NADPH phosphatase activity of Mesh1 controls sleep in Drosophila

Recent findings have shown that metazoans accumulate the bacterial second messenger guanosine tetraphosphate (ppGpp) and possess Mesh1, a gene encoding a ppGpp hydrolase domain. Mesh1 deficiency affects sleep behavior and eclosion under starvation in Drosopila, and ferroptosis in human cells. However, human Mesh1 also exhibits NADPH/NADP+ phosphatase activity in vitro, making it unclear whether these phenotypes result from loss of ppGpp hydrolysis, NADPH/NADP+ dephosphorylation, or both. To address this, we performed biochemical and genetic analysis of Drosophila Mesh1. We first found that Drosophila Mesh1 dephosphorylates NADPH, but not NADP+, in vitro. We subsequently sought to generate Drosophila Mesh1 point mutants specifically impaired in NADPH phosphatase activity. Based on structural data, we mutated W138 and R142, residues that are predicted to interact with NADPH but not ppGpp. W138 was replaced with phenylalanine results in complete loss of NADPH phosphatase activity with retained ppGpp hydrolase activity. Flies carrying the W138F mutation, introduced via genome editing, exhibited shortened total sleep and increased sleep fragmentation in behavioral assays, without changes in intracellular ppGpp levels. These results indicate that the NADPH phosphatase activity of Mesh1, and specifically the W138 residue, is essential for normal sleep regulation in Drosophila.