DIFFERENTIAL ACTIVATION OF NAD KINASE BY PLANT CALMODULIN ISOFORMS - THE CRITICAL ROLE OF DOMAIN-I

NAD kinase is a Ca2+/calmodulin (CaM)-dependent enzyme capable of conv erting cellular NAD to NADP. The enzyme purified from pea seedlings ca n be activated by highly conserved soybean CaM, SCaM-1, but not by the divergent soybean CaM isoform, SCaM-4 (Lee, S. H., Kim, J. C., Lee, M . S., Heo, W. D., Seo, H. Y., Yoon, H. W., Hong, J. C., Lee, S. Y., Ba hk, J. D., Hwang, I., and Cho, M. J. (1995) J. Biol. Chem. 270, 21806- 21812). To determine which domains were responsible for this different ial activation of NAD kinase, a series of chimeric SCaMs were generate d by exchanging functional domains between SCaM-4 and SCaM-1. SCaM-411 1, a chimeric SCaM-1 that contains the first domain of SCaM-4, was sev erely impaired (only 40% of maximal) in its ability to activate NAD ki nase. SCaM-1444, a chimeric SCaM-4 that contains the first domain of S CaM-1 exhibited nearly full (similar to 70%) activation of NAD kinase. Only chimeras containing domain I of SCaM-1 produced greater than hal f-maximal activation of NAD kinase. To define the amino acid residue(s ) in domain I that were responsible for this differential activation, seven single residue substitution mutants of SCaM-1 were generated and tested for NAD kinase activation. Among these mutants, only K30E and G40D showed greatly reduced NAD kinase activation. Also a double resid ue substitution mutant, K30E/G40D, containing these two mutations in c ombination was severely impaired in its NAD kinase-activating potentia l, reaching only 20% of maximal activation. Furthermore, a triple muta tion, K30E/M36I/G40D, completely abolished NAD kinase activation. Thus , our data suggest that domain I of CaM plays a key role in the differ ential activation of NAD kinase exhibited by SCaM-1 and SCaM-4. Furthe r, the residues Lys(30) and Glu(40) of SCaM-1 are critical for this fu nction.