Differential Stimulation of NAD Kinase and Binding of Peptide Substrates by Wild-Type and Mutant Plant Calmodulin Isoforms

Calmodulin fromArabidopsis thalianaconsists of at least four isoforms, which differ in their deduced amino acid sequences by as many as six conservative substitutions. To determine whether these differences are biochemically significant, cDNAs encoding three of the four isoforms were engineered to produce recombinant proteins inEscherischia coli,purified to apparent homogeneity, and assayed for their abilities to activate pea leaf NAD kinasein vitro.The CaM-2 isoform was a significantly more efficient activator of NAD kinase compared with the CaM-4 and -6 isoforms based on both the apparentVmaxit elicited and theK0.5activation. These results are consistent with the hypothesis that theArabidopsisCaM isoforms have evolved to optimize the proteinʹs interaction with different Ca2+/CaM-regulated target enzymes. The ability to activate NAD kinase was also investigated for a carboxy-terminal nonsense mutant of CaM-6 (CaM-6M), which substituted 14 hydrophilic amino acids for a region of seven amino acids that normally form an exposed hydrophobic surface whenwtCaM-6 binds Ca2+. CaM-6M-activated NAD kinase displayed an apparentVmaxthat was reduced 40% and aK0.5that was an order of magnitude greater than the CaM-6-activated enzyme. The Ca2+-dependence of CaM-6M to activate NAD kinase was identical to that of CaM-6, but CaM-6M bound synthetic peptide substrates with lower apparent affinity than did CaM-6. Thus, the carboxy-terminal hydrophobic domain of CaM appears to be critical for its interaction with NAD kinase. In contrast, amino-terminal fusions of a hydrophilic, α-helical 12-residuec-mycepitope tag to CaM-2 and -4 yielded proteins that activated NAD kinase to apparentVmaxvalues within 10% of those obtained with the wild-type CaM proteins.