The crystal structure of putative precorrin isomerase CbiC
in cobalamin biosynthesis
Yanyan Xuea,b, Zhiyi Weia,b, Xu Lib, Weimin Gonga,b,*
aNational Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, PR China
bSchool of Life Sciences, University of Science and Technology of China, Hefei, Anhui 230026, PR China
Received 28 July 2005; received in revised form 24 November 2005; accepted 29 November 2005
Available online 27 December 2005
The leptospira cbiC encodes the enzyme catalyzing the methyl rearrangement reaction of the cobalamin biosynthesis pathway. The
protein has been cloned and overexpressed as a His-tagged recombinant protein in Escherichia coli. The crystal structures have been
solved in two crystal forms (P42212 and P3121) diffracting to 3.0 and 2.3 A˚resolution, respectively. The structures are similar to the
precorrin-8x methyl mutase (CobH), an enzyme of the aerobic pathway to vitamin B12.
? 2005 Elsevier Inc. All rights reserved.
Keywords: Leptospira interrognans; Leptospirosis; Vitamin B12; Precorrin isomerase; Crystal structure
Cobalamin (vitamin B12) is one of the most structurally
complex protein cofactors of the nature. Most prokaryotic
organisms as well as animals (including humans) and pro-
tists have enzymes that require cobalamin as cofactor,
whereas plants and fungi are thought not to use it. Among
the cobalamin-utilizing organisms, only some bacterial and
archaeal species are able to synthesize cobalamin de novo
(Rodionov et al., 2003). Studies over the past decade have
demonstrated that there are at least two quite distinct
routes for cobalamin synthesis, representing oxygen-depen-
dent (aerobic) and oxygen-independent (anaerobic) path-
ways (Scott and Roessner, 2002). The two pathways
differ in their timing of cobalt insertion and the require-
ment for molecular oxygen (Raux et al., 1999; Schubert
et al., 1999).
All the intermediates between uro’gen III and cobina-
mide are known for the aerobic pathway, in contrast, few
intermediates on the anaerobic pathway are known. The
genes encoding the aerobic pathway enzymes are prefixed
Cob, whereas the genes encoding the anaerobic pathway
are prefixed Cbi (Roper et al., 2000). Many of the aerobic,
Cob, enzymes share a high degree of similarity with the
anaerobic, Cbi, suggesting that although independent, the
two pathways are broadly similar (Roth et al., 1993).
Leptospira interrognans is most common in tropical
areas and infects animals. Infection can cause mild symp-
toms or more serious disease, known as leptospirosis. Here,
we report the crystal structure of the L. interrognans puta-
tive CbiC (CbiC_LEPIN) with two different crystal forms,
P42212 and P3121. CbiC, a cobalt-precorrin 8 isomerase,
catalyzes cobalt-precorrin 8 to cobyrinic acid by methyl
rearrangement in anaerobic pathway (Roessner et al.,
1992; Roth et al., 1993). The specific homology of CbiC
in aerobic pathway is CobH, which shows high similarity
to CbiC despite the divergence between the two pathways.
Structure analysis suggests that CbiC_LEPIN are highly
similar with CbiC/CobH from other sources in fold and
has small differences in surrounding of cobalt-precorrin 8
binding. The CbiC_LEPIN structure will provide clues
for understanding the function of CbiC/CobH family in
the biosynthesis pathway.
1047-8477/$ - see front matter ? 2005 Elsevier Inc. All rights reserved.
E-mail address: email@example.com (W. Gong).
Journal of Structural Biology 153 (2006) 307–311
2. Materials and methods
2.1. Protein expression and purification
The gene cbiC from L. interrogans was cloned into a
pET-22b (Novagen) expression plasmid to yield pET-His-
cbiC. The plasmid was transformed into Escherichia coli
strain BL21 (DE3) for protein expression. Five milliliter
aliquots of an overnight culture were subcultured into
400 ml fresh Luria–Bertani medium (10 g Bacto tryptone,
5 g yeast extract, and 10 g NaCl per liter of solution) con-
taining ampicillin (50 lg/ml) and allowed to grow at 310 K
until OD600= 0.6. At this stage isopropyl-b-D-thiogalacto-
side was added to a final concentration of 0.2 mM and the
cells were grown for a further 20 h at 289 K. Then cells
were harvested by centrifugation at 8000 rpm for 10 min.
The cell pellet was resuspended in binding buffer (20 mM
Tris/HCl, pH 7.5, 0.2 M NaCl) and sonicated using a
JY92-II sonic dismembrator. The solution was centrifuged
again (16000 rpm, 4.5 min) and the supernatant was
applied in a Ni-affinity column equilibrated with binding
buffer. The column was then washed with 10 column vol-
umes of binding buffer, 6 column volumes of wash buffer
(50–100 mM imidazole, 0.2 mM NaCl, and 20 mM Tris/
HCl, pH 7.5), and finally the protein was eluted in 6 col-
umn volumes of elution buffer (200 mM imidazole,
0.2 mM NaCl, and 20 mM Tris/HCl, pH 7.5). The frac-
tions were checked by SDS–PAGE and those containing
CbiC_LEPIN protein were concentrated by ultrafiltration
equipment. The final protein concentration was measured
with a Bio-Rad Protein Assay kit (Bio-Rad Pacific, USA).
2.2. Crystallization and preliminary data collection
Preliminary crystallization conditions were screened
using Crystal Screen I and II (Hampton Research, CA,
USA) with the handing-drop vapour-diffusion technique
at 277 K. Drops containing equal volumes (1 ll) of protein
(15 mg/ml) and reservoir solution were equilibrated against
400 ll reservoir solution. Two conditions ([10%(v/v)
PEG6000, 2.0 M sodium chloride] and [20%(v/v) ethanol,
0.1 M Tris, pH 8.5]) produced CbiC_LEPIN protein crys-
tals with P42212 and P3121 crystal forms, respectively.
More suitable crystals for diffracting experiment were
obtained in [15%(v/v) ethanol and 0.1 M Tris, pH 8.0] after
optimizing the second condition. Preliminary diffraction
data were collected on a MAR Research image-plate sys-
tem with a local X-ray source at 100 K with 1.5418 A˚wave-
length. All data were processed and scaled with the
DENZO and SCALEPACK (Otwinowski and Minor,
1997). The processing statistics of the two different crystal
forms are summarized in Table 1.
2.3. Structure determination
The crystal structure of CbiC_LEPIN with the P42212
crystal form was solved using Molrep (Vagin and
Teplyakov, 1997). The monomer structure of Precorrin-
8x Methyl Mutase from Thermus thermophilus (PDB code:
1V9C) was used as a search model for the molecular
replacement. The model consisting of a dimer was refined
in 20–3.0˚ Aresolution range by using CNS (Brunger
et al., 1998) with maximum-likelihood amplitude targets
and manually adjusted and rebuilt of the model using the
program O (Jones et al., 1991) with 2Fo? Fcand Fo? Fc
electron-density maps as references. Water molecules were
added to the model and individual atomic B factor was
refined at latter stage of refinement. NCS restraints were
applied through all stages of refinement. The final Rcryst
and Rfreefactors are 20.4 and 25.1%, respectively. A mono-
mer structure of this refined model was used as a search
model to find solutions for the P3121 crystal form with
one molecule per asymmetric unit. The refinement strategy
Data collection and structure refinement statistics of two forms crystal of
Unit cell parameters
a = b = 113.08,
c = 114.50
a = b = 55.89,
c = 142.55
1Molecules per asymmetric
Resolution range (˚ A
) 20–3.0 (3.07–3.00)50–2.3
No. of total reflections
No. of unique reflections
Resolution (˚ A
No. of reflections
Rmsd from ideal values
Bond length (˚ A
Bond angels (?)
Average B-factor (˚ A
No. of atoms
) 20–3.0 (3.19–3.0)
20.4 (30.2)/25.1 (38.1) 22.4 (20.5)/28.4 (26.8)
71 (including 8
Most favored regions (%) 85.1
Additionally allowed (%) 14.4
Generously allowed (%)
Numbers in parentheses are for the highest resolution shell.
aRmerge=P|Ii? Im|/PIi, where Ii is the intensity of the measured
and calculated structure factors. Rfree=P
and set aside prior to refinement.
cValues for the two different monomers (A and B) respectively in an
reflection and Imis the mean intensity of all symmetry-related reflections.
P||Fobs| ? |Fcalci/P|Fobs|, where Fobsand Fcalcare observed
T is a test data set of about 10% of the total reflections randomly chosen
TiFobs| ? |Fcalci/P
Y. Xue et al. / Journal of Structural Biology 153 (2006) 307–311
of the P3121 structure is similar to the P42212 structure
with the final Rcryst and Rfree factors 22.4 and 28.4%,
respectively, in 50–2.3˚ A
resolution range. The stereochem-
ical qualities of the final models of two crystal forms were
checked by PROCHECK (Laskowski et al., 1993), and the
final refinement statistics and geometry are listed in Table
1. The relatively high Rfreefactor value of the P3121 struc-
ture is mainly dues to existing obvious ice rings during data
3. Results and discussion
3.1. Protein preparation and crystallization
The recombinant protein of CbiC_LEPIN was overex-
pressed at 310 K at first, but the protein was insoluble. Var-
ious expression conditions were tried to increase solubility
of the protein. Finally, the recombinant protein was
expressed solubly in higher yield under the following condi-
tions: 289 K, 0.2 mM IPTG, 20 h induction in strain BL21
(DE3). The protein was purified using the nickel-affinity
column and the sample was monitored by SDS–PAGE
after purification and concentration. The purified His-
tagged putative CbiC protein was used for crystallization.
After an initial screen, two crystal forms of the protein
were observed. The polyhedral crystal form was only
obtained a 7–8 A˚resolution data at first. After optimiza-
tion and using a cryoprotectant solution (10% glycerol)
before flash freezing the crystal to liquid nitrogen, the res-
olution was improved greatly. The maximum resolution
was 2.3 A˚and the space group was hexagonal P3121, with
a = b = 55.89 A˚,
a = b = 90?, c = 120?. The rod-shaped crystal form was
obtained a 3.0 A˚resolution data set. The space group
a = b = 113.08 A˚, c = 114.50 A˚, a = b = c = 90?.
c = 142.55 A˚,
3.2. Structure overview
The final models contain 215 residues in one molecule of
P3121 crystal form and 434 residues (217 residues each
molecule) in two molecules (forming a dimer) of P42212.
N-terminal residues, Met1–Gln5 (in P3121) and Met1–
Asp3 (in P42212), the C-terminal residues, Glu222–
Arg223 (in P3121) and Gly221–Arg223 (in P42212), as well
as the His?tag were not visible in electron-density maps.
The three monomer structures (one in P3121 and two in
P42212) are almost identical with the average root-mean-
square deviation (rmsd) about 0.46 A˚(for 215 Ca between
P3121 and any one of P42212 monomer structures) or
about 0.26 A˚(for 217 Ca between two P42212 monomer
structures). The CbiC_LEPIN structure contains 11 a-heli-
ces and a six-stranded mixed b-sheet in which the five
strands (b2–b6) are parallel and the edge strand b1 run
anti-parallel to b6. The b-sheet is surrounded by four
a-helices in one side and seven in another (Fig. 1A).
CbiC_LEPIN forms a dimer in both of the two crystal
forms with a crystallographic (in P3121 crystal) or a non-
crystallographic (in P42212 crystal) 2-fold symmetry
between the two monomers (Fig. 1B). The dimer is formed
by both hydrophobic and hydrophilic interactions (includ-
ing some salt bridges.) The dimerization form of CbiC_LE-
PIN is in agreement with other CbiC/CobH structures. It
suggests that dimerization is necessary for having biologi-
cal function in CbiC/CobH family. Two identical cavities
formed by two CbiC/CobH molecules in a dimer are also
essential for their substrates binding (Shipman et al., 2001).
3.3. Comparison with other CbiC/CobH
Sequence and structural alignment reveal that Cbi-
C_LEPIN is fairly similar to other CbiC/CobH (Figs. 2A
and B). Farther structural analyses show that CbiC/CobH
Fig. 1. The ribbons diagram of the monomer and the dimer structure of CbiC_LEPIN. (A) The protein is color-coded by secondary structure types. (B)
The two monomers are related by a crystallographic (in P3121 structure) or a non-crystallographic (in P42212 structure) 2-fold screw axis where is showed
as ‘‘2.’’ This figure and Fig. 2B were prepared with RIBBONS (Carson, 1997).
Y. Xue et al. / Journal of Structural Biology 153 (2006) 307–311
family members also have high similarity in shape of the
two identical cavities (Fig. 2B) and centralized distribu-
tions of conserved residues on and around the surface of
the cavities (Figs. 2A and C). Recently, the complex struc-
ture of Pseudomonas denitrificans CobH (CobH_PSEDE)
binding with hydrogenobyrinic acid (HBA), which is the
product of the reaction catalyzed by CobH, has been
solved (Shipman et al., 2001). This structure shows that
the cavities supply the binding surface for the substrates.
And comparing with the complex structure, conformation
of residues on the cavities of the CobH_PSEDE native
structure does not show obvious changes. Thus, although
without substrates binding, the similarity of the cavities
in CbiC/CobH family suggests that cobalt-precorrin 8,
Fig. 2. (A) Sequence alignment of CbiC_LEPIN and other CbiC/CobH protein. The sequences of CbiC/CobH from Leptospira interrognans (LEPIN),
Pseudomonas denitrificans (PSEDE), Methanococcus jannaschii (METJA), Synechocystis sp. strain PCC 6803 (SYNY3), Salmonella typhimurium (SALTY),
Methanobacterium thermoautotrophicum (METTH), Mycobacterium tuberculosis (MYCTU), Thermus thermophilus (THETH), and Thermoplasma
acidophilum (THEAC) were aligned using ClustalW (Notredame et al., 2000). The last two protein sequences come from PDB, no related information of
these two proteins can be obtain from other protein sequence databases; thus, the two protein are putative CbiC/CobH protein. The alignment was edited
with referenced to structural alignment results to avoid gaps inside the conserved secondary structural elements. The secondary structure of CbiC_LEPIN,
which is defined by the analysis of the structure using DSSP program (Kabsch and Sander, 1983), is indicated above the alignment. Residues in the
alignment that are identical are shown in red boxes; those that are similar are shown in yellow boxes. The residues that are involved in hydrophobic
interactions or hydrophilic interactions with HBA in structure of CobH_PSEDE (PDB id: 1I1H) are marked by solid circles or hollow circles, respectively.
This figure was prepared with ESPript (Gouet et al., 1999). (B) The superposition of the structure of CbiC_LEPIN (red), CobH_PSEDE (blue), and other
CbiC/CobH from Thermus thermophilus (pink, PDB id: 1V9C) and Thermoplasma acidophilum (yellow, PDB id: 1OU0). The structural alignment was
performed using O (Jones et al., 1991). The two black arrows denote that the locations of halves of the two identical cavities, respectively, in monomer
structures. (C) Surface distributions of conserved residues in CbiC_LEPIN. The two identical cavities are marked by circles on the left picture. The right
picture is a 90? rotated view around a vertical axis in which only one cavity can be seen.
Y. Xue et al. / Journal of Structural Biology 153 (2006) 307–311
which is the substrate of CbiC, is highly similar to the sub- Download full-text
strate of CobH, precorrin 8.
Although the residues that composed the cavities are
mostly conserved, there are still some non-conserved resi-
dues distribute on the surface of the cavities. These non-
conserved residues might respond for different substrates
(cobalt-precorrin 8 or precorrin 8) binding. In addition,
N-terminal helices functioning to cap the active site of cav-
ity in CbiC/CobH structures are probably disordered in a
precorrin-8x methylmutase related protein from Thermo-
plasma acidophilum (Fig. 2B), which is a putative CbiC/
CobH family member.
There is little biochemical characterization of CbiC_LE-
PIN and consequently the information that can be derived
from the structure reported here is limited. Whether bind-
ing mode of cobalt-precorrin 8 is highly similar to that of
precorrin 8 and what the cobalt-precorrin 8 is, need to be
This work was supported by National Foundation of
Talent Youth (Grant No. 30225015), The 973 programs
(Grant Nos. 2004CB720000 and 2004CB520801), the Key
Important Project and other projects from the National
Natural Science Foundation of China (Grant Nos.
10490913, 30121001, and 30130080) and Chinese Academy
of Sciences (KSCX2-SW-224). We thank Prof. Peng Liu
and Yuhui Dong in Institute of High Energy Physics,
and Yi Han in Institute of Biophysics for diffraction data
collection. The atomic coordinates and structure factors
of the CbiC structure of the two crystal forms (P3121
and P42212) have been deposited in the Protein Data Bank
as 2AFR and 2AFV, respectively.
Brunger, A.T., Adams, P.D., Clore, G.M., DeLano, W.L., Gros, P.,
Grosse-Kunstleve, R.W., Jiang, J.S., Kuszewski, J., Nilges, M., Pannu,
N.S., Read, R.J., Rice, L.M., Simonson, T., Warren, G.L., 1998.
Crystallography & NMR system (CNS): a new software suite for
macromolecular structure determination. Acta. Crystallogr. D 54,
Carson, M., 1997. Ribbons. Methods Enzymol. 277, 493–505.
Gouet, P., Courcelle, E., Stuart, D.I., Metoz, F., 1999. ESPript: multiple
sequence alignments in PostScript. Bioinformatics 15, 305–308.
Jones, T.A., Zou, J.Y., Cowan, S.W., Kjeldgaard, M., 1991. Improved
methods for building protein models in electron density maps and the
location of error in these models. Acta Crystallogr. A 47, 110–119.
Kabsch, W., Sander, C., 1983. Dictionary of protein secondary structure:
pattern recognition of hydrogen-bonded and geometrical features.
Biopolymers 22, 2577–2637.
Laskowski, R.A., MacArthur, M.W., Moss, D.S., Thornton, J.M., 1993.
PROCHECK: a program to check the stereochemical quality of
protein structures. J. Appl. Cryst. 26, 283–291.
Notredame, C., Higgins, D.G., Heringa, J., 2000. A novel algorithm for
multiple sequence alignment. J. Mol. Biol. 302, 205–217.
Otwinowski, Z., Minor, W., 1997. Processing of X-ray diffraction data
collected in oscillation mode. In: Carter, C.W., Sweet, R.M. (Eds.),
Methods in Enzymology 276, Macromolecular Crystallography, part
A, Academic Press, New York, pp. 307–326.
Raux, E., Schubert, H.L., Roper, J.M., Wilson, K.S., Warren, M.J., 1999.
Vitamin B12: insights into biosynthesis’s mount improbable. Bioorg.
Chem. 27, 100–118.
Rodionov, D.A., Vitreschak, A.G., Mironov, A.A., Gelfand, M.S., 2003.
Comparative genomics of the vitamin B12 metabolism and regulation
in prokaryotes. J. Biol. Chem. 278, 41148–41159.
Roessner, C.A., Warren, M.J., Santander, P.J., Atshaves, B.P., Ozaki, S.,
Stolowich, N.J., Iida, K., Scott, A.I., 1992. Expression of 9 Salmonella
typhimurium enzymes for cobinamide synthesis. Identification of the
11-methyl and 20-methyl transferases of corrin biosynthesis. FEBS
Lett. 301, 73–78.
Roper, J.M., Raux, E., Brindley, A.A., Schubert, H.L., Gharbia, S.E.,
Shah, H.N., Warren, M.J., 2000. The enigma of cobalamin (vitamin
B12) biosynthesis in Porphyromonas gingivalis. J. Biol. Chem. 275,
Roth, J.R., Lawrence, J.G., Rubenfield, M., Kieffer-Higgins, S., Church,
G.M., 1993. Characterization of the cobalamin (vitamin B12)
biosynthetic genes of Salmonella typhimurium. J. Bacteriol. 175,
Schubert, H.L., Raux, E., Wilson, K.S., Warren, M.J., 1999. Common
chelatase design in the branched tetrapyrrole pathways of heme and
anaerobic cobalamin synthesis. Biochemistry 38, 10660–10669.
Scott, A.I., Roessner, C.A., 2002. Biosynthesis of cobalamin (vitamin
B(12)). Biochem. Soc. Trans. 30, 613–620.
Shipman, L.W., Li, D., Roessner, C.A., Scott, A.I., Sacchettini, J.C., 2001.
Crystal structure of precorrin-8x methyl mutase. Structure 9, 587–596.
Vagin, A., Teplyakov, A., 1997. MOLREP: an automated program for
molecular replacement. J. Appl. Cryst. 30, 1022–1025.
Y. Xue et al. / Journal of Structural Biology 153 (2006) 307–311