D-ribulose 1,5-bisphosphate carboxylase from Paracoccus denitrificans

Abstract

The facultative chemolithottopkic hydrogen bacterium Paracoccu~ denitrificans is capable of growing autotrophically with hydrogen and CO2 as well as heterotrophieally on a variety of organic substrates in the presence of oxygen or nitrate as electron accepters [ 1 ]. 14CO2 incorporation experiments provided indirect evidence that autotrophicaUy grown ceils of this bacterium contain l~ribulose 5.phosphate kinase and D-ribulose 1,5-hisphosphate (RuBP) carboxylase (RuBPCase) which catalyze the primary steps of CO2 fixation in autotrophs. However. in cells grown heterotrophically on acetate the activity of these enzymes was not detectable [2]. in the present work, s(,me properties of purified RuLPCase from P. denitnficans have been examined. The molecular weight and quaternary structure of the enzyme are typical of the large carboxylases from other prokaryotic and eukaryotic organisms. Materials and Methods Organism, growth conditions and harvesting of cellsP. denltrlficans strain 381 (ATCC 17741, DSM 65), formerly Mlcrococcus denltrificans [3] was kindly provided by Dr. ICA. Malik (G6ttingen). Bacteria were grown au :otrophicaily i~l the mineral salts medium of Schlegel et al. [4] at 30°C in a fermenter sapplied with a gas mixture of 80% H2, 10% O= and 10% CO2. Cells were harvested in mid-exponential phase, washed twice in 20 mM Tris-Ht.'l buffer, pH 7.8, containing 50 mM NaHCOa, 10 mM MgCl=, I mM EDTA and l mM dithioerythritol (enzyme buffer), and stored at -2o0c. Enzyme purificationAll purification steps were carried out at 0--4~C. 20 g of frozen cells were thawed by suspension in 2 vol. of em:yme buffer and passed through a French pressure c ell at 1500 kp/cm z. The preparation was centrifuged at 40000 X g for 20rain and the s~pernatant respun at 140 000 × g for 60 rain. The resulting supemata~t was brought to 25% saturation by ~he addition of a saturated ammonium sulphate solution (pH 7.8) and c,.,ntrifuged at 20000 X g for 15 min. The supematar.t obtained was taken to 45% ammonium sulphate saturation, the precipitate collected by centrifugation and dissolved ~,! enzyme buffer then dial. yzed against this buffer for 12 h. RuBPCase in this fraction wa.~ subjected to sedimentation into a linear sucrose density gradient (0.2-0.8 M) as detailed previ. ously [5,6]. After centrifugation 1 ml fractions were collected from the top of the tubes. The protein from the five fractions containing the highest RuBPCase activity was precipitated between 30 and 40% anunonium sulphate saturation, collected by cenUifugation and dissolved in enzy:ne buffer fo~owed b) dialysis asbefore. In a final step the protein was adsorbed onto ~ g of DEAE-Sephadex A-50 equilibrated with enzFme buffer in a beaker. By stepwise elution witb enzyme buffer containing increasing concemrations of KC! RuBPCase was desorbed with 0.25 M KCL '~:~e enzyme precipitated at 50% ammonimn sulphate .~turation was redissolved in enzyme buffer and dialyzed .~gahm this buffer.

Ribulose bisphosphate carboxylase from methanol-grown Paracoccus denitrificans

“…The average mole ratio of large and small subunits was 1.07 ± 10%. From the native molecular weight of 525,000, we infer that the Paracoccus carboxylase has eight large and eight small subunits, which is in agreement with findings of Bowien for the enzyme isolated from P. denitrificans grown on H2 and N03 (2).…”

Molecular and cellular regulation of autotrophic carbon dioxide fixation in microorganisms.

“…Dissociation of the Aphanothece enzyme occurs at low ionic strength (339), another effective method for preparing large and small subunits for structure-function studies. In the chemolithoautotrophic bacteria, a number of reports have led to the general impression that RuBPC/O from all of these organisms is composed of the usual L8S8 structure (34,39,41,52,212,251,252,255,307). Past suggestions of L6S6 (181,218,343), L8 (254), or alternative structures (174, 217) have not been substantiated, and part of the inaccuracies of these studies may be due to problems similar to those delineated above for the cyanobacterial enzyme.…”

The Carboxysomes (Polyhedral Bodies) of Autotrophic Prokarygtes

“…Tabita & McFadden, 1974b;Bowien et al, 1976;Bowien, 1977;Purohit, Becker & Evans, 1982). RuBisCO purification from bacteria without carboxysomes has almost invariably been performed from supernatants obtained after high-speed differential centrifugation of broken cells (e.g.…”

“…RuBisCO purification from bacteria without carboxysomes has almost invariably been performed from supernatants obtained after high-speed differential centrifugation of broken cells (e.g. Tabita & McFadden, 1974b;Bowien et al, 1976;Bowien, 1977;Purohit, Becker & Evans, 1982). When performed with bacteria lacking carboxysomes this procedure clearly permits purification of RuBisCO, which is essentially in the cytoplasm.…”