Synthesis of catalytically active form III ribulose 1,5-bisphosphate carboxylase/oxygenase in archaea

Abstract

Ribulose 1,5 bisphosphate carboxylase/oxygenase (RubisCO) catalyzes the biological reduction and assimilation of carbon dioxide gas to organic carbon; it is the key enzyme responsible for the bulk of organic matter found on earth. Until recently it was believed that there are only two forms of RubisCO, form I and form II. However, the recent completion of several genome-sequencing projects uncovered open reading frames resembling RubisCO in the third domain of life, the archaea. Previous work and homology comparisons suggest that these enzymes represent a third form of RubisCO, form III. While earlier work indicated that two structurally distinct recombinant archaeal RubisCO proteins catalyzed bona fide RubisCO reactions, it was not established that the rbcL genes of anaerobic archaea can be transcribed and translated to an active enzyme in the native organisms. In this report, it is shown not only that Methanococcus jannaschii, Archaeoglobus fulgidus, Methanosarcina acetivorans, and Methanosarcina barkeri possess open reading frames with the residues required for catalysis but also that the RubisCO protein from these archaea accumulates in an active form under normal growth conditions. In addition, the form III RubisCO gene (rbcL) from M. acetivorans was shown to complement RubisCO deletion strains of Rhodobacter capsulatus and Rhodobacter sphaeroides under both photoheterotrophic and photoautotrophic growth conditions. These studies thus indicate for the first time that archaeal form III RubisCO functions in a physiologically significant fashion to fix CO(2). Furthermore, recombinant M. jannaschii, M. acetivorans, and A. fulgidus RubisCO possess unique properties with respect to quaternary structure, temperature optima, and activity in the presence of molecular oxygen compared to the previously described Thermococcus kodakaraensis and halophile proteins.

FIG. 1.

Purity of protein fractions eluted from Superose 12 gel filtration columns of recombinant RubisCO preparations from M. jannaschii (A), A. fulgidus (B), and M. acetivorans (C). Protein standards are shown in the leftmost lanes of panels A and B. SDS-PAGE was performed as described in Materials and Methods. Purified proteins were used to prepare antibodies of each enzyme as described in Materials and Methods.

FIG. 2.

Sequence alignment showing that the key catalytic residues important for RuBP-dependent CO₂ fixation are maintained in the archaeal RubisCO proteins used in this study. The numbers represents the amino acid residues corresponding to the Synechococcus PCC 6301 form I protein. Residues known to be important for either catalysis (C) or RuBP binding (R) are indicated. I-Syn 6301, Synechococcus PCC 6301 form I; II-Rr, R. rubrum form II; III-Mj, Methanocaldococcus (Methanococcus) jannaschii form III; III-Af RbcL-2, A. fulgidus RbcL-2 form III; III-Ma, M. acetivorans form III; III-Mb, M. barkeri form III; III-Mm, M. mazei form III; III-Ph, Pyrococcus horikoshii form III; III-Tk, T. (Pyrococcus) kodakaraensis form III.

FIG. 3.

Western immunoblot showing RubisCO protein accumulation in M. jannaschii with respect to growth temperature. The antiserum used in Western immunoblotting was generated against M. jannaschii recombinant RubisCO protein purified from E. coli. (A) Purified recombinant M. jannaschii RubisCO (3 μg) from E. coli grown at 37°C; (B) crude extract (49 μg) prepared from M. jannaschii grown at 65°C; (C) crude extract (49 μg) prepared from M. jannaschii grown at 85°C.

FIG. 4.

Activity time course for purified recombinant M. jannaschii RubisCO assayed at 65°C (♦) and 85°C (▪) (A) and A. fulgidus RubisCO assayed at 90°C (▴), 65°C (▪), and 37°C (♦) (B). Each assay contained 5 μg of protein and was performed under anaerobic conditions; no activity was detected at 37°C for the M. jannaschii enzyme.

FIG. 5.

Exposure of recombinant M. jannaschii, A. fulgidus, and M. acetivorans RubisCO to oxygen (2.5 μg of protein in all cases). M. jannaschii (A) and A. fulgidus (B) RubisCO were assayed at 83°C, while M. acetivorans RubisCO (C) was assayed at 37°C. The aerobic assay (♦) was performed in vials in the presence of ambient air, whereas the anaerobic assay (▪) was carried out under an argon atmosphere. The recovered assay (▴) represents an assay in which a continuous stream of oxygen was allowed to permeate the assay vial headspace for 45 min; the oxygen-exposed enzyme was then injected into an anaerobic vial and assayed for the indicated times.

FIG. 6.

Complementation and growth of R. capsulatus cbbLS/cbbM knockout strain (form I [cbbLS] and II [cbbM] RubisCO genes deleted) SBI-II⁻ (22) at the expense of the M. acetivorans RubisCO (rbcL) gene in plasmid pRPSMCS3MA. (A) Using malate as the carbon source, photoheterotrophic cultures were grown in glass bottles continuously bubbled with argon (without scrubbing the gas through a heated-copper-filings system) to remove trace amounts of oxygen. ▪, R. capsulatus strain SBI-II⁻; ♦, R. capsulatus strain SBI-II⁻ containing plasmid pRPSMCS3MA; ▴, wild-type R. sphaeroides strain HR containing plasmid pRPSMCS3MA. (B) Photoautotrophic growth of R. capsulatus knockout strain SBI-II⁻ (cbbLS/cbbM) (▪) along with wild-type R. sphaeroides HR (♦) and R. sphaeroides knockout strain 16 (cbbLS/cbbM) (▴), all containing plasmid pRPSMCS3MA. The cells were grown in sealed and crimped tubes pressurized with oxygen-free 20% CO₂ balanced with H₂. The gas was exchanged every 24 h.

FIG. 7.

Western immunoblot of extracts of R. capsulatus SBI-II⁻ and R. sphaeroides strain 16 complemented with plasmid pRPSMCS3MA (containing M. acetivorans rbcL). The immunoblot was tested using antibodies directed against purified recombinant M. acetivorans RubisCO. All lanes contained soluble crude extract prepared from stationary phase cultures from the following strains: photoheterotrophically grown wild-type R. sphaeroides strain HR (lane A); photoautotrophically grown wild-type R. sphaeroides strain HR (lane B); purified recombinant M. acetivorans RubisCO (lane C); photoheterotrophically grown R. capsulatus SBI-II⁻ complemented with plasmid pRPSMCS3MA (containing M. acetivorans rbcL) (lane D); photoautotrophically grown R. capsulatus SBI-II⁻ complemented with plasmid pRPSMCS3MA (lane E); photoautotrophically grown R. sphaeroides strain 16 complemented with plasmid pRPSMCS3MA (lane F); photoheterotrophically grown R. sphaeroides strain 16 complemented with plasmid pRPSMCS3MA (lane G); photoheterotrophically grown wild-type R. capsulatus strain SB1003 (lane H); and purified recombinant M. acetivorans RubisCO (lane I). Each lane received approximately 15 μg of protein.