Coenzyme A has most recently been identified with a function in carrying endogenous nitric oxide (NO) in the form of an S-nitrosothiol (SNO; SNO-CoA)⁵. Here the thiol of CoA is uniquely activated via a thionitrite linkage (as opposed to a thioester), providing a basis for NO group transfer to target proteins in cellular signaling (aka S-nitrosylation). Thus conceived, SNO-CoA participates in a reversible chemical equilibrium with SNO-protein substrates to determine steady-state levels of cellular SNOs ^{5– 8}. This is well-exemplified in the functions of SNO-CoA reductases (SCoRs), enzymes that degrade SNO-CoA ^{5, 6}. Deletion of SCoRs leads to elevated SNO-CoA, thereby increasing SNO-protein levels ^{5, 6, 9}. Yet protein S-nitrosylation in this model remains exclusively non-enzymatic (i.e., SNO-CoA acts as a non-specific NO donor), and no enzymes are known to utilize SNO-CoA as a cofactor for any purpose.

As principal mediators of signal transduction, protein S-nitrosylation and phosphorylation have been suggested to share features ¹⁰, including conservation across phylogeny, global control over cellular function and site-directed modification through sequence motif recognition ^{10 – 12}. In addition, excessive or dysregulated S-nitrosylation plays causal roles in many of the same diseases as dysregulated phosphorylation, including Alzheimer’s, cancers, muscular dystrophy, heart failure and diabetes ^{13 – 17}. But whereas phosphorylation of proteins is enzymatically driven, convention holds that dysregulated S-nitrosylation is mediated chemically by high amounts of NO and of LMW SNOs (like SNO-CoA) that modify proteins indiscriminately ^18,19. More recently, the idea has been raised that NO synthase-derived NO may be transferred between proteins, constituting ‘nitrosylase activity’, and heterotrimeric machinery for S-nitrosylation analogous to that used for ubiquitinylation has been identified in bacteria ^{10 – 12,20 – 25}. However, a basic catalytic mechanism that would solidify an enzymatic basis for S-nitrosylation has been elusive, and no S-nitrosylases are known to mediate physiology or disease.

Here we describe the prototype for a class of enzyme, SCAN (for SNO-CoA-Assisted Nitrosylase) that uses SNO-CoA as its cofactor to S-nitrosylate specific target proteins, and reveal core mechanistic parallels with acyltransferases, firmly establishing enzymatic function. Separate domains in SCAN are involved in SNO-CoA and target protein binding, allowing SCAN to selectively catalyze NO group transfer from SNO-CoA to SCAN to multiple target proteins, including the insulin receptor β-subunit (INSRβ) and insulin receptor substrate 1 (IRS1). SCAN activity acts physiologically to regulate insulin signaling on the one hand, while aberrant activity contributes to diabetes on the other hand. While SCAN activity is dependent on NO derived from NOS, the major role of NOS is to generate the LMW SNO cofactor. Thus, our characterization of SCAN unveils enzymatic principles shared with other enzymatic mediators of signal transduction and supports a new paradigm for NO biology.