Radical SAM-Dependent Adenosylation Involved in Bacteriohopanepolyol Biosynthesis?

Article abstract of DOI:10.1002/cjoc.201900399

Bacteriohopanepolyols are a group of triterpenoids that play important roles in regulating bacterial cell membrane function. As an intermediate in bacteriohopanepolyol biosynthesis, adenosylhopane production is related to a putative Fe-S protein HpnH, but the exact role of this enzyme remains unsolved. Here we report characterization of HpnH as a novel radical S-adenosylmethionine (SAM) superfamily enzyme. In contrast to almost all the members in the superfamily, HpnH does not initiate the reaction by a hydrogen atom abstraction process. Instead, it catalyzes the adenosylation of hopene via a radical addition reaction to produce adenosylhopane, representing the second example of radical SAM-dependent adenosylation involved in natural product biosynthesis.

Full text of DOI:10.1002/cjoc.201900399

Chinese Journal
of Chemistry
CJC
中 国 化 学 - An International Journal
Accepted Article
Title: Radical SAM-dependent adenosylation involved in bacteriohopanepolyol
biosynthesis
Authors: Yuting Zhong, Xinjian Ji, and Qi Zhang*
This manuscript has been accepted and appears as an Accepted Article online.
This work may now be cited as: Chin. J. Chem. 2019, 37, 10.1002/cjoc.201900399.
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published online in Early View: http://dx.doi.org/10.1002/cjoc.201900399.
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Radical SAM-dependent adenosylation involved in bacteriohopanepolyol biosynthesis
Yuting Zhong, Xinjian Ji, and Qi Zhang*
Department of Chemistry, Fudan University, Shanghai, 200433, China
*E-mail: qizhang@sioc.ac.cn
Abstract
Bacteriohopanepolyols are a group of triterpenoids that play important role in regulating
bacterial cell membrane function. As an intermediate in bacteriohopanepolyol biosynthesis,
adenosylhopane production is related to a putative Fe-S protein HpnH, but the exact role of this
enzyme remains unsolved. Here we report characterization of HpnH as a novel radical S-
adenosylmethionine (SAM) superfamily enzyme. In contrast to almost all the members in the
superfamily, HpnH does not initiate the reaction by a hydrogen atom abstraction process.
Instead, it catalyzes the adenosylation of hopene via a radical addition reaction to produce
adenosylhopane, representing the second example of radical SAM-dependent adenosylation
involved in natural product biosynthesis.
The radical S-adenosylmethionine (SAM) superfamily is thus far the largest known enzyme
superfamily with more than 220 000 members found in all three domains of life.1-4 These
enzymes typically have a CX3CX2C motif that binds a [4Fe–4S] cluster via the three Cys thiols.
The fourth iron of the [4Fe–4S] cluster is vacant and can bind a SAM molecule in a bidentate
fashion via the Met moiety of SAM. Such a binding allows an inner-sphere electron transfer
from the [4Fe-4S] cluster to SAM to generate a 5′-deoxyadenosyl (dAdo) radical, which can
form a transient organometallic intermediate Ω with the [4Fe–4S] cluster.5,6 Homolysis of the
C-Fe bind of Ω would regenerate the dAdo radical, which leads to a highly diverse array of
reactions.
For almost all the known radical SAM enzymes, the reactions are initiated by a dAdo radical-
mediated hydrogen atom abstraction process, which produces a substrate radical intermediate
as well as 5′-deoxyadenosine (dAdoH) as a byproduct (Figure 1A). Due to the high energetic
barrier of hydrogen abstraction from an sp2 carbon and the propensity of radical addition to
double bonds, the canonical radical SAM chemistry can be easily switched to an adenosylation
reaction to result in a nucleoside-containing product. Such a strategy was early used in the study
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of pyruvate formate-lyase activase (PFL) to capture the dAdo radical7 and has been recently
developed for several radical SAM enzymes, including L-Trp lyase NosL,8-10 the class C radical
SAM methyltransferase NosN,11 7-carboxy-7-deazaguanine synthase QueE,12 and three L-Tyr
lyases ThiH, HydG and FibC13 (Figure 1B). To date, the menaquinone biosynthesis enzyme
MqnE represents the only biochemically characterized enzymes that naturally catalyzes a
radical SAM-dependent adenosylation reaction (Figure 1C).14,15 Because of the high efficiency
of dAdo radical addition, it is expected that such an adenosylation strategy should also be
involved in other biosynthesis systems.
NH2
NH2
N
NH2
N
N
A
N
N
N
N
N
R
O
O
N
N
N
S
S
H
N
HO
HO
O
O
O
NH2
NH2
4Fe-4S 2+
R
4Fe-4S +
OH OH
OH OH
Ado
OH OH
dAdoH
R
H
SAM
L-methinine
NH2
NH2
N
dAdo
B
N
N
N
N
N
NosN
NosL
COOH
N
N
Ado
+
dAdo
+
Ado
S
S
COOH
O
O
N
H
N
H
OH OH
OH OH
ThiH
HydG
FbiC
O
N
O
O
dAdo
R2
R2
O
N
N
QueE
N
dAdo
HN
H2N
O
HN
H2N
Ado
Ado
R1
O
R1
N
N
=
=
R1 OH, R2 OH
=
=
R1 NH2 R2 OH
,
C
COOH
COOH
O
=
=
R1 OH, R2 OCH3
MqnE
Ado
dAdo
COOH
e
CO2
O
Figure 1. Reactions catalyzed by radical SAM enzymes. (A) Canonical radical SAM-dependent
reactions involving a dAdo radical-mediated hydrogen abstraction. (B) Radical SAM-
dependent adenosyltion involving unnatural substrate analogues. (C) MqnE-catalyzed reaction,
which prior to this study represents the only known naturally occurred radical SAM-dependent
adenosylation reaction.
，
Bacteriohopanepolyols are a group of pentacyclic triterpenoids biosynthesized by many
bacteria to perform a regulating and rigidifying function in membranes, in a way analogous to
that of the sterols in eukaryotes.16,17 These compounds are ubiquitous in sedimentary deposits,
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and the diverse range and degree of taxonomic specificity enables their use as molecular
biomarkers in organic geochemistry.18 The model actinomycete strain Streptomyces coelicolor
A(3)2 produces aminobacteriohopanetriol (ATBH) as the only detected elongated hopanoid,
and a gene cluster was identified to be responsible for ATBH biosynthesis (Figure 2A).19 It has
been proposed that HpnH, a putative radical SAM enzyme, catalyzes the coupling of hopene
and SAM to produce adenosylhopane, which is subsequently converted to ribosylhopane by a
nuclesidase HpnG, and the latter compound is then converted to ATBH by a putative
aminotransferase HpnO (Figure 2B).20,21 Deletion of hpnG in Streptomyces coelicolor resulted
in the exclusive production of adenosylhopane,20 whereas deletion of hpnH in various strains
resulted in the production of the C30 hopanoids such as hopene, but no C35 hopanoids such as
ATBH.22-24 These results are consistent with the proposed role of HpnH in catalyzing the
adenosylaiton of hopene to produce adenosylhopane, but to date the exact role of HpnH in
ATBH biosynthesis remains unclear.
Figure 2. Biosynthesis of bacteriohopanepolyol. (A) The biosynthetic gene cluster in S.
coelicolor. Annotation of the genes is according to the SCO numbers and as described by
Poralla et al.19 The key enzymes in the pathway (shown in B) are highlighted in blue.
orf08/SCO6759, phytoene or squalene synthase; orf09/SCO6760, phytoene or squalene
synthase; orf10/SCO6761, unknown function; orf11/SCO6762, phytoene dehydrogenase;
orf12/SCO6763, polyprenyl diphosphate synthase or farnesyl diphosphate synthase;
orf13/SCO6764, squalene-hopene cyclases (SHC); orf14/SCO6765, nucleosidase (hpnG);
orf15/SCO6766, adenosylhopane synthase (hpnH); orf16/SCO6767, putative 4- hydroxy-3-
methylbut-2-en-1-yl diphosphate synthase; orf17/SCO6768, putative 1-deoxy-d-xylulose 5-
phosphate synthase; orf18/SCO6769, putative aminotransferase (hpnO); orf19/SCO6770,
DNA-binding protein; orf20/SCO677, unknown function. (B) The proposed biosynthetic
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pathway of ATBH in S. coelicolor A(3)2.
To investigate the catalytic activity of HpnH, hpnH gene was amplified from the genomic DNA
of S. coelicolor and expressed in Escherichia coli with an N-terminal hexa-histidine tag, and
the protein was purified by Ni2+ affinity chromatography under strictly anaerobic conditions.
Analysis of the reaction mixture containing SAM, sodium dithionite, and the reconstituted
HpnH showed that dAdoH was produced in the assay mixture (Figure 3A, trace ii), suggesting
that HpnH is a radical SAM enzyme.
We next synthesized hopene from squalene by using the cell lysate of the E. coli cells that
overexpress the squalene-hopene cyclase SHC from S. coelicolor.25 Hopene was then fed to the
E. coli cells that overexpresses hpnH. Liquid chromatography (LC)-high resolution (HR)-mass
spectrometry (MS) analysis of the resulting cell extract revealed a compound exhibited a
positive charged molecular ion at m/z = 662.5002 (0.3 ppm for a molecular formula of
C40H63N5O3, [M+H]+ = 662.5004) (Figure 3B, trace ii, and Figure S3A), and this compound
was not observed with the control E. coli cells carrying an empty vector (Figure 3B, trace i).
HR-MS/MS analysis of this compound showed a major fragment ion at m/z = 136.06, which is
characteristic of the adenosine-containing compounds (Figure 3C and Figure S3B). To further
validate the identity of the compound, we followed the procedure of Rohmer et al by deleting
hpnG in S. coelicolor.20 The hpnG-knockout mutant was grown using R2YE solid culture and
adenosylhopane was obtained by extracting the cell medium. LC-HRMS analysis showed that
the compound obtained in the feeding experiment coeluted with adenosylhopane produced from
the hpnG-knockout mutant, supporting the compound produced in feeding experiment is
adenosylhopane (Figure 3B, trace iii).
We next performed the in vitro reaction of HpnH with hopene, SAM and sodium dithionite. To
increase the solubility of hopene, the nonionic surfactant triton X-100 was added in the reaction
to a final concentration of 1%. LC-HRMS analysis of the reaction mixture showed that
adneosylhopane was indeed produced in the reaction as expected (Figure 3B, trace v), which
was absent in the control assays either with the supernatant of boiled enzyme (Figure 3B, trace
vi). Although dAdoH was also produced in the reaction, the yield decreased 3-4 folds compared
to that without hopene (Figure 3A), supporting that the newly generated dAdo radical was
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trapped by hopene. Moreover, SAM consumption also increased apparently in the assay with
hopene (Figure 3A, trace i). These results strongly support that HpnH catalyzes a radical SAM-
dependent adenosylation of hopene to produce adenosylhopane.
Figure 3. Characterization of HpnH reaction. (A) LC-MS analysis of SAM cleavage assays, the
multiple selected ion monitoring (SIM) mode includes [M+H]+ = 399.1 (corresponding to
SAM), and 252.1 (corresponding to dAdoH), showing i) the reaction with the supernatant of
boiled HpnH, ii) the HpnH reaction in which hopene was omitted, and iii) HpnH with all the
required components. (B) LC-MS analysis of adenosylhopane, showing the extracted ion
chromatograms (EICs) of [M+H]+= 662.5 for i) control experiments with E. coli cells carrying
an empty vector, ii) the extract of cell culture in which hopene was fed to the hpnH-expressing
E. coli cells, iii) the authentic adenosylhopane produced in the hpnG-knockout mutant of
Streptomyces coelicolor, iv) control reaction with the supernatant of boiled HpnH, and vi) in
vitro assay of HpnH with all the required components. (C) The Hpn-catalyzed reaction.
We previously showed that S-guanosylmethionine (SGM) and S-cytidinylmethionine (SCM),
two SAM analogues containing a guanine and a cytosine moiety, respectively, were able to
support the catalysis of the tryptophan lyase NosL, allowing for the generation of different
nucleoside adducts in NosL reaction with a tryptophan structural analogue with an olefin
moiety.8,9 Aiming to produce novel hoponoids with a different nucleoside moiety, we ran the
HpnH assay with SGM and SCM, respectively, and similar reactions with NosL were also
performed as positive control assays. However, although significant amounts of 5′-
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deoxyguanosine (dGuoH) and 5′-deoxycytidine (dCydH) were produced in NosL reaction,
neither of them was produced in HpnH reaction. These observations suggested that HpnH has
a more stringent substrate specificity, which can only recognize SAM, but not SGM and SCM.
In summary, we have shown that HpnH in the biosynthetic pathway of bacteriohopanepolyols
is a novel radical SAM enzyme. In contrast to most members of the radical SAM superfamily
that initiates the reaction by a hydrogen abstraction mediated by the dAdo radical, the dAdo
radical generated in HpnH catalysis adds to the double bond of hopene to produce
adenosylhopane, representing the second example of naturally-occurred radical SAM-
dependent adenosylation reaction. Owing to the enormous number of the radical SAM
superfamily and the fact that most of the members are functionally unknown, we expect that
such a type of adenosylation reaction could be more general and awaits characterization by
future biochemical studies.
Acknowledgements
This work is supported in part by grants from the National Key Research and Development
Program (2016 Y F A0501302), and from National Natural Science Foundation of China
(31670060 to Q.Z.).
References
(1) Gerlt, J. A.; Bouvier, J. T.; Davidson, D. B.; Imker, H. J.; Sadkhin, B.; Slater, D. R.;
Whalen, K. L. Enzyme Function Initiative-Enzyme Similarity Tool (EFI-EST): A web tool for
generating protein sequence similarity networks. Biochim Biophys Acta 2015, 1854, 1019-1037.
(2) Frey, P. A.; Hegeman, A. D.; Ruzicka, F. J. The radical SAM superfamily. Crit. Rev.
Biochem. Mol. Biol. 2008, 43, 63-88.
This article is protected by copyright. All rights reserved.

(3) Broderick, J. B.; Duffus, B. R.; Duschene, K. S.; Shepard, E. M. Radical S-
adenosylmethionine enzymes. Chem Rev 2014, 114, 4229-4317.
(4) Landgraf, B. J.; McCarthy, E. L.; Booker, S. J. Radical S-Adenosylmethionine
Enzymes in Human Health and Disease. Annu Rev Biochem 2016, 85, 485-514.
(5) Horitani, M.; Shisler, K.; Broderick, W. E.; Hutcheson, R. U.; Duschene, K. S.; Marts,
A. R.; Hoffman, B. M.; Broderick, J. B. Radical SAM catalysis via an organometallic intermediate
with an Fe-[5'-C]-deoxyadenosyl bond. Science 2016, 352, 822-825.
(6) Byer, A. S.; Yang, H.; McDaniel, E. C.; Kathiresan, V.; Impano, S.; Pagnier, A.; Watts,
H.; Denler, C.; Vagstad, A. L.; Piel, J.; Duschene, K. S.; Shepard, E. M.; Shields, T. P.; Scott,
L. G.; Lilla, E. A.; Yokoyama, K.; Broderick, W. E.; Hoffman, B. M.; Broderick, J. B. Paradigm
Shift for Radical S-Adenosyl-l-methionine Reactions: The Organometallic Intermediate Omega
Is Central to Catalysis. J Am Chem Soc 2018, 140, 8634-8638.
(7) Wagner, A. F. V.; Demand, J.; Schilling, G.; Pils, T.; Knappe, J. A dehydroalanyl
residue can capture the 5 '-deoxyadenosyl radical generated from S-adenosylmethionine by
pyruvate formate-lyase-activating enzyme. Biochemical
Communications 1999, 254, 306-310.
&
Biophysical Research
(8) Ji, X.; Li, Y.; Xie, L.; Lu, H.; Ding, W.; Zhang, Q. Expanding Radical SAM Chemistry
by Using Radical Addition Reactions and SAM Analogues. Angew Chem Int Ed Engl 2016, 55,
11845-11848.
This article is protected by copyright. All rights reserved.

(9) Ji, X.; Zhang, Q. Using radical SAM chemistry to access nucleoside-containing
compounds. Synlett 2017, 28, 143-147.
(10) Bhandari, D. M.; Fedoseyenko, D.; Begley, T. P. Tryptophan Lyase (NosL): A
Cornucopia of 5'-Deoxyadenosyl Radical Mediated Transformations. J Am Chem Soc 2016,
138, 16184-16187.
(11) Ji, X.; Mandalapu, D.; Cheng, J.; Ding, W.; Zhang, Q. Expanding the Chemistry of the
Class C Radical SAM Methyltransferase NosN by Using an Allyl Analogue of SAM. Angew
Chem Int Ed Engl 2018, 57, 6601-6604.
(12) Wilcoxen, J.; Bruender, N. A.; Bandarian, V.; Britt, R. D. A Radical Intermediate in
Bacillus subtilis QueE during Turnover with the Substrate Analogue 6-Carboxypterin. J Am
Chem Soc 2018, 140, 1753-1759.
(13) Wu, Y.; Wu, R.; Mandalapu, D.; Ji, X.; Chen, T.; Ding, W.; Zhang, Q. Radical SAM-
dependent adenosylation catalyzed by l-tyrosine lyases. Org Biomol Chem 2019, 17, 1809-
1812.
(14) Mahanta, N.; Fedoseyenko, D.; Dairi, T.; Begley, T. P. Menaquinone biosynthesis:
formation of aminofutalosine requires a unique radical SAM enzyme. J Am Chem Soc 2013,
135, 15318-15321.
(15) Joshi, S.; Fedoseyenko, D.; Mahanta, N.; Ducati, R. G.; Feng, M.; Schramm, V. L.;
Begley, T. P. Antibacterial Strategy against H. pylori: Inhibition of the Radical SAM Enzyme
This article is protected by copyright. All rights reserved.

MqnE in Menaquinone Biosynthesis. ACS Med Chem Lett 2019, 10, 363-366.
(16) Sahm, H.; Rohmer, M.; Bringer-Meyer, S.; Sprenger, G. A.; Welle, R. Biochemistry
and physiology of hopanoids in bacteria. Adv Microb Physiol 1993, 35, 247-273.
(17) Kannenberg, E. L.; Poralla, K. Hopanoid biosynthesis and function in bacteria.
Naturwissenschaften 1999, 86, 168-176.
(18) Cooke, M. P.; Talbot, H. M.; Farrimond, P. Bacterial populations recorded in
bacteriohopanepolyol distributions in soils from Northern England. Org Geochem 2008, 39,
1347-1358.
(19) Poralla, K.; Muth, G.; Hartner, T. Hopanoids are formed during transition from
substrate to aerial hyphae in Streptomyces coelicolor A3(2). Fems Microbiology Letters 2000,
189, 93-95.
(20) Liu, W.; Sakr, E.; Schaeffer, P.; Talbot, H. M.; Donisi, J.; Hartner, T.; Kannenberg, E.;
Takano, E.; Rohmer, M. Ribosylhopane, a novel bacterial hopanoid, as precursor of C35
bacteriohopanepolyols in Streptomyces coelicolor A3(2). Chembiochem 2014, 15, 2156-2161.
(21) Challis, G. L. Exploitation of the Streptomyces coelicolor A3(2) genome sequence for
discovery of new natural products and biosynthetic pathways. Journal of Industrial Microbiology
& Biotechnology 2014, 41, 219-232.
(22) Bradley, A. S.; Pearson, A.; Saenz, J. P.; Marx, C. J. Adenosylhopane: The first
intermediate in hopanoid side chain biosynthesis. Org Geochem 2010, 41, 1075-1081.
This article is protected by copyright. All rights reserved.

(23) Welander, P. V.; Doughty, D. M.; Wu, C. H.; Mehay, S.; Summons, R. E.; Newman,
D. K. Identification and characterization of Rhodopseudomonas palustris TIE-1 hopanoid
biosynthesis mutants. Geobiology 2012, 10, 163-177.
(24) Schmerk, C. L.; Welander, P. V.; Hamad, M. A.; Bain, K. L.; Bernards, M. A.;
Summons, R. E.; Valvano, M. A. Elucidation of the Burkholderia cenocepacia hopanoid
biosynthesis pathway uncovers functions for conserved proteins in hopanoid-producing
bacteria. Environ Microbiol 2015, 17, 735-750.
(25) Hammer, S. C.; Marjanovic, A.; Dominicus, J. M.; Nestl, B. M.; Hauer, B. Squalene
hopene cyclases are protonases for stereoselective Bronsted acid catalysis. Nat Chem Biol
2015, 11, 121-126.
This article is protected by copyright. All rights reserved.