Comparative analysis of cGAS catalytic mechanisms across species via combined biochemical assays and computational...

Cyclic GMP-AMP synthase (cGAS) is a DNA-sensing enzyme that synthesizes cyclic GMP-AMP (cGAMP) to trigger innate immune responses. Although functionally conserved, cGAS exhibits marked sequence divergence across vertebrate species. To investigate how this diversity affects enzyme performance, we expressed and purified cGAS from six representative species and measured their specific activities. Biochemical assays revealed striking interspecies variation: mouse and bovine cGAS displayed high catalytic efficiency, whereas human and bombina cGAS were substantially less active. CAVER analysis indicated that the width of the substrate-access pathway within the active-site pocket correlates with catalytic activity. Notably, the peripheral β5-β6 loop exhibits strong interspecies variability, and loop-swap experiments demonstrated that this region critically influences catalytic activity, likely via electrostatic modulation. Together, these results enhance our understanding of the evolutionary tuning of cGAS activity across species.