low MCM activity (10–12 ϫ
Ϫ3
10 ␮mol minϪ1 mgϪ1) in crude extracts of B. xenovorans, an
organism that lacks the gene encoding a stand-alone MCM
(16). However, when we cloned and purified the B. xenovorans
IcmF, we found it to be devoid of MCM activity, which in fact,
prompted a closer inspection of the protein sequence and
therefore its predicted activity.
Myxalamids are inhibitors of the eukaryotic electron transfer
chain that are produced by the myxobacteria, Myxococcus xan-
thus and Stigmatella aurantiaca (36). In studies on M. xanthus
and S. aurantiaca mutants in which the branched-chain
ketoacid dehydrogenase was disrupted (bkd mutants), it was
shown that isobutyryl-CoA is incorporated into the final
product (37). These results were unexpected because the bkd
mutants are unable to form isobutyryl-CoA starter units from
valine. The authors suggested that fatty acid degradation by ␣-
and ␤-oxidation of iso-odd fatty acid could be responsible for
isobutyryl-CoA synthesis (37). We
Our findings expand our view of the distribution of B12-de-
pendent mutases. We find that ICM activity is much more
widely distributed in nature than previously suspected and
raises questions about the metabolic pathways in which this
activity is involved.
speculate that the ICM activity of
IcmF found in both these bacteria
might play a role in this process
instead.
Another interesting implication
of our study stems from the identi-
fication of stand-alone ICMs in a
number of archaea and bacteria
(supplemental Table S2 and supple-
mental Fig. S2). Recently, Fuchs and
co-workers (17, 38) have reported
the discovery of a novel CO2-fixa-
tion pathway in several archaea.
They have characterized the 16
enzymes in the 3-hydroxypropi-
onate/4-hydroxybutyrate pathway
in Metallosphaera sedula. In this
pathway, two CO2 molecules are
fixed with acetyl-CoA and reduc-
FIGURE 7. Binding isotherms for AdoCbl binding to IcmF. A, representative isothermal titration calorimetry
data set for the binding of AdoCbl (250 ␮M stock solution) to 15.4 ␮M apo-IcmF in 50 mM sodium phosphate
buffer, pH 7.5, 250 mM NaCl, 2 mM TCEP at 20 °C. B, titration curves for the binding of AdoCbl to apo-IcmF alone
(E) and in the presence of 1 mM GDP (ƒ) or 1 mM GMPPNP (ꢂ). Data were fitted to a two-site model and yielded
the parameters reported in Table 1.
tively converted to succinyl-CoA.
An intermediate step in this path-
way is the conversion of methylma-
TABLE 1
Thermodynamic parameters for the binding of AdoCbl to IcmF
All experiments were performed in 50 mM NaPi, pH 7.5, 250 mM NaCl, 1–2 mM TCEP at 20 °C as described under “Experimental Procedures.” The data represent the
mean Ϯ S.D. of three independent experiments.
Ligand
Site
n
Kd
⌬H
T؉⌬S
⌬G
␮M
kcal/mol
kcal/mol
kcal/mol
None
1
2
1
2
1
2
0.9 Ϯ 0.1
1.0 Ϯ 0.2
0.9 Ϯ 0.1
1.2 Ϯ 0.1
1.0 Ϯ 0.1
1.0 Ϯ 0.1
0.081 Ϯ 0.014
1.98 Ϯ 0.42
Ϫ6.2 Ϯ 0.2
Ϫ2.0 Ϯ 0.9
Ϫ8.7 Ϯ 0.7
Ϫ2.4 Ϯ 0.3
Ϫ9.6 Ϯ 0.1
Ϫ0.5 Ϯ 0.7
ϩ3.4 Ϯ 0.3
ϩ5.6 Ϯ 1.0
ϩ0.6 Ϯ 0.8
ϩ5.1 Ϯ 0.4
Ϫ0.4 Ϯ 0.4
ϩ7.4 Ϯ 0.5
Ϫ9.5 Ϯ 0.1
Ϫ7.7 Ϯ 0.1
Ϫ9.2 Ϯ 0.1
Ϫ7.5 Ϯ 0.2
Ϫ9.2 Ϯ 0.3
Ϫ7.9 Ϯ 0.2
GDP
0.132 Ϯ 0.009
2.77 Ϯ 1.27
GMPPNP
0.154 Ϯ 0.071
1.30 Ϯ 0.51
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IcmF Is an Isobutyryl-CoA Fusion Protein
and N. pharaonis stand-alone ICMs
rather than MCMs are predicted to
exist, raising questions about the
presence of an intact 3-hydroxypro-
pionate/4-hydroxybutyrate path-
way in these organisms (supple-
mental Fig. S2). In contrast, in A.
fulgidus and Halorubrum lacuspro-
fundi both copies of the mcm and
icm genes are present. In M. sedula,
only one copy of MCM is present
(supplemental Fig. S2). Fuchs and
co-workers (17) noted that in some
organisms the enzymes from the
first half of the cycle are missing
and proposed that in this situa-
tion reversal of the second half
of the pathway might be important
for acetyl-CoA assimilation into
succinyl-CoA. Interestingly, the
first three reactions of this reverse
sequence (acetoacetyl-CoA ␤-keto-
thiolase, 3-hydroxybutyryl-CoA de-
hydrogenase, and crotonyl-CoA
hydratase) are identical to those in
the acetyl-CoA assimilation path-
way described for Streptomyces col-
linus, which converts acetyl-CoA to
crotonyl-CoA (33). The latter, via
the action of crotonyl-CoA reduc-
tase, is converted to butyryl-CoA,
which is isomerized to isobutyryl-
CoA by the action of a stand-alone
FIGURE 8. Kinetic and spectroscopic characterization of IcmF. A, Michaelis-Menten analysis of the IcmF
ICM. Isobutyryl-CoA can be con-
verted to succinyl-CoA. Thus, in or-
ganisms lacking enzymes in the first
half of the 3-hydroxypropionate/4-
hydroxybutyrate pathway, ICM
may afford an alternative route for
assimilation of acetyl-CoA.
reaction as determined in the coupled enzyme assay. The specific activity (S.A.) of IcmF, analyzed alone (E) or
in the presence of GDP (ƒ) or GTP (ꢂ), yielded the kinetic parameters reported in Table 2. B, UV-visible spectra
of holo-IcmF under steady-state turnover conditions. Holo-IcmF (10.3 ␮M, solid line) was incubated at 20 °C for
2 min with 3.4 mM isobutyryl-CoA (dotted line) in the presence of 2 mM GDP (dashed line) or GMPPNP (dashed-
dotted line). C, EPR spectra of IcmF (28 ␮M) reconstituted with 20 ␮M cob(II)alamin (spectrum 1) in the presence
of 10 mM 5Ј-deoxyadenosine (spectrum 2) and 8 mM isobutyryl-CoA (spectrum 3). An EPR spectrum of free
“base-on” cob(II)alamin (spectrum 4) is shown for comparison. D, Michaelis-Menten analysis of the GTPase
activity of IcmF determined as described under “Experimental Procedures.”
It is interesting how MCM-like enzymes have evolved dis-
tinct substrate specificities by virtue of very limited changes in
their active site residues. Muller and co-workers (39, 40)
described a B12-dependent enzyme that is involved in the path-
way of degradation of fuel oxygenates. This enzyme in Methyl-
ibium petroleiphilum PM1 was shown to convert 2-hydroxy-
isobutyryl-CoA into 3-hydroxybutyryl-CoA. The remarkable
feature of this enzyme is that it resembles ICM and has two
subunits, IcmA and IcmB. However, in the active site of IcmA,
Phe is substituted by Ile, whereas Gln is conserved (see Fig. 5 in
TABLE 2
Kinetic parameters for IcmF
All experiments were performed in 50 mM NaPi, pH 7.5, 250 mM NaCl at 37 °C as
described under “Experimental Procedures.” The data represent the mean Ϯ S.D. of
three independent experiments.
Nucleotide
None
GDP
GTP
K
m (isobutyryl-CoA),
20.1 Ϯ 1.3
45.3 Ϯ 4.0
50.8 Ϯ 2.6
␮M
Ϫ1
k
cat, s
3.10 Ϯ 0.05
1.96 Ϯ 0.04
1.88 Ϯ 0.08
Ϫ1
k
cat/Km, M Ϫ1 s
(1.5 Ϯ 0.1) ϫ 105 (4.3 Ϯ 0.5) ϫ 104 (3.7 Ϯ 0.4) ϫ 104
lonyl-CoA to succinyl-CoA, which is catalyzed by MCM. The Ref. 39). It is interesting that M. petroleiphilum also has a copy
majority of MCMs in bacteria are heterodimers, in which one of of the icmF and mcm genes (based on amino acid substitutions
the subunit binds the substrate and the cofactor. Although M. in the active site sequences). Another example of subtle alter-
sedula clearly encodes MCM in its genome, some of the puta- ations in substrate specificity is seen in ethylmalonyl-CoA
tive mutases in other organisms that were identified as MCMs mutase from Rhodobacter sphaeroides (41). This enzyme inter-
(see supplemental Table S1 in Ref. 17) are predicted to be stand- converts ethylmalonyl-CoA and methylsuccinyl-CoA. Like
alone ICMs based on the Tyr 3 Phe/Arg 3 Gln substitutions MCM, ethylmalonyl-CoA mutase is predicted to have Tyr and
in their active sites. Thus, in H. marismortui, Halobacterium sp., Arg residues in the active site. However, to utilize the larger
JANUARY 1, 2010•VOLUME 285•NUMBER 1
JOURNAL OF BIOLOGICAL CHEMISTRY 665

IcmF Is an Isobutyryl-CoA Fusion Protein
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FIGURE 9. Phylogenetic tree of MeaBs that is located in operons with
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the same operon with MCM are as follows: MeaB(AAL86727), B. halodurans
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Geobacter metallireducens (YP_385162), Leptospira borgpetersenii (YP_
799393), M. xanthus (YP_630483), and N. farcinica (YP_119677). MeaI
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