Anaerobic 5-Hydroxybenzimidazole Formation from Aminoimidazole Ribotide: An Unanticipated Intersection of Thiamin and Vitamin B12 Biosynthesis

Article abstract of DOI:10.1021/jacs.5b03576

Comparative genomics of the bacterial thiamin pyrimidine synthase (thiC) revealed a paralogue of thiC (bzaF) clustered with anaerobic vitamin B₁₂ biosynthetic genes. Here we demonstrate that BzaF is a radical S-adenosylmethionine enzyme that catalyzes the remarkable conversion of aminoimidazole ribotide (AIR) to 5-hydroxybenzimidazole (5-HBI). We identify the origin of key product atoms and propose a reaction mechanism. These studies represent the first step in solving a long-standing problem in anaerobic vitamin B₁₂ assembly and reveal an unanticipated intersection of thiamin and vitamin B₁₂ biosynthesis.

Full text of DOI:10.1021/jacs.5b03576

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Anaerobic 5-Hydroxybenzimidazole formation from aminoimidaz-
ole ribotide: an unanticipated intersection of thiamin and vitamin
B12 biosynthesis
.
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Angad P. Mehta1, Sameh H. Abdelwahed1,4, Michael K. Fenwick2, Amrita B. Haz-
ra3, Michiko E. Taga3, Yang Zhang2, Steven E. Ealick2 and Tadhg P. Begley1*
.
1Department of Chemistry, Texas A&M University, College Station, TX-77843, USA. *begley@chem.tamu.edu
2 Department of Chemistry and Chemical Biology, Cornell University, 120 Baker Lab, Ithaca, New York 14853,
USA
3 Department of Plant and Microbial Biology, University of California, Berkeley, USA. 4Therapeutic chemistry de-
partment, National research center, Dokki, Cario, Egypt.
Supporting Information Placeholder
ABSTRACT: Comparative genomics of the bacterial
thiamin pyrimidine synthase (thiC) revealed a paralog
of thiC (bzaF) clustered with anaerobic vitamin B12
biosynthetic genes. Here we demonstrate that BzaF is
a radical S-adenosylmethionine enzyme that catalyz-
es the remarkable conversion of aminoimidazole ribo-
tide (AIR) to 5-hydroxybenzimidazole (5-HBI). We
identify the origin of key product atoms and propose a
reaction mechanism. These studies represent the first
step in solving a long-standing problem in anaerobic
vitamin B12 assembly and reveal an unanticipated
intersection of thiamin and vitamin B12 biosynthesis.
The formation of dimethylbenzimidazole (DMB, 3),
Figure 1: The oxygen-dependent and oxygen-
independent biosynthesis of the DMB ligand of vita-
min B12. (A) The structure of vitamin B12 1 showing
the DMB ligand in red. (B) Oxygen-dependent DMB
formation from FMN 2. (C) Labeling studies showing
the oxygen-independent biosynthetic origin of the at-
oms of DMB in Eubacterium limosum. 5-
Hydroxybenzimidazole (HBI) and 5-hydroxy-6-
methylbenzimidazole have been identified as precur-
sors.11
the lower axial ligand of vitamin B12 (cobalamin), 1,
(Figure 1A) is the last remaining major unsolved puz-
zle in B12 biosynthesis. In aerobic organisms, DMB is
biosynthesized from flavin mononucleotide (FMN, 2)
in a remarkable reaction catalyzed by the BluB gene
product (Figure 1B). This oxygen requiring DMB syn-
thase has been biochemically and structurally charac-
terized, but its mechanism is not yet known.1,2 The
anaerobic pathway to DMB involves different chemis-
try and labeling studies demonstrate that FMN is not
the precursor (Figure 1C).3-6 The anaerobic DMB syn-
thase has not been yet identified.
Our approach to elucidating the anaerobic biosyn-
thesis of DMB was indirect and derived from our stud-
ies on the bacterial thiamin pyrimidine synthase
(ThiC). This radical SAM enzyme catalyzes a remark-
able rearrangement of aminoimidazole ribotide (AIR,
10) to the thiamin pyrimidine (12, Figure 2).7-10
Figure 2: The remarkable, ThiC-catalyzed, conver-
sion of AIR 10 to the thiamin pyrimidine 12.
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2
While looking for a ThiC paralog, to facilitate our
of H- and 13C-AIR isotopologs to HBI followed by the
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mechanistic studies on this complex reaction, we no-
ticed that some anaerobes in the SEED database
(http://theseed.uchicago.edu/FIG/) had a thiC paralog
(bzaF) clustered with vitamin B12 biosynthetic genes
(Figure 3). This suggested the possibility that BzaF
might catalyze the conversion of AIR to a DMB pre-
cursor. Previous in vivo labeling studies in E. limo-
sum, using 4-aminoimidazole, eliminated this hetero-
cycle as a DMB precursor.12 In this communication we
report the successful reconstitution of the BzaF-
identification of the site of labeling by NMR and MS
analysis. The results of these experiments are shown
in Figure 5 and in Table S1 (See also Figures S20-
S29). The key findings, summarized in Figure 5A are
as follows: 1) The proS hydrogen at C5 of AIR is ab-
stracted by the adenosyl radical (Table S1, lines 2-4).
2) The C7 hydrogen of HBI is derived from the C5′
proR hydrogen of AIR (Table S1, line 3 and Figure
5F). 3) The C6 hydrogen of HBI is derived from the
C4′ hydrogen of AIR (Table S1, line 5 and Figure 5E).
4) The C1′ carbon of AIR is lost as formate (Table S1,
lines 6-9 and Figure 5GHI). 5) The C5 amino group
nitrogen of AIR is partially retained in the product (Ta-
ble S1, line 10, 62% retention, see legend to Table
S1). 6) The C4 carbon of HBI is derived from the C2′
carbon of AIR (Figure 5D). From these experiments,
we can deduce the origin of all of the atoms of HBI as
shown in Figure 6.
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catalyzed
conversion
of
AIR
to
5-
hydroxybenzimidazole (HBI), we identify the hydrogen
atom initially abstracted by the 5′-deoxyadenosyl rad-
ical and the origin, in AIR, of key HBI atoms. These
results are integrated into a mechanistic hypothesis
for the BzaF (HBI synthase) catalyzed reaction.
- Full reaction
(A)
5’-deoxyadenosine
5’+deoxyadenosine*
Recons' tu' on*+*HPLC*(290nm)*
- No AIR
Figure 3: The bzaF gene cluster in Desulfuromonas
acetoxidans. cobCUT, cobSD and cbiBC have estab-
lished functions in vitamin B12 biosynthesis. BzaF has
38% sequence identity and 53.6% sequence similarity
to the D. acetoxidans ThiC. (Bza is the IUPAC-IUB
abbreviation for "benzimidazolyl".)
- No SAM
) *++#,- . /012#
- No Dithionite
31#4. 5678# - No HBI synthase
5-HBI
31#9:; #
31#<9= #
31#47>67127>- #
! "#$%
! ( #
! &%
31#
! &#$%
31.5# 32#
! ' %
! "#
! $#
20# 21# 22#
! ! #
! %#
23#
! &#
! ' #
25#
24#
! " #$%" &' ($
! " #$%" &' ($
Time (min)
The bzaF gene from D. acetoxidans was synthe-
sized with codon optimization (for gene details - Fig-
ure S33), cloned in the THT vector and co-expressed
in the presence of a plasmid encoding the suf operon
for in vivo [4Fe-4S] reconstitution in Escherichia coli
BL21 (DE3) (See supporting information - page 13).
The enzyme was then purified under anaerobic condi-
tions using Ni-NTA chromatography (see SI for exper-
imental details). The enzyme yield was 3 mg/L of cul-
ture. BzaF has a molecular mass of 48 kDa, and con-
tains 3 irons and 2.6 sulfides/monomer. The UV-
Visible spectrum of the purified protein showed the
long wavelength absorption characteristic of the [4Fe-
4S]+2 cluster (Figure 4B).
(B)
0.6
(C)
-
-
135.05&
0.6"
135.05&
135.0554
Full reaction
(D)
0.5
0.5"
0.4"
mA
5-HBI,
0.4
reference
standard
0.3
mA#0.3"
0
0
.
.
2
2"
1"
0"
-
Full reaction
+ Reference
standard
0
0
.
.
1
175.1197
150.1097
115.0375
0
250"
250
350"
450"
550"
550
350
wavelength#(nm)#
450
! "&$
! %&$ ! %#$
130& ! "#$ 140&
145&
135&
20#
21#
22#
23#
24#
24
22 23
20
21
Wavelength (nm)
! me$(min)$
Time (min)
m/z$
Figure 4: Characterization of the product of the
BzaF catalyzed reaction. (A) HPLC (254 nm) analysis
of the HBI synthase reaction mixture showing a new
product eluting at 22.7 min and 5′-dA eluting at 31
min (blue trace). This product was not formed in re-
action mixtures lacking dithionite (red trace). (See
Figure S31 for all controls). (B) UV-Vis spectrum of
HBI synthase as isolated. (C) Product identification by
HPLC analysis (red trace – HBI, blue trace – full en-
zymatic reaction, green trace - co-injection of HBI and
the reaction product). (D) MS analysis of the 22.7 min
enzymatic product showing [M+H]+ = 135.05 Da, con-
sistent with HBI formation.
Anaerobic incubation of AIR and SAM with dithio-
nite-reduced BzaF (RT, 60 min) resulted in the for-
mation of 5′-deoxyadenosine (5′-dA) and a new prod-
uct as determined by HPLC analysis (Figure 4A). This
product had an [M+H]+ of 135.05 Da and co-migrated
with an authentic standard of HBI (Figure 4C and D).
This identification was confirmed by scale-up of the
reaction followed by NMR analysis of the purified
product. The ratios of HBI and 5′-dA to BzaF were 0.7
(HBI:BzaF) and 1.1 (5′dA:BzaF) respectively. The
corresponding ratios for ThiC are 0.4 (HMP-
P:Caulobacter crescentus ThiC) and 0.6 (5′dA:C.
crescentus ThiC).
These labeling studies allow us to propose a mech-
anistic hypothesis for the HBI-synthase-catalyzed re-
action (Figure 7A). As expected from the sequence
similarity with ThiC, HBI formation is initiated by the
abstraction of the C5′ proS hydrogen of AIR by the 5′-
deoxyadenosyl radical to give 15. C-O bond cleavage
followed by N-glycosyl bond cleavage gives 17. At
this point, the chemistry diverges from the proposed
ThiC pathway (See Figure S32). Electrophilic addition
As a first step in elucidating the mechanism of HBI
synthase, we have determined the origin of key atoms
of HBI. Our strategy involved the conversion of a set
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trace of unlabeled AIR in the sample (See Figure S30
of the ribose C4′ to the aminoimidazole leads to the
for 13C-NMR).
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5
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thiamin pyrimidine while electrophilic addition of the
ribose C5′ gives 19 which leads to DHBI. Loss of
phosphate followed by deprotonation gives 21, which
is quenched by hydrogen atom donation from an un-
known source. A ring opening/closing sequence gives
24. Loss of ammonia from 24 gives imine 25 in which
38% of the imine nitrogen is derived from N1 of AIR
and the remainder is derived from the C5 amino
group. This is consistent with the partial scrambling of
the two amidine nitrogens in 23 by C-C bond rotation.
Loss of formate, ring closure and a series of dehydra-
tions and tautomerizations complete the formation of
HBI 14.
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Figure 6: Origin of all of the atoms of HBI based on
the labeling studies shown in Figure 5 and in Table
S1.
A homology model for HBI synthase was construct-
ed using the structure of the Arabidopsis thaliana
ThiC/AIR/SAH/Fe complex as a template.13 HBI syn-
thase lacks the chloroplast-targeting sequence specif-
ic to the Arabidopsis ThiC as well as the N-terminal
ThiC domain.7,13,14 This N-terminal domain is also
missing from ThiCs from anaerobic organisms. HBI
synthase is predicted to have the noncanonical active
site architecture recently reported for ThiC with an
additional iron chelating two conserved histidine resi-
dues and the amino and carboxylate groups of
SAM.13 The homology model was consistent with the
observed abstraction of the 5’-ProS hydrogen atom of
the substrate and showed remarkable conservation of
active site residues with only four differences out of
21 residues in the first shell when compared to ThiC
(Figure 7B - 7C). Three of the differences involve con-
tacts with AIR. In ThiC, Asn228 hydrogen bonds to
AIR O3′ and is replaced by Ser66 in HBI synthase.
Thr320 hydrogen bonds to Glu422, which in turn hy-
drogen bonds to AIR O2′, and is replaced by Ala161
in HBI synthase; Glu244 is equivalent to Glu422 and
still hydrogen bonds to AIR O2′. Asp383 (if protonat-
ed) hydrogen bonds to AIR N3 (2.8 Å) and is replaced
by Asn227 in HBI synthase. The fourth difference is
near SAM, where Arg343 hydrogen bonds to N7 of
SAM and is replaced by Lys187, which is also pre-
dicted to hydrogen bond to N7, in HB synthase.
BLAST searches15 starting with either D. acetoxidans
HBI synthase or A. thaliana ThiC suggest that these
four differences alone distinguish the two paralogs.
(A)
13
(B)
4
7
6
(C)
(D)
x
x
x
(E)
(F)
x
7.4$
7.0$
7.6$
7.2$
ppm$
Full reaction +
formate
(G)
(H)
Full reaction
No Dithionite
(I)
ppm$
174$
172$
170$
Conserved active sites suggest similar catalytic
mechanisms for most enzymatic reactions. This is
clearly not the case for ThiC and BzaF. These pro-
teins reveal unique features of enzymatic catalysis of
reactions that proceed via high-energy intermediates.
For such reactions, it is likely that the high-energy
substrate radical 15 is converted to HMP-P 12 or to
HBI 14 in a complex multistep sequence in which
several steps along the reaction coordinate do not
require catalytic assistance from the enzyme. This
allows for similar active sites to produce different
products. This catalytic motif has previously been ob-
served in terpene cyclases where similar active sites
can catalyze the conversion of high-energy carbo-
Figure 5: Labeling studies to determine the origin of
key HBI atoms. (A) Summary of the labeling studies.
(B) Partial 1H NMR for authentic HBI. (C)-(F): 1H NMR
of HBI purified from enzymatic reactions using (C)
unlabeled AIR (D) 2′-13C-AIR (E) 4-2H-AIR and (F)
5′,5′-2H2-AIR. (G) – (I): 13C DEPT 90 analysis of the
reaction mixture. (G) HBI synthase reaction with
1′,2′,3′,4′,5′-13C5-AIR spiked with formate (10 mM)
after 2hrs. (H) HBI synthase reaction with
1′,2′,3′,4′,5′-13C5-AIR. (I) No Dithionite – control. X is
an unknown impurity. The doublet at 7.6 and the dou-
blet of doublets at 7.05 in spectrum E are due to a
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cations to different products. For example, the D100E vitamin B12 biosynthesis. The biosynthesis involves
1
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5
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mutant of trichodiene synthase generates six ter-
penes16 and native baruol synthase produces a library
of 23 terpenes.17
an unanticipated intersection of thiamin and vitamin
B12 biosynthesis and uses chemistry that exceeds in
complexity any of the enzymatic reactions used in the
assembly of the corrin ring of vitamin B12. Mechanistic
and structural studies are in progress to elucidate the
details of this remarkable reaction.
Our studies on the identification and reconstitution
of the vitamin B12 HBI synthase represent the first
steps in solving a long-standing problem in anaerobic
N
N
N
PO
PO
PO
HO
O
O
(A)
N
N
N
(B)
+
H
9
H
H
NH2
NH2
NH2
Ad
OH
OH
OH
HO
Ad
HO
HO
16
15
10
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13
11
10
N
H2N
N
H2N
NH
N
N
HN
BzaF
NH
HN
N
N
H
O
+
+
H
PO
O
O
O
+
PO
PO
N
Pi
OH
HO
19
OH
HO
OH
HO
OH
HO
17
17
20
ThiC
HN
N
N
O
NH
NH
NH
HN
NH2
HN
NH
O
O
HN
O
O
PO
22
18
OH
HO
23
OH
HO
OH
HO
21
(C)
OH
O
O
O
O
HO
HO
HO
NH2
NH
NH2
NH2
O
O
O
NH
O
O
O
N
H
N
N
H
24
25
23
H
HO
HO
HO
HO
H
N
O
O
O
O
O
N
N
N
NH2
N
OH
O
H2N
12
N
N
H
N
N
H
26
27
28
32
29
H
HO
HO
HO
O
N
N
N
N
OP
N
N
N
H
N
30
31
14
H
Figure 7: (A) Mechanistic proposal for the radical-triggered conversion of AIR to HBI catalyzed by HBI syn-
thase. (B) Homology model for HBI synthase (BzaF). (C) X-ray structure of the ThiC active site. The [4Fe-4S]
cluster, additional iron, SAM, AIR, and key active site residues are shown. SAM was generated by adding a me-
thyl group in the S-configuration to SAH. The additional iron is also chelated to two conserved side chains
(His270 and His334) in HBI synthase (BzaF) and His426 and His490 in ThiC.
(2) Cooper, A. J. L.; Pinto, J. T. Chemtracts 2006, 19, 474.
(3) Scott, A. I. J. Org. Chem. 2003, 68, 2529.
ASSOCIATED CONTENT
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1992, 204, 679.
(5) Vogt, J. R. A.; Lamm-Kolonko, L.; Renz, P. Eur. J. Biochem. 1988,
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(7) Chatterjee, A.; Li, Y.; Zhang, Y.; Grove, T. L.; Lee, M.; Krebs, C.;
Booker, S. J.; Begley, T. P.; Ealick, S. E. Nat. Chem. Biol. 2008, 4, 758.
(8) Chatterjee, A.; Hazra, A. B.; Abdelwahed, S.; Hilmey, D. G.; Begley,
T. P. Angew. Chem., Int. Ed. 2010, 49, 8653.
Supporting Information Available: Detailed experi-
mental procedures are provided in the supporting infor-
mation. This material is available free of charge via the In-
ternet at http://pubs.acs.org
AUTHOR INFORMATION
Corresponding Author
(9) Lawhorn, B. G.; Mehl, R. A.; Begley, T. P. Org. Biomol. Chem. 2004,
2, 2538.
Prof. Tadhg P. Begley
(10) Yamada, K.; Kumaoka, H. J Nutr Sci Vitaminol (Tokyo) 1983, 29,
389.
Department of Chemistry, Texas A&M University, Col-
(11) Renz, P.; Endres, B.; Kurz, B.; Marquardt, J. Eur. J. Biochem.
1993, 217, 1117.
(12) Endres, B.; Weurfel, A.; Volgler, B.; Renz, P. Biol. Chem. Hoppe-
Seyler 1995, 376, 595.
(13) Fenwick, M. K.; Mehta, A., P.; Zhang, Y.; Abdelwahed, S. H.;
Begley, T. P.; Ealick, S. E. Nature Commun 2015, 6, 6480.
(14) Coquille, S.; Roux, C.; Mehta, A.; Begley, T. P.; Fitzpatrick, T. B.;
Thore, S. J Struct Biol, 184, 438.
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Mol Biol 1990, 215, 403.
lege
Station,
TX-77843,
USA.
*begley@chem.tamu.edu
ACKNOWLEDGMENT
This research was supported by the Robert A. Welch
Foundation (A-0034 to TPB) and the National Insti-
tutes of Health (DK44083 to TPB; DK067081 to SEE).
(16) Cane, D. E.; Xue, Q.; Fitzsimons, B. C. Biochemistry 1996, 35,
12369.
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