The activity of the enzyme and of several active center mutants was deter-mined using an assay based on O-acetylserine and thio-nitrobenzoate TNB.. Abbreviations CysK, O-acetylserine sul
Trang 1sulfhydrylase isozyme B from Escherichia coli
Georg Zocher, Ulrich Wiesand and Georg E Schulz
Institut fu¨r Organische Chemie und Biochemie, Albert-Ludwigs-Universita¨t, Freiburg im Breisgau, Germany
In bacteria, archaea and plants, the biosynthesis of
l-cysteine involves l-serine and inorganic sulfur
com-pounds [1–5] In higher animals, however, l-cysteine
is derived from l-methionine [1] The bacterial
path-way starts with a transferase that uses acetyl-CoA to
modify serine The resulting O-acetylserine (OAS) is
then converted to cysteine by a sulfhydrylase (OASS,
EC 2.5.1.47), which in general uses hydrogen sulfide
In a number of bacteria, the second step of synthesis
is performed by the two isozymes A and B, named
CysK and CysM, respectively CysK uses mostly
hydrogen sulfide, which is produced in a reduction
pathway that begins with sulfate and requires
dioxy-gen In contrast, CysM has a characteristic main
chain variation around position 210 that opens the active center for larger thiol-carrying compounds, in particular for thiosulfate [2,6] The reaction with thio-sulfate results in S-sulfo-cysteine, which can be easily converted to cysteine and sulfate Consequently, the use of thiosulfate is of particular importance in an anaerobic environment, because it does not require dioxygen for the reduction of sulfate to hydrogen sulfide The isozyme CysM is of technical interest because it processes compounds much larger than hydrogen sulfide, and is therefore a promising candi-date for the production of novel b-substituted
l-amino acids as building blocks for the synthesis of pharmaceuticals and agrochemicals [7–9]
Keywords
biosynthesis of L -cysteine; enzymatic assay;
homodimer asymmetry; nonstandard
L -amino acids; X-ray diffraction
Correspondence
G E Schulz, Institut fu¨r Organische Chemie
und Biochemie, Albert-Ludwigs-Universita¨t,
Albertstr 21, 79104 Freiburg im Breisgau,
Germany
Fax: +49 761 203 6161
Tel: +49 761 203 6058
E-mail: georg.schulz@ocbc.uni-freiburg.de
Website: http://www.structbio.
uni-freiburg.de
(Received 24 July 2007, revised 22 August
2007, accepted 23 August 2007)
doi:10.1111/j.1742-4658.2007.06063.x
The crystal structure of the dimeric O-acetylserine sulfhydrylase isozyme B from Escherichia coli (CysM), complexed with the substrate analog citrate, has been determined at 1.33 A˚ resolution by X-ray diffraction analysis The C1-carboxylate of citrate was bound at the carboxylate position of O-acetylserine, whereas the C6-carboxylate adopted two conformations The activity of the enzyme and of several active center mutants was deter-mined using an assay based on O-acetylserine and thio-nitrobenzoate (TNB) The unnatural substrate TNB was modeled into the reported struc-ture The substrate model and the observed mutant activities may facilitate future protein engineering attempts designed to broaden the substrate spec-trum of the enzyme A comparison of the reported structure with previ-ously published CysM structures revealed large conformational changes One of the crystal forms contained two dimers, each of which comprised one subunit in a closed and one in an open conformation Although the homodimer asymmetry was most probably caused by crystal packing, it indicates that the enzyme can adopt such a state in solution, which may be relevant for the catalytic reaction
Abbreviations
CysK, O-acetylserine sulfhydrylase (EC 2.5.1.47) isozyme A; CysM, O-acetylserine sulfhydrylase (EC 2.5.1.47) isozyme B from Escherichia coli; CysM(K268A), surface mutant K268A of CysM; CysM(RKE), triple surface mutant E57R-Y148K-R184E of CysM; CysM(salmo),
isozyme B from Salmonella typhimurium; DTNB, S,S¢-bis(5-thio-2-nitrobenzoate); TNB, thio-nitrobenzoate; OAS, O-acetylserine; OASS, O-acetylserine sulfhydrylase; PLP, pyridoxal 5¢-phosphate.
Trang 2Five structures of CysK-type enzymes from bacteria
[10–14], archaea [15] and plants [16,17], and two
struc-tures of bacterial CysM [6,18], have been published
The differences between the isozymes CysK and CysM
have been described [6,18] In this article, we present
the structure of CysM complexed with the substrate
analog citrate at high resolution, together with
enzy-matic activity data of several mutants Moreover, we
provide a model of the substrate thio-nitrobenzoate
(TNB) bound at the active center, which may be a
guide for future enzyme engineering studies
Results and Discussion
CysM structures
In solution, CysM from E coli is a dimer of
2· 32 893 Da consisting of 303 amino acid residues
per subunit An earlier study [6] yielded a medium
quality structure of the wild-type enzyme in crystal
form I at 2.7 A˚ resolution [P6522, four subunits per
asymmetric unit; reservoir: 0.1 m ammonium sulfate,
0.1 m citrate pH 5.6 with poly(ethyleneglycol)] An
improved structure was derived from crystal form II of
the triple surface mutant CysM(RKE) that diffracted
to 2.1 A˚ resolution, but was completely twinned,
decreasing the effective resolution [I41, four
subun-its per asymmetric unit; reservoir: 0.15 m CaCl2, 0.1 m
Hepes pH 7.6 with poly(ethyleneglycol)] [6] In this
article, we report the structure of the surface mutant
CysM(K268A) at 1.33 A˚ resolution in crystal form III
(Table 1) Crystal form III was grown essentially under
the same conditions as form I, except for the absence
of ammonium sulfate The surface mutation K268A
was at the rim of a packing contact and was not
required for crystallization, but was essential for the
superior packing order and for reproducible crystal
growth
The structure of crystal form III was determined by
the molecular replacement method In contrast with
the other crystal forms, form III contained only one
subunit per asymmetric unit and a lower solvent
content, both of which are typical prerequisites for
high resolution X-ray diffraction (Table 1) Although
crystal forms I and III were grown from the same
citrate buffer, only form III showed a citrate molecule
bound to the active center Apparently, the high ionic
strength of ammonium sulfate prevented citrate
binding in form I The structure of CysM in crystal
form III is shown in Fig 1 Citrate was bound in
two conformations with occupancies of 60% and
40%, as revealed by the electron density depicted in
Fig 2 Binding in multiple conformations indicates
low affinity, which, in turn, agrees with our observa-tion that citrate does not inhibit the enzyme (see below)
In order to identify established structures of related enzymes, we searched the Protein Data Bank for sequence homologs and detected 11 entries with sequence identity above 30%, all of which were OASSs Lowering the threshold, the next entries were two cystathione b-synthases with 29% and 24% iden-tity Ten of the entries were CysK-type enzymes, which showed around 40% sequence identity with isozyme CysM and are not considered in the following analysis One entry was CysM from Salmonella typhimurium [CysM(salmo)] [18], which has 94% sequence identity and is closely related to the enzyme CysM from E coli presented here
Enzymatic activity and reaction geometry
In order to obtain data on enzyme engineering for the synthesis of novel compounds [7–9], we produced active center mutants and determined their catalytic activity using TNB as the nucleophile TNB seems to
be most appropriate for guiding enzyme engineering intended for the synthesis of compounds of similar size The activities of wild-type CysM and of the crys-tallized mutant K268A were identical, and only the wild-type value is given in Table 2 This agreement
Table 1 Structure analysis Values in parentheses are for the high-est resolution shell The data were collected at 0.9050 A ˚ wave-length at beamline PX-II of the Swiss Light Source (SLS, Villigen, Switzerland) The crystal belonged to space group P6522 with unit cell axes a ¼ b ¼ 76.6 A˚ and c ¼ 209.8 A˚ containing one CysM subunit per asymmetric unit and 55% solvent.
Data collection
Refinement Number of atoms, protein (residues 1–294)
2290 Number of atoms, glycerol ⁄ citrate 12 ⁄ 26
Rcryst⁄ R free (2% test set) 0.158 ⁄ 0.172 Average isotropic B-factors (A˚2 )
main chain ⁄ side chains 16.6 ⁄ 20.4 glycerol ⁄ citrate ⁄ water 24.2 ⁄ 16.6 ⁄ 33.0
Rmsdbond lengths (A ˚ ) ⁄ angles () 0.016 ⁄ 1.68 Ramachandran: most
favorable ⁄ allowed (%)
98.0 ⁄ 2.0
Trang 3was expected because position 268 is at the surface
dis-tant from the active center and from the dimer
inter-face (Fig 1)
The reported CysM structure contains a substrate-like active center ligand, which is the bound citrate molecule depicted in Fig 2 A comparison with the four known external aldimine complexes of CysK-type enzymes [11,13,14,16] showed clearly that the C1-boxylate of citrate occupies the binding site of the car-boxylate of OAS Whereas the C1-carcar-boxylate is well fixed at loop 69 (residues 68–72), the distal C6-carbox-ylate of citrate adopts two conformations The hydro-xyl group of citrate points towards the internal aldimine, as is expected for the amino group of OAS (Fig 2) In view of the bound citrate molecule, we determined the enzyme activity in the presence of up
to 25 mm citrate, but observed no change Therefore, citrate is not an inhibitor This agrees with the two observed citrate conformations, because multiple bind-ing is usually weak
The observed kinetic parameters of wild-type CysM from E coli are in general agreement with those of the homolog CysM(salmo) [18,19] Of the active center mutants produced, the deletion of a methyl group near
Fig 1 Stereo ribbon plot of the high resolu-tion structure of the CysM dimer, including the molecular twofold axis (black), which is crystallographic The position of the surface mutation K268A is shown as a yellow sphere 25 A ˚ away from the active center The cofactor PLP covalently linked to Lys41, the bound citrate molecule in its major con-formation and the mutated residues Thr68, Gln140 and Arg210 in the active center are depicted as ball-and-stick models The sub-units have different colors The mobile loops defined in Fig 5 are labeled using gray spheres The active center pocket opening
is indicated by a yellow stick.
Fig 2 Detailed stereoview of the active center of CysM The covalently bound PLP and the associated citrate are shown in orange Citrate was bound with 100% occu-pancy The minor conformation of citrate is gray The (Fo) F c ) electron density map of citrate is outlined at the 3.0 r contour level The mutated residues are cyan Hydrogen bonds to the citrate molecule are indicated
by broken lines Chain cuts are marked by halos.
Table 2 Enzymatic activity of CysM from Escherichia coli The
esti-mated relative errors are about 20% The OAS concentration was
always 10 m M ; the TNB concentration varied from 10 to 1000 l M
The temperature was 37 C The values in parentheses were
mea-sured at 25 C.
kcat
(s)1)
KM(TNB) (m M )
kcat⁄ K M (TNB) (%)
Temperature dependence a
a
The temperature dependence is defined here as k cat ⁄ K M (TNB)
measured at 37 C relative to the value measured at 25 C b The
absolute kcat⁄ K M (TNB) value at 37 C was 3.5 · 10 4
M )1Æs)1 This
value was set to 100% c The absolute k cat ⁄ K M (TNB) value at 25 C
was 1.4 · 10 4
M )1Æs)1.
Trang 4pyridoxal 5¢-phosphate (PLP) in mutant T68S caused
the smallest disturbance (Table 2) Given the high
activity of this mutant, we determined the KM(TNB)
value, which was essentially identical to that of the
wild-type (Table 2) We conclude that the missing
methyl group of T68S decreases the activity only
slightly and does not affect TNB binding A decisive
decrease to merely 2% catalytic efficiency was
observed with mutant R210A Even stronger decreases
were caused by the removal of a carboxamide in
mutant Q140A, and by the deletion of a hydroxyl
group in mutant T68A The enzyme was inactive when
a carboxylate was introduced at position 140 (Q140E)
The moderate activity reduction of T68S and the
strong effects of mutations Q140A, T68A and Q140E
agree well with the data derived for the corresponding
mutants of the CysK-type enzyme from Arabidopsis
thaliana[16]
In a second series of experiments, we determined the
kcat⁄ KM(TNB) values at 25C The results were similar
to those at 37C, except for a 2.3-fold decrease for the
wild-type and for mutants T68S and R210A (Table 2)
The 2.3-fold decrease relates well to the decrease in kcat
expected from the ‘rule-of-thumb’ factor of two for a
10 K temperature drop [20], showing that the
activa-tion energy of the catalyzed reacactiva-tion lies in the usual
range and does not change for T68S and R210A In
contrast, mutants Q140A and T68A showed much
higher temperature dependence factors, corresponding
to an appreciable increase in the activation energy [20]
We conclude that Q140A and T68A, which are close
to PLP, directly affect the reaction In contrast, the
activity decrease of R210A, which is rather distant from PLP, is probably a result of inefficient TNB binding, causing a large increase in KM(TNB) The proposed binding deficiency agrees with our TNB model (see below) and also with an earlier thiosulfate model [6] The mutants were also checked with respect to their
A280⁄ A412 ratio A photometric measurement of CysM(K268A) yielded a ratio of 4.3, which agrees well with the ratio of 4.0–4.2 established for the closely homologous CysM(salmo) [18] It also agrees with the theoretical value calculated from the absorption spec-tra of the tryptophans, tyrosines and PLP The mutants showed A280⁄ A412 ratios in the range 4.3–4.5, except for mutant Q140E with a ratio of 5.5 This deviation was significant It corresponds to a PLP occupancy of about 75% Mutant Q140E showed no enzymatic activity (Table 2) It is conceivable that the newly introduced glutamate adjacent to PLP made a salt bridge to Lys41, prohibiting the formation of the internal aldimine (see Fig 2)
In order to model the reaction geometry, we used the established external aldimine structure of a related CysK structure [11] and transferred it to CysM, where
it could be accommodated without steric collision (Fig 3) The expected reaction geometry at the exter-nal aldimine intermediate [11] defines the thiol position
of TNB to a small region above the plane of the acry-late double bond As a result of this constraint and of the spacious active center pocket of CysM, TNB was placed rather easily In our model, the carboxylate of TNB is fixed by Arg210 and the nitro group points to the solvent (Fig 3) The thiolate is located above the
Fig 3 Stereoview of the reaction geometry based on the structure of CysM(K268A) The observed internal aldimine with Lys41 is given in a transparent mode (gray) The external aldimine structure has been transferred from a CysK-type enzyme [11] It is shown together with a manually placed model of the bound substrate TNB, the carboxylate of which is fastened to Arg210 The thiolate of TNB is approximately at the same position as the attacking sulfur of thiosulfate in a previous model [6], which is well suited for the nucleophilic attack (red dotted line) on the amino acrylate double bond (green spheres) Hydrogen bonds are given as black broken lines All van der Waals distances between TNB and its environment are above 3.0 A ˚ The two shortest contacts are marked by green dotted lines.
Trang 5acrylate plane forming an S–Cb–Ca angle of about
90 This is an ideal position for attacking the double
bond In summary, our negative experience with
muta-tions close to PLP suggests that this region should not
be touched when trying to produce novel l-amino
acids [7–9] Rather, such engineering attempts should
follow the TNB model, which suggests residues
Met119, Phe141, Thr175, Pro207 and Arg210 as the
main targets
Induced fit
A comparison between the E coli CysM structures in
the three crystal forms revealed several characteristic
features, which are also valid for the crystal structure
of CysM(salmo) [18] In order to establish the intrinsic
mechanical properties of CysM, we superimposed the
observed chain folds in Fig 4 Deviations occurred at
the N- and C-termini and at the four surface loops at
positions 21, 60, 190 and 271, far away from the active
center and also from the dimer interface These
dif-ferences are of low significance, because they are at
positions that are usually mobile More interesting
variations occurred near the active center
As shown in Fig 1, the opening of the active center
pocket to the solvent is rather distant from the dimer
interface The opening can be considered as a mouth
with two lips One lip consists of loops 69, 94 and
helix a4(118–132), and the other is formed by
loops 202 and 215 (Fig 4) The lip positions vary
greatly between the structures Similar changes have
been reported for the other isozyme, CysK, for which
the extreme lip positions have been named ‘open’ and
‘closed’ [11] The chain fold of CysM(K268A) with the
bound citrate is ‘half-closed’ (Fig 4) Although the
variations in Fig 4 are probably caused by more or
less random crystal contacts, they still outline the available conformational space and, most probably, the induced fit motions during the reaction
The conformational changes are also reflected in the B-factor distributions that report the polypeptide chain mobility As the B-factor level is strongly dependent
on the quality of the crystal order, the B-factor distri-butions have been normalized by referring them to the average B-factors of the respective chains They are displayed in Fig 5 The distribution of CysM(K268A) shows nine characteristic mobility peaks Of these, the loops at peak positions 94, 116, 132, 202 and 215 form the lips of the mouth of the active center pocket (Fig 1) and are therefore important for catalysis The other peaks correspond to loops at the surface that are usually mobile (Fig 1) Interestingly, loop 69 is close
to PLP and not mobile (Fig 5), although it partici-pates in the induced fit (Fig 4)
The mobility distributions of CysM(K268A), wild-type CysM and CysM(salmo), and those of subunits B and D of CysM(RKE), resemble each other closely (Fig 5) However, a most surprising deviation of the B-factor distribution occurs in subunits A and C of CysM(RKE) [6] The CysM(RKE) crystal contains dimers A–B and C–D, providing four independent sub-unit structures Dimer A–B is asymmetric with respect
to mobility and also with respect to structure The B-factor distribution of subunit A is exceptional, as it shows almost no mobility peak In contrast, the respective distribution of subunit B shows the common mobility peaks, including those of the active center lips (Fig 5) The same asymmetry is observed with sub-units C and D of the other dimer As the three muta-tions of CysM(RKE) are all at the surface distant from the active center, they are unlikely to affect the internal stability of the protein Consequently, the
Fig 4 Stereoview of a superposition of five distinct CysM chain folds showing wild-type CysM in blue [6], CysM(RKE) subunit A in green, CysM(RKE) subunit D in orange [6], CysM(K268A) in red and CysM(salmo) in gray [18] The highly mobile regions are labeled using gray spheres (see Fig 5).
Trang 6observed asymmetry should reflect a general property
of CysM
The asymmetry of CysM(RKE) is probably caused
by crystal packing contacts Such contacts are usually
weak, so that they can only switch between
conforma-tions that are connected via low energy barriers As a
consequence, the observation of two independent
asymmetric homodimers in a crystal indicates that this
asymmetric state can be easily adopted in solution
Therefore, it is conceivable that the ‘closed’
tion of subunit A corresponds to the CysM conforma-tion after substrate binding, whereas the ‘open’ conformation of subunit B shows CysM when releas-ing the products after the reaction has taken place Such a see-saw system is discussed as ‘half-site reactiv-ity’ [21] We conclude that the observed asymmetry suggests that CysM is a suitable candidate for explor-ing the half-site reactivity hypothesis
Experimental procedures
Mutagenesis and activity assay
The mutants were produced with the QuikChange method (Stratagene, Heidelberg, Germany), verified by DNA sequencing (SeqLab, Go¨ttingen and GATC, Konstanz, Germany) and expressed and purified as described previ-ously [6] They were stored at )20 C in a 12 mgÆmL)1 solution containing 10 mm Tris⁄ HCl pH 8.0 For the assay, we incubated 950 lL of buffer A (100 mm Hepes
pH 7.0, 10 mm OAS, 10–1000 lm TNB) at 37C (or
25C) for 3 min, and started the reaction by adding
50 lL of a solution containing 0.5–80 lg of the enzyme The enzyme solution was always freshly prepared from stored protein so that the exposure time to 37C (or
25C) was minimized This was important for the low activity mutants at positions 68 and 140 near PLP TNB was always freshly prepared in 50 mm Hepes pH 7.0
by adding 2 mm dithiothreitol and 0.5 mm S,S¢-bis(5-thio-2-nitrobenzoate) (DTNB) to yield 1 mm TNB The absorption of TNB was monitored at 412 nm using e412¼
13 600 m)1Æcm)1 [19], as well as at 500 nm using e500¼
970 m)1Æcm)1, which was established in a separate experi-ment The measurement at 500 nm was necessary in order
to reach TNB concentrations beyond the Michaelis con-stant of 0.7 mm The cysteine-nitrobenzoate produced has its absorption maximum at 312 nm and does not absorb light at 412 nm The values for kcat and KM(TNB) were obtained from reciprocal plots; the values for
kcat⁄ KM(TNB) were derived from linear plots
Crystallization, structure determination, refinement and modeling
The surface mutant K268A was produced and purified as described previously [6] and then crystallized using the hanging drop method The drops contained 2 lL of an
8 mgÆmL)1 enzyme solution mixed with 2 lL of reservoir buffer [100 mm sodium citrate pH 5.4, 18% (w⁄ v) poly(eth-yleneglycol) 3000] Crystals of CysM(K268A) grew within about 10 days at 20C to sizes of up to 1000 lm ·
400 lm· 400 lm The crystals were transferred in four steps to 28% (v⁄ v) glycerol in reservoir buffer and flash-frozen in a 100 K nitrogen gas stream
Fig 5 Relative B-factor distributions of CysM subunits in four
dif-ferent crystal forms The B-factors were referred to the respective
subunit averages in order to eliminate differences arising from
crys-tal packing quality variations All distributions were smoothed by
sliding a three-residue-averaging window along the chain The top
diagram K268A refers to the reported high resolution structure with
labels at nine high mobility peaks (see Figs 1 and 4) Distribution
WT is an average of the four closely related subunit chains of the
wild-type structure [6] The distribution of CysM(salmo) is from
unit A, which is virtually the same as those of the other seven
sub-units [18] The two distributions at the bottom are from dimers
A–B and C–D of CysM(RKE) [6] which, however, were split into an
average of the closely related subunits B and D and the equally
well-related subunits A and C.
Trang 7The X-ray data were collected at the Swiss Light Source
(Villigen, Switzerland) (Table 1) and processed with
pro-grams xds and xscale [22] Using phaser [23] and the
wild-type CysM structure [6], the phases were established
by molecular replacement To avoid model bias, the CysM
structure and the water structure were completely rebuilt
using arp⁄ warp [24] The structure was manually
com-pleted using coot [25] and then refined with refmac5 [26]
Finally, we performed a translation libration screw
refine-ment with refmac5 using the 12 translation libration screw
groups (1–22, 23–65, 66–84, 85–98, 99–114, 115–131, 132–
164, 165–188, 189–208, 209–221, 222–249, 250–294)
pro-posed by the program tlsmd [27] The CysM structure was
validated with rampage [28] The rigid TNB molecule was
positioned manually into the active center Numerous
options were checked visually using coot [25], and
inter-preted with respect to the quality of all contacts The
short-est distance to the adjacent residues was maximized in
order to avoid steric hindrance as much as possible Figures
were drawn using povscript+ [29] and povray (http://
www.povray.org) The coordinates and structure factors
have been deposited in the Protein Data Bank under
acces-sion code 2v03
Acknowledgements
We thank the team of beamline PX-II at the Swiss
Light Source (Villigen, Switzerland) for their help with
data collection, and Wacker-Chemie (Munich,
Ger-many) for support of the project
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