Identification of O-acetylhomoserine sulfhydrylase, a putative enzyme responsible for methionine biosynthesis in Clostridioides difficile: Gene cloning and biochemical characterizations

Article abstract of DOI:10.1002/iub.2139

O-acetylhomoserine sulfhydrylase (OAHS) is a pyridoxal 5′-phosphate-dependent enzyme involved in microbial methionine biosynthesis. In this study, we report gene cloning, protein purification, and some biochemical characteristics of OAHS from Clostridioides difficile. The enzyme is a tetramer with molecular weight of 185 kDa. It possesses a high activity in the reaction of L-homocysteine synthesis, comparable to reported activities of OAHSes from other sources. OAHS activity is inhibited by metabolic end product L-methionine. L-Propargylglycine was found to be a suicide inhibitor of the enzyme. Substrate analogue N^γ-acetyl-L-2,4-diaminobutyric acid is a competitive inhibitor of OAHS with K_i = 0.04 mM. Analysis of C. difficile genome allows to suggest that the bacterium uses the way of direct sulfhydrylation for the synthesis of L-methionine. The data obtained may provide the basis for further study of the role of OAHS in the pathogenic bacterium and the development of potential inhibitors.

Full text of DOI:10.1002/iub.2139

Received: 11 April 2019
DOI: 10.1002/iub.2139
Accepted: 16 July 2019
R E S E A R C H C O M M U N I C A T I O N
Identification of O-acetylhomoserine sulfhydrylase, a putative
enzyme responsible for methionine biosynthesis in
Clostridioides difficile: Gene cloning and biochemical
characterizations
1
1
1
Vitalia V. Kulikova
| Svetlana V. Revtovich | Natalia P. Bazhulina
|
1
1
1
1
Natalya V. Anufrieva | Mikhail I. Kotlov | Vasiliy S. Koval | Elena A. Morozova
|
2
3
1
Hideyuki Hayashi | Yury F. Belyi | Tatyana V. Demidkina
1
Engelhardt Institute of Molecular Biology
Abstract
of the Russian Academy of Sciences,
Moscow, Russia
0
O-acetylhomoserine sulfhydrylase (OAHS) is a pyridoxal 5 -phosphate-dependent
enzyme involved in microbial methionine biosynthesis. In this study, we report gene
cloning, protein purification, and some biochemical characteristics of OAHS from
Clostridioides difficile. The enzyme is a tetramer with molecular weight of 185 kDa. It
possesses a high activity in the reaction of L-homocysteine synthesis, comparable to
reported activities of OAHSes from other sources. OAHS activity is inhibited by meta-
bolic end product L-methionine. L-Propargylglycine was found to be a suicide inhibitor
2
Department of Chemistry, Osaka Medical
College, Japan
3
Gamaleya Research Center for
Epidemiology and Microbiology, Russian
Ministry of Public Health, Moscow, Russia
Correspondence
Vitalia V. Kulikova, Engelhardt Institute of
Molecular Biology of the Russian Academy
of Sciences, Vavilova str., 32, Moscow
γ
of the enzyme. Substrate analogue N -acetyl-L-2,4-diaminobutyric acid is a competitive
inhibitor of OAHS with K = 0.04 mM. Analysis of C. difficile genome allows to sug-
i
1
19991, Russia.
gest that the bacterium uses the way of direct sulfhydrylation for the synthesis of L-
methionine. The data obtained may provide the basis for further study of the role of
OAHS in the pathogenic bacterium and the development of potential inhibitors.
Email: vitviku@yandex.ru
Funding information
Russian Foundation for Basic Research,
Grant/Award Number: 18-34-00669-mol-a
K E Y W O R D S
Clostridioides difficile, methionine biosynthesis, O-acetylhomoserine sulfhydrylase
1
| INTRODUCTION
(CGS, EC 2.5.1.48) and cysteine-S-conjugate β-lyase (CBL,
EC 4.4.1.13) are pivotal enzymes in the transsulfuration path-
0
Pyridoxal 5 -phosphate (PLP)-dependent enzymes catalyze
chemically diverse reactions of amino acids conversions, thus
playing key roles in numerous metabolic processes. This can
be seen for the two pathways of methionine biosynthesis in
microorganisms called direct sulfhydrylation and trans-
sulfuration (Figure 1). PLP-dependent cystathionine γ-synthase
way, while O-acetylhomoserine sulfhydrylase (OAHS, EC
2.5.1.49) is involved in methionine biosynthesis in the
sulfhydrylation pathway. Data on methionine biosynthesis in
bacteria are still rather limited. However, as a rule, bacteria use
either transsulfuration or direct sulfhydrylation pathways. Up
to now Corynebacterium glutamicum was shown to be the
only bacterium that uses both pathways for methionine
γ
Abbreviations: ADAB, N -acetyl-L-2,4-diaminobutyric acid;
1
synthesis.
CBL, cysteine-S-conjugate β-lyase; CGS, cystathionine γ-synthase;
DTT, dithiothreitol; EDTA, ethylenediaminetetraacetic acid; OAHS,
O-acetylhomoserine (OAH) sulfhydrylase; OSH, O-succinylhomoserine;
Clostridioides difficile is an anaerobic gram-positive spore-
forming gut pathogen that causes antibiotic-associated diarrhea
on the worldwide scale. According to the UniProt database in
0
PLP, pyridoxal 5 -phosphate; PPG, L-propargylglycine.
©
2019 International Union of Biochemistry and Molecular Biology
IUBMB Life. 2019;1–9.
wileyonlinelibrary.com/journal/iub
1

2
KULIKOVA ET AL.
FIGURE 2 The γ-substitution reaction catalyzed by O-
acetylhomoserine sulfhydrylase
synthesized from L-homoserine as described in Reference 7.
ADAB was synthesized from L-2,4-diaminobutyric acid and
4-nitrophenyl acetate using the selective acetylation condition
described by Reference L-2,4-Diaminobutyric acid
8
dihydrochloride (2.30 g, 12.0 mmol) was dissolved in 20 mL
H O, and the pH was adjusted to 11.0 with 1 M NaOH. The
2
solution was placed on ice and stirred for 10 min. Then 4.25 g
(24.0 mmol) of 4-nitrophenyl acetate was added, and the solu-
tion was stirred at room temperature for 4 hr. Meanwhile, the
pH of the solution was maintained between 11.0 and 11.2 with
1
M NaOH. The solution was concentrated to 50 mL, washed
with ethyl acetate three times to remove the unreacted
-nitrophenyl acetate and 4-nitrophenol. Ethanol was added to
FIGURE 1 Biosynthesis of L-methionine in bacteria. Adapted
from BioCyc Database Collection
4
the genome of C. difficile, the sequence Q187D4 is annotated
as OAHS. Considering the role of OAHS in methionine
biosynthesis, the inhibitors of C. difficile OAHS could be anti-
biotics against the pathogen. Meantime OAHS is not a well-
studied member of PLP-dependent enzymes family. There are
the solution until precipitation was observed, and was left
ꢀ
overnight at 4 C. The crystals were dissolved in a minimal
+
amount of H O, and applied to an AG50 column (H form,
2
1
5 mm × 170 mm). The column was washed with H O.
2
γ
N -acetyl-L-2,4-diaminobutyric acid was eluted in pass-
through fractions, and separated well from the unreacted
L-2,4-diaminobutyric acid. The fractions containing the prod-
uct was combined and evaporated to 20 mL. Ethanol and
2
preliminary biochemical data on the enzyme from Neurospora
3
and Corynebacterium acetophilum. Some characteristics of
4
5
the enzyme from Aspergillus nidulans, C. glutamicum, and
6
recombinant OAHS from Thermus thermophilus have been
2
-propanol were added until crystallization started, and left
published. Structural data on the enzyme are rather limited as
well. The coordinates of the enzyme from T. thermophilus
ꢀ
overnight at 4 C. The crystals were collected and washed with
ice-cold 2-propanol. The identity of the synthesized compound
was confirmed by MALDI-MS: m/z = 161 [M + H] .
+
(PDB entry 2CB1), Wolinella succinogenes (PDB entry 3RI6)
have been deposited. We cloned the gene of C. difficile, pro-
posed to code OAHS, purified the recombinant enzyme from
E. coli cells, and determined its substrate and reaction specific-
ities, quaternary structure, and steady-state parameters of the
physiological reaction of L-homocysteine synthesis (Figure 2).
The ionic and tautomeric composition of the external Schiff
base of OAHS was determined. To elucidate the mechanism of
OAHS, the substrate analogue, N -acetyl-L-2,4-diaminobutyric
acid (ADAB) was synthesized and interaction of OAHS with it
and some other substrate analogues was studied.
2
.2 | The search of PLP-dependent enzymes
involved in methionine biosynthesis in the
C. difficile genome
The search for C. difficile enzymes annotated as CBL, CGS,
and OAHS was performed in the UniProt database. Addi-
tional search was performed by BLAST based on the amino
acid sequences of these enzymes with proven functions
according to BRENDA database. The selected amino acid
sequences were aligned using the AlignX program complex
γ
(
Vector NTI). Sequences with homology less than 50% were
2
2
| EXPERIMENTAL PROCEDURES
.1 | Materials
excluded from the analysis.
2
.3 | Gene cloning
O-succinyl-L-homoserine, L-methionine, PLP, dithiothreitol
DTT), ethylenediaminetetraacetic acid (EDTA), protamine
(
Chromosomal DNA of strain C. difficile D1/11 (Bacterial col-
lection of the laboratory of molecular pathogenesis, Gamaleya
Research Centre) was used as a template in polymerase chain
sulfate were purchased from Sigma. Na S and L-
propargylglycine were from Fluka, O-acetyl-L-homoserine was
2

KULIKOVA ET AL.
3
reaction (PCR) to synthesize the gene encoding OAHS. Gene
a Sonicator (Qsonica Sonicators). The sonified solution was
diluted to a protein concentration of 10 mg/mL and nucleic
acids were precipitated with 2% solution of protamine sul-
fate for 30 min at room temperature with gentle stirring. The
0
was amplified using the pair of primers: 5 -GGGGATG
0
0
CCATGGATAATAAAGA-3 (forward) and 5 -CAACTGG
0
AGCTCCTGTTATTATA-3 (reverse). To clone the
ꢀ
amplicon, the restriction nuclease sites NcoI and SacI were
inserted in forward and reverse primers. After the PCR, the
amplicon was isolated from the reaction mixture using the
DNA isolation kit (Fermenats, Latvia). The purified DNA
was treated with the restriction endonucleases NcoI and SacI
and ligated with the pET28a vector (Novagen), which was
treated with the same enzymes. The resulting mixtures were
incorporated by electroporation into the Escherichia coli
strain DH5α (Thermo Scientific). The plasmid containing the
clone was selected after growing the transformed mixture on
the solid medium (LB agar with 50 μg/mL of kanamycin).
The colonies were transferred to a liquid LB medium with
kanamycin and cultured for 15–18 hr. Then, the cells were
separated by centrifugation and used to isolate plasmid DNA
using the GeneJET Plasmid Miniprep Kit (Thermo Scientific).
The plasmid was identified by restriction analysis with appro-
priate restriction endonucleases. The final control of the clon-
ing accuracy was performed by sequencing the DNA inserts
lysate was clarified by centrifugation for 30 min at 4 C. The
clarified solution was syringe-filtered through a 0.45 mm
cellulose acetate filter (Corning Life Sciences, Lowell, MA).
The enzyme was purified by ion exchange chromatography
using a HiPrep DEAE FF 16/10 column (GE Healthcare,
Sweden) equilibrated in binding buffer (50 mM potassium
phosphate, pH 7.5, containing 1 mM DTT, 1 mM EDTA,
and 0.1 mM PLP) and was eluted with 200 mM KCl in the
same buffer. The peak fractions containing OAHS were
pooled and concentrated. The enzyme concentration was
10
determined by the Bradford method. The purity was
11
assayed by SDS–PAGE. The specific activity of 95% pure
enzyme preparations was 50 units/mg for the γ-substitution
reaction of OAHS with the cosubstrate Na S.
2
2
.5 | Determination of the oligomeric
composition of the enzyme
(
NBI Genom, Moscow). The sequence has been submitted to
The molecular weight of the enzyme was determined by gel
filtration on a Superose 6 Increase 10/300 GL column (GE
Healthcare, Sweden). A 50 mM potassium phosphate buffer,
pH 7.5, containing 1 mM DTT, 1 mM EDTA, and 0.1 mM
PLP was used for elution.
the GenBank nucleotide sequence database with code
KX371589 (genomic DNA translation APU51341, Figure 3).
2
.4 | Purification of recombinant OAHS
The protein was expressed in E. coli BL21 (DE3) cells in
2
.6 | Enzyme assays
9
ꢀ
auto-induction medium at 37 C for 24 hr, after which the
ꢀ
harvested cells were stored at −20 C. The frozen cell pellet
The OAHS reaction was carried out in closed tube with
OAH as the substrate at 25 C for 4 min. The reaction mix-
ꢀ
was thawed and resuspended in 0.05 M potassium phosphate
buffer, pH 7.5, containing 1 mM DTT, 1 mM EDTA, and
ture (0.2 mL) contained 50 mM potassium phosphate buffer
0.1 mM PLP. The cell suspension was disrupted on ice by
pH 7.5, 0.1 mM PLP, 10 mM OAHS, and 5 mM Na S. The
2
sonication for 15 min with 5 s pulses at 70% amplitude using
reaction was started by the addition of approximately 0.5 μg
FIGURE 3 Alignment of
Clostridioides difficile O-
acetylhomoserine sulfhydrylase sequences
APU51341 (strain D1/11) and Q187D4
(strain 630)

4
KULIKOVA ET AL.
of the protein. After incubation, the concentration of homo-
cysteine formed was estimated by the method of Kredich
and Becker. One unit of the enzyme was defined as the
amount producing 1 μmol of the product per min. The
steady-state parameters of the γ-substitution reaction of
OAHS were determined by the method of Kredich and
2.9 | Determination of the dissociation
constant of PLP
12
The K value of PLP was measured by spectrophotometric
d
titration method. Aliquots of a PLP solution were added to
the solution of the apoenzyme (9.7 × 10− M) in 50 mM
potassium phosphate buffer, pH 7.5, containing 1 mM DTT.
6
Becker with varying Na S or OAHS concentrations. The
−6
2
The concentration of PLP was varied from 5.0 × 10 M to
data were processed according to the Michaelis–Menten
equation using the Enzfitter software program. The molecu-
lar mass of the enzyme subunit 46.35 kDa for OAHS from
C. difficile was used in the calculations. The γ-substitution
reaction with O-succinylhomoserine (OSH) was carried out
under the same conditions.
−4
ꢀ
1
.2 × 10 M. After 30 min incubation at 30 C, free PLP
was removed with a 30,000-Da molecular weight cut-off
concentrator (Sartorius, UK) by centrifugation at 5,000g at
ꢀ
4
C for 5 min. The content of PLP in each fraction was
determined in 0.1 M NaOH. The data were fitted to the
17
Scatchard plot.
The activity of the enzyme in the γ-elimination reaction
of OAHS was determined by the rate of formation of
α-ketobutyrate in coupled reaction with D-2-hydroxyis-
ocaproate dehydrogenase in a 50 mM potassium phos-
phate buffer, pH 7.5, 0.1 mM PLP.
3
3
| RESULTS AND DISCUSSION
1
3
.1 | The search of PLP-dependent enzymes
involved in methionine biosynthesis in the
Inhibition of the γ-substitution reaction by amino acids
was studied under the conditions described previously by
varying the concentrations of substrate and inhibitors in the
reaction mixture. The values of inhibition constants were
determined using the Enzfitter software. The data were
C. difficile genome
We analyzed C. difficile genome to find genes of CBL
and CGS, involved in the biosynthesis of methionine in
transsulfuration pathway, and OAHS involved in the
direct sulfhydrylation pathway (Table 1). All the enzymes
(CBL, CGS, and OAHS) belong to the structural Class I
of PLP-dependent enzymes and the subclass of CBL,
which includes the enzymes involved in the metabolism
of sulfur-containing amino acids.
sequences of these enzymes share high degree of
similarity.
1
4
processed using Dixon coordinates.
To analyze the inactivation by L-propargylglycine
PPG), normalized activities, remaining after given times of
(
incubation, were plotted against the relative concentrations
of PPG and enzyme active sites.
1
8,19
For this reason, the
2
0
2
.7 | Preparation of the holoenzyme and the
apoenzyme
3.2 | Transsulfuration pathway
Holoenzyme was obtained by adding a 50-fold molar excess of
PLP to an enzyme solution in 50 mM potassium phosphate
buffer, 0.5 mM DTT, pH 7.5. After incubation for 1 hr at 25 C,
We found several dozen of sequences annotated as
C. difficile CBL in UniProt database. Degree of similarity
with the sequence of the well-characterized E. coli CBL
(P06721) proved to be about 32%. Degree of similarity with
the sequences of CBL from BRENDA database does not
exceed 57% with similarity for Lactobacillus casei CBL
(A6YH85). The data are presented in Table 1.
Only the one fragment of 90 amino acids annotated as
C. difficile CGS is placed in UniProt database. It has 72%
degree of similarity with the C-terminal fragment of
Haemophilus influenza CGS (P44502) representing only a
quarter of the full-length amino acid sequences of CGS from
different sources. By BLAST search of CGS sequences from
BRENDA within the C. difficile (taxid 1496) nucleotide col-
lection database, the most similar protein sequence Q181I0
(≤54% degree of similarity) was found. Earlier we have
demonstrated that the sequence Q181I0 related to methio-
nine γ-lyase in the genome of the bacterium does not contain
characteristic for methionine γ-lyase active site residues and
ꢀ
the excess of PLP was removed by dialysis against the same
buffer. The apoenzyme was obtained using D,L-penicillamine
15
as described.
The PLP content in enzyme preparations was determined
in 0.1 M NaOH on the basis of 6,600 M− cm as the molar
absorption coefficient of PLP at 390 nm.
1
−1
1
6
2
.8 | Spectrophotometric measurements
Absorption spectra of the holoenzyme and the apoenzyme
ꢀ
were recorded at 25 C in a solution containing 50 mM
potassium phosphate buffer, 0.5 mM DTT, and 1 mM
EDTA, pH 7.5, at a protein concentration of 1.5 × 10− mM
2
(0.7 mg/mL) with Cary-50 spectrophotometer (Agilent
Technologies, Santa Clara).

KULIKOVA ET AL.
5
TABLE 1 The amino acid sequences of PLP-dependent enzymes involved in methionine biosynthesis: the best similarity of the sequences of
Clostridioides difficile enzymes with the sequences of the same enzymes from BRENDA database
The best
Enzyme
UniProt database (C. difficile)
BRENDA database
similarity
Transsulfuration pathway
Cystathionine γ-synthase
b
A0A160XRW0 (90 aa fragment),
Arabidopsis thaliana (P55217) ,
Escherichia coli (P00935) ,
—
b
0
A449YKL4 (92 aa fragment),
A0A450HRH7 (92 aa fragment)
Helicobacter pylori (Q1M0P5), Lens
culinaris (L0EKR2), Mycobacterium
ulcerans (A0PKT3), Solanum
tuberosum (Q9MT29)
b
Cysteine-S-conjugate
β-lyase
A0A031WAA6, A0A069ADS8, Q184L6,
A0A069AJU0, A0A125V982,
Arabidopsis thaliana (P53780) , Brassica
57% (T4HBK5 vs.
A6YH85)
a
b
oleracea (Q84UD0) , Escherichia coli
(P06721) , Homo sapiens (Q6YP21),
Lactobacillus casei (B3TNN8,
B3TNN9, A6YH85) , Lactococcus
lactis (A2RM21) , Neurospora crassa
(Q1K8G0) , Saccharomyces cerevisiae
(E3VL26, P43623) , Solanum
tuberosum (Q9MT31), Streptomyces
venezuelae (Q9EYM7)
b
A0A161KUL1, A0A1M2Y4M0,
A0A160XPA5, A0A160XN97,
A0A171AJQ8, A0A1R5LSM2,
A0A160Y2D0, A0A1R5J0N3, T4ITF1,
T4HBK5, C9YQJ7, A0A0H3N6V8,
A0A381KMM0, A0A381IDR8,
A0A381KL77, A0A381KKP8,
A0A0N1ICC2, A0A0N1HQP1,
A0A0N0NV10, A0A2A3K3N2,
A0A2A3K4S5, A0A381KJ14
b
b
b
b
Direct sulfhydrylation pathway
b
O-acetylhomoserine
sulfhydrylases
Q187D4, A0A125V6A2, A0A0H3N2Q1,
C9YMB5
Leptospira meyeri (P94890) ,
67% (Q187D4 vs.
P94890)
Mycobacterium tuberculosis
L7N4M1), S. cerevisiae (P06106) ,
b
(
Thermus thermophilus (Q76K51,
b
Q93I77) , Wolinella succinogenes
(Q7M844)
a
At present time, formerly cystathionine β-lyase.
The enzymes with confirmed reaction specificity.
b
speculated that it might be the sequence of O-succi-
for Q187D4 sequence, we can conclude that the identifica-
tion of the protein Q187D4 as OAHS is correct.
2
0
nylhomoserine sulfhydrylase.
We cloned the gene,
expressed it in E. coli, purified Q181I0 protein and evaluated
that it contains PLP, but does not possess activities charac-
teristic for PLP-dependent enzymes involved in methionine
biosynthesis: the enzyme has no activity of CGS, CBL, and
OAHS (unpublished data). Thus, given our results, we
hypothesize that transsulfuration pathway is not active in
C. difficile, because it requires both CBL and CGS.
So, the presence of enzymes necessary for the trans-
sulfuration pathway in the C. difficile genome is not obvi-
ous, because 57% degree of similarity does not allow to
assign all analyzed sequences (Table 1) to CBL and we did
not find full-length C. difficile CGS sequences in UniProt
database. Thus, one may assume that C. difficile uses the
direct sulfhydrylation pathway for methionine biosynthesis.
3
.3 | Direct sulfhydrylation pathway
3.3.1 | Purification of the enzyme and its
characteristics
Besides the sequence Q187D4 (strain 630), UniProt database
contains three C. difficile sequences completely identical to
it. The best value of sequence similarity (67%) of the
Q187D4 with the sequences of OAHSes in BRENDA data-
base was found for Leptospira meyeri OAHS (P94890). In
combination with the analysis of the characteristic amino
acid residues for PLP-dependent enzymes catalyzing the
After purification, a homogeneous enzyme preparation
(APU51341) was obtained that had an OAHS specific activ-
ity of 50 U/mg. The subunit molecular mass of the enzyme
was estimated by SDS-PAGE to be approximately 46 kDa
(Figure 4). This coincided well with the value calculated
from the deduced amino acid sequence (46.355 kDa). The
molecular mass of the protein was estimated to be about
2
0
conversion of sulfur containing amino acids, performed

6
KULIKOVA ET AL.
TABLE 2 Kinetic parameters of the γ-substitution reaction
cat, s−
1
kcat/K
m
, M−1 s−1
Substrate
K
m
, mM
k
5
OAH
0.6
0.4
94.12
96.65
1.6 × 10
2.4 × 10
5
Na S
2
OSH, Na
2
S
ND
Abbreviation: ND, not detectable.
6
the enzyme from T. thermophilus (0.72 U/mg), Neurospora
2
(
(
less than 0.04 U/mg), and Pseudomonas aeruginosa
21
0.012 U/mg). The enzyme activity showed an optimum
pH of 7.5 and higher activity was shown in potassium phos-
phate buffer (compared to sodium phosphate and tris–HCl
buffers). The Km for OAH was determined to be 0.6 mM,
which is lower as compared with OAHS from C. glu-
5
6
tamicum (2 mM) and T. thermophilus (6.8 mM), the K
m
for Na S was 0.4 mM that is smaller than K of Na S for
2
m
2
5
the enzyme from C. glutamicum. The k values were sev-
cat
eral times higher than those for previously reported
enzymes. The enzyme does not catalyze the γ-elimination
reaction with OAH and OSH. Also it does not produce
L-cystathionine in the γ-substitution reaction of OSH with
L-cysteine (CGS activity) and does not catalyze the
β-elimination reaction of L-cystathionine (CBL activity).
1
FIGURE 4 SDS-page of OAHS from Clostridioides difficile,
molecular weight markers
2
3.3.3 | Inhibition of OAHS
We studied the inhibition of OAHS in the γ-substitution
reaction (Figure 2) by some amino acids. The end product of
the biosynthetic pathway L-Met inhibited the reaction with
K = 9 mM. The inhibitor ADAB was designed as analog of
i
the natural substrate OAH that does not contain leaving
γ-group and therefore stops the catalytic reaction at the
enamine intermediate. ADAB was found to be reversible
inhibitor of OAHS. The inhibition was competitive with
K = 0.04 mM.
i
PPG has been described to be an active site-directed
inhibitor of transsulfuration-catalyzing enzymes, such as
22
cystathionine γ-lyase (CGL, EC 4.4.1.1), CBL, and CGS.
We determined the activity levels remaining after a 10-min
incubation of OAHS with varying amounts of PPG and rela-
tive activities were plotted against the ratios of the amount
of PPG against the enzyme active sites. As can be seen from
the Figure 6, almost every PPG molecule leads to the inacti-
vation of a single OAHS active site, confirming an
extremely strong inhibition of the enzyme by PPG. Consid-
ering that the dialysis against inhibitor-free buffer did not
lead to a recovery of the enzymatic activity, the inactivation
of the enzyme by PPG seems to be irreversible. Suicide inhi-
FIGURE 5 Gel filtration of Clostridioides difficile O-
acetylhomoserine sulfhydrylase. The column was calibrated using
molecular weight markers (Sigma-Aldrich): 66 kDa—bovine serum
albumin; 150 kDa—alcohol dehydrogenase from yeast; 200 kDa—
β-amylase from sweet potato
180 kDa by gel filtration (Figure 5). Therefore, OAHS from
C. difficile exists in a solution as a tetramer.
3
.3.2 | Catalytic properties of OAHS
bition of CGS (K = 45 μM) and CGL by PPG has been
i
22,23
The enzyme reacted specifically with OAH and did not cata-
lyze the γ-substitution reaction with OSH (Table 2). Most
OAHSes do not catalyze the reaction with OSH, except for
demonstrated.
For OAHS, however, the inactivation rate
was too rapid to determine K and k (inactivation rate con-
i
i
stant). In the structures of methionine γ-lyase from

KULIKOVA ET AL.
7
FIGURE 6 Analysis of suicide inhibition of O-acetylhomoserine
sulfhydrylase by L-propargylglycine. OAHS (100 μM) were incubated
for 10 min with 10–100 μM PPG
FIGURE 7 Resolution of absorption spectrum of O-
acetylhomoserine sulfhydrylase internal aldimine (pH 7.5) onto bands
of individual electron transitions of its tautomers and conformers;
points present experimental data, bold lines are calculated data, and
thin lines present bands corresponding to individual electron
transitions. The structures are labeled as in Table 3
24
Pseudomonas putida (PDB entry 1E5E) and human CGL
complexed with PPG, Cγ of PPG is covalently bound to the
active site Tyr114 and Tyr115, respectively, which form the
basis for irreversible inhibition of the enzymes. Structural
basis for the inhibition mechanism of OAHS by PPG proba-
bly analogous because there is Tyr107 in the same position
in the OAHS sequence.
3
.3.4 | Spectral characteristics of OAHS
PLP-dependent enzymes and the intermediates of the reac-
tions they catalyze have remarkable spectral properties that
are used in studies of diverse mechanisms of enzymatic
amino acids conversions. The absorption spectrum of the
holoenzyme at pH 7.5 in the region of 300–500 nm have the
form peculiar for the internal aldimines of PLP-dependent
enzymes (Figure 7) with a major band in the region of
4
15–416 nm corresponding to ketoenamine tautomer of the
FIGURE 8 Equilibrium of tautomers of an internal aldimine
cationic form
internal aldimines. The spectrum is similar to that of
T. thermophilus OAHS holoenzyme that showed absorption
maximum at 425 nm. In the spectrum there is also a shoul-
der with lower intensity in the 325–340 nm region
corresponding to enolimine tautomer of the internal
aldimines. The binding constant of the coenzyme was deter-
mined equal to 1.3 × 10 M, which is comparable with Kd
value of PLP (2.94 × 10 M), reported for OAHS from
6
26
seven were used. The method has been described in detail
27
earlier. The correctness of the model used for description
of the resolved spectrum was mainly assessed by correlation
of the total amounts of tautomers and conformers to the
amount of PLP bound. The coincidence or closeness of
parameters of the experimental absorption bands with
parameters of the corresponding absorption bands for model
aldimines was used as another test. Data obtained by resolu-
tion using the two models were analyzed, and in the cationic
model the amount of PLP bound in the enzyme was in corre-
lation with the total amount of tautomers and conformers
−5
−
5
25
yeast.
To determine the ionization state of the internal aldimine
of OAHS necessary for understanding mechanism of the
enzymatic reaction, the absorption spectrum recorded at
pH 7.5 were resolved into the bands corresponding to the
individual electron transitions of PLP aldimines with amino
acids. The absorption bands, logarithmically normal func-
tions, and parameters of bands of the tautomers and con-
formers of two ionic forms of model aldimines, cationic and
(ΣC = 1.00 ± 0.02). For the neutral model there was a pro-
i
nounced discrepancy (ΣC = 1.31 ± 0.04).
i
Consequently, the internal aldimine of OAHS can be ade-
quately described by the cationic model (Figure 8), which
contains two tautomers, ketoenamine (Structure II) and
neutral, with the transition pK between them no more than
a

8
KULIKOVA ET AL.
TABLE 3 Absorption spectrum band parameters of internal aldimine of O-acetylhomoserine sulfhydrylase
ν × 10− (cm−1
23.88 ± 0.02
26.46 ± 0.01
29.60 ± 0.01
30.85 ± 0.01
35.04 ± 0.01
35.81
3
)
λ (nm)
ε × 10−3 (M−1ꢁcm−1)
10.90 ± 0.03
8.50 ± 1.93
W × 10−3 (cm−1
)
ρ
f
n (%)
Structure
E (eV)
2.96
3.28
3.67
3.80
4.34
1
II
418.8 ± 0.3
377.9 ± 0.2
337.8 ± 0.1
326.3 ± 0.1
285.4 ± 0.1
279.3
3.53
3.90
3.73
3.47
5.05
4.50
1.45
1.47
1.22
1.29
1.20
1.59
0.20 ± 0.01
0.04 ± 0.01
0.04
59 ± 1
14 ± 1
Ii∠
I
II┴
9.43 ± 0.58
13.0 ± 1
14 ± 3
8.50 ± 1.67
0.04 ± 0.01
0.29 ± 0.03
0.70 ± 0.29
2
a
II
11.08 ± 1.25
17.09 ± 2.86
a
4
.44
Note: E, electron transition energy; ν, wave number; λ, wavelength; ε, molar absorption coefficient; W, half-width; ρ, asymmetry; f, oscillation strength; n, the tautomer
and conformer contents. Above-line indices (1, 2) correspond to the first and second electron transitions of Structure II. Above-line indices (⊥,∠) correspond to two
conformers of Structure II (the conformer with the aldimine group in the plane perpendicular to the pyridine cycle plane and the conformer with the aldimine bond
0
released from the coenzyme ring plane but with retained coupling and hydrogen bond between the aldimine nitrogen atom and the coenzyme 3 -hydroxygroup).
a
Experimental information about these bands is insufficient.
enolimine (Structure I). Figure 7 presents the resolution of
the absorption spectrum of the holoenzyme at pH 7.5
according to the model. Experimental parameters of the
bands are presented in Table 3.
Resolution of the spectrum has shown that, in order to
sufficiently describe it, it is necessary to introduce into the
model two structures with absorptions in the regions of
3
(
10–335 and 366–400 nm, in addition to ketoenamine
Figure 8, Structure II) and enolimine (Figure 8, Structure I).
In the case of model aldimines of PLP with amino acids,
2
8
such structures can be aldimine conformers. A conformer
with the aldimine group completely released from coupling
with π-electrons of the coenzyme pyridine ring, that is, the
conformer with the aldimine group located in the plane per-
┴
FIGURE 9 Absorption spectra of the holoenzyme (0.78 mg/mL)
pendicular to the pyridine cycle is designated as “II .” The
and the enzyme complexed with L-Met (90 mM, dashed line), L-
propargylglycine (2 mM, dashed-dotted line), and N -acetyl-L-
conformer with aldimine bond released from the coenzyme
γ
ring plane but with retained coupling and hydrogen bond
2
,4-diaminobutyric acid (0.4 mM, dotted line) in 0.05 М potassium
0
between the nitrogen atom of the aldimine and 3 -oxygroup
phosphate buffer, рН 7.5
∠
of the coenzyme is designated as “II .” Our findings indicate
inhibitors. Changes in absorption at 440–500 nm may be
due to formation of reaction intermediates (quinonoids,
aminocrotonate) proposed on the way of the γ-substitution
that the internal aldimine of OAHS at pH 7.5 exists as
one ionic form with predominance of tautomer II (both
0
heteroatoms are protonated, whereas the 3 -oxygroup is
29
reaction. The increase in the absorption intensity in the region
of 320–340 nm may be associated with changes of ionic state
of external aldimines and their tautomerism, as well as with for-
deprotonated and bound by a hydrogen bond with the nitro-
gen atom of the aldimine group). Parameters of the dominant
band with λmax = 418.8 nm with molar absorption coeffi-
0
cient 10,900 M− cm coincided with the corresponding
parameters for the absorption band of Structure II of the
model compounds and with parameters of the absorption
band of ketoenamine of Citrobacter freundii tyrosine
1
−1
mation of intermediates with the tetrahedral C4 -atom of PLP.
The spectral characteristics of OAHS holoenzyme and its com-
plexes with the inhibitors will be used in the future pre-steady-
state stopped-flow studies of OAHS mechanism.
2
7
phenol-lyase holoenzyme.
The absorption spectra of the enzyme complexed with
inhibitors were collected (Figure 9). In the spectra the intensity
of ketoenamine band decreased, absorption in the region of
4
| CONCLUSION
4
3
40–500 nm increased, and absorption in the region of
20–360 nm significantly increased. The absorption in the
To our knowledge this is the first case of obtaining and
characterization of OASH from a pathogenic species of
Clostridioides. Our results indicate that most likely C. difficile
utilizes direct sulfhydrylation pathway for methionine
region of 415 nm should be assigned to the absorption of
ketoenamine of the external aldimines formed OAHS with the

KULIKOVA ET AL.
9
biosynthesis. Inhibitors of the enzyme may be potential antibi-
otics against C. difficile.
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ACKNOWLEDGMENTS
18. Kack H, Sandmark J, Gibson K, Schneider G, Lindqvist Y. Crystal
structure of diaminopelargonic acid synthase: Evolutionary rela-
The work was supported by the Russian Foundation for
Basic Research 18-34-00669-mol-a.
0
tionships between pyridoxal-5 -phosphate-dependent enzymes.
J Mol Biol. 1999;291:857–876.
1
9. Tran TH, Krishnamoorthy K, Begley TP, Ealick SE. A novel mecha-
nism of sulfur transfer catalyzed by O-acetylhomoserine sulfhydrylase
in the methionine-biosynthetic pathway of Wolinella succinogenes.
Acta Crystallogr D Biol Crystallogr. 2011;67:831–838.
ORCID
Vitalia V. Kulikova https://orcid.org/0000-0003-0618-
2
0. Revtovich SV, Morozova EA, Anufrieva NV, Kotlov MI,
Belyi YF, Demidkina TV. Identification of methionine γ-lyase in
genomes of some pathogenic bacteria. Dokl Biochem Biophys.
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