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))

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) . E(3) complex formation as a prerequisite for the C-3 deoxyenation 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 the two-hybrid system which scores for interactions between two proteins coexpressed in yeast, the E(1) . 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) . 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) . E(3) complex was sensitive to the salt concentration in the buffer, 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) . E(3) complex formation for catalysis and, consequently, corroborate the mechanism proposed for the overall deoxygenation process.