Biochemical Characterization of an Arginine 2,3-Aminomutase with Dual Substrate Specificity

Article abstract of DOI:10.1002/cjoc.202000119

The radical S-adenosylmethionine (SAM) aminomutases represent an important pathway for the biosynthesis of β-amino acids. In this study, we report biochemical characterization of BlsG involved in blasticidin S biosynthesis as a radical SAM arginine 2,3-aminomutase. We showed that BlsG acts on both L-arginine and L-lysine with comparable catalytic efficiencies. Similar dual substrate specificity was also observed for the lysine 2,3-aminomutase from Escherichia coli (LAM_EC). The catalytic efficiency of LAM_EC is similar to that of BlsG, but is significantly lower than that of the enzyme from Clostridium subterminale (LAM_CS), which acts only on L-lysine rather than on L-arginine. Moreover, we showed that enzymes can be grouped into two major phylogenetic clades, each corresponding to a certain C3 stereochemistry of the β-amino acid product. Our study expands the radical SAM aminomutase members and provides insights into enzyme evolution, supporting a trade-off between substrate promiscuity and catalytic efficiency.

Full text of DOI:10.1002/cjoc.202000119

Chinese Journal
of Chemistry
CJC 中 国 化 学 - An International Journal
Accepted Article
Title: Biochemical Characterization of an Arginine 2,3-aminomutase with Dual
Substrate Specificity
Authors: Junfeng Zhao, Wenjuan Ji, 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. 2020, 38, 10.1002/cjoc.202000119.
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Report
biosynthesis, isomerization, nucleoside, enzyme evolution, enzyme
kinetics
CJC
Biochemical Characterization of an Arginine 2,3-aminomutase with Dual
Substrate Specificity
Junfeng Zhao, Wenjuan Ji, Xinjian Ji, and Qi Zhang*
Department of Chemistry, Fudan University, 2005 Songhu Road, Shanghai, 200438, China
Cite this paper: Chin. J. Chem. 2020, 38, XXX—XXX. DOI: 10.1002/cjoc.202000XXX
Summary of main observation and conclusion The radical S-adenosylmethionine (SAM) aminomutases represent an important pathway for the
biosynthesis of β-amino acids. In this study, we report biochemical characterization of BlsG involved in blasticidin S biosynthesis as a radical SAM arginine
2
,3-aminomutase. We showed that BlsG acts on both L-arginine and L-lysine with comparable catalytic efficiencies. Similar dual substrate specificity was
also observed for the lysine 2,3-aminomutase from Escherichia coli (LAMEC). The catalytic efficiency of LAMEC is similar to BlsG, but is significantly lower
than the enzyme from Clostridium subterminale (LAMCS), the latter acts only on L-lysine and not on L-arginine. Moreover, we showed that enzymes can
be grouped into two major phylogenetic clades, each corresponding to a certain C3 stereochemistry of the β-amino acid product. Our study expands the
radical SAM aminomutase members and provides insights into enzyme evolution, supporting a trade-off between substrate promiscuity and catalytic
efficiency.
Background and Originality Content
C-C bond fragmentation and a re-addition process.[3,4] The third
β-amino acids serve as building blocks for a wide range of
type of aminomutase is a group of radical S-adenosyl-L-
methionine (SAM) enzymes, which are pyridoxal-5′-phosphate
(PLP)-dependent and utilize SAM as a catalytic cofactor to
catalyze the amino group rearrangement.[5,6]
bioactive natural products, including antibiotics, insecticides, and
antifungal, antitumor, and antiviral agents.[1] These amino acids
have similar polarity with α-amino acids but offer additional
properties, such as resistance to proteolysis. β-amino acids are
biosynthesized via various pathways, including decarboxylation of
aspartate and aspartate derivatives, Michael-type addition of
ammonia/amino groups onto α, β-unsaturated carboxylates,
transamination of β-ketocarboxylates, and aminomutase-
catalyzed reactions with α-amino acids.[1] Thus far, three types of
aminomutases have been identified. The first type is a family of
enzymes that use 4-methylideneimidazol-5-one (MIO) as a key
cofactor, which is produced autocatalytically from an Xxx-Ser-Gly
The radical SAM superfamily enzymes represent the largest
known enzyme family consisting of more than 125,000 members
found in all three domains of life.[5-8] These enzymes contain a
[
4Fe-4S] cluster to bind SAM and reductively cleave its carbon-
sulfur bond to produce L-methionine and a highly reactive 5ˊ-
deoxyadenosyl (dAdo) radical. The dAdo radical then abstracts a
hydrogen from the substrate to produce 5ˊ-adenosine (dAdoH)
and a substrate-based radical, thereby initiating diverse reactions
relevant to the modification of nucleic acids and proteins, and the
biosynthesis of cofactors and natural products.[5-8] Recently, a
new type of radical SAM chemistry is emerging, in which the dAdo
(
Xxx represents an Ala or a Thr) motif at the enzyme active site.[2]
The second type is the adenosylcobalamin-dependent glutamate
mutase, which produces 3-methylaspartate via a radical-mediated
2
radical is added to an sp carbon to result in an adenosylation
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First author et al.
reaction.[9] This type of radical SAM chemistry has been found
naturally in menaquinone biosynthesis and bacteriohopanepolyol
biosynthesis,[10-13] and can be achieved with unnatural
A
NH2
COOH
LAMCS
LAMEC
2 H O 2 NH3
COOH
2
(
S)
+
H
2
N
COOH
2
H N
-
α
(3S) β-lysine NH
CH COOH
3
L- -lysine
2
NH2
B
NH
2
EF-P
(R)
H
2
N
-
COOH
substrates.[14-17] Very recently, it has been shown that the dAdo
2
H N
COOH
(R)
NH
2
α
β
NH
2
L- -lysine
(3R) -lysine
O
EF-P
NH
radical-based adenosylation can even be achieved via homolytic
C
NH
NH
NH
NH
2
substation reactions.[18]
COOH BlsG
NH
COOH
NH
2
H N
2
N
H
H N
2
N
H
-
OH
N
α
2
L- -arginine
(3S) β-arginine
O
N
O
2
O
O
Lysine 2,3-aminomutase (LAM), which catalyzes the
interconversion of L-α-lysine to β-lysine, represents the first
biochemically characterized member of the radical SAM
superfamily.[19,20] In contrast to the adenosylation reactions and
most other radical SAM-dependent reactions, in which SAM is
consumed stoichiometrically, LAM uses SAM as a catalytic
cofactor and the dAdo radical is regenerated from dAdoH in the
end of each reaction cycle, making LAM a highly efficient enzyme
in terms of bioenergy and atom economy. The LAM from
Clostridium subterminale (LAMCS) catalyzes the conversion of L-α-
lysine to (3S)-β-lysine,[21] which is the first step of lysine
catabolism by clostridia (Figure 1A).[22] LAMcs has a turnover
number around 2000 min-1,[20,23] representing the most efficient
H
2
N
N
N
H
CH
3
blasticidin S
Figure 1. Reactions catalyzed by the radical SAM-dependent
aminomutases. (A) The LAMCS-catalyzed reaction is the first step in lysine
catabolism by clostridia. (B) (3R)-β-lysine produced by LAMEC is utilized for
post-translational of elongation factor P (EF-P). (C) (3S)-β-arginine
produced by BlsG is an intermediate in blasticin S biosynthesis.
Results and Discussion
Blasticidin S, produced by Streptomyces griseochromogenes,
is a peptidyl nucleoside antibiotic that exhibits potent activity
against both prokaryotic and eukaryotic cells.[28] Blasticidin S
contains a (3S)-β-arginine moiety, which has been shown to be
derived from L-α-arginine by isotope labeling experiments (Figure
enzyme among the radical SAM superfamily. In contrast to LAMCS
the LAM from Escherichia coli (LAMEC) produces (3R)-β-lysine,[24]
which is utilized for post-translational β-lysylation of elongation
factor P (Figure 1B).[25,26] Besides LAM, an L-glutamate 2,3-
aminomutase from Clostridium difficile (EMACD) was also
biochemically characterized, which is homologous to LAM and
have a turnover number around 400 min-1.[27] LAM-homologous
enzymes have been found in various organisms, and the exact
functions of these enzymes remained to be determined.
,
1
C).[29] The biosynthetic gene cluster of Blasticidin S encodes an
LAM-homologous enzyme BlsG,[30,31] which is likely responsible
for the production of (3S)-β-arginine. To investigate the function
of this enzyme, the blsG gene was amplified from the genomic
DNA of S. griseochromogenes and expressed in E. coli with an N-
terminal hexa-histidine tag, and the protein was purified by Ni2+
affinity chromatography under strictly anaerobic conditions. After
chemical reconstitution of the [4Fe-4S] cluster, the reaction was
performed by incubation of the reconstituted protein with SAM,
L-α-arginine, and sodium dithionite. The reaction mixture was
then treated with phenyl isothiocyanate (PITC) to derivatize the
amino acids, and analyzed by liquid chromatography high
resolution mass spectrometry (LC-HRMS), which clearly revealed
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A
Running title: Radical SAM L-arginine 2,3-aminomutase
Chin. J. Chem.
a new product in the BlsG reaction. Compared with the PITC-
derived L-α-arginine, this compound exhibited exactly the same
molecular weight but was eluted slightly earlier in LC analysis
Kcat/Km
-
1
Substrate
enzyme
k
cat (min )
Km (mM)
(
mM/min)
L-α-arginine
BlsG
LAMEC
BlsG
13.8 ± 0.8
9.6 ± 0.9
2.9 ± 0.3
4.7 ± 0.8
5.7 ± 1.3
5.2 ± 1.0
4.8 ± 0.6
3.2 ± 0.1
4.8
2.0
(
Figure 2, trace i), supporting production of β-arginine in the BlsG
L-α-lysine
40.9 ± 5.2
7.2
reaction.
LAMEC
LAMcs
EAMCD
34.9 ± 5.0
6.7
2208.1 ± 122.0
417.2 ± 1.5
460.0
130.3
L-α-glutamate
To quantitatively analyze the reaction efficiency, kinetic
studies were performed for the reaction with BlsG and LAMEC. The
-
1
results showed that BlsG has a turnover number of 13.8 min ,
-1
which is slightly higher than LAMEC (9.6 min ), and the Km values
of BlsG and LAMEC are 2.9 mM and 4.7 mM, respectively (Table 1).
These results demonstrate that both BlsG and LAMEC are L-
Arginine 2,3-aminomutases, and the catalytic efficiency of BlsG is
Figure 2. HPLC analysis of the PITC-treated reactions of L-α-arginine with
four aminomutases. (i) BlsG, (ii) LAMEC, (iii) LAMCS and (iv) EAMCD
.
about 2-fold higher than that of LAMEC
.
We also expressed and purified LAMCS, LAMEC, and EMACD
for parallel assays. LC-HRMS analysis of the reaction mixture of
LAMCS suggest that production of β-arginine was not observed
The comparable efficiencie of BlsG and LAMEC on L-α-
arginine inspired us to further investigate the BlsG reaction with
L-α-lysine. To this end, we performed the reaction by incubation
of BlsG with L-α-lysine, SAM, and sodium dithionite, and the
reaction mixture was analyzed by LC-HRMS after derivatization
(
Figure 2, trace ii), and this is consistent with the previous study
by Frey et al showing that LAMCS has a very stringent substrate
specificity and does not act on L-α-arginine.[32] In contrary, the
with PITC. Parallel reactions were also performed on LAMCS
,
parallel assay with LAMEC showed that β-arginine was apparently
produced in the LAMEC reaction (Figure 2, trace iii), suggesting
that LAMEC can act both on L-α-lysine and L-α-Arginine.
Production of β-arginine was not observed in the reaction with
EAMCD (Figure 2, trace iv).
LAMEC, and EAMCD. This analysis showed that production of β-
lysine was observed for all the enzymes except for EAMCD (Figure
3). Subsequent kinetic analysis showed that LAMCS has a kcat of
-1
2
208.0 min and a Km of 4.8 mM, which are very close to those
reported previously.[20,23] For LAMEC, the Km is 5.2 mM and the
-
1
k
cat is 34.2 min , the latter value is much higher than that
reported previously but still within an order of magnitude.[24]
Table 1. Kinetic parameters of the four radical SAM 2,3-
aminomutases investigated in this study. See Figure S1-S6 for the
detailed information of the kinetic analysis.
-1
Notably, for BlsG the kcat is 40.9 min , which is about 3-fold
-1
higher than its kcat on L-α-arginine (13.8 min ). The Km value on L-
α-lysine (5.7 mM) is about 2-fold higher than that on L-α-Arginine
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(
2.9 mM). These results suggest that although BlsG has higher
affinity toward L-α-arginine than L-α-lysine, the latter compound
actually has higher reactivity in BlsG reaction, raising a possibility
that BlsG may have evolved from a LAM progenitor. These
observations reveal that unlike LAMCS, which is highly specific and
efficient toward L-α-lysine, BlsG and LAMEC have dual activity and
can act both on L-α-arginine than L-α-lysine, but only have
moderate catalytic efficiencies. We also tested the BlsG activity
on L-α-glutamate, but no activity was observed. In the positive
control assay with EAMCD, the kinetic study revealed a kcat of
Figure 4. Classification of radical SAM aminomutases. (A) The maximum
likelihood phylogeny of the four aminomutases investigated in this study
together with other 60 homologous enzymes. See Figure S7 for the full
phylogenetic tree. (B) HPLC analysis of the FADD-treated L-α-lysine reactions
-1
4
17.2 min and a Km of 3.2 mM (Table 1), which are close to that
with (i) BlsG, (ii) LAMCS and (iii) LAMEC.
reported previously.[27]
To confirm that the β-lysine produced by BlsG has a (3S)-
configuration, we treated the BlsG reaction mixture with N-(5-
fluoro-2,4-dinitrophenyl)-L-alaninamide (FDAA) and performed
HPLC and LC-HRMS analysis. For comparison, parallel assays were
also performed for the reactions from LAMCS and LAMEC. The
results showed that the FDAA-derivatized product from the BlsG
reaction has exactly the same elution time with that in the LAMCS
reaction, and is different from that produced in the LAMEC
reaction (Figure 4B). This observation suggests that BlsG produces
the corresponding (3S)-β-amino acids for both L-α-arginine and L-
α-lysine, which is different from LAMEC that produces β-amino
acids with an opposite C3 stereochemistry. Although the C3 of β-
glutamate is achiral, the stereospecific hydrogen abstraction by
the dAdo radical in EAMCD catalysis is likely similar to that of
LAMCS and BlsG and is from the 3-pro-R position,[24] as EAMCD falls
into the same major clade with LAMCS and BlsG (Figure 4A and
Figure S7).
Figure 3. HPLC analysis of the PITC-treated reactions of L-α-lysine with
four aminomutases. (i) BlsG, (ii) LAMEC, (iii) LAMCS and (iv) EAMCD
.
To provide further functional and evolutionary insights into
the radical SAM-dependent aminomutases, we selected 64 LAM-
homologous proteins from bacteria and constructed a
phylogenetic tree by using maximum likelihood reference (Figure
4
A).[33] This analysis revealed the proteins fall into two major
polyphyletic clades that both contain Gram-positive and Gram-
negative sequences. BlsG, EAMCD, and LAMCS fall into the same
major clade, whereas LAMEC falls into another major clade. This
observation is consistent with the fact that LAMCS and BlsG
produce (3S)-β-amino acids (i.e. (3S)-β-lysine by LAMCS and (3S)-β-
arginine by BlsG), whereas LAMEC produces (3R)-β-lysine.
Conclusions
In summary, this study characterized BlsG as a radical SAM
arginine 2,3-aminomutase and showed it acts both on L-α-lysine
and L-α-arginine with similar catalytic efficiencies. Similar dual
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Running title: Radical SAM L-arginine 2,3-aminomutase
Chin. J. Chem.
Gruber, K.; Kratky, C. Coenzyme B(12) dependent glutamate
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substrate specificity and catalytic efficiencies are also observed
for LAMEC, although the latter enzyme is evolutionarily distinct
and produces β-amino acids with a different stereochemistry. In
contrast to these two promiscuous enzymes, LAMCS has a very
strict substrate specificity and a significantly higher catalytic
efficiency. These observations are consistent with the early
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trade-off of catalytic efficiency.[34] It appears that BlsG has
evolved from a progenitor of LAMCS, and because the selection
pressure toward arginine activity (which is required for the
biosynthesis of blasticidin S, a secondary metabolite) has not
been strong enough, the promiscuous activity of BlsG retained.
Because enzymes homologous to BlsG are widespread in nature,
both in prokaryotes in eukaryotes, it is anticipated that many of
these homologous enzymes act on different substrates and/or
have new activities, which could be a treasure trove for
biocatalysis and bioengineering applications.
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Supporting Information
The supporting information for this article is available on the
WWW under https://doi.org/10.1002/cjoc.2018xxxxx.
Zhong, Y.; Ji, X.; Zhang, Q. Radical SAM‐dependent adenosylation
involved in bacteriohopanepolyol biosynthesis. Chin. J. Chem 2020,
Acknowledgement
This work is supported in part by grants from National Natural
Science Foundation of China (21822703, 31670060, and
3
8, 39-42.
Zhong, Y.; Ji, X. Adenosylhopane biosynthesis by the Radical SAM
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1921003), and from the National Key Research and
enzyme HpnH. Chin. J. Chem 2020, 38, 218-219.
Development Program (2018Y F A0900402 and 2016 Y F
A0501302).
Bruender, N. A.; Grell, T. A.; Dowling, D. P.; McCarty, R. M.;
Drennan, C. L.; Bandarian, V. 7-Carboxy-7-deazaguanine Synthase: A
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Manuscript accepted: XXXX, 2020
Accepted manuscript online: XXXX, 2020
Version of record online: XXXX, 2020
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