Key result
Murine inducible nitric-oxide synthase expressed without calmodulin exhibited significantly reduced NO synthesis activity (10.8 vs 94 min-1 per μg of protein) and compromised heme incorporation.
CaM-free murine iNOS retains partial heme incorporation, dimerization, and catalytic activity, challenging previous reports that it is completely inactive and monomeric.
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Questions absolute CaM dependence for murine iNOS activity; challenges prior inactivity assumptions but remains hypothesis-generating without human validation.
Nitric-Oxide Synthase (NOS), that produces the biological signal molecule Nitric-Oxide (NO), exists in three different isoforms called, neuronal (nNOS), endothelial (eNOS) and inducible (iNOS). All NOS isoforms require post-translational interaction with the calcium-binding protein, calmodulin (CaM) for manifesting their catalytic activity. However, CaM has been suggested to control the translational assembly of the enzyme as well, particularly in helping its inducible isoform, iNOS assume a stable, heme-replete, dimeric and active form. Expression of recombinant murine iNOS in E.coli in the absence of CaM has been previously shown to give extremely poor yield of the enzyme which was claimed to be absolutely heme-free, devoid of flavins, completely monomeric and catalytically inactive when compared to the heme-replete, active, dimeric iNOS, generated through co-expression with CaM. In contrast, we found that although iNOS expressed without CaM does produce significantly low amounts of the CaM-free enzyme, the iNOS thus produced, is not completely devoid of heme and is neither entirely monomeric nor absolutely bereft of catalytic activity as reported before. In fact, iNOS synthesized in the absence of CaM undergoes compromised heme incorporation resulting in extremely poor dimerization and activity compared to its counterpart co-expressed with CaM. Moreover, such CaM-free iNOS has similar flavin content and reductase activity as iNOS co-expressed with CaM, suggesting that CaM may not be as much required for the functional assembly of the iNOS reductase domain as its oxygenase domain. LC-MS/MS-based peptide mapping of the CaM-free iNOS confirmed that it had the same full-length sequence as the CaM-replete iNOS. Isothermal calorimetric measurements also revealed high affinity for CaM binding in the CaM-free iNOS and thus the possible presence of a CaM-binding domain. Thus CaM is essential but not indispensible for the assembly of iNOS and such CaM-free iNOS may help in elucidating the role of CaM on iNOS catalysis.
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Nagpal et al. (2015) studied this question. Calmodulin-free expression of iNOS vs. Calmodulin co-expression with iNOS was evaluated on NO synthesis activity (min-1 per μg of protein). Murine inducible nitric-oxide synthase expressed without calmodulin exhibited significantly reduced NO synthesis activity (10.8 vs 94 min-1 per μg of protein) and compromised heme incorporation.
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