A point mutation in a plant calmodulin is responsible for its inhibition of nitric-oxide synthase.
The calcium/calmodulin-dependent activation of nitric-oxide synthase (NOS) and its production of nitric oxide (NO) play a key regulatory role in plant and animal cell function. SCaM-1 is a plant calmodulin (CaM) isoform that is 91% identical to mammalian CaM (wild type CaM (wtCaM)) and a selective competitive antagonist of NOS (Cho, M. J., Vaghy, P. L., Kondo, R., Lee, S. H., Davis, J. P., Rehl, R., Heo, W. D., and Johnson, J. D. (1998) Biochemistry 37, 15593-15597). We have used site-directed mutagenesis to show that a point mutation, involving the substitution of valine for methionine at position 144, is responsible for SCaM-1's inhibition of mammalian NOS. An M144V mutation in wild type CaM produced a mutant (M144V) which exhibited nearly identical inhibition of NOS's NO production and NADPH oxidation, with a similar K(i) (approximately 15 nM) as SCaM-1. A V144M back mutation in SCaM-1 significantly restored its ability to activate NOS's catalytic functions. The length of the hydrophobic amino acid side chain at position 144 appears to be critical for NOS activation, since M144L and M144F activated NOS while M144V and M144C did not. Despite their competitive antagonism of NOS, M144V, like SCaM-1, exhibited a similar dose-dependent activation of phosphodiesterase and calcineurin as wtCaM. SCaM-1 and M144V produced greater inhibition of NOS's oxygenase domain function (NO production) than its reductase domain functions (NADPH oxidation and cytochrome c reduction). Thus, CaM's methionine 144 plays a critical role the activation of NOS, presumably by influencing the function of NOS's oxygenase domain.
Kondo
R
R
Department of Molecular and Cellular Biochemistry, The Ohio State University Medical Center, Columbus, Ohio 43210, USA.
Tikunova
S B
SB
Cho
M J
MJ
Johnson
J D
JD
D002147:Calmodulin / Q000235:genetics* / Q000378:metabolism; D004789:Enzyme Activation / Q000235:genetics; D015971:Gene Expression Regulation, Enzymologic; D018506:Gene Expression Regulation, Plant; D017343:Genes, Plant; D019001:Nitric Oxide Synthase / Q000235:genetics* / Q000378:metabolism; D010944:Plants; D017354:Point Mutation
D002147:Calmodulin; D019001:Nitric Oxide Synthase