Using rationally designed search motifs, we have revealed several classes of proteins with additional molecular signatures that we have shown to be functional. A subset of these moonlighting proteins contains catalytic switch centres that acts to alter the microenvironment surrounding the protein. Using examples from plant and mammalian systems we show with mutational studies that the moonlighting catalytic centres are critical for biological function. Our archetypical example of a switch is a guanylate cyclase (GC) catalytic centre embedded within the kinase domain. For instance, kinase and GC activity are reciprocally regulated by changes in calcium in the phytosulfokine receptor (PSKR1). Importantly, in both the PSKR1 and brassinosteroid receptor (BRI1), GC and kinase are regulated by phosphorylation and cGMP inhibits kinase activity. These observations led us to consider that enrichment of cGMP within the protein microenvironment may have an auto-regulatory function necessary to enable biological function. This concept was enhanced by our identification of another moonlighting guanylate cyclase that is modulated by a moonlighting H-NOX centre. Importantly, these effects can be contained within the microenvironment of a protein complex thereby keeping a response to micro-cues separated from major cellular responses. Our findings will add to understanding of micro-regulation of protein function and also how spatial and temporal positioning influences cellular (and potentially bodily) function.