We have previously determined the crystal structure of a novel pentagonal ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) from the hyperthermophilic archaeon, Thermococcus kodakaraensis KOD1. Here we have carried out biochemical studies to identify the necessities and/or advantages of this intriguing pentagonal structure. The structure indicated the presence of three neighboring residues (Glu-63, Arg-66, and Asp-69), participating in ionic interactions within unique dimer-dimer interfaces. We constructed three single mutant proteins (E63S, R66S, and D69S) and one triple mutant protein (E63S/R66S/D69S) by replacing the charged residues with serine. The wild type (WT) and all mutant proteins were purified and subjected to gel permeation chromatography at various temperatures. WT and D69S proteins were decameric at all temperatures examined between 30 and 90 degrees C. The majority of E63S and R66S were decamers at 30 degrees C but were found to gradually disassemble with the elevation in temperature. E63S/R66S/D69S was found in a dimeric form even at 30 degrees C. An interesting correlation was found between the subunit assembly and thermostability of the proteins. Circular dichroism and differential scanning calorimetry analyses indicated that the denaturation temperatures of dimeric enzymes (E63S, R66S, and E63S/R66S/D69S) were approximately 95 degrees C, whereas those of the enzymes retaining a decameric structure (WT and D69S) were approximately 110 degrees C. Disassembly into tetramer or dimer units did not alter the slopes of the Arrhenius plots, indicating that the decameric structure had no effect on catalytic performance per se. The results indicate that the decameric assembly of Tk-Rubisco contributes to enhance the thermostability of the enzyme. Taking into account the growth temperature of strain KOD1 (65-100 degrees C), the decameric structure of Tk-Rubisco can be considered essential for the stable presence of the enzyme in the host cells. This study provides an interesting example in which the thermostability of a protein can be enhanced by formation of a unique quaternary structure not found in mesophilic enzymes.