Dataset Information

Structure of Pisum sativum Rubisco with bound ribulose 1,5-bisphosphate.

ABSTRACT: The first structure of a ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) from a pulse crop is reported. Rubisco was purified from Pisum sativum (garden pea) and diffraction-quality crystals were obtained by hanging-drop vapour diffusion in the presence of the substrate ribulose 1,5-bisphosphate. X-ray diffraction data were recorded to 2.20?Å resolution from a single crystal at the Canadian Light Source. The overall quaternary structure of non-activated P. sativum Rubisco highlights the conservation of the form I Rubisco hexadecameric complex. The electron density places the substrate in the active site at the interface of the large-subunit dimers. Lys201 in the active site is not carbamylated as expected for this non-activated structure. Some heterogeneity in the small-subunit sequence is noted, as well as possible variations in the conformation and contacts of ribulose 1,5-bisphosphate in the large-subunit active sites. Overall, the active-site conformation most closely correlates with the `closed' conformation observed in other substrate/inhibitor-bound Rubisco structures.

SUBMITTER: Loewen PC

PROVIDER: S-EPMC3539695 | biostudies-literature | 2013 Jan

REPOSITORIES: biostudies-literature

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Structure of Pisum sativum Rubisco with bound ribulose 1,5-bisphosphate.

Loewen Peter C PC Didychuk Allison L AL Switala Jacek J Perez-Luque Rosa R Fita Ignacio I Loewen Michele C MC

Acta crystallographica. Section F, Structural biology and crystallization communications 20121225 Pt 1

The first structure of a ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) from a pulse crop is reported. Rubisco was purified from Pisum sativum (garden pea) and diffraction-quality crystals were obtained by hanging-drop vapour diffusion in the presence of the substrate ribulose 1,5-bisphosphate. X-ray diffraction data were recorded to 2.20 Å resolution from a single crystal at the Canadian Light Source. The overall quaternary structure of non-activated P. sativum Rubisco highlights the ...[more]

PMID: 23295478

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Project description:The sequences classified as genes for various ribulose-1,5-bisphosphate (RuBP) carboxylase/oxygenase (RuBisCO)-like proteins (RLPs) are widely distributed among bacteria, archaea, and eukaryota. In the phylogenic tree constructed with these sequences, RuBisCOs and RLPs are grouped into four separate clades, forms I-IV. In RuBisCO enzymes encoded by form I, II, and III sequences, 19 conserved amino acid residues are essential for CO(2) fixation; however, 1-11 of these 19 residues are substituted with other amino acids in form IV RLPs. Among form IV RLPs, the only enzymatic activity detected to date is a 2,3-diketo-5-methylthiopentyl 1-phosphate (DK-MTP-1-P) enolase reaction catalyzed by Bacillus subtilis, Microcystis aeruginosa, and Geobacillus kaustophilus form IV RLPs. RLPs from Rhodospirillum rubrum, Rhodopseudomonas palustris, Chlorobium tepidum, and Bordetella bronchiseptica were inactive in the enolase reaction. DK-MTP-1-P enolase activity of B. subtilis RLP required Mg(2+) for catalysis and, like RuBisCO, was stimulated by CO(2). Four residues that are essential for the enolization reaction of RuBisCO, Lys(175), Lys(201), Asp(203), and Glu(204), were conserved in RLPs and were essential for DK-MTP-1-P enolase catalysis. Lys(123), the residue conserved in DK-MTP-1-P enolases, was also essential for B. subtilis RLP enolase activity. Similarities between the active site structures of RuBisCO and B. subtilis RLP were examined by analyzing the effects of structural analogs of RuBP on DK-MTP-1-P enolase activity. A transition state analog for the RuBP carboxylation of RuBisCO was a competitive inhibitor in the DK-MTP-1-P enolase reaction with a K(i) value of 103 mum. RuBP and d-phosphoglyceric acid, the substrate and product, respectively, of RuBisCO, were weaker competitive inhibitors. These results suggest that the amino acid residues utilized in the B. subtilis RLP enolase reaction are the same as those utilized in the RuBisCO RuBP enolization reaction.

Project description:The ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO) enzyme found in plants, algae, and an array of autotrophic bacteria is also encoded by a subset of methanotrophs, but its role in these microbes has largely remained elusive. In this study, we showed that CO2 was requisite for RubisCO-encoding Methylococcus capsulatus strain Bath growth in a bioreactor with continuous influent and effluent gas flow. RNA sequencing identified active transcription of several carboxylating enzymes, including key enzymes of the Calvin and serine cycles, that could mediate CO2 assimilation during cultivation with both CH4 and CO2 as carbon sources. Marker exchange mutagenesis of M. capsulatus Bath genes encoding key enzymes of potential CO2-assimilating metabolic pathways indicated that a complete serine cycle is not required, whereas RubisCO is essential for growth of this bacterium. 13CO2 tracer analysis showed that CH4 and CO2 enter overlapping anaplerotic pathways and implicated RubisCO as the primary enzyme mediating CO2 assimilation in M. capsulatus Bath. Notably, we quantified the relative abundance of 3-phosphoglycerate and ribulose-1,5-bisphosphate 13C isotopes, which supported that RubisCO-produced 3-phosphoglycerate is primarily converted to ribulose-1-5-bisphosphate via the oxidative pentose phosphate pathway in M. capsulatus Bath. Collectively, our data establish that RubisCO and CO2 play essential roles in M. capsulatus Bath metabolism. This study expands the known capacity of methanotrophs to fix CO2 via RubisCO, which may play a more pivotal role in the Earth's biogeochemical carbon cycling and greenhouse gas regulation than previously recognized. Further, M. capsulatus Bath and other CO2-assimilating methanotrophs represent excellent candidates for use in the bioconversion of biogas waste streams that consist of both CH4 and CO2. IMPORTANCE The importance of RubisCO and CO2 in M. capsulatus Bath metabolism is unclear. In this study, we demonstrated that both CO2 and RubisCO are essential for M. capsulatus Bath growth. 13CO2 tracing experiments supported that RubisCO mediates CO2 fixation and that a noncanonical Calvin cycle is active in this organism. Our study provides insights into the expanding knowledge of methanotroph metabolism and implicates dually CH4/CO2-utilizing bacteria as more important players in the biogeochemical carbon cycle than previously appreciated. In addition, M. capsulatus and other methanotrophs with CO2 assimilation capacity represent candidate organisms for the development of biotechnologies to mitigate the two most abundant greenhouse gases, CH4 and CO2.

Dataset Information

Structure of Pisum sativum Rubisco with bound ribulose 1,5-bisphosphate.

Publications

Structure of Pisum sativum Rubisco with bound ribulose 1,5-bisphosphate.

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