A mechanistic study of the non-oxidative decarboxylation catalyzed by the radical S-adenosyl-l-methionine enzyme BlsE involved in blasticidin S biosynthesis

Article abstract of DOI:10.1039/c7cc04286h

Decarboxylation is a fundamentally important reaction in biology and involves highly diverse mechanisms. Here we report a mechanistic study of the non-oxidative decarboxylation catalyzed by BlsE, a radical S-adenosyl-l-methionine (SAM) enzyme involved in blasticidin S biosynthesis. Through a series of biochemical analysis with isotopically labeled reagents, we show that the BlsE-catalyzed reaction is initiated by the 5′-deoxyadenosyl (dAdo) radical-mediated hydrogen abstraction from a sugar carbon of the substrate cytosylglucuronic acid (CGA), and does not involve a carboxyl radical as has been proposed for 4-hydroxyphenylacetate decarboxylase (HPAD). Our study reveals that BlsE represents a mechanistically new type of radical-based decarboxylase.

Full text of DOI:10.1039/c7cc04286h

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DOI: 10.1039/C7CC04286H
Mechanistic study of the non‐oxidative decarboxylation catalyzed
by the radical S‐adenosyl‐L‐methionine enzyme BlsE involved in
blasticidin S biosynthesis
Received 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
www.rsc.org/
Lei Liu1,2# Xinjian Ji,2# Yongzhen Li,2,3 Wenjuan Ji,2 Tianlu Mo,2 Wei Ding,2 and Qi Zhang2*
Decarboxylation is a fundamentally important reaction in biology catalyzes a radical SAM‐dependent decarboxylation reaction
and involves highly diverse mechanisms. Here we report that converts CGA to cytosylarabinopyranose (CAP) (Fig. 1A).10
mechanistic study of the non‐oxidative decarboxylation catalyzed
by BlsE, a radical S‐adenosyl‐L‐methionine (SAM) enzyme involved
O-
O
A
O
N
O
BlsE
SAM
N
O
N
O
in blasticidin S biosynthesis. By a series of biochemical analysis
with isotopically labeled reagents, we show that the BlsE‐
catalyzed reaction is initiated by the 5ˊ‐deoxyadenosyl (dAdo)
radical‐mediated hydrogen abstraction from a sugar carbon of the
substrate cytosylglucuronic acid (CGA), and does not involve a
carboxyl radical as has been proposed for 4‐hydroxyphenylacetate
decarboxylase (HPAD). Our study reveals that BlsE represents a
mechanistically new type of radical‐based decarboxylase.
NH2
HO
HO
COO-
N
NH2
HO
OH
CO2
OH
HO
COO-
CGA
CAP
B
HemN
2 SAM
HN
HN
HN
NH
NH
NH
NH
COO-
COO-
2 CO2 + 2 H+ + 2 e-
HN
Blasticidin S (BLS) is a peptidyl nucleoside antibiotic produced
by Streptomyces griseochromogenes,1 which is biosynthesized
from the primary precursors cytosine, glucose, arginine, and
methionine.2 The cytidine moiety of blasticidin S binds tightly
to the corresponding guanine base at the P site of the
ribosome 50S subunit and forms a Watson‐Crick base pair,3
thereby inhibiting peptide bond formation and causing
translation termination. It has also been shown that BLS can
rapidly inhibit DNA synthesis.4 As a result, this compound
exhibits potent activity against both prokaryotic and
eukaryotic cells,5 and has been widely used in protecting rice
plants from infection by the fungus that causes rice blast.
Recently, BLS has also been used as a selectable marker in
biological research to select transformed cells that carry a
resistance gene.6
COO-
protoporphyrinogen IX
COO-
coproporphyrinogen III
C
HPAD-AE
dAdo
SH
SAM
SH
+ L-Met
OH
C503
p-cresol
dAdoH
-OOC
H+
OOC
e-
CO2
SH
C503
C503
S
C503
O-
O-
O
H
E505
E505
E505
OH
HPA
OH
OH
O
O
O
Fig. 1. Radical‐SAM dependent decarboxylations. (A) The BlsE‐
catalyzed non‐oxidative decarboxylation. (B) Oxidative
decarboxylation catalyzed by HemN. (C) The Kolbe‐type
decarboxylation reaction catalyzed by 4‐hydroxyphenylacetate
decarboxylase (HPAD). The dAdo radical produced by the
HPAD activating enzyme (HPAD‐AE) produces a glycyl radical in
HPAD, which, upon substrate binding, generates a thiyl radical
that initiates the Kolbe‐type reaction. Residue numbers are
shown for the HPAD from Clostridium scatologenes.
The BLS biosynthetic gene cluster was first identified in
8
1998 by Zabriskie and coworkers,7,
which expands
approximately 20 kilobase pairs (kbs) and encodes a series of
proteins, including a unique radical S‐adenosyl‐L‐methionine
(SAM) enzyme BlsE. Disruption of blsE completely abolished
BLS production and resulted in accumulation of
cytosylglucuronic acid (CGA) (Fig. 1A) in the culture of the
mutant strain.9 He and coworkers later showed that BlsE
Radical SAM superfamily is the largest known enzyme
superfamily that currently consists of more than 16,500
members found in all three domains of life.11‐13 These enzymes
utilize a [4Fe–4S] cluster to bind SAM and reductively cleave its
carbon‐sulfur bond to produce
deoxyadenosyl (dAdo) radical, which initiates a highly diverse
variety of reactions.11‐13
well‐known radical SAM
decarboxylase is the oxygen‐independent coproporphyrinogen
III oxidase HemN, which catalyzes protoporphyrinogen IX
production by sequential decarboxylation of the two
propionate side chains of coproporphyrinogen III (Fig. 1B).14‐17
This decarboxylation reaction is oxidative, in which a carboxyl
a highly reactive 5ˊ‐
1.College of Life Science & Biotechnology, Mianyang Normal University, Mianyang
621000, PR China.
2.Department of Chemistry, Fudan University, Shanghai, 200433, China.
3.Medical College of Qinghai University, Xining, 810016, China
#These authors contribute equally to this work.
A
*To whom correspondence should be addressed. E‐mail: qizhang@sioc.ac.cn.
†Electronic Supplementary Information (ESI) available: [details of any
supplementary information available should be included here]. See
DOI: 10.1039/x0xx00000x
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group is released as a carbon dioxide with a simultaneous loss oxidative decarboxylation might also be a Koble‐type, involving
View Article Online
DOI: 10.1039/C7CC04286H
of two electrons. Similar radical‐mediated oxidative a carboxyl radical resulting from the dAdo radical‐mediated
decarboxylation is also found for other radical‐based enzymes, hydrogen abstraction from the carboxyl group. Because the
such as the heme‐containing enzyme OleTJE.18‐21
carboxyl‐associated hydrogen is readily exchangeable with the
Unlike HemN, however, the BlsE‐catalyzed decarboxylation solvent, we reasoned that, if the BlsE‐catalyzed reaction is
is non‐oxidative and no electron is released externally (Fig. 1A). indeed a Kolbe‐type, deuterium incorporation into dAdoH
The radical‐mediated non‐oxidative decarboxylations are should be observed when the reaction is performed in D2O.
relatively rare in biochemistry. A well‐characterized example is Such a strategy has been extensively used in mechanistic
the
reaction
catalyzed
by
4‐hydroxyphenylacetate investigation of radical SAM enzymes, such as HydG, CofH, and
decarboxylase (HPAD), an enzyme involved in the last step of NosL.24‐28 To this end, we synthesized CGA and performed the
tyrosine fermentation in clostridia, which converts 4‐ reaction with reconstituted BlsE, SAM, and dithionite in 90%
hydroxyphenylacetate (HPA) to 4‐methylphenol (p‐cresol) (Fig. D2O. Liquid chromatography with high‐resolution mass
1C).22, 23 In this reaction, the dAdo radical generated by the spectrometry (LC‐HR‐MS) analysis of the reaction mixture
radical SAM HPAD activating enzyme (HPAD‐AE) abstracts a showed that deuterium incorporation into dAdoH was not
hydrogen from HPAD to produce a stable glycyl radical, which, apparent (Fig. 2A), suggesting that unlike HAPD, the BlsE‐
upon substrate binding, abstracts a hydrogen from the catalyzed reaction is not a Kolbe‐type. Furthermore, we
neighbouring Cys to produce a key thiyl radical. This thiyl observed apparent deuterium incorporation into the substrate
radical then abstracts the hydrogen from the 4‐ CGA (Fig. 2B), which likely resulted from quenching of a
hydroxyphenylacetate carboxyl group, resulting in a carboxyl carbon‐centered substrate radical by
a solvent‐derived
radical and a Kolbe‐type decarboxylation reaction (Fig. 1C).
hydrogen equivalent. Careful HR‐MS/MS analysis of the
deuterated CGA (m/z = 289.1) shows that the deuterium atom
is on the sugar moiety (Fig. S1, ESI†). It is noteworthy that
similar observations of substrate deuteration have been made
in many cases, including the radical SAM dehydratase AprD4,29
and NosL and DesII with unnatural substrates.30‐33 Together,
this analysis suggests that the dAdo radical‐mediated
hydrogen abstraction in BlsE catalysis does not occur on the
carboxyl group, but on a site on which the hydrogen is not
exchangeable with solvent.
To further interrogate the hydrogen abstraction site in BlsE
catalysis, we synthesized CGA‐D5, which contains five
deuterium atoms on the sugar moiety (Scheme 1). LC‐HR‐MS
analysis of the BlsE reaction with CGA‐D5 showed that
deuterium was apparently incorporated into dAdoH (Fig. 2C),
suggesting that the dAdo radical‐mediated hydrogen
abstraction occurs on a sugar carbon of CGA. We then carried
out a detailed time course analysis of the assays with either
CGA or CGA‐D5 as a substrate. This analysis showed that the
reaction with CGA is apparently faster than that with CGA‐D5,
Fig. 2. Mechanistic investigation of the BlsE‐catalyzed non‐
oxidative decarboxylation of CGA. (A) Mass spectrum of dAdoH
produced in BlsE reaction with CGA in 90% D2O. We noted that
a very small proportion of dAdoH was deuterated, which is
likely a result of hydrogen abstraction from the newly formed
deuterated CGA (see Fig. 2B). (B) Deuterium incorporation into
CGA when the reaction was performed in D2O. For the HR‐
MS/MS spectrum of the deuterated CGA (highlighted by a
green arrow), see Fig. S1, ESI†. (C) Mass spectrum of dAdoH
produced in BlsE reaction with CGA‐D5 in H2O. (D) Time course
analysis of CAP production in BlsE reactions. The assays were
showing a kinetic isotope effect (KIE) of
～2.3 (Fig. 2D). On the
contrary, the enzyme activity did not obviously decrease when
the reaction was performed in 90% D2O (Fig. 2D). These
analyses strongly support that the BlsE‐catalyzed reaction is
not a Kolbe‐type decarboxylation but is initiated by the dAdo
radical‐mediated hydrogen abstraction from a sugar carbon of
CGA.
performed by incubating 250 μM CGA or CGA‐D5 with
～50 μM
reconstituted BlsE, 1 mM SAM, 2mM sodium dithionite in 40
mM Tris‐HCl buffer (pH 8.0 in H2O or pH/pD 8.0 in 90% D2O).
Reactions were maintained at room temperature (
～
25oC)
before quenching at different time points by addition of formic
acid to a final concentration of 5% (v/v), and the product CAP
was quantified by HPLC. Because the reactions were not
performed in parallel (reaction in D2O involves an additional
buffer exchange step), whether there is a small solvent KIE in
the reaction remains to be determined.
Scheme 1. Chemical synthesis of CGA‐D5 from perdeuterated D‐
glucose.
Because radical SAM‐dependent reactions are in most
cases initiated by
abstraction process, it appears that the BlsE‐catalyzed non‐
a dAdo radical‐mediated hydrogen
The results presented above reveal that BlsE reaction is a
new type of enzymatic non‐oxidative decarboxylation.
2 | J. Name., 2012, 00, 1‐3
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Although the hydrogen atom abstracted by the dAdo radical it is apparent that the microenvironment of enzyme active site
View Article Online
DOI: 10.1039/C7CC04286H
has not yet been definitely established, we proposed that it is plays a key role in fine‐tuning the reactivity of the radical
very likely from the carboxyl β positions (i.e. the C4ˊ of CGA) intermediates to achieve distinct catalytic outcomes, as has
(Fig. 3A). Such a hydrogen abstraction process results in a β been recently reported in the catalysis of several other radical
28, 34‐39
carbon‐centered radical intermediate
1
, whose Cα‐C bond is SAM enzymes.27,
Further detailed mechanistic
cleaved heterolytically with the assistance of a proton transfer investigation on the protein/substrate interaction and
process. The resulting decarboxylated radical is then reduced thermodynamics that govern BlsE catalysis is currently in
by a hydrogen equivalent to afford the non‐oxidative progress.
2
decarboxylated product CAP (Fig. 3A). Because when running
This work is supported by grants from the National Key
the assay in 90% D2O, a significant proportion of CGA was di‐ Research and Development Program (2016 Y F A0501302), from
deuterated (Fig. S2, ESI†), the hydrogen equivalent that National Natural Science Foundation of China (1500028 and
reduces the decarboxylated radical
2 should derive from the 31670060), from the Open Fund of Key Laboratory of
solvent (Fig. 3A) and not from dAdoH, because otherwise, Glycoconjugate Research, Fudan University, Ministry of Public
mono‐deuterated CAP is expected.
Health, and from the Chemical Structure Association Trust.
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