Analyzing of RuBisCO from the extremely halophilic archaeon for applications

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We performed to clone the gene for RuBisCO from the extremely halophilic Archaeon, Haloferax volcanii in order to analyze the molecular mechanism for salt-resistance of this RuBisCO. The gene for RuBisCO of H. volcanii was higher similar to other Archaeal RuBisCO than that of photosynthetic organisms. Archaeal RuBisCO is called RuBisCO-like protein (RLP) and predicted that this protein does not catalyze the CO_2 fixing reaction in the Calvin cycle because Archaea does not possess the ability of photostnthesis. Non-photosynthetic bacteria including Bacillus subtilis also possess genes for RLP. The function of RLP in non-photosynthetic bacteria and Archaea is unknown yet. The gene for the RLP of B. subtilis is encoded by ylaW, which lies at the first position in the ykrWXYZ operon, close to the ykr7S operon. It was reported that the ykrWXYZ and ykrTS operons were involved in the methionine salvage pathway, but enzymes and catalyzing steps in the methionine salvage pathway of B. subtilis … More are unknown. The reaction steps and enzymes in this pathway were identified to elucidate the catalyzing reaction of RLP by 1H-NMR and UV-visible spectroscopy using recombinant proteins from those operons. This analysis revealed that RLP catalyzes the 2,3-diketo-5-methylthiopentyl-l-phosphate enolase reaction in the fourth step in the methionine salvage pathway. Furthermore, we identified that YkrS is methylthioribose-l-phosphate isomerase, YkrY is methylthioribulose-1-phosphate dehydratase, YkrX is 2-hydmxy-3-keto-5-methylthiopentenyl-1-phosphate phosphatase and YkrZ is 1,2-dihydroxy-3-keto-5-methylthiopentene dioxygenase in this pathway. Wild type B. subtilis grew on methylthioadenosine (MTA), an intermediate in the methionine salvage pathway, as the sole sulfur compound. A mutant with the RLP gene disrupted did not grow on MTA. Interestingly, the mutant was rescued for growth on MTA by introducing the gene of photosynthetic RuBisCO from a photosynthetic bacterium. This result indicates that photosynthetic RuBisCO also catalyzes the same reaction step as RLP. Less