Thus, although the sulfur atom of the thioester moiety is important for the reaction mechanism of thioester-dependent enzymes, its specific properties can also affect the affinity and comp
Trang 1catalytic cysteine are required for a productive mode
of substrate binding in bacterial biosynthetic thiolase,
a thioester-dependent enzyme
Gitte Merila¨inen1,2, Werner Schmitz3, Rik K Wierenga1,2and Petri Kursula1
1 Department of Biochemistry, University of Oulu, Finland
2 Biocenter Oulu, University of Oulu, Finland
3 Biozentrum der Universita¨t, Wu¨rzburg, Germany
The reaction mechanisms of many enzymes depend on
thioester chemistry For example, enzymes involved in
lipid metabolism, functioning in both degradative
and synthetic pathways, use as substrates fatty acid
molecules, conjugated via a reactive thioester moiety
to the SH group of pantetheine This pantetheine
moiety is part of either CoA or acyl carrier protein
(ACP) All members of the thiolase superfamily of
enzymes, including, for example, thiolases as well as
the related 3-ketoacyl-ACP-synthases (KAS) [1], have
a reactive cysteine in the active site, playing a key role
in the reaction cycle by accepting the fatty-acyl moiety from either acyl-CoA (thiolases) or from acyl-ACP (KAS)
The kinetic and structural properties of the bacterial Zoogloea ramigera thiolase have been studied in detail [2–11] This biosynthetic thiolase is a condensing enzyme that catalyses the formation of acetoace-tyl(AcAc)-CoA from two molecules of acetyl(Ac)-CoA, utilizing the unique chemistry of thioester compounds This reaction consists of two chemical conversions via
a ping-pong mechanism [12]: an acetyl transfer and a
Keywords
active site; calorimetry; coenzyme A;
thiolase; X-ray crystallography
Correspondence
P Kursula, Department of Biochemistry,
University of Oulu, PO Box 3000,
FIN-90014, Oulu, Finland
Fax: +358 8 5531141
E-mail: petri.kursula@oulu.fi
(Received 1 July 2008, revised 2 September
2008, accepted 10 October 2008)
doi:10.1111/j.1742-4658.2008.06737.x
Thioesters are more reactive than oxoesters, and thioester chemistry is important for the reaction mechanisms of many enzymes, including the members of the thiolase superfamily, which play roles in both degradative and biosynthetic pathways In the reaction mechanism of the biosynthetic thiolase, the thioester moieties of acetyl-CoA and the acetylated catalytic cysteine react with each other, forming the product acetoacetyl-CoA Although a number of studies have been carried out to elucidate the thio-lase reaction mechanism at the atomic level, relatively little is known about the factors determining the affinity of thiolases towards their substrates
We have carried out crystallographic studies on the biosynthetic thiolase from Zoogloea ramigera complexed with CoA and three of its synthetic analogues to compare the binding modes of these related compounds The results show that both the CoA terminal SH group and the side chain
SH group of the catalytic Cys89 are crucial for the correct positioning of substrate in the thiolase catalytic pocket Furthermore, calorimetric assays indicate that the mutation of Cys89 into an alanine significantly decreases the affinity of thiolase towards CoA Thus, although the sulfur atom of the thioester moiety is important for the reaction mechanism of thioester-dependent enzymes, its specific properties can also affect the affinity and competent mode of binding of the thioester substrates to these enzymes
Abbreviations
Ac, acetyl; AcAc, acetoacetyl; ACP, acyl carrier protein; CT, cytosolic thiolase; ITC, isothermal titration calorimetry; KAS, 3-ketoacyl-ACP-synthase; MPD, 2-methyl-2,4-pentanediol; PDB, Protein Data Bank; PP, pantetheine-11-pivalate.
Trang 2Claisen condensation (Fig 1) During acetyl transfer,
the C1 atom of Ac-CoA reacts electrophilically with
the reactive SH of Cys89, forming a covalent
acety-lated intermediate [13] In the subsequent Claisen
con-densation, the C2 atom of the second Ac-CoA attacks
the Ac-enzyme intermediate nucleophilically; the
nucle-ophile is generated by proton abstraction from the C2
of Ac-CoA (Fig 1) It has been shown that two
oxyanion holes in the catalytic cavity play a key role
in this reaction mechanism [2] Oxyanion hole I is formed by a water molecule, referred to as Wat82, and Ne2(His348) This oxyanion hole binds the Ac-CoA thioester oxygen atom, facilitating the nucleophilic attack of the C2 atom of Ac-CoA to the carbonyl carbon atom of the acetyl moiety of the acetylated enzyme The electrophilic reactivity of the latter atom
Fig 1 The thiolase reaction mechanism and the compounds used (A) The thiolase reaction In the biosynthetic direction, the overall reac-tion uses two molecules of Ac-CoA to generate AcAc-CoA and CoA Cys89 is activated for nucleophilic attack by His348 (B) Comparison of the covalent structures of CoA (top), SPP (middle) and OPP (bottom) SPP is different from CoA, such that the pantetheine moiety has an ester linkage to a pivalate group instead of 3¢-phospho-ADP In OPP, the reactive SH group of SPP is further replaced by an OH group In CoA, certain atoms of the pantetheine moiety are numbered.
Trang 3is increased by oxyanion hole II, being formed by
N(Cys89) and N(Gly380) and binding the thioester
oxygen atom of the Cys89-bound acetyl group;
oxyan-ion hole II is similar to the ‘classical’ oxyanoxyan-ion hole
seen in, for example, serine proteases
Although mutation of the reactive Cys89 to serine
still allows for transacetylation at low efficiency [6], the
exchange of the substrate thioester sulfur atom to an
oxygen apparently prevents the reaction from taking
place [4–7] The compounds used in such studies have
consisted of thio- and oxoesters of a CoA analogue,
pantetheine-11-pivalate (PP) [3], which lacks the
3¢-phospho-ADP moiety of CoA, having a pivalate
group at the 11-hydroxyl moiety of pantetheine instead
(Fig 1) The acetyl and acetoacetyl thioesters of PP
(Ac-SPP and AcAc-SPP) have been found to be
substrates of the bacterial biosynthetic thiolase [3–5]
Furthermore, 3-pentynoyl-SPP was used to identify
Cys378 as a catalytic residue [7,8]
The oxoesters of PP are nonreactive [5–7,14] Lower
reactivity of the oxoester compared to the thioester is
also seen when comparing the kinetic properties of
crotonyl-CoA and crotonyl-oxyCoA: the hydration
rate of the oxoester by crotonase is 330-fold lower
[15] Due to different resonance properties of the
thio-and oxoesters [16], the pKa of the a carbon of a
thio-ester is approximately 21, whereas the pKa for the
corresponding atom in an oxoester is 26 [17] This will
make Claisen condensation more difficult with an
oxo-ester because the nucleophilic carbanion is harder to
generate (Fig 1) Furthermore, the reactivity of the
carbonyl carbon of thioesters and oxoesters towards
nucleophilic attack is different [18,19], with oxoesters
being less reactive Thus, from the chemical properties
of oxoesters, it can be postulated that they are less
reactive in both halves of the thiolase reaction than
thioesters No previous studies have addressed the
pos-sibility that the poor reactivity of the esters of OPP in
the thiolase reaction could also be, at least partially,
related to the preferred nonproductive binding modes
of these oxoesters
Members of the thiolase superfamily, being
struc-turally and mechanistically related [1], are of
consider-able interest in the field of biotechnology; the
applications utilizing their potential in biosynthetic
reactions are diverse [20–25] The Z ramigera thiolase
is a very close homologue of the human cytosolic
thiolase (CT) [11], which is a key enzyme in the
cho-lesterol synthesis pathway [26–28], and a detailed
understanding of the binding determinants of its
substrate could also help in the development of
suit-able drugs towards CT, with the aim of lowering high
cholesterol levels Additionally, other human thiolases
have been found to be drug targets for the treatment
of heart failure [29–32]
In the present study, we used a combination of crys-tallography and calorimetry to analyse the binding determinants of CoA to the bacterial biosynthetic thio-lase from Z ramigera, concentrating specifically on the
SH groups of the substrate and the catalytic Cys89, which is conserved throughout the thiolase superfam-ily The results obtained indicate that the sulfur atoms
of both the enzyme and the substrate are important for the correct productive mode of binding of CoA in the thiolase active site, improving our understanding
of substrate recognition by thioester-dependent enzymes
Results and Discussion
The biosynthetic thiolase from Z ramigera is a tetra-meric 160 kDa enzyme, consisting of four identical subunits of 392 residues (Fig 2) Three domains of approximately equal lengths have been identified in the thiolase fold The core of the monomer is formed by the N-terminal domain (with the catalytic cysteine) and the C-terminal domain The third domain, referred
to as the loop domain, protrudes out of the N-terminal domain The loop domain covers the catalytic site and provides the binding site for the 3¢-phospho-ADP moiety of CoA (Fig 2)
In the present study, we aimed to analyse in detail, using X-ray crystallography and isothermal titration calorimetry, the factors that influence the productive mode of binding of a CoA substrate to the active site
of biosynthetic thiolase We solved five new liganded crystal structures of Z ramigera thiolase (Table 1; see also Fig S1) and compared these with the previously available structures of liganded complexes of this and other thiolases The results obtained, as discussed in detail below, indicate a crucial role for sulfur–sulfur interactions in defining the catalytically competent binding mode of CoA in the active site
The pantetheine binding tunnel of biosynthetic thiolase
When CoA binds to Z ramigera biosynthetic thiolase, its pantetheine moiety enters the narrow pantetheine binding tunnel and its SH group closes off the catalytic cavity, near the catalytically important residues Cys89, His348 and Cys378 The residues forming the walls of the tunnel include the side chains of Leu148, His156, Met157, Ala234, Phe235, Ala243, Ala246, Ser247, Gly248 and Leu249 of the loop domain, as well as Ala318 and Phe319 from the C-terminal domain
Trang 4b-strand Cb2, and Met134 of the neighboring subunit.
These residues encircle the pantetheine moiety The
two peptide moieties of the pantetheine unit are both
similarly tightly wedged between Phe235 and Leu249
(the outermost peptide bond, near the bulk solvent)
and between Phe319 and Leu148 (the innermost
pep-tide bond, near the catalytic site)
The atoms shaping the binding environment of the
terminal sulfur atom of CoA are listed in Table 2 and
shown in Fig 2 Apart from the sulfur-containing
resi-dues Cys89, Met157, Met288 and Cys378, these also
include side chain atoms of Ala318 and Phe319; the
latter two residues are a part of the highly conserved
NEAF sequence motif [1] Within 4.8 A˚ from the CoA
sulfur atom, there are also the catalytic water (Wat82)
and Ne2(His348) (Table 2) (i.e the atoms forming
oxyanion hole I) Wat82 is hydrogen bonded to
Nd1(Asn316) and Wat49 Wat82 and Wat49 are pres-ent in each of the five new structures (Fig 3)
A prominent feature of the binding pocket for the CoA sulfur atom is the presence of the four sulfur atoms from Cys89, Met157, Met288 and Cys378 Due
to the high polarizability of sulfur atoms [33], it is expected that sulfur–sulfur interactions will contribute significantly to the van der Waals binding energy Cys89 and Cys378 are catalytic residues, but also Met157 and Met288 are highly conserved in the thio-lase family An interesting exception is thiothio-lase T2; in this case, Met288 is replaced by a phenylalanine This
is a unique feature of the T2 thiolase sequence, which correlates with its unique substrate specificity: T2 is able to use not only acetoacetyl-CoA, but also the branched 2-methylacetoacetyl-CoA molecule as a substrate [34]
C
Fig 2 The active site of thiolase (A) The overall shape of the biosynthetic thiolase tetramer The four individual active sites of the tetramer are indicated by the bound CoA molecules The catalytic site of the yellow domain is marked by an arrowhead (B) The binding mode of CoA
to the thiolase monomer The three domains are coloured yellow (N-terminal domain), light-green (loop-domain) and light gray (C-terminal domain), respectively The NEAF motif (including Phe319) is coloured red, the loop 231–240 (shifted in the OPP⁄ Ac-OPP structures and con-taining Phe235) is coloured purple and the catalytic Cys89 is coloured dark blue His156 at the entrance of the pantetheine-binding cavity is coloured orange Note how the CoA interacts mainly with the loop domain and Cys89 (C) A detailed view of the surroundings of the termi-nal moiety of CoA in the thiolase active site, as seen in the complex of unmodified thiolase with CoA (a similar view to that in B) Contacts
of the terminal sulfur are coloured green, hydrogen bonds are coloured red, and interactions between hydrophobic side chains and the planar amide bonds of CoA are coloured yellow.
Trang 5Only two hydrophilic side chains point into the
pantetheine binding tunnel: Ser247 and His156 Ser247
is known to adopt two different conformations; in the
unliganded form, it points away from the tunnel, and,
in the liganded conformation, it points into the tunnel,
interacting with the N4 atom of the CoA substrate
[2,10] His156 has previously been observed in only
one conformation, having van der Waals contacts to the pantetheine moiety of CoA at the outer edge of the tunnel Water molecules are also present in the pantetheine binding tunnel, both in the unliganded and liganded states [2,11] There is one conserved water near the bottom of the pantetheine binding tunnel (Wat149), interacting with O(Gly248) and O(His348)
Table 2 Distances between the terminal sulfur or oxygen atom of the pantetheine moiety of the active site ligand and surrounding atoms.
As a reference, the structure of the unmodified Z ramigera thiolase complexed with CoA (PDB entry 1DLV) has been used, considering all atoms within 4.8 A ˚ of the CoA sulfur The thiolases are from Z ramigera, unless otherwise specified In the oxidized structures, the active site cysteine, Cys89 or its equivalent, has been oxidized to a sulfenic acid In the acetylated structures, the active site cysteine has been acetylated.
Wild-type oxidized
Wild-type oxidized
Human CT oxidized
Human T2 oxidized
Human T2 acetylated
Wild-type acetylated
Table 1 Data processing and refinement statistics The numbers in parentheses refer to the highest resolution shell.
CoA (oxidized
Ac-OPP (oxidized Cys89)
Data processing
Resolution range (A ˚ ) 20–2.65
(2.72–2.65)
20–1.51 (1.55–1.51)
20–1.87 (1.95–1.87)
20–2.30 (2.40–2.30)
20–2.07 (2.20–2.07)
Unit cell parameters (A ˚ , ) 84.3, 79.2, 150.8
90, 92.9, 90
84.3, 78.7, 148.3
90, 92.9, 90
84.4, 79.1, 148.8
90, 92.7, 90
84.2, 79.6, 148.9
90, 92.1, 90
84.4, 79.0, 148.8
90, 93.0, 90 Refinement
rmsd B factors for bonded atoms
(main chain, side chain) (A˚2 )
Trang 6This water is present in all of the new structures (Fig 3)
The mode of binding of SPP closely resembles that of CoA
The crystal structure of Z ramigera thiolase was solved in complex with SPP, a functional CoA ana-logue SPP and its thioesters are known to be func-tional substrates for thiolase, but kinetic constants have been reported only for AcAc-SPP [3]; for exam-ple, Km and kcat are 73 lm and 469⁄ s for AcAc-SPP, whereas they are 24 lm and 465⁄ s, respectively, for AcAc-CoA In the crystal structure, the binding modes
of the reactive sulfur moieties of SPP and CoA are highly similar, explaining the reactive nature of SPP For example, the distance between the sulfur atoms of SPP and the reactive Cys89 is 4.1 A˚, whereas the cor-responding distance for the CoA complex is 3.9 A˚ (Table 2)
The pantetheine moiety of SPP binds in the pant-etheine binding tunnel in a similar (but not identical) way to that seen for CoA (Fig 3A) The pivalate head group points outwards from the pantetheine binding tunnel, overlapping with the pyrophosphate moiety of CoA It lies on a hydrophobic surface comprising Leu249, Phe18 and Met134; the latter comes from the tetramerization loop of an opposing subunit These results indicate that the 3¢-phospho-ADP group of CoA is not crucial for the correct positioning of the reactive end group of the substrate in the thiolase cata-lytic cavity However, it could be important for increasing the affinity of binding, as well as the solub-ility of the substrate
Interestingly, the side chain of Ser247 points away from SPP, as previously seen in unliganded thiolase; in the case of a bound CoA ligand, it points towards the ligand This difference correlates with minor conforma-tional differences between the pantetheine moieties of CoA and SPP The highly similar mode of binding of the reactive moieties of CoA and SPP in the active site
of thiolase provides a structural basis for the observa-tion that SPP compounds are funcobserva-tional substrates of thiolase
The binding mode of OPP is unproductive The structure of Z ramigera biosynthetic thiolase was also solved in the presence of OPP and its acetylated analogue The complex with OPP was refined at a res-olution of 1.51 A˚ The structure indicates a surprising binding mode (Fig 3A); OPP is bound further away from the catalytic cavity than SPP and CoA
A
B
C
Fig 3 Comparison of the active site ligand binding modes to
bio-synthetic thiolase (A) The binding modes of SPP (magenta), OPP
(cyan), Ac-OPP (orange) and CoA (gray) A water molecule (I) in the
OPP complex is bound to oxyanion hole I His156 has a double
con-formation in the Ac-OPP complex (B) The binding mode of CoA to
thiolase harboring a modified active-site Cys89 is identical to that
seen for unmodified thiolase The CoA complexes of unmodified
(gray), oxidized (yellow) and acetylated (green) Z ramigera thiolase;
the human oxidized CT CoA complex (dark gray); and the human
acetyl-T2 CoA complex (pink) are shown Oc(Ser247) points
towards the pantetheine moiety, except for the complex between
oxidized Z ramigera thiolase and CoA, in which Ser247 has a
dou-ble conformation (C) Superposition of CoA complexes of wild-type
(gray) and C89A (brown) thiolase In the CoA C89A complex, there
are two extra water molecules in the active site cavity; one water
(II) is bound to oxyanion hole II and another nearby water molecule
(e) is hydrogen bonded to it.
Trang 7The catalytic cavity lies at the bottom of the
pant-etheine-binding tunnel The terminal oxygen of OPP is
5.2, 7.4, 6.2 and 7.6 A˚ away from the sulfur atoms of
Cys89, Met288, and Cys378 and Met157, respectively
(Table 2) OPP has a terminal hydroxyl group, which
can form hydrogen bonds with water when in solution,
and with the protein once bound in the complex From
the structure, it is apparent that the hydroxyl group of
OPP is in hydrogen bonding interaction with
Ne2(His348) (at 3.2 A˚) and O(Ala318) (at 3.3 A˚),
being inserted into a pocket between these atoms This
pocket would be too tight for a sulfur atom, such as
those in CoA and SPP This mode of binding places
the terminal oxygen atom further away from the center
of the catalytic cavity and, in this structure, oxyanion
hole I has a water molecule bound (Fig 3)
In the CoA mode of binding [10], there is a
water-mediated hydrogen bond between Ne2(His156) and
O9(CoA), as well as a hydrogen bond between
Oc(Ser247) and N4(CoA), and the carbonyl O(Ser247)
is hydrogen bonded to N8(CoA) For OPP, these
hydrogen bonds are not observed The side chain of
Ser247 points away from the bound OPP, and there
only exists a hydrogen bond between O(Ser247) and
N4(OPP) because the pantetheine group has moved
away from the bottom of the tunnel compared to SPP
and CoA (Fig 3) Consequently, the terminal t-butyryl
moiety of OPP is in a different conformation than that
of SPP Because this group is in a different
conforma-tion in SPP and OPP, it is not specifically recognized
by thiolase, which is as expected for a synthetic
ana-logue The t-butyryl group of OPP is bound in a
pocket created by a rotation of the His156 side chain
around v1, away from its position observed in all
earlier structures The residues forming the walls of
this hydrophobic pocket include Met143, Ile144,
Leu148, His156, Ala234, Phe235, Leu249 and Met134
(from the opposing subunit) The side chains of Ile144
and Met134 are also in a slightly different
conforma-tion compared to all previous structures Moreover,
the entire loop containing residues 231–240 (Fig 2)
moves away by approximately 1 A˚ in the presence of
OPP; at the same time, the B factors for this loop
indi-cate a more rigid structure than in the presence of
CoA (data not shown) These changes are relatively
extensive compared to those seen upon CoA binding,
in which case it has been noted that the Sc atom of
Cys89 and the Oc atom of Ser247 are the only
non-hydrogen protein atoms that move detectably during
the thiolase reaction cycle [2,9,10]
OPP differs from SPP by only one atom, and the
replacement of the sulfur atom of SPP by an oxygen
changes the interaction such that OPP binds in a
differ-ent way to the thiolase active site This mode of binding
of OPP to biosynthetic thiolase is unproductive, and not competent for catalysis Nonproductive binding is a well-known phenomenon in enzymological studies [33] Classic examples from structural studies include the binding of (NAG)3to lysozyme [35] and that of the ind-olyl acryloyl moiety in the active site of chymotrypsin [36] More recent examples concern crystallographic binding studies of elastase [37], dihydrofolate reductase [38] and methylmalonyl CoA decarboxylase [39]
We also solved the structure of the complex of thio-lase with Ac-OPP; from the electron density map and the subsequent structure comparison, it can be seen that the binding mode is the same as that for OPP; furthermore, the structural changes of the protein part described for the OPP complex are seen as well in the Ac-OPP complex Due to low occupancy of the com-pound in the structure, further detailed analysis of the exact binding mode has not been performed, but the mode of binding of the acetyl moiety is clearly differ-ent from the mode of binding of the acetyl moiety of Ac-CoA Apparently, the common carbonyl oxygen atom of the acetyl group of these two molecules does not provide enough binding energy to favour a similar mode of binding for Ac-OPP and Ac-CoA
Binding mode of CoA to thiolases oxidized or acetylated at Cys89
The structure of CoA-complexed Z ramigera thiolase,
in which the catalytic Cys89 was oxidized, was also determined This was carried out to allow a detailed comparison of the binding mode of CoA to thiolase when the sulfur atom of Cys89 is modified covalently The oxidation of the catalytic cysteine to a cysteine sulfenic acid has been observed in complexes of the human cytosolic thiolase [11] and the human mito-chondrial thiolase T2 [34] The extra oxygen atom points away from the ligand binding pocket into oxy-anion hole II and, in these complexes, the mode of binding of CoA is unaffected by the different oxidation state of the cysteine sulfur (Fig 3B) The same applies
to thiolases complexed with CoA in their acetylated form, as seen in the structures of human T2 [Protein Data Bank (PDB) entry 2F2S] and Z ramigera thio-lase [9] For the Z ramigera thiothio-lase, the mode of binding of CoA is unaffected by the oxidation (or acet-ylation) state of the active site cysteine (Fig 3B) Indeed, the mode of binding of the CoA sulfur atom is conserved also in the structures with acetyl-CoA com-plexed with the acetylated active site [10] In the OPP complexes, Cys89 is oxidized to a cysteine sulfenic acid, but the above results indicate that the different
Trang 8modes of binding of OPP and Ac-OPP, when
com-pared with the corresponding SPP or CoA compounds,
cannot be attributed to the oxidation state of the
cata-lytic cysteine
Binding mode of CoA to the C89A thiolase
variant
To better understand the importance of the sulfur–
sulfur interactions in the catalytic cavity of thiolase,
the CoA binding properties of its C89A variant were
also studied In the crystal structure, CoA binding
essentially follows the wild-type mode, apart from the
reactive terminus of CoA The terminal sulfur is
inserted deeper into the catalytic cavity by
approxi-mately 1.7 A˚, being sandwiched between His348 and
Cys378 In the wild-type complex, the reactive
termi-nus of CoA is apparently in a somewhat strained
con-formation (Fig 3C) This indicates that the sulfur
atom of Cys89 is crucial for the correct positioning of
the reactive moiety of CoA In addition, in the
com-plex of the C89A thiolase mutant with CoA, a water
molecule is placed in oxyanion hole II, formed by the
backbone nitrogens of residues Cys89 and Gly380, and
a second water is observed hydrogen bonded to
O(Gly147) (Fig 3C) The tight fit of CoA into the
active site in this case is illustrated by the distances
from the S atom of CoA to Ne2(His348) (3.3 A˚),
Cb(Ala89) (3.6 A˚), a water bound to oxyanion hole II
(3.3 A˚) and water 82 (3.5 A˚) The distances to the
active site sulfurs are: Sc(Cys378) (4.3 A˚), Sd(Met288)
(4.7 A˚) and Sd(Met157) (5.7 A˚), reflecting the overall
weakening of the sulfur–sulfur van der Waals interac-tions in the CoA binding mode of the C89A variant, especially concerning Met157 (Table 2), in addition to the absence of the sulfur–sulfur interaction between CoA and Cys89
Calorimetric analysis of the binding of CoA by wild-type thiolase and the C89A variant The detailed crystallographic binding studies described above show that the sulfur–sulfur interactions in the active site of thiolase are important for competent binding of the substrate Therefore, a calorimetric study was carried out to further investigate the impor-tance of sulfur–sulfur interactions for the affinity of thiolase towards its substrate Isothermal titration calorimetry (ITC) was used to compare the affinity
of CoA to wild-type thiolase and its C89A variant The results obtained (Fig 4 and Table 3) indicate that the affinity is significantly higher for the wild-type enzyme compared to the C89A variant, although only one sulfur atom is missing in the mutant The differ-ence in affinity corresponds to a loss of free energy of binding (DDG) of 0.8 kcalÆmol)1, whereas the loss of enthalpy of binding (DDH) is 3.7 kcalÆmol)1 The key difference in the crystal structures is the absence of the sulfur atom of Cys89, generating small structural dif-ferences (Fig 3); the binding cavity for the reactive moiety of the substrate is less compact in the C89A mutant, which is consistent with the favorable differ-ence binding entropy term [D(-TDS)] The magnitude
of the unfavorable DDH term indicates that the sulfur–
Fig 4 Calorimetric analysis of CoA binding
by (A) wild-type and (B) C89A thiolase.
Curve fitting in both cases was carried out
by setting the binding stoichiometry to 1.
Note the different scales on the y-axis,
which are related to the binding enthalpy.
Trang 9sulfur interactions provide a significant contribution to
the binding energy of CoA [33]
Concluding remarks
Cys89 is the catalytic residue in Z ramigera thiolase,
and its mutation to alanine leads to a complete loss of
activity [2] A corresponding catalytic cysteine residue
is conserved in all members of the thiolase superfamily
In the present study, we have shown that, apart from
the lack of activity, the mutation C89A also lowers the
affinity of biosynthetic thiolase towards CoA
signifi-cantly In complexes between biosynthetic thiolase and
CoA or SPP, a functional substrate analogue, the sulfur
atom of the substrate is always closely embraced by
four sulfur atoms from the enzyme When OPP is used,
being identical to SPP except for the replacement of the
terminal sulfur atom by oxygen, the binding mode of
the ligand also changes, resulting in a nonproductive
binding mode Our data indicate an important role for
the interactions between the CoA substrate sulfur
group and the thiolase active site in assuring an optimal
affinity, as well as a competent mode of binding
There exists considerable interest in the properties
and engineering possibilities of enzymes in the thiolase
superfamily, both within biotechnology and
pharma-cology, due to their involvement in, for example,
dif-ferent natural product synthesis pathways, as well as
in lipid and cholesterol metabolism The results
obtained in the present study indicate that the sulfur
atom of the thioester moiety is not only important
because of the high intrinsic reactivity of thioester
sub-strates, but also that it can play a key role in achieving
the proper affinity and competent mode of binding of
the substrates in the active site cavities of
thioester-dependent enzymes This should be taken into
consid-eration when designing, for example, new substrates or
inhibitors for these enzymes
Experimental procedures
Protein expression, purification and
crystallization
Z ramigera thiolase and its C89A mutant were expressed
and purified as previously described [2,9] Crystallization of
wild-type thiolase and its C89A variant were carried out at
22C using vapour diffusion in a mother liquour contain-ing 1 m Li2SO4, 0.9 m (NH4)2SO4, 0.1 m sodium citrate (pH 5), 1 mm EDTA, 1mM NaN3and 1 mm dithiothreitol
Synthesis of OPP and SPP All intermediates and products were purified using silica gel chromatography, and their purity was checked by TLC The identity of the compounds was demonstrated by 1
H-NMR All chemicals were obtained from Sigma-Aldrich (Taufkirchen, Germany)
Synthesis of ethanolamino-tert.butyl-diphenyl-silane
A solution of 10 mmol tert.butyl-diphenyl-silyl-chloride in
20 mL of tetrahydrofurane was added dropwise with stirring
to 50 mmol ethanolamine in 30 mL of tetrahydrofurane at
0C After stirring for 2 h at room temperature (RT), the solution was evaporated Yield: 8.7 mmol ethanolamino-tert.butyl-diphenyl-silane
Purification of pantothenoic acid
A solution of 20 mmol d(+)-calcium pantothenate in 5 mL
of 4 m HCl was extracted once with 40 mL of chloro-form⁄ methanol (2 : 1) and three times with 40 mL of chlo-roform⁄ methanol (9 : 1) The organic layers were evaporated Yield: 10 mmol pantothenoic acid
Synthesis of tert.butyl-diphenyl-silyl-pantetheinate Dicyclohexyl-carbodiimide (11 mmol) was added to
10 mmol pantothenoic acid in 50 mL of tetrahydrofurane The suspension was stirred until dicyclohexyl-carbodiimide dissolved completely; 8.7 mmol ethanolamino-tert.butyl-diphenyl-silane was then added The solution was stirred for 4 h at RT Yield: 2.6 mmol tert.butyl-diphenyl-silyl-pantetheinate 1H-NMR: C2: 3.48 p.p.m., s.; C11: 3.75 p.p.m., t.; phenyl: 7.65 p.p.m., d
Synthesis of tert.butyl-silyl-OPP
A solution of 4 mmol pyridine was added to a solution of
2 mmol tert.butyl-diphenyl-silyl-pantetheinate in 50 mL of dichlormethane; 2 mmol pivaloylchloride in 20 mL of dic-hlormethane was then added dropwise with stirring After
Table 3 Calorimetric analysis of CoA binding to the Z ramigera biosynthetic thiolase at 25 C The values and error estimates are calculated from separate measurements (three for the wild-type and two for C89A) K a and K d are the association and dissociation constants, respectively.
d (l M )
Trang 10stirring for 24 h at RT, the residue was dissolved in 20 mL
of acetoacetate and extracted with 25 mL each of 0.1 m
HCl, saturated CuSO4 in water, and 2 m NaCl Yield:
1.3 mmol tert.butyl-silyl-OPP 1H-NMR: C2: 3.67 p.p.m.,
s.; C11: 3.92 p.p.m., t.; phenyl: 7.82 p.p.m., d.;
pivalate-methyl: 1.26 p.p.m., s
Synthesis of OPP
A solution of 1 mL of hydrogen fluoride was added to
1.3 mmol tert.butyl-silyl-O-pantetheine-11-pivalate in
30 mL of acetonitrile After stirring for 30 min at RT, the
solution was evaporated The residue was suspended in
20 mL of water and extracted three times with 20 mL of
ethylacetate Yield: 1.2 mmol OPP 1H-NMR: C2:
3.78 p.p.m., s.; C11: 4.03 p.p.m., t.; pivalate-methyl: 1.43, s
Synthesis of Ac-OPP
A solution of 1 mmol pyridine and 0.5 mmol acetyl
chlo-ride was added to a solution of 0.1 mmol OPP in 2 mL of
chloroform and stirred at RT for 15 min The suspension
was extracted three times with 2 mL of brine Yield:
89 lmol Ac-OPP 1H-NMR: C2: 3.59 p.p.m., s.; C11:
4.03 p.p.m., t.; pivalate-methyl: 1.23, s.; acetyl-methyl:
2.17 p.p.m., s
Synthesis of SPP
Bis(N-pantothenylamidoethyl) disulfide was converted into
Bis(N-pantothenylamidoethyl-11-pivalate) disulfide
accord-ing to the synthesis of tert.butyl-silyl-OPP The resultaccord-ing
disulfide was cleaved by treatment with mercaptoethanol
Crystal handling
Crystal structures were determined for the complexes of
thiolase with OPP, Ac-OPP and SPP OPP, Ac-OPP and
SPP were poorly soluble in aqueous buffer solutions In the
OPP soaking experiment, OPP could be dissolved in the
cryoprotectant solution, containing, in addition to the
con-stituents of the well solution, 12% 2-methyl-2,4-pentanediol
(MPD), 12% glycerol and 100 mm OPP Prior to data
collection, the crystal was transferred to this cryosolution
A second crystal was soaked similarly in a solution
contain-ing Ac-OPP instead of OPP In both soakcontain-ing experiments,
the crystals started suffering during the soak under all
tested conditions; thus, the soaking time was approximately
1 min
SPP was dissolved in MPD, at an approximate
concen-tration of 100 mm For soaking SPP into thiolase crystals,
the crystals were transferred to drops of mother liquor
con-taining one-fifth of the SPP stock solution This soaking, in
approximately 20% MPD and 20 mm SPP in mother
liquor, was allowed to continue for 4 days prior to data collection This experiment was performed for both a wild-type thiolase crystal and a C89A mutant crystal Analysis
of the data collected from these crystals showed the mode
of binding of SPP to wild-type thiolase, but no binding was observed when using the C89A crystals
Two more structures were determined and analysed: the structure of a complex of CoA with the C89A variant and
a complex of CoA bound in the active site with an oxidized Cys89 The latter structure was obtained from a soaking experiment of a wild-type thiolase crystal with 5 mm b-hy-droxybutyryl-CoA for 1 min During refinement, it was seen that the structure contained only CoA and the active site Cys89 was oxidized The soaking of CoA into a C89A thiolase crystal was performed at 5 mm CoA
Data collection, structure solution and refinement
Data were collected on beamlines BW7B, X11 and X13
at the EMBL-Hamburg Outstation⁄ DESY (Hamburg, Germany), except for the data from the Ac-OPP complex, which were collected on a Nonius FR591 rotating anode source (Bruker AXS, Delft, the Netherlands) Data process-ing (Table 1) was carried out with xds [40] and xdsi [41] Five percent of all reflections were used for calculating the free R factor [42] The structures were refined using refmac5 [43] tls parameters [44] were applied, and water molecules were added using arp⁄ warp [45] CoA, OPP, Ac-OPP and SPP were built when their electron densities were strong and continuous Model building and analysis were performed using o [46] and coot [47] The refinement statistics are given in Table 1
The coordinates and structure factors were deposited to the PDB under the following accession codes: 2VU2 (thio-lase-SPP complex), 2VU1 (thiolase-OPP complex), 2VU0 (oxidized CoA complex), 2VTZ (C89A thiolase-CoA complex) and 1OU6 (Ac-OPP complex)
Structure analysis The A and B subunits of the biosynthetic thiolase tetra-mer are always best defined in this crystal form of Z ram-igera thiolase due to the layer-like packing of the thiolase tetramers in the crystal lattice, and the B subunit has been used for previous analyses [2,9,10] In the case of SPP and OPP, however, the ligand is slightly better defined in the
A subunit, and this subunit has mainly been analysed in the present study with respect to these ligands All dis-cussed features can, however, be seen in the B subunit Cys89 was oxidized in the structures of the complexes with OPP and Ac-OPP, and it was built as a cysteine sulfenic acid, with the oxygen atom pointing into oxyan-ion hole II