Differential activation of NAD kinase by plant calmodulin isoforms. The critical role of domain I. J Biol Chem 1997 Apr 04;272(14):9252-9
Date
04/04/1997Pubmed ID
9083059DOI
10.1074/jbc.272.14.9252Scopus ID
2-s2.0-0001281471 (requires institutional sign-in at Scopus site) 67 CitationsAbstract
NAD kinase is a Ca2+/calmodulin (CaM)-dependent enzyme capable of converting cellular NAD to NADP. The enzyme purified from pea seedlings can 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., Bahk, J. D., Hwang, I., and Cho, M. J. (1995) J. Biol. Chem. 270, 21806-21812). To determine which domains were responsible for this differential activation of NAD kinase, a series of chimeric SCaMs were generated by exchanging functional domains between SCaM-4 and SCaM-1. SCaM-4111, a chimeric SCaM-1 that contains the first domain of SCaM-4, was severely impaired (only 40% of maximal) in its ability to activate NAD kinase. SCaM-1444, a chimeric SCaM-4 that contains the first domain of SCaM-1 exhibited nearly full ( approximately 70%) activation of NAD kinase. Only chimeras containing domain I of SCaM-1 produced greater than half-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 residue substitution mutant, K30E/G40D, containing these two mutations in combination was severely impaired in its NAD kinase-activating potential, reaching only 20% of maximal activation. Furthermore, a triple mutation, K30E/M36I/G40D, completely abolished NAD kinase activation. Thus, our data suggest that domain I of CaM plays a key role in the differential activation of NAD kinase exhibited by SCaM-1 and SCaM-4. Further, the residues Lys30 and Glu40 of SCaM-1 are critical for this function.
Author List
Lee SH, Seo HY, Kim JC, Heo WD, Chung WS, Lee KJ, Kim MC, Cheong YH, Choi JY, Lim CO, Cho MJAuthor
Sang H. Lee PhD Professor in the Pharmacology and Toxicology department at Medical College of WisconsinMESH terms used to index this publication - Major topics in bold
3',5'-Cyclic-AMP PhosphodiesterasesAmino Acid Sequence
Calmodulin
Electrophoresis, Polyacrylamide Gel
Enzyme Activation
Models, Molecular
Molecular Sequence Data
NADP
Phosphotransferases (Alcohol Group Acceptor)
Plant Proteins
Recombinant Fusion Proteins
Structure-Activity Relationship
