A mathematical genomics perspective on the moonlighting role of glyceraldehyde-3-phosphate dehydrogenase (GAPDH)

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a highly conserved enzyme across Archaea, Bacteria, and Eukarya, known for its canonical role in glycolysis and diverse moonlighting functions in transcription regulation, host-pathogen interactions, and immune modulation. To investigate how sequence-level variation contributes to this functional versatility, we performed a comparative analysis of 165 GAPDH proteins from 158 species using a diverse suite of computational descriptors. By integrating amino acid composition, poly-string profiling, polarity transition patterns, intrinsic disorder propensity, and fractal spatial metrics, we present a comprehensive quantitative framework for examining GAPDH evolution. Our results show that while core catalytic residues are strongly preserved, selective enrichment of small nonpolar residues (valine, alanine) and a balanced distribution of order- and disorder-promoting residues support both structural stability and regulatory flexibility. These findings suggest that GAPDH multifunctionality arises from a composite of conserved and divergent sequence signatures. Unlike previous single-parameter studies, this work introduces a unified, multi-parametric strategy that uncovers hidden evolutionary patterns and offers a generalizable framework for decoding moonlighting behavior in proteins. Beyond reinforcing known aspects of GAPDH biology, this integrative analysis positions GAPDH as a model for exploring broader principles of protein multifunctionality and highlights the power of quantitative profiling in revealing hidden functional layers.