Unusual stereoselectivity of methionine-γ-lyase from Citrobacterfreundii toward diastereomeric (S)-methionine S-oxide

Article abstract of DOI:10.1016/j.mencom.2021.03.030

Using a diastereomeric mixture of (S)-methionine S-oxide as an example, kinetic preference of methionine-γ-lyase toward a stereogenic center at the γ-sulfur atom of the (2S, R_S) diastereomer was discovered for the first time.

Full text of DOI:10.1016/j.mencom.2021.03.030

Mendeleev
Communications
Mendeleev Commun., 2021, 31, 236–238
Unusual stereoselectivity of methionine-g-lyase
from Citrobacterfreundii toward diastereomeric (S)-methionine S-oxide
Nikolai G. Faleev,*a Marina A. Tsvetikova,a Mikhail M. Ilyin,a Vyacheslav S. Yufryakov,a
Natal’ya G. Kolotyrkina,b Viktoria V. Kulikova,c Tatiana V. Demidkinac and Konstantin A. Kochetkova,d
a A. N. Nesmeyanov Institute of Organoelement Compounds, Russian Academy of Sciences,
119991 Moscow, Russian Federation. E-mail: ngfal@ineos.ac.ru
b N. D. Zelinsky Institute of Organic Chemistry, Russian Academy of Sciences, 119991 Moscow,
Russian Federation
c V. A. Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, 119991 Moscow,
Russian Federation
d D. I. Mendeleev University of Chemical Technology of Russia, 125047 Moscow, Russian Federation
DOI: 10.1016/j.mencom.2021.03.030
O
S
O
Using a diastereomeric mixture of (S)-methionine S-oxide as
an example, kinetic preference of methionine-g-lyase toward
a stereogenic center at the g-sulfur atom of the (2S,RS)
diastereomer was discovered for the first time.
enzyme
(2S,SS)-stereoisomer
HO
Me
NH2
(2S,SS) and (2S,RS)
Keywords: methionine-g-lyase, stereoselectivity, kinetic resolution, diastereomers, (S)-methionine S-oxide.
The biocatalytic cleavage of racemates has been intensely
studied in recent years.1,2 Lipases are widely used in organic
synthesis to separate racemic amines and diamines,3,4 alcohols,5
diols6 and hydroxy esters,7 and to perform stereoselective
reactions, e.g., the Michael reaction.8 For example, previously
we used PPL lipase for hydrolytic cleavage of esters of a number
of amino acids9,10 and for enantioselective acylation of
venfalaxine with vinyl acetate,11 and utilized methionine-g-lyase
for the cleavage of phosphorus analogues of sulfur-substituted
amino acids.12,13
The successful stereoselective cleavage of unusual substrates
comprising a few chiral centers by enzymes is much less
common. As a rule, the known examples include the preliminary
separation of racemic diastereomers by standard methods, most
commonly by chromatography, followed by enzymatic cleavage
of the substrates into individual enantiomers.6,14–16 In this case,
the cleavage of both diastereomers usually occurs with the same
result and with similar stereospecificity.
Methionine-g-lyase (EC 4.4.1.11, MGL) used in this work
belongs to the extensive family of pyridoxal-5'-phosphate (PLP)
dependent lyases that have a wide substrate specificity and
absolute kinetic preference toward the stereogenic center at the
substrate α-carbon atom. It catalyzes the reversible α,g-elimi-
nation of l-methionine and its analogues, S-alkyl-l-methionines,
as well as b-elimination of l-cysteine and analogues to give
ammonia, α-keto acids and thiols.17 It was revealed that MGL
also catalyzed the α,b-elimination of l-cysteine S-alk(en)yl
sulfoxides to give thiosulfinates that were found to have in vitro
antimicrobial activity against gram-negative and gram-positive
bacteria.18 The mutant form of the recombinant enzyme from
Citrobacterfreundii with replacement of histidine by Cys115
cleaves sulfoxides more efficiently than wild-type MGL.19
Studies of the reaction of the enzyme with (2S,SS) and (2S,RS)
l-cysteine S-alk(en)yl sulfoxides disclosed that C115H MGL
decomposed both diastereomers to an equal extent,20 i.e., that
process was nonselective with respect to the configuration of the
leaving group in the b-elimination reaction. Similar experiments
with α,g-elimination were not carried out before, and the
question of the kinetic preference of the enzyme toward a
stereogenic center of a certain configuration in the g-leaving
group remained open thus far.
Our goal was to explore the reaction of MGL with a new
substrate, i.e., a diastereomeric mixture of (2S,SS)- and (2S,RS)-
(S)-methionine S-oxide 1. This is important because MGL is
considered as a promising anticancer agent. The defect of
metabolism known as the ‘methionine dependence’ is the
inability of tumor cells to synthesize methionine.21 To date, the
antitumor efficiency of the enzyme has been shown in vitro and
in vivo.22,23
The experiments were carried out at room temperature by
taking aliquots of the reaction mixture at certain time intervals.†
An accurately weighed amount of the standard (R)-Phe was
added to each aliquot and the amount of each stereoisomer in the
reaction mixture was determined. The experimental results and
chromatographic analysis of the reaction mixtures demonstrated
that the concentration of the major (2S,SS) diastereomer did not
change in the course of the reaction with MGL, whereas the
concentration of the minor (2S,RS) diastereomer gradually
decreased (Table 1). Accordingly, the ratio of diastereomers also
changed from 63:37 to 84:16 during the reaction time studied.
†
The enzymatic process was carried out in a solution of potassium
phosphate buffer (10 ml, pH 8.1) containing pyridoxal phosphate
(1 mmol), l-methionine sulfoxide (40 mg, 0.24 mmol), and methionine-
g-lyase (39 mg) at room temperature with stirring. During the reaction,
aliquots (0.2 ml) of the reaction mixture were taken at regular intervals,
in which the relative content of diastereomers was determined. For this
purpose, standard (R)-Phe solution (0.2 ml, 0.24 mmol) was added to
each aliquot, and the mixture was refluxed at 100 °C for 15 min, separated
from the protein precipitate and evaporated. The residue was analyzed by
NMRcombinedwithHPLConachiralcolumn(seeOnlineSupplementary
Materials). The reaction products were further analyzed by IR spectro-
scopy and mass spectrometry.
© 2021 Mendeleev Communications. Published by ELSEVIER B.V.
on behalf of the N. D. Zelinsky Institute of Organic Chemistry of the
Russian Academy of Sciences.
– 236 –

Mendeleev Commun., 2021, 31, 236–238
O
O
S
stereoselectivity toward the chiral leaving groups in the reactions
in question.
The use of wild-type MGL allowed us to isolate one of the
stereoisomers by a relatively rare approach utilizing the high
enzyme selectivity toward one of the substrate’s diastereomers.
As a result, MGL consumes only (2S,RS)-1 diastereomer as the
substrate in the g-elimination reaction while the other one
remains unchanged. This allows one to isolate the major
(2S,SS)-1 diastereomer with high enantiomeric purity.
O
O
i
S
+
HO
Me
HO
Me
HO
Me
NH2
O
(2S,RS)-1
decay products
retention
O
O
i
S
HO
Me
NH2
(2S,SS)-1
Enzymatic technique for the preparation of chiral sulfoxides
that is enantio-complementary to the known (S)-enatiomer-
reducing activity of methionine sulfoxide reductase A was
described previously.25 Kinetic resolution has been successfully
accomplished with high enantioselectivity, and (R)-configured
chiral sulfoxides were prepared in ~50% yield.26 Such examples
of the separation of diastereomers due to their different resistance
to external effects, including chiral ones to which enzymes may
be attributed,1 are quite rare. In fact, diastereomeric ferrocene27
and paracyclophane28 complexes were previously separated due
to their different stability on the carrier surface, which resulted in
the complete decomposition of one of the diastereomers by silica
gel chromatography and isolation of pure stereomeric products
in each of the examples studied.
Scheme 1 Reagents and conditions: i, methionine-g-lyase, phosphate
buffer (pH 8.1), room temperature, 24 h.
Table 1 Action of methionine-g-lyase on (S)-methionine S-oxide 1 SS/RS
diastereomeric mixture.
HPLC data/a mmol (%)
t/h
(2S,SS)-1
(2S,RS)-1
0
5
15.1 (63)
16.2 (74)
14.4 (84)
8.9 (37)
5.8 (26)
2.8 (16)
24
a Chirobitic TAG column 250×4.6 mm; MeOH/H2O (60:40) + 0.2% DEA,
0.70 ml min–1, 210 nm UV detector.
It is of note that such a high stereoselectivity of this enzyme
to the configuration of the chiral center on the g-sulfur atom in
the presence of one more chiral atom at the α-position was
observed for the first time. This fact expands the prospects of
MGL utilization in processes involving the isolation of individual
stereoisomers of physiologically active compounds with central
chirality not only in the α-position of amino acids12,13 but also
additionally in the g-position.
Thus, the enzyme catalyzes the stereoselective cleavage of
only one of the stereoisomers in the diastereomeric mixture,
while the other one remains unchanged. The latter was isolated
after 48 h processing with ee > 92%. Such a high stereoselectivity
of this enzyme toward the configuration of the chiral center on
the sulfur atom in the presence of one more chiral atom at the
g-position has been discovered for the first time. All the reaction
1
products were identified. The H NMR spectra of the starting
This study was supported by the Ministry of Science and
Higher Education of the Russian Federation and by the Program
of fundamental research for state academies for
2013–2020 years (no. 01201363820). This work was performed
using scientific equipment of the Center for the Study of the
Structure of Molecules at INEOS RAS (Institute of
Organoelement Compounds of the Russian Academy of
Sciences).
compound containing a superposition of the spectra of the two
stereoisomers are transformed on treatment with MGL into
distinct spectra of the major (2S,SS)-1 diastereomer. The positive
sign specific rotation values of the original stereoisomer mixture
([a]D20 = +3.74) remains positive ([a]D = +2.02) in the final
reaction mixture. The decomposition products of the minor
diastereomer include α-ketobutyric and methanesulfinic acids
identified by IR, 1H NMR spectroscopy and mass spectrometry.
Thus, the IR spectrum of the product mixture isolated after the
reaction contains, in addition to the signals of the pure
diastereomer, also intense bands at 1090 and 980 cm–1
characteristic of methanesulfinic acid, as well as signals of the
methylthio group at 1317 cm–1. New bands corresponding to the
carbonyl group of α-ketobutyric acid appear at 1630 and
1600 cm–1. The mass spectral decomposition of the mixture is
also consistent with the presence of an (S)-methionine S-oxide
stereoisomer, α-ketobutyric and methanesulfinic acids. MALDI
data (103.9545) additionally indicate the presence of ketobutyric
acid. The formation of sulfinic acid in the reaction products can
be explained by the autoxidation of monomethyl sulfoxide,
which is formed as an intermediate upon decomposition.
The mechanism of elimination reactions catalyzed by MGL
implies proton transfer to the leaving group as one of the stages.
It was suggested according to X-ray diffraction analysis,24 that
side group of active site Lys (Lys210 of C. freundii MGL) is the
proton donor in the case of α,b-elimination reaction of sulfur
containing amino acids catalyzed by the PLP-dependent
enzymes, whereas active site Tyr (Tyr113 of C. freundii MGL) is
the donor in α,g-elimination reaction. These groups are located
on the opposite sides of the cofactor’s pyridoxal ring plane.
Thus, the chiral environment of the leaving group should
principally differ in α,b- and α,g-elimination reactions. This
may have been the reason for the differences in the enzyme
Online Supplementary Materials
Supplementary data associated with this article can be found
in the online version at doi: 10.1016/j.mencom.2021.03.030.
References
1 U. T. Bornscheuer and R. J. Kazlauskas, Hydrolases in Organic
nd
Synthesis, 2 edn., Wiley-VCH, Weinhein, 2006.
2 P.-Y. Stergiou, A. Foukis, M. Filippou, M. Koukouritaki, M. Parapouli,
L. G. Theodorou, E. Hatziloukas, A. Afendra, A. Pandey and
E. M. Papamichael, Biotech. Adv., 2013, 31, 1846.
3 A. Kamal, G. B. R. Khanna, R. Ramu and T. Krishnaji, Tetrahedron
Lett., 2003, 44, 4783.
4 A. Luna, I. Alfonso and V. Gotor, Org. Lett., 2002, 4, 3627.
5 T. Sakai, Y. Miki, M. Tsuboi, H. Takeuchi, T. Ema, K. Uneyama and
M. Utaka, J. Org. Chem., 2000, 65, 2740.
6 R. Liu, P. Berglund and H.-E. Hogberg, Tetrahedron: Asymmetry,
2005, 16, 2607.
7 P. Borowiecki and M. Bretner, Tetrahedron: Asymmetry, 2013, 24, 925.
8 O. M. Rodrigo, A. de Souza, L. M. C. Matos, K. M. Gonzalves,
I. C. R. Costa, I. Babics, S. G. F. Leite, E. G. Oestreicher and
O. A. C. Antunesa, Tetrahedron Lett., 2009, 50, 2017.
9 F. M. Plieva, K. A. Kochetkov, I. Singh, V. S. Parmar, Yu. N. Belokon’
and V. I. Lozinsky, Biotechnol. Lett., 2000, 22, 551.
10 E. A. Markvicheva, S. V. Kuptsova, T. Yu. Mareeva, A. A. Vikhrov,
T. N. Dugina, S. M. Strukova, Yu. N. Belokon, K. A. Kochetkov,
E. N. Baranova, V. P. Zubov, D. Poncelet, V. S. Parmar, R. Kumar and
L. D. Rumsh, Appl. Biochem. Biotechnol., 2000, 88, 145.
– 237 –

Mendeleev Commun., 2021, 31, 236–238
11 K.A. Kochetkov, M.A. Galkina and O. M. Galkin, Mendeleev Commun.,
21 J. O. Mecham, D. Rowitch, C. D.Wallace, P. H. Stern and R. M. Hoffman,
Biochem. Biophys. Res. Commun., 1983, 117, 429.
2010, 20, 314.
12 K. V. Alferov, N. G. Faleev, E. N. Khurs, Yu. N. Zhukov and
R. M. Khomutov, Mendeleev Commun., 2002, 12, 2.
13 K. V. Alferov, N. G. Faleev, E. N. Khurs, Yu. N. Zhukov and
R. M. Khomutov, Mendeleev Commun., 2003, 13, 127.
14 O. O. Kolodiazhna, A. O. Kolodiazhna and O. I. Kolodiazhnyi,
Tetrahedron: Asymmetry, 2013, 24, 37.
15 S. Gogoi and N. P. Argade, Tetrahedron: Asymmetry, 2006, 17, 927.
16 T. Saravanan, R. Selvakumar, M. Doble and A. Chadha, Tetrahedron:
Asymmetry, 2012, 23, 1360.
22 R. M. Hoffman, Expert Opin. Biol. Ther., 2015, 15, 21.
23 K. Kawaguchi, Q. Han, S. Li,Y. Tan, K. Igarashi, T. Murakami, M. Unno
and R. M. Hoffman, Cells, 2019, 8, 410.
24 A. Messerschmidt, M. Worbs, C. Steegborn, M. C. Wahl, R. Huber,
B. Laber and T. Clausen, Biol. Chem., 2003, 384, 373.
25 V. Nosekand and J. Míšek, Chem. Commun., 2019, 55, 10480.
26 J. Yang, Y. Wen, L. Peng, Y. Chen, X. Cheng and Y. Chen, Org. Biomol.
Chem., 2019, 17, 3381.
27 V. V. Dunina, E. D. Razmyslova, O. N. Gorunova, M. V. Livantsov and
Y. K. Grishin, Tetrahedron: Asymmetry, 2005, 16, 817.
28 V. V. Dunina, E. I. Turubanova, M. V. Livantsov, K. A. Lyssenko,
N. V. Vorontsova, D. Yu. Antonov and Y. K. Grishin, Tetrahedron:
Asymmetry, 2009, 20, 1661.
17 H. Tanaka, N. Esaki and K. Soda, Enzyme Microb. Technol., 1985, 7,
530.
18 E. A. Morozova, V. V. Kulikova, A. N. Rodionov, S. V. Revtovich,
N. V. Anufrieva and T. V. Demidkina, Biochimie, 2016, 128–129, 92.
19 V. V. Kulikova, N. V. Anufrieva, S. V. Revtovich, A. S. Chernov,
G. B. Telegin, E. A. Morozova and T. V. Demidkina, IUBMB Life, 2016,
68, 830.
20 V. Kulikova, E. Morozova, A. Rodionov, V. Koval, N. Anufrieva,
S. Revtovich and T. Demidkina, Biochimie, 2018, 151, 42.
Received: 20th October 2020; Com. 20/6343
– 238 –