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 (E1) and its reductase (E3)

Abstract

CDP-6-deoxy-L-threo-D-glycero-4-hexulose 3-dehydrase (E1), together with its reductase (E3), catalyzes a novel deoxygenation reaction essential for the biosynthesis of 3,6-dideoxyhexoses. In an attempt to gain evidence substantiating the E1.E3 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 E1 and E3 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 E1 upon its coupling with E3. 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 E1.E3 complex formation in vivo was also firmly established. These results, when considered with the incompatibility of other electron transfer proteins as replacements for E3 in this electron relay, nicely demonstrated the specificity of the E1-E3 recognition. The apparent dissociation constant of the E1.E3 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 E3 and E1 of this complex was deduced as 1.7. Interestingly, while the conformation of the E1.E3 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 E3. In conjunction with early mechanistic studies, the present data establish the significance of the E1.E3 complex formation for catalysis and, consequently, corroborate the mechanism proposed for the overall deoxygenation process.