Biosynthesis of 3,6-Dideoxyhexoses: In Vivo and in Vitro Evidence for Protein--Protein Interaction between CDP-6-deoxy-L-threo-D-glycero-4-hexulose 3-Dehydrase (E_1) and Its Reductase (E_3)

摘要

CDP-6-deoxy-L-threo-D-glycero-4-hexulose 3-dehydrase (E_1). together with its reductase (E_3), catalyzes a novel deoxygenation reaction essential for the biosynthesis of 3,6-dideoxyhexoses. In an attempt to gain evidence substantiating the E_1 centredot E_3 complex formation as a prerequisite for the C-3 deoxygenation activity, we have carried out experiments to study the interaction between these two proteins. The detection of a new species when a mixture of E_1 and E_3 was analyzed by size-exclusion chromatography was the initial indication supporting the proposed complex formation. Additional evidence for the expected complex formation was provided by the change of the CD spectrum of E_1 upon its coupling with E_3. The fact that the catalytic efficiency of this system is limited by the quantity of one enzyme, which becomes catalytically competent only after coupling with the second enzyme, further illustrated the importance of such a complex formation to the deoxygenation activity. By using thetwo-hybrid system which scores for interactions between two proteins coexpressed in yeast, the E_1 centre dot E_3 complex formation in vivo was also firmly established. These results, when considered with the incompatibility of other electron transfer proteins as replacements for E_3 in this electron relay, nicely demonstrated the specificity of the E_1--E_3 recognition. The apparent dissociation constant of the E_1 centre dot E_3 complex formed in rapid equilibrium was estimated to be 288 ± 22 nM from the correlation between the initial rate of the overall reaction and the concentration of one protein component, and the stoichiometry between E_3 and E_1 of this complex was deduced as 1.7. Interestingly, while the conformation of the E_1 centre dot E_3 complex was sensitive to the salt concentration in the buffet, the decrease in the catalytic activity at high ionic strength was most likely due to the retardation of the electron transfer mediated by E_3. In conjunction with early mechanistic studies, the present data establish the significance of the E_1 centre dot E_3 complex formation for catalysis and, consequently, corroborate the mechanism proposed for the overall deoxygenation process.