Crystallization and preliminary X-ray analysis of binary and ternary complexes of Haloferax mediterranei glucose dehydrogenase

Abstract

Haloferax mediterranei glucose dehydrogenase (EC 1.1.1.47) belongs to the medium-chain alcohol dehydrogenase superfamily and requires zinc for catalysis. In the majority of these family members, the catalytic zinc is tetrahedrally coordinated by the side chains of a cysteine, a histidine, a cysteine or glutamate and a water molecule. In H. mediterranei glucose dehydrogenase, sequence analysis indicates that the zinc coordination is different, with the invariant cysteine replaced by an aspartate residue. In order to analyse the significance of this replacement and to contribute to an understanding of the role of the metal ion in catalysis, a range of binary and ternary complexes of the wild-type and a D38C mutant protein have been crystallized. For most of the complexes, crystals belonging to space group I222 were obtained using sodium/potassium citrate as a precipitant. However, for the binary and non-productive ternary complexes with NADPH/Zn, it was necessary to replace the citrate with 2-methyl-2,4-pentanediol. Despite the radical change in conditions, the crystals thus formed were isomorphous.

Full text

crystallization communications
Acta Cryst. (2005). F61, 743–746 doi:10.1107/S1744309105019949 743
Acta Crystallographica Section F
Structural Biology
and Crystallization
Communications
ISSN 1744-3091
Crystallization and preliminary X-ray analysis of
binary and ternary complexes of Haloferax
mediterranei glucose dehydrogenase
Julia Esclapez,
a
K. Linda Britton,
b
Patrick J. Baker,
b
Martin Fisher,
b
Carmen Pire,
a
Juan Ferrer,
a
Marı
´a Jose
´Bonete
a
and
David W. Rice
b
*
a
Departamento de Agroquı
´mica y Bioquı
´mica,
Facultad de Ciencias, Universidad de Alicante,
Ap. 99 Alicante 03080, Spain, and
b
Krebs
Institute for Biomolecular Research, Department
of Molecular Biology and Biotechnology,
The University of Sheffield, Sheffield S10 2TN,
England
Correspondence e-mail: d.rice@sheffield.ac.uk
Received 6 May 2005
Accepted 23 June 2005
Online 8 July 2005
Haloferax mediterranei glucose dehydrogenase (EC 1.1.1.47) belongs to the
medium-chain alcohol dehydrogenase superfamily and requires zinc for
catalysis. In the majority of these family members, the catalytic zinc is
tetrahedrally coordinated by the side chains of a cysteine, a histidine, a cysteine
or glutamate and a water molecule. In H. mediterranei glucose dehydrogenase,
sequence analysis indicates that the zinc coordination is different, with the
invariant cysteine replaced by an aspartate residue. In order to analyse the
significance of this replacement and to contribute to an understanding of the
role of the metal ion in catalysis, a range of binary and ternary complexes of the
wild-type and a D38C mutant protein have been crystallized. For most of the
complexes, crystals belonging to space group I222 were obtained using sodium/
potassium citrate as a precipitant. However, for the binary and non-productive
ternary complexes with NADPH/Zn, it was necessary to replace the citrate with
2-methyl-2,4-pentanediol. Despite the radical change in conditions, the crystals
thus formed were isomorphous.
1. Introduction
Extremely halophilic archaea are found in highly saline environments
such as natural salt lakes or saltern pools. These microorganisms
require between 2.5 and 5.2 Msalt for optimal growth (Kamekura,
1998) and they can balance the external salt concentration by accu-
mulating intracellular KCl close to saturation. The biochemical
machinery of these microorganisms has therefore been adapted in the
course of evolution to be able to function at salt concentrations at
which most biochemical systems cease to function.
Comparison of the amino-acid compositions and structures of
halophilic proteins and their mesophilic counterparts has shown that
a significant difference in the characteristics of the surface of halo-
philic proteins is an excess of acidic over basic residues (Lanyi, 1974;
Bo
¨hm & Jaenicke, 1994; Dym et al., 1995; Frolow et al., 1996; Britton et
al., 1998, 2005).
The extremely halophilic archaeon Haloferax mediterranei (ATCC
33500/R4) is able to grow in a minimal medium with glucose as the
sole carbon source, which is catabolized by a modified Enter–
Doudoroff pathway. Glucose dehydrogenase (GlcDH) catalyses the
first step in this pathway, the oxidation of -d-glucose to gluconic
acid, preferentially using NADP
+
as a coenzyme. Sequence analysis
has shown that GlcDH belongs to the zinc-dependent medium-chain
alcohol dehydrogenase (MDR) superfamily (Bonete et al., 1996; Pire
et al., 2001). Biochemical studies have established that the protein is
dimeric, with a subunit molecular weight of 39 kDa, and have
confirmed the requirement of zinc for catalysis. In previous work, the
crystallization of recombinant GlcDH in the presence of NADP
+
has
been reported under conditions which closely mimic those experi-
enced by the enzyme in the cell of the halophile (Ferrer et al., 2001);
more recently, the structure has been determined to 1.6 A
˚resolution
(Britton et al., 2005). This structure has revealed that the surface of
the enzyme is not only decorated with acidic residues, but also
displays a significant reduction in the fraction of exposed hydro-
phobic surface compared with non-halophilic glucose dehydro-
genases. This reduction in hydrophobic surface predominately arises
from the loss of the exposed alkyl component of lysine side chains as
a result of the reduction of lysine content in the enzyme (Britton et
#2005 International Union of Crystallography
All rights reserved

al., 2005). Moreover, genome comparisons have shown that there
appears to be a general reduction in the frequency of lysine in
halophilic proteins (Kennedy et al., 2001).
At the active site of the prototypical MDR superfamily member,
horse liver alcohol dehydrogenase (HLADH), the essential catalytic
zinc ion is coordinated by three protein ligands (Cys46, His67 and
Cys174), with the coordination shell of the zinc being completed by a
water molecule (Eklund et al., 1982). The mechanism of enzymes of
the MDR superfamily is commonly proposed to involve the exchange
of the zinc-bound water molecule with the hydroxyl of the substrate,
from which a proton is then removed by a base to generate an
alkoxide intermediate, which subsequently collapses to a carbonyl
with concomitant reduction of NAD(P)
+
. In this mechanism, the zinc
is proposed to remain tetrahedrally coordinated throughout (Eklund
et al., 1982; Ehrig et al., 1991; Ramaswamy et al., 1999). In a recent
alternative proposal, it has been suggested the zinc ion cycles
between different four- and five-coordinate intermediates, the iden-
tities of which are not yet clear (Makinen et al., 1983; Kleifeld et al.,
2003).
Of the three protein ligands to the zinc in enzymes of the MDR
superfamily, the first cysteine (Cys46 in HLADH) and the histidine
(His67 in HLADH) are very strongly conserved in the sequence. In
some family members, the second cysteine (Cys174 in HLADH) is
replaced by a glutamate [for example, Glu155 in Thermoplasma
acidophilum GlcDH (John et al., 1994) and Glu153 in rat sorbitol
dehydrogenase (Johansson et al., 2001)]. This sequence pattern is also
seen in the structure of H. mediterranei GlcDH, where a glutamate
residue (Glu64) occupies the equivalent position to the second
cysteine (Britton et al., 2005). However, the structure of H. medi-
terranei GlcDH has shown that there is an additional difference, with
an aspartate residue (Asp38) occurring in a structurally equivalent
position to the highly conserved first cysteine of this motif (Britton et
al., 2005). This is a sequence change rarely seen in the MDR family,
but that has also been observed in the sequence of other halophilic
glucose dehydrogenases [for example, the Halobacterium sp1 (Ng et
al., 2000) and Haloferax volcanii GlcDHs; http://zdna2.umbi.umd.edu]
and raises the intriguing question as to whether the presence of this
aspartate residue in the active site is a halophilic adaptation. In order
to investigate the role of the metal and its ligands in catalysis and
to enhance our understanding of the reaction mechanism in
H. mediterranei GlcDH, we have explored a range of crystallization
conditions of complexes of both the wild-type and the mutant D38C
enzymes. In this paper, we report the preliminary crystallographic
analysis of these various enzyme–substrate complexes.
2. Materials and methods
2.1. Site-directed mutagenesis, expression and purification
The gene encoding the halophilic GlcDH was cloned into pGEM-
11Zf(+) and site-directed mutagenesis was performed using the
GeneEditor in vitro site-directed mutagenesis system (Promega). The
protocol supplied with the kit was followed except that the length of
the DNA-denaturation stage was increased from 5 min at room
temperature to 20 min at 310 K. The expression, renaturation and
purification of the recombinant wild-type and mutant proteins were
performed as described previously (Pire et al., 2001). The purified
D38C GlcDH was dialyzed against 50 mMphosphate buffer pH 7.3
containing 2 MNaCl at 277 K overnight. Prior to crystallization,
protein samples were concentrated to approximately 20 mg ml
1
using a Vivaspin concentrator (30 kDa molecular-weight cutoff).
2.2. Crystallization and diffraction data collection
Crystals of the wild-type and D38C mutant of GlcDH grew after
4–6 d in the presence of different substrates using the hanging-drop
vapour-diffusion method, mixing small volumes (2–3 ml) of protein
sample with an equal volume of a precipitant solution at 290 K. For
the wild-type protein, crystals of the free enzyme and the binary
complex with NADP
+
, using sodium citrate as the precipitate, have
already been reported (Ferrer et al., 2001), but using these conditions
crystals of the binary complex with NADPH could not be obtained
either in the presence or absence of zinc. However, crystals of the
NADPH–Zn complex could be produced by preparing the protein
with 1 mMNADPH and 1 mMZnCl
2
and using 67–72%(v/v)
2-methyl-2,4-pentanediol (MPD) in 100 mMHEPES pH 7.5 as the
precipitant. The D38C mutant protein behaved in the same manner
as the wild type, with crystals of the apoenzyme and various NADP
+
complexes forming in the presence of citrate and the NADPH
complexes in the presence of MPD. For the D38C protein, the protein
sample was mixed with 1 mMNADP
+
or 1 mMNADPH, 1 mM
ZnCl
2
,10mMglucose and 10 mMgluconate as appropriate. For the
free enzyme and the complexes containing NADP
+
the precipitant
used was 1.4–1.6 Msodium citrate in 100 mMHEPES pH 7.0 and for
the complexes containing NADPH the precipitant was 62–72%(v/v)
MPD in 100 mMHEPES pH 7.5. Crystals of D38C GlcDH were also
grown in the presence of 2 MKCl, 1 mMZnCl
2
and 1 mMNADP
+
using 1.4–1.6 Mpotassium citrate as precipitant in 100 mMHEPES
buffer pH 7.0.
The crystals of the wild-type and D38C free enzyme grown in the
presence of sodium citrate showed a hexagonal bipyramidal
morphology (maximum dimensions 0.25 0.40 0.25 mm), whereas
crystals with a rod-like morphology (Fig. 1; maximum dimensions
0.6 0.6 0.4 mm) were obtained for all the wild-type and mutant
complexes with NADP
+
or NADPH, irrespective of whether the
precipitant was sodium citrate or MPD.
Crystals grown with sodium citrate as the precipitant were
mounted in X-ray-transparent capillaries. Preliminary data sets were
collected at 290 K by the rotation method with 1rotations per frame
using a MAR 345 detector, with double-mirror-focused Cu KX-rays
produced by a Rigaku RU-200 rotating-anode generator. The crystals
grown in the presence of MPD were flash-cooled in a cold nitrogen-
gas stream and data were collected using the same method at 100 K.
Data from the crystals of the D38C GlcDH–NADP
+
–Zn complex
crystallization communications
744 Esclapez et al. Glucose dehydrogenase Acta Cryst. (2005). F61, 743–746
Figure 1
A crystal of the binary complex of D38C GlcDH with NADPH and zinc.

grown in the presence of KCl and potassium citrate were collected at
the SRS Daresbury synchrotron on station PX14.2 at a wavelength of
0.97 A
˚with 1rotations using an ADSC Q4 detector. The data for
each crystal were processed and analysed using the HKL suite of
programs (Otwinowski & Minor, 1997) and subsequently handled
using the CCP4 suite (Collaborative Computational Project, Number
4, 1994).
3. Results and discussion
Analysis of the various data sets using the autoindexing routine of
DENZO showed that D38C GlcDH had crystallized in two different
forms, designated I and II, which are the same as those seen for the
wild-type enzyme (Ferrer et al., 2001). The crystals of the D38C free
enzyme (form I) belong to a hexagonal lattice P622, with unit-cell
parameters a=b= 89.4, c= 211.5 A
˚,== 90, = 120and a unit-
cell volume of 1.46 10
6
A
˚
3
. Given the GlcDH subunit molecular
weight of 39 kDa, the V
M
for a monomer in the asymmetric unit is
3.1 A
˚
3
Da
1
, which lies within the normal range for proteins
(Matthews, 1977). The crystals of the binary complexes and non-
productive ternary complexes (form II) were all isomorphous,
whether they were grown using citrate or MPD as the precipitant, and
diffracted to up to 1.5 A
˚resolution. These crystals belong to one of
the special pair of space groups I222 or I2
1
2
1
2
1
, with unit-cell para-
meters as shown in Table 1 and a unit-cell volume of 1.0 10
6
A
˚
3
.
The asymmetric unit appears to contain a monomer (V
M
=
3.2 A
˚
3
Da
1
). Data-collection statistics for the form I and form II
crystals are given in Table 1.
A cross-rotation function and translation function were calculated
in both I222 or I2
1
2
1
2
1
space groups on the form II data for the D38C
NADPH–Zn–glucose complex at a resolution of 20–3.0 A
˚, using a
single subunit of the wild-type H. mediterranei GlcDH as a search
model and the program AMoRe (Navaza, 1994). A clear translation-
function solution of correlation coefficient 67.6% and Rfactor 36.9%
was only seen in space group I222, indicating that this is the correct
space group. For the data from the form I crystals a similar process
was undertaken. In this case, a clear translation-function solution was
only seen in space group P6
2
22 (correlation coefficient 71.6%, R
factor 38.7%), identifying this as the correct space group.
Previously, we have obtained form II crystals from the wild-type
enzyme in complex with NADP
+
and also from the D38C mutant in
complex with NADP
+
and zinc (PDB codes 1ss0 and 1ss5, respec-
tively). All the D38C GlcDH complex crystals reported here are
isomorphous with the form II crystals. However, it is interesting to
note that the crystals of the NADP
+
complexes grow using citrate as
the precipitant, whereas those complexes containing NADPH only
grow when MPD is used as the precipitant. Despite these radically
different conditions, the crystals thus produced are isomorphous.
Analysis of these structures is now under way and given the
successful crystallization of this wide range of complexes of the wild-
type and mutant D38C GlcDH, we will be able to provide new
insights into the structure–function relationships of the enzyme and
perhaps shed light on the question of whether the aspartate residue at
position 38 in the H. mediterranei GlcDH is a halophilic adaptation or
purely serendipitous.
We thank the support staff at CCLRC Synchrotron Radiation
Source, Daresbury for assistance with station alignment. This work
was supported by the BBSRC, the Wellcome Trust, The Woolfson
Foundation and MCYT (BIO2002-03179) and by FPI fellowships
from Generalitat Valenciana (Spain) to JE. The Krebs Institute is a
designated BBSRC Biomolecular Sciences Centre and a member of
the North of England Structural Biology Centre.
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Acta Cryst. (2005). F61, 743–746 Esclapez et al. Glucose dehydrogenase 745
Table 1
X-ray data-collection statistics.
Values in parentheses refer to the highest resolution shell.
Protein sample Wild type D38C
Complex NADPH/Zn Free enzyme NADP
+
/Zn NADP
+
/Zn/gluconate NADPH/Zn/glucose NADPH/Zn NADPH
Source/wavelength Cu KCu KSRS† (0.97 A
˚)CuKCu KCu KCu K
Resolution (A
˚) 2.01 3.55 1.50 2.2 2.0 1.84 2.0
Highest resolution shell (A
˚) 2.08–2.01 3.63–3.55 1.55–1.50 2.27–2.22 2.05–2.0 1.89–1.84 2.05–2.00
Space group I222 P6
2
22 I222 I222 I222 I222 I222
Unit-cell parameters (A
˚)
a60.6 89.4 60.5 61.6 60.1 60.5 60.7
b107.7 89.4 109.3 112.2 106.3 108.9 108.1
c153.6 211.5 151.9 150.5 151.9 151.2 152.6
Unique reflections 32173 6269 66981 24766 30873 44001 32792
Completeness (%) 94.9 (82.5) 96.7 (100) 94.4 (93.5) 94.5 (92.9) 92.8 (89.6) 94.5 (91.5) 95.7 (99.3)
Multiplicity 3.9 (3.4) 8.74 (3.4) 5.1 (4.6) 9.34 (3.96) 8.03 (2.16) 7.1 (2.23) 5.4 (2.62)
hI/(I)i30.1 (3.7) 11.71 (2.4) 1.37 (1.01) 14.3 (3.3) 10.0 (1.8) 16.3 (1.8) 14.58 (2.41)
R
merge
‡ 0.059 (0.354) 0.10 (0.50) 0.051 (0.709) 0.10 (0.42) 0.095 (0.45) 0.047 (0.49) 0.092 (0.447)
† SRS: CCLRC Daresbury Synchrotron Radiation Source. ‡ R
merge
=PhPijIðh;iÞhIðhÞij=PhPiIðh;iÞ.

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crystallization communications
746 Esclapez et al. Glucose dehydrogenase Acta Cryst. (2005). F61, 743–746