The so-called substrate-binding loop residues 187–210 was partially disordered in several subunits, in both the presence and absence of NAD+.. However, in two subunits, this loop was com
Trang 1dehydrogenase from Pseudomonas putida
Karthik S Paithankar1, Claudia Feller2, E Bartholomeus Kuettner1, Antje Keim1, Marlis Grunow2 and Norbert Stra¨ter1
1 Center for Biotechnology and Biomedicine, Institute of Bioanalytical Chemistry, Faculty of Chemistry and Mineralogy,
University of Leipzig, Germany
2 Institute of Biochemistry, Faculty of Biosciences, Pharmacy, and Psychology, University of Leipzig, Germany
Short-chain dehydrogenases⁄ reductases (SDRs)
consti-tute a large protein family that now includes more
than 1000 enzymes in humans, mammals, insects and
bacteria [1] The dehydrogenases act on a wide variety
of substrates, including steroids, retinoids,
prostaglan-dins, sugars and alcohols The name SDR is based on
their smaller subunit size of about 250 residues com-pared with the medium-chain dehydrogenase⁄ reductase family that has a subunit size of about 350 residues The SDR enzymes exhibit a sequence identity of 15–30% and share two signature motifs: a GxxxGxG motif involved in coenzyme binding; and a YxxxK
Keywords
crystal structure; loop closure; protein
dynamics; SDR
Correspondence
M Grunow, Institute of Biochemistry,
Faculty of Biosciences, Pharmacy, and
Psychology, University of Leipzig,
Bru¨derstraße 34, D-04103 Leipzig, Germany
Fax: +49 341 9736998
Tel: +49 341 9736907
E-mail: gru@rz.uni-leipzig.de
N Stra¨ter, Center for Biotechnology and
Biomedicine, Institute of Bioanalytical
Chemistry, Faculty of Chemistry and
Mineralogy, University of Leipzig, Deutscher
Platz 5, D-04103 Leipzig, Germany
Fax: +49 341 9731319
Tel: +49 341 9731311
E-mail: strater@bbz.uni-leipzig.de
(Received 5 July 2007, revised 8 August
2007, accepted 10 September 2007)
doi:10.1111/j.1742-4658.2007.06102.x
D-3-Hydroxybutyrate dehydrogenase from Pseudomonas putida belongs to the family of short-chain dehydrogenases⁄ reductases We have determined X-ray structures of the D-3-hydroxybutyrate dehydrogenase from Pseudo-monas putida, which was recombinantly expressed in Escherichia coli, in three different crystal forms to resolutions between 1.9 and 2.1 A˚ The so-called substrate-binding loop (residues 187–210) was partially disordered in several subunits, in both the presence and absence of NAD+ However,
in two subunits, this loop was completely defined in an open conformation
in the apoenzyme and in a closed conformation in the complex structure with NAD+ Structural comparisons indicated that the loop moves as a rigid body by about 46 However, the two small a-helices (aFG1 and aFG2) of the loop also re-orientated slightly during the conformational change Probably, the interactions of Val185, Thr187 and Leu189 with the cosubstrate induced the conformational change A model of the binding mode of the substrate D-3-hydroxybutyrate indicated that the loop in the closed conformation, as a result of NAD+ binding, is positioned compe-tent for catalysis Gln193 is the only residue of the substrate-binding loop that interacts directly with the substrate A translation, libration and screw (TLS) analysis of the rigid body movement of the loop in the crystal showed significant librational displacements, describing the coordinated movement of the substrate-binding loop in the crystal NAD+ binding increased the flexibility of the substrate-binding loop and shifted the equi-librium between the open and closed forms towards the closed form The finding that all NAD+-bound subunits are present in the closed form and all NAD+-free subunits in the open form indicates that the loop closure is induced by cosubstrate binding alone This mechanism may contribute to the sequential binding of cosubstrate followed by substrate
Abbreviations
PfHBDH, Pseudomonas fragi D-3-hydroxybutyrate dehydrogenase; PpHBDH, Pseudomonas putida D-3-hydroxybutyrate dehydrogenase; SDR, short-chain dehydrogenase ⁄ reductase.
Trang 2motif in the active site Crystal structures are now
known for about 60 SDR members and it is now clear
that SDRs are single-domain enzymes; by contrast,
medium-chain dehydrogenases⁄ reductases consist of a
cosubstrate-binding domain and a substrate-binding
domain [2,3] SDR enzymes exist as monomers, dimers
or tetramers The tetrameric enzymes exhibit 222
point-group symmetry Conventionally, the three
mutually perpendicular two-fold axes are named P, Q
and R [4]
One of the most variable parts of the different SDR
enzymes is a loop consisting of two a-helices that
pro-trudes out of the compact tetramer This so-called
sub-strate-binding loop, which is located between b-strand
F and a-helix G, is involved in recognition of the
structurally different substrates The substrate-binding
loop differs significantly in length and sequence and
also adopts different conformations when comparing
open or closed forms of different SDR enzymes It has
also been shown in some crystal structures that the
loop undergoes a conformational change upon
sub-strate binding [4–6] Subsub-strate-induced conformational
changes from an open to a closed conformation, or
from a disordered (conformationally flexible) to an
ordered structure, have also been observed Nakamura
et al recently demonstrated by X-ray crystallography
and CD spectroscopy that coenzyme binding to
3a-hy-droxysteroid dehydrogenase alone induces a transition
of the loop from a disordered structure to a
conforma-tion consisting of two a helices [7] Spectroscopic
studies on 17-b-hydroxysteroid dehydrogenase by
fluo-rescence energy transfer also indicated that a
confor-mational change might occur upon coenzyme binding
[8] To the best of our knowledge, a coenzyme-induced
conformational change to a closed conformation of
the substrate-binding loop has, to date, not been
ana-lyzed by crystallographic means
D-3-Hydroxybutyrate dehydrogenase from
Pseudo-monas putida (PpHBDH) (EC 1.1.1.30, GenBank
accession number AJ310211.2) catalyzes the reversible
and stereospecific oxidation of D-3-hydroxybutyrate
to acetoacetate using NAD+ as a coenzyme One
subunit contains 256 amino acids with a calculated
molecular weight of 26.6 kDa [9] X-ray structures
of the homologous enzyme from Pseudomonas fragi
(PfHBDH) in the presence of NAD+ and inhibitor
(Protein Data Bank accession code: 1X1T) and
with-out cosubstrate (Protein Data Bank accession code:
1WMB) have been recently determined [10]
Interest-ingly, in these structures the substrate-binding loop
was ordered in the absence of NAD+ and disordered
in the complex structure with bound NAD+ In
addi-tion, a crystal structure of a human cytosolic HBDH
(DHRS6) with bound NAD+ is available [11] Based
on homology modelling, substrate and inhibitor dock-ing studies, and site-directed mutagenesis, residues Gln91, His141, Lys149, Tyr152 and Gln193 were found to be involved in substrate binding in PpHBDH [9] With the exception of Gln193, these residues are located in the deep active-site cleft The exact boundaries of the flexible loop differ among enzymes Based on the structure of PpHBDH, the loop runs from residue 187 to residue 210 We will refer to the rest of the one-domain protein as the cat-alytic subdomain
In this study we determined the structure of PpHBDH using three different crystal forms The X-ray structures showed a completely ordered confor-mation of the substrate-binding loop in at least one subunit in the open and closed forms A comparison
of these conformers, and an analysis of the mobility of the loop in the crystals, allowed a detailed description
of the enzyme dynamic properties and conformational change during HBDH catalysis
Results and Discussion Monomer and tetramer structure Three different crystal forms of PpHBDH were obtained in the presence or absence of NAD+ (Table 1) In crystal form I the asymmetric unit was found to contain one tetramer and two dimers (the tetrameric structure is generated by a crystallographic two-fold axis) In crystal form II the asymmetric unit was found to contain two dimers, and in crystal
for-m III one tetrafor-mer was present in the asyfor-mfor-metric unit In crystal form I, two of the eight subunits (designated as chains A and B) contained no bound NAD+, as shown by the electron density maps In crystal form II, only subunit A was NAD+ free Therefore, crystal forms I and II contain tetramers with all four binding sites occupied and tetramers with two bound NAD+molecules In crystal form III the enzyme was completely devoid of NAD+ cosub-strates The comparison of several independent subun-its, with and without bound NAD+, allowed an analysis to be made of cosubstrate-induced move-ments within the enzyme, in particular of the sub-strate-binding loop
Each subunit of HBDH has an a⁄ b doubly wound structure with the characteristic dinucleotide-binding motif known as the Rossmann fold (Fig 1) The sub-unit structure was made of a core b-sheet composed of seven parallel b-strands (bA, bB, bC, bD, bE, bF and bG) buried between three a-helices (aB, aC and aG,
Trang 3or aD, aE and aF) located on both sides of the
b-sheet The substrate-binding loop consisted of two
helices, designated aFG1 and aFG2
With the exception of the substrate-binding loop,
the catalytic subdomains formed a compact, flat
tetra-meric structure of dimensions 70· 80 · 40 A˚ along
the Q, P and R axes, respectively Only the
substrate-binding loop protruded from the main body of the
tetramer along the R axis (Fig 1) This loop was
partially disordered in most subunits (Table 2)
How-ever, the loop was completely defined in subunit A of
crystal form II and in subunit B of crystal form III,
both in the absence of NAD+, as well as in subunit D
of crystal form I in the presence of NAD+
Table 1 Crystal and refinement data of crystal forms I, II and III.
au, Asymmetric unit.
Unit-cell
dimensions (A ˚ )
Resolution (A ˚ ) a
30–2.0 (2.09–2.02)
30–1.9 (1.97–1.9)
30–2.1 (2.2-2.12) Completeness (%) a 96.6 (74.4) 99.5 (98.3) 99.1 (94.4)
R sym (%)a 8.2 (59.2) 4.8 (15.1) 6.7 (36.9)
Redundancy a 7.5 (4.8) 11.3 (11.0) 9.9 (7.8)
Wilson B factor (A ˚ 2 ) 36.1 18.6 34.4
R ⁄ R free (%) 20.2 ⁄ 27.4 16.8 ⁄ 21.8 18.1 ⁄ 24.6
No of water
molecules
< B protein > (A˚2 ) 42.7 18.6 33.0
< B waters > (A˚2) 43.5 20.7 33.8
a The values given in parentheses refer to the highest resolution
shell.
A
B
C
Fig 1 Crystal structure of P putida D-3-hydroxybutyrate
dehydro-genase (PpHBDH) (A) Fold of one subunit in the closed
conforma-tion The substrate-binding loop is colored red and the bound NAD +
is colored yellow (B) View of the tetramer structure along the R
axis (blue) The P and Q axes are marked in red and green,
respec-tively Shown are subunits A, B, C and D of crystal form I Only in
subunits C and D (green) is an NAD + molecule (yellow) bound In
subunits A and B (blue) the coenzyme-binding site is not occupied.
The substrate-binding loops are depicted in red (C) View along the
P axis.
Trang 4Comparison of the subunit structures Crystal form I
Figure 2A shows a superposition of the eight subunits
in crystal form I It demonstrates that subunits with and without NAD+ adopted different conformations for regions in the substrate-binding loop, helix aC and the residues Ala88 and Gly89 after b-strand D Those regions that showed conformational variability in the superposition also displayed significantly higher B fac-tors (Fig 2B) The average B factor for the atoms of helix C was 47.1 A˚2 (average over all molecules) and its residues superimposed with an rmsd of 0.6 A˚, indi-cating an increased mobility of this helix Whereas the subunits without NAD+ had their substrate-binding loop in an open conformation, all subunits with NAD+exhibited a closed conformation Interestingly,
in subunit H (with bound NAD+), residues 186–189 corresponded to the open conformation of the sub-strate-binding loop, whereas residues 204–208 (at the end of the loop) were in a position that is similar to the position of this region in the closed conformation Residues 190–203 were disordered This finding indi-cated that the loop can also change to the open con-formation in the presence of NAD+
Crystal form II
A superposition of the subunits from crystal form II showed that the substrate-binding loop of subunit A (NAD+ free) is in an open conformation and com-pletely ordered whereas the corresponding loops in the NAD+-complexed subunits B to D are in a closed conformation and partially disordered (Fig 2C) Besides this, the largest variability was seen again in helix aC and in the region after b-strand D Compared with the average B factor of 19.7 A˚2 for all four pro-tein molecules, helix aC had a somewhat higher B fac-tor, of about 29 A˚2, also in this crystal form In the NAD+-free subunit A, residues 88 and 89 of the cen-tral b-strand had a conformation different from the NAD+-containing subunits, similar to the situation in subunit IB Both residues shifted up to 2 A˚ upon NAD+binding
Crystal form III
In crystal form III (all subunits are NAD+free), sub-unit B possessed a completely ordered substrate-bind-ing loop, whereas this loop was partially disordered in the other subunits All four subunits in crystal form III were in an open conformation with respect to the sub-strate-binding loop (Fig 2D) In contrast to crystal
2 )
2 )
+ )(A˚
2 )
2 )
a The
Trang 5forms I and II, residues 88 and 89 adopted a
confor-mation similar to that observed in the subunit
struc-tures with bound NAD+ As in the other crystal
forms, helix C showed a higher variability, with an
rmsd of 0.4 A˚, and a higher B factor, of 46 A˚2,
com-pared with the average B factor of 40.4 A˚2
Crystal packing interactions
As in other crystal structures of HBDH enzymes, the
exposed substrate-binding loop, which forms two faces
on opposite sides of the HBDH tetramer (Fig 1), was
involved in crystal contacts in almost all subunits
(Table 2) Interestingly, only in the NAD+-bound
sub-units IIC and IID, where the loop is in a closed
con-formation, it was not involved in crystal contacts
Furthermore, the loop was in a closed conformation in
all subunits with bound NAD+ and in an open
con-formation in all cosubstrate-free subunits These
find-ings suggest that the closed conformation of the loop
is a result of cosubstrate binding
Comparison of PpHBDH with PfHBDH and DHRS6
The major differences between the PfHBDH and
PpHBDH structures are in the substrate-binding loop,
in residues of the central b-strand D and in helix aC
(Fig 3) The substrate-binding loop in the
cosubstrate-free PfHBDH structure has an intermediate position
between the open and closed forms of this loop in
PpHBDH The loop is completely disordered in
the structure of PfHBDH in complex with NAD+
The conformational change of residues 88 and 89 in
the central b-strand of PpHBDH upon NAD+binding
was not observed in PfHBDH In both structures of
the latter enzyme, the b strand was in exactly the same
conformation as in the NAD+-bound form of
PpHBDH
In addition to these conformational differences, helix
aC is four residues shorter in PpHBDH because of a
deletion of residues Moreover, the substrate-binding
loop of PpHBDH is one residue shorter that that of
PfHBDH, because of a deletion at the end of helix
aFG1
In human DHRS6, a cytosolic type 2 human HBDH
enzyme [11], the substrate-binding loop is present in a
A
B
C
D
Fig 2 Superposition of subunit structures (A) Crystal form I: A,
green; B, black; C, yellow; D, red; E, magenta; F, blue; G, cyan; H,
brown (B) C a traces of the eight monomers of crystal form I
col-ored by the B factor (from blue at B < 30 A ˚ 2 to red at B > 60 A ˚ 2 ).
(C) Crystal form II: A, green; B, red; C, blue; D, black (D) Crystal
form III: A, black; B, red; C, blue; D, green.
Trang 6closed conformation (Fig 3) A sulfate molecule from
the crystallization solution is bound to the active site
at the presumed binding site for the substrate The
loop closure may be caused by the sulfate ion, as
dis-cussed by the authors, and also by interactions with
NAD+, as described below The substrate-binding
loop of DHRS6 is six residues shorter, as in the
Pseu-domonas HBDH enzymes Consequently, there is a
shortening of helix aFG1 and a slight relocation of
helix aFG2
NAD+binding
The conformation of NAD+ and the structure of the
cosubstrate-binding pocket of PpHBDH are very
simi-lar to those of PfHBDH, with a distance of about
14 A˚ between the C2 of nicotinamide and the C6 of
the adenine ring As indicated in the superpositions of
Figs 2 and 3, the main difference is the displacement
of Ala88 and Gly89 by about 2 A˚ in the NAD+-free
subunits IB (chain B of crystal form I) and IIA The B
value for these residues is around 10 A˚2 higher than
the average B factor of the subunit In the subunits
with bound NAD+, the B factor of the two residues is
comparable with the average B value of the subunit
Nevertheless, the conformation of residues 88 and 89
is clearly defined in all electron density maps The
main chain torsion angles of residues 87–90 all
corre-spond to allowed regions of the Ramachandran plot
In crystal form III, residues 88 and 89 adopted a
con-formation similar to that of the NAD+-bound
sub-units in crystal forms I and II In this crystal form, the
B factor of residues 88 and 89 was 5–10 A˚2 higher
than the average B value of the corresponding subunit
The two residues are thus more mobile than in the presence of NAD+, but less than in the alternate con-formations observed in subunits IB and IIA
NAD+interacts, via a hydrogen bond of one of its ribose hydroxyl oxygens, with the peptide carbonyl group of Asn87 (Fig 4) The loss of this interaction may be an important factor for the change of main chain conformation of this residue in the absence of NAD+ The present data thus indicate that residues 87–90 have a higher mobility in the absence of bound cosubstrate (as indicated by higher B factors) and they can adopt alternative conformations
Translation, libration and screw (TLS) refinement
At the end of the refinement procedure, anisotropic displacement parameters were determined by a TLS refinement [12,13] Translation (T) and libration (L) tensors describe the anisotropic motion of groups in the crystal Besides improving the fit of the model to the observed data, the TLS tensors may allow a description of correlated motions in the crystal It must be stressed, however, that a fit of the TLS model
to the observed structure factor amplitudes implies nothing about the relative phases of the atomic dis-placements within the group [13]
Gln91
Gly89
Ala88 Asn87
lle90
Fig 4 Conformational change of residues 87–91 upon NAD+ bind-ing The carbon atoms are colored grey in the conformation in the presence of NAD + (yellow) and green in the NAD + -free subunit A
of crystal form I.
Fig 3 Comparison of P putida D-3-hydroxybutyrate
dehydroge-nase (PpHBDH) (open form green, closed form red), Pseudomonas
fragi D-3-hydroxybutyrate dehydrogenase (PfHBDH) (open form
blue) and DHRS6 (black) The structure of the NAD+-bound form of
PfHBDH (not shown) with the disordered substrate-binding loop
superimposes closely with the open form.
Trang 7All three crystal forms were subject to TLS
refine-ment with the substrate-binding loop region (186–212)
and the catalytic domain (2–185, 213–256) in each
sub-unit as two distinct TLS groups By TLS refinement,
the R factors (Rfreefactors) improved by 2.2% (3.3%),
0.7% (1.0%) and 1.7% (2.9%) for crystal forms I, II
and III, respectively
The total B factor (Btotal) of each atom is the sum of
the TLS contribution (BTLS, from the rigid body
motion) and the residual B factor (Bresidual, the
individ-ual mobility independent of the rigid bodies) The
residual B factors of the substrate-binding loop were
relatively constant and had values similar to those
of other regions (data shown in the supplementary
Fig S1) The significantly higher B factors of the
sub-strate-binding loop are predominantly caused by a
rigid body motion of the loop For all crystal forms, a
significant librational movement is present for the
sub-strate-binding loop (data shown in the supplementary
Table S1) Furthermore, the libration is quite
aniso-tropic in nature (Fig 5)
How does the anisotropic librational motion of the
substrate-binding loop compare with the closing
motion of this loop? A superposition of the main axis
of the L tensors for all subunits showed that all L ten-sors roughly describe a similar libration movement in which the main rotation component is an axis approxi-mately parallel to the two helices of the loop (Fig 5, superposition not shown) The second largest rota-tional component describes the closure motion of the loop
Movement of the substrate-binding loop
In order to characterize the nature of the movement of the substrate-binding loop, the structures of open and closed forms were compared using program dyndom
to determine dynamic regions that move as pseudo-rigid structures, termed ‘dynamic domains’ (Fig 6) For stretches of five amino acid residues, the rotational movement between two enzyme conformations was analyzed Residues that belong to one rigid body show
a similar rotation in the superposition and thus form a cluster, as shown in Fig 6B The analysis revealed two dynamic domains: residues 4–183 and 212–254 form one domain; and residues 187–210 of the substrate-binding loop form the other domain However, within the substrate-binding loop, three subclusters were defined: residues 187–199 of helix aFG1; residues 204–
210 of helix aFG2; and residues 200–203 of the loop connecting the two helices Thus, the substrate-binding loop moves largely as a rigid body; however, the inter-nal structure of the loop changes slightly by a re-orien-tation of the two helices Residues 184–186 and 211–
213 are the bending residues that connect the two dynamic domains (Fig 6)
The substrate-binding loops in the open and closed conformations superimposed with an rmsd of 1.4 A˚ The movement corresponded to a rotation by 46 with
a small translational component of 0.06 A˚ It is typical for hinge-bending movements that the rotation axis passes near the bending residues, which thus act as a mechanical hinge (Fig 6) [14] An analysis of the main chain conformations in the different loop structures showed that several small changes of main-chain tor-sion angles of the bending residues allowed the move-ment of the substrate-binding loop, but no large changes of the main chain conformation were observed (data not shown)
A movement of the substrate-binding loop upon co-substrate binding has not been observed before for other SDR enzymes Only for human estrogenic 17b-hydroxysteroid dehydrogenase [5,15], Drosophila lebanonensis alcohol dehydrogenase [16], Datura stra-monium tropinone reductase [17] and Escherichia coli b-keto acyl carrier protein reductase [18,19] have struc-tures of the apoenzyme and of the binary complex
Fig 5 Principal axis (green) of the libration tensor of the
substrate-binding loop of subunit IIIA The anisotropic movement of the Ca
atoms, as derived from the TLS tensors, is depicted by thermal
ellipsoids for the loop (red) and for the catalytic subdomain (blue).
For orientation, a superimposed NAD + molecule is shown in yellow,
although it is not bound in this crystal form.
Trang 8with cosubstrate been determined, without significant
differences in the position of the substrate-binding
loops In the crystal structure of 3a-hydroxysteroid
dehydrogenase⁄ carbonyl reductase from Comamonas
testosteroni the substrate-binding loop is largely
dis-ordered in the absence and presence of bound
NAD+[20] There are, however, crystal structures of
SDR enzymes in the closed form available for binary
complexes with cosubstrate, such as the structure of
human DHRS6 in complex with NAD [11] A struc-ture of the DHRS6 apoenzyme is not yet available
Residues inducing closure movement
In an analysis of ligand-induced domain movements in other proteins, it was shown that a small number of residues from the closing domain interact with the ligand bound to the binding domain in the open
200
200
201
202
203
205
206
208
209
210
207
204
211
212
213
185
184
186
198
199
192 191
190
195
194
193
189
196
188
197
187
203
A
B
213 211
186
184
200
203
213 211
186
184
Fig 6 Dynamic domains of P putida D-3-hydroxybutyrate dehydrogenase (PpHBDH)
on the basis of a comparison of conformers
ID and IIA, which contain a fully defined substrate-binding loop (A) The two dynamic domains are colored blue and red for the fold of conformer ID (closed) and the bend-ing residues are shown in green (B) Cluster-ing of the rotational movements of stretches of five residues, on which the assignment of the domains moving as rigid bodies is based in program DYNDOM [31] Each sphere represents the rotation vector
of a five-residue stretch For the substrate-binding loop and the bending residues, the rotation vectors are labeled according to the residue in the center of the stretch.
Trang 9conformation to initiate and drive domain closure [21].
In this model, specific interactions of the ligand with
residues on the coenzyme-binding domain produce a
torque about the hinge axis driving the domain
clo-sure Residues are assumed to induce closure
move-ment if they satisfy specific conditions These residues
are usually located in the bending regions or in the
closing domain Additionally, thermal motions may
also contribute to the closure movement
A large number of interactions are involved in the
binding of NAD+ to the catalytic subdomain of
PpHBDH However, there are only a few interactions
of the substrate-binding loop with NAD+ that might
cause the conformational change A crucial residue for
the coenzyme-induced conformational change might be
Thr187, which forms hydrogen bonds to the
nicotin-amide NH2 and to a phosphate oxygen of NAD+via
its hydroxyl group (Fig 7) In the absence of NAD+,
the alcoholic oxygen is displaced by about 1.5 A˚, such
that it is 4 A˚ distant from the position of its putative
hydrogen-bonding partners This interaction might
trigger the closure motion of the substrate-binding
loop in the presence of NAD+ because Thr187 is
located just behind the bending residues 184–186 and
the torque produced by the interaction of Thr187 with
NAD+ drives or supports the loop movement about
the hinge residues A further, nonpolar interaction of
the substrate-binding loop with the coenzyme is
medi-ated by the side chain of Leu189, which makes
hydro-phobic contacts with the ribose group and
nicotinamide ring of NAD+ in the closed
conforma-tion but is faced towards the solvent in the open
con-formation (Fig 7) Both residues are conserved in the
bacterial HBDHs as part of a ‘TPLV’ motif Also, the
interaction of the main chain NH and CO groups of
Val185 might contribute to the conformational change
by binding to the NAD+ nicotinamide group There are no further interactions that might explain the con-formational change of the substrate-binding loop upon NAD+binding The rest of the substrate-binding loop
is also quite diverged, with the exception of Gln193 In the human enzyme DHRS6, Thr187 is conserved and makes a polar contact to NAD+, as in the bacterial enzyme Leu189 is replaced by a serine, which is in hydrogen-bonding distance to a phosphate oxygen atom of NAD+and may thus also be involved in the induction of closure movement via cosubstrate bind-ing The interaction of Val186 (Val185 in PpHBDH) and Thr188 (Thr187) of 3a-hydroxysteroid dehydroge-nase with the cofactor has also been discussed to stabi-lize the substrate-binding loop in the loop–helix transition observed in this enzyme [7]
Substrate binding
A model for the binding mode of the substrate D-3-hy-droxybutyrate to HBDH has been suggested based on
a homology model of PpHBDH and molecular model-ling techniques [9] In this model, the side chain of Gln193 belonging to the substrate-binding loop forms hydrogen bonds to the carboxylate group of the sub-strate Figure 8 shows the modelled substrate in the
Leu189
Val185 Thr187
Fig 7 Interactions of NAD + with P putida D-3-hydroxybutyrate
dehydrogenase (PpHBDH), which might drive a cosubstrate-induced
conformational change of the substrate-binding loop The closed
conformation is depicted in green and the open conformation in
grey Also shown is the rotation axis in blue.
Lys149
Leu189 Tyr152
His141
Gln193 Gln91
Fig 8 A model for the binding mode of substrate D-3-hydroxy-butyrate (cyan) to P putida D-3-hydroxyD-3-hydroxy-butyrate dehydrogenase (PpHBDH) Shown are the enzyme conformations in the open (green) and closed (red) forms, and selected residues, as discussed
in the main text The cosubstrate NAD + is shown with yellow car-bon atoms Hydrogen car-bonds are shown as dashed lines, and the red dashed line between the nicotinamide C4 atom and the sub-strate carbon atom bound to the subsub-strate alcohol group marks the distance between the reactive centers.
Trang 10crystal structure of PpHBDH in the closed form The
side chain of Gln193 was shown to contribute
signifi-cantly to substrate binding because the Km value
increased from 0.6 mm for the wild-type enzyme to
about 70 mm in a Gln193Ala mutant, whereas the kcat
value decreased from 432Æs)1 in the wild-type enzyme
to 215Æs)1 in the same mutant Gln193 was in
hydro-gen-bonding distance to the carboxylate group of the
substrate in the closed conformation of the
substrate-binding loop In all subunits where this residue was
defined in subunits with bound NAD+ (D, E and G
of crystal form II as well as B and C of crystal
for-m II), this residue was positioned to interact with the
substrate This finding indicates that the loop is indeed
in a position competent for catalysis upon NAD+
binding, even in the absence of the bound substrate
Gln193 is the only residue of the substrate-binding
loop that forms polar interactions with the substrate
In addition, the side chain of Leu189 has hydrophobic
contacts to the methyl group of the substrate model
Further polar interactions to the substrate are formed
by Lys149, Gln91, His141 and Tyr152 Of these
resi-dues, the important function for substrate binding has
been demonstrated for a Gln91Ala mutant with Km
51 mm and kcat 411Æs)1 and for a Lys149Ala mutant
that was essentially inactive [9] His141 appears to be
also important for efficient catalysis because in a
His141Ala mutant the Km increased to only 4 mm,
whereas the kcat decreased significantly to 13Æs)1
Tyr152 is known to be a core catalytic residue It is
assumed to be present as a tyrosinate and to accept a
proton from the alcohol group in order to facilitate H–
transfer to NAD+ Our crystallographic study
con-firmed the repositioning of the substrate-binding loop
in the closed conformation, as obtained from a
mole-cular dynamics simulation of a PpHBDH model [9]
A superposition of the active-site structures of
PpHBDH and DHRS6 showed that the human
enzyme developed a different environment for substrate
binding: His141 was replaced by an alanine, Lys149 by
an arginine, Gln91 by a valine and Gln193 (from the
substrate-binding loop) by an arginine (data not
shown) These replacements might account for the
sig-nificant differences in the kinetic data of both enzymes
Figure 9 shows the molecular surface of HBDH in
the open conformation, together with the model for
the substrate binding mode The NAD+ coenzyme
taken from the structures of PpHBDH in the closed
form was included in the calculation of the surface
Thus, the surface represented the protein in a state
where NAD+ has just bound, but the loop is still in
the open conformation The substrate was bound in a
deep groove formed between the substrate-binding
loop and the catalytic subdomain The carboxylate group of the substrate was located at a region with positive potential, which is mainly caused by Lys149 Also shown is the substrate-binding loop in the closed conformation In particular, residues Leu189 and Gln193 would block the entrance to the substrate-binding pocket in the closed conformation A molecu-lar surface drawn for the enzyme in the closed form revealed no access to the buried substrate-binding pocket (data not shown) Kinetic studies demonstrated that HBDH, similarly to other NAD+-dependent de-hydrogenases, has an ordered sequential binding mech-anism of cosubstrate binding followed by substrate binding (M Grunow, unpublished results) Therefore, although the binding of the coenzyme obviously induces a change of the enzyme to the closed form, the loop must exist in an equilibrium with the open form
to enable binding of the substrate and release of the products This flexibility is demonstrated by a partial disorder of the substrate-binding loop in some subunits
of NAD+-bound subunits of PpHBDH and also by the strong disorder of the loop in the NAD+– PfHBDH complex [10]
In conclusion, the crystallographic analysis of PpHBDH in different crystal forms and in the pres-ence and abspres-ence of bound NAD+ showed that the presence of the cosubstrate alone is able to induce a conformation of the substrate-binding loop that is competent for catalysis Such a conformational change of the substrate-binding loop has not been observed in previous crystallographic studies on other SDR enzymes Our results are in agreement with the
Fig 9 Molecular surface of P putida D-3-hydroxybutyrate dehydro-genase (PpHBDH) in the open conformation (conformer IIA) colored
by the electrostatic potential Positive potential is depicted in blue and negative potential in red Also shown are the model for sub-strate binding in green and the subsub-strate-binding loop conformation
in the closed form in yellow.