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Crystallization and preliminary crystallographic study of glucose dehydrogenase from the archaebacterium Thermoplasma acidophilum
J. Mol. Biol. (1991) 222, 143-144
Crystallization and Preliminary Crystallographic Study of Glucose Dehydrogenase from the Archaebacterium Thermoplasma acidophilum Jeremy R. Bright, Ruth Mackness, Michael J. Danson David W. Hough, Garry L. Taylort, Paul Towner Department of Biochemistry, University of Bath Claverton Down, Bath BA2 7A Y, U.K.
and David Byrom ICI
Biological Cleveland,
(Received 24 July
Products, Billingham TS23 ILB, U.K.
1991; accepted 12 August
1991)
Thermoplasma of glucose dehydrogenase from the archaebacterium were obtained using the hanging-drop vapour diffusion method and polyethylene glycol as a precipitant in the presence of NADP+ at pH 5.4. The crystals belong to the hexagonal space group PfS122 or P6,22, with unit cell dimensions a= b= 121.9 a, c= 229% A and with two molecules in the asymmetric unit.
Single
crystals
acidophilum
Keywords: archaebacteria;
glucose dehydrogenase:
Thermoplasma acidophilum is a thermophilic archaebacterium growing at 55 to 6O”C, pH 1 to 2. During aerobic growth, it catabolizes glucose via a non-phosphorylated Entner-Doudoroff pathway (Budgen & Danson, 1986; Danson, 1989), the first enzyme of which is an NAD(P)-dependent glucose dehydrogenase. We have purified this enzyme to homogeneity (Smith et al., 1989) and its gene has now been cloned, sequenced and expressed in Escherichia coli (Bright et al., 1991). The high level of thermostability of the expressed glucose dehydrogenase has permitted its rapid purification and we now report the crystallization and a preliminary crystallographic st)udy of this archaebacterial enzyme. Crystals were obtained by the hanging-drop vapour diffusion method (McPherson, 1990). Trials indicated that a protein concentration of 8 mg/ml was bett#er than 15 mg/ml. Best crystals were grown using 2 9/, (w/v) polyethylene glycol6000 (pH 53) as the precipitant solution in the reservoir. Typically 6 ~1 of protein solution at 8 mg/ml concentration was mixed with an equal volume of the precipitant solution and left to equilibrate at room temperature. Crystals of hexagonal prism habit appeared wit’hin five days, with maximum dimensions of t Author addressed.
to whom
002%2836/91/220143-02
all correspondence
$03.00/O
should
crystallography
0.3 mm x 0.15 mm x 0.15 mm. These crystals showed diffraction to only 12 a (1 L& = 0.1 nm) resolution. Further trials involved adding NADP to the hanging drops. Similar hexagonal prisms were obtained at pH 5.4 in the presence of O-4 mMNADP+ and 2% (w/v) PEG 6000. These diffracted out to 3.0 A resolution on a laboratory rotating anode X-ray source. The NADP+ appears to have conferred greater order on the crystals. At @4 mMNADP+ only 70% of binding sites are occupied, so further trials were carried out with increasing concentrations of NADP+. However: precipitation of the protein was observed during attempts to saturate the enzyme with its cofactor. Diffraction data were collected on a Siemens area detector mounted on a Siemens rotating anode X-ray source operating at 45 kV and 80 mA. Frames of data were recorded while the crystal was oscillated through 0.25 degree steps. The data were processed with the XENGEN suite of software (A. Howard, 1988, Genex Corporation, Gaithersburg, MD, U.S.A.). The hexagonal prism crystals belong to space group P6,22 or P6,22, as judged by systematically absent or weak (F < 3.50) 001 reflections. The unit cell parameters are a = b = 121.9 A, c = 229.6 A, c( = /3 = 90”, y = 120”. With a molecular mass of 40 kDa for the monomer, V,,, values of 6.16 A3/Da, 3.08 b3/Da and 1.54 A3/Da are obtained assuming a
be
143
0 1991 Academic Press Limited
144
J. R. Bright
monomer. dimer or tetramer in the asvmmetric unit giving solvent contents of 80 yjO. 60”:1~ and 20y0 _ respectively. The asymmetric unit is most likely to be a, dimer, as its solvent content falls within the reported range for protein crystals (Matthews. 1968). The active glucose dehydrogenase molecule from Thermoplasma acidophilum is a tetramer of identical 40 kI>a monomers (Smith et al.. 1989: Bright et al.. 1991). Assuming the asymmetric unit contains a dimer. the other dimer can be generated by a cry”tallographic 2-fold axis to produce a tetramer with 222 point symmetry. The only other reported crystallization of a glucose dehydropenase is that from Bacillus megaterium, M1286, which is also tetrameric. but belongs tjo the “short” family of dehydrogenases with a molecular mass of 30 kDa (Pal et al.. 1987). In this preliminary crystallization report of the eubacterial enzyme. the point group symmetry of the t’etramer was unresolved. A more complete analysis of the cofa.c*tordependence on ordered crystal formaCon, as well as a search for suitable heavy atom derivatives, are in progress.
et al.
References Bright, J. K., Byrom. I).. Danson. M. ,I.. Hougll. I). 11’. B Towner. I’. (1991). Cloning and sequence determirration of the gem encoding glucose dehydrqenase t’ron~ the thermophilic archaebacterium Th,rrrrloplt~,srrLa acidophilum,. Eur. J. Niochem. In the J)re’ss. Rudgen, N. & Danson. M. ,J. (19%). Metabolism of glucose c,icl a modified Entner J)oudoroff J’athway in 7’hwmoplosmo the thermophilic archaebactrrium aridophilum. FE&S’ Lettws, 196. dO7-210. Jjanson. M. J. (1989). (‘entral metaholistn of t,hr archarhactrrium: an overview. (‘an. J. Microhiol. 35. !iXMl. Matkhewa. 1% LV. (1968). SolvtAnt (*ontent of’ Jtrc)tein crystals. .I. !Mol. Hiol. 33. 4!)JLk97. ,McPherson, A. .J. (I 990). (lurrent approacahrs t)o ma~t~omolecular c~rvstallieation. Eur. J. Bioch,rm. 189. I -21. Pal, I’..
Edited by A. Klug
,I.
261, 975977.
Crystallization and Preliminary Crystallographic Study of Glucose Dehydrogenase from the Archaebacterium Thermoplasma acidophilum Jeremy R. Bright, Ruth Mackness, Michael J. Danson David W. Hough, Garry L. Taylort, Paul Towner Department of Biochemistry, University of Bath Claverton Down, Bath BA2 7A Y, U.K.
and David Byrom ICI
Biological Cleveland,
(Received 24 July
Products, Billingham TS23 ILB, U.K.
1991; accepted 12 August
1991)
Thermoplasma of glucose dehydrogenase from the archaebacterium were obtained using the hanging-drop vapour diffusion method and polyethylene glycol as a precipitant in the presence of NADP+ at pH 5.4. The crystals belong to the hexagonal space group PfS122 or P6,22, with unit cell dimensions a= b= 121.9 a, c= 229% A and with two molecules in the asymmetric unit.
Single
crystals
acidophilum
Keywords: archaebacteria;
glucose dehydrogenase:
Thermoplasma acidophilum is a thermophilic archaebacterium growing at 55 to 6O”C, pH 1 to 2. During aerobic growth, it catabolizes glucose via a non-phosphorylated Entner-Doudoroff pathway (Budgen & Danson, 1986; Danson, 1989), the first enzyme of which is an NAD(P)-dependent glucose dehydrogenase. We have purified this enzyme to homogeneity (Smith et al., 1989) and its gene has now been cloned, sequenced and expressed in Escherichia coli (Bright et al., 1991). The high level of thermostability of the expressed glucose dehydrogenase has permitted its rapid purification and we now report the crystallization and a preliminary crystallographic st)udy of this archaebacterial enzyme. Crystals were obtained by the hanging-drop vapour diffusion method (McPherson, 1990). Trials indicated that a protein concentration of 8 mg/ml was bett#er than 15 mg/ml. Best crystals were grown using 2 9/, (w/v) polyethylene glycol6000 (pH 53) as the precipitant solution in the reservoir. Typically 6 ~1 of protein solution at 8 mg/ml concentration was mixed with an equal volume of the precipitant solution and left to equilibrate at room temperature. Crystals of hexagonal prism habit appeared wit’hin five days, with maximum dimensions of t Author addressed.
to whom
002%2836/91/220143-02
all correspondence
$03.00/O
should
crystallography
0.3 mm x 0.15 mm x 0.15 mm. These crystals showed diffraction to only 12 a (1 L& = 0.1 nm) resolution. Further trials involved adding NADP to the hanging drops. Similar hexagonal prisms were obtained at pH 5.4 in the presence of O-4 mMNADP+ and 2% (w/v) PEG 6000. These diffracted out to 3.0 A resolution on a laboratory rotating anode X-ray source. The NADP+ appears to have conferred greater order on the crystals. At @4 mMNADP+ only 70% of binding sites are occupied, so further trials were carried out with increasing concentrations of NADP+. However: precipitation of the protein was observed during attempts to saturate the enzyme with its cofactor. Diffraction data were collected on a Siemens area detector mounted on a Siemens rotating anode X-ray source operating at 45 kV and 80 mA. Frames of data were recorded while the crystal was oscillated through 0.25 degree steps. The data were processed with the XENGEN suite of software (A. Howard, 1988, Genex Corporation, Gaithersburg, MD, U.S.A.). The hexagonal prism crystals belong to space group P6,22 or P6,22, as judged by systematically absent or weak (F < 3.50) 001 reflections. The unit cell parameters are a = b = 121.9 A, c = 229.6 A, c( = /3 = 90”, y = 120”. With a molecular mass of 40 kDa for the monomer, V,,, values of 6.16 A3/Da, 3.08 b3/Da and 1.54 A3/Da are obtained assuming a
be
143
0 1991 Academic Press Limited
144
J. R. Bright
monomer. dimer or tetramer in the asvmmetric unit giving solvent contents of 80 yjO. 60”:1~ and 20y0 _ respectively. The asymmetric unit is most likely to be a, dimer, as its solvent content falls within the reported range for protein crystals (Matthews. 1968). The active glucose dehydrogenase molecule from Thermoplasma acidophilum is a tetramer of identical 40 kI>a monomers (Smith et al.. 1989: Bright et al.. 1991). Assuming the asymmetric unit contains a dimer. the other dimer can be generated by a cry”tallographic 2-fold axis to produce a tetramer with 222 point symmetry. The only other reported crystallization of a glucose dehydropenase is that from Bacillus megaterium, M1286, which is also tetrameric. but belongs tjo the “short” family of dehydrogenases with a molecular mass of 30 kDa (Pal et al.. 1987). In this preliminary crystallization report of the eubacterial enzyme. the point group symmetry of the t’etramer was unresolved. A more complete analysis of the cofa.c*tordependence on ordered crystal formaCon, as well as a search for suitable heavy atom derivatives, are in progress.
et al.
References Bright, J. K., Byrom. I).. Danson. M. ,I.. Hougll. I). 11’. B Towner. I’. (1991). Cloning and sequence determirration of the gem encoding glucose dehydrqenase t’ron~ the thermophilic archaebacterium Th,rrrrloplt~,srrLa acidophilum,. Eur. J. Niochem. In the J)re’ss. Rudgen, N. & Danson. M. ,J. (19%). Metabolism of glucose c,icl a modified Entner J)oudoroff J’athway in 7’hwmoplosmo the thermophilic archaebactrrium aridophilum. FE&S’ Lettws, 196. dO7-210. Jjanson. M. J. (1989). (‘entral metaholistn of t,hr archarhactrrium: an overview. (‘an. J. Microhiol. 35. !iXMl. Matkhewa. 1% LV. (1968). SolvtAnt (*ontent of’ Jtrc)tein crystals. .I. !Mol. Hiol. 33. 4!)JLk97. ,McPherson, A. .J. (I 990). (lurrent approacahrs t)o ma~t~omolecular c~rvstallieation. Eur. J. Bioch,rm. 189. I -21. Pal, I’..
Edited by A. Klug
,I.
261, 975977.