Abstract
Despite the exponential growth in genome sequencing, the functional annotation of genes, particularly the discovery of novel enzymatic activities, remains a formidable challenge. This gap is exacerbated by annotation biases that propagate assumptions about enzyme function across homologous sequences. Here, we report the identification of an unannotated enzymatic activity within a presumed well-characterized enzyme family. The gene rv2531c, under strong purifying selection across Mycobacterium tuberculosis strains, has been annotated as a member of the lysine-ornithine-arginine (KOR) decarboxylase superfamily, which includes over 26,000 sequences. Contrary to this annotation, we show that Rv2531c does not decarboxylate KOR substrates. Instead, using an integrative approach combining bioinformatics, microbiology, metabolomics, and enzymology, we demonstrate that Rv2531c is a L-glutamate decarboxylase that sustains carbon flux through the γ-aminobutyric acid shunt in M. tuberculosis. This newly identified subfamily of enzymes is conserved across Bacteria, Archaea, and Eukarya and exhibits distinct structural and kinetic features, including an additional domain, hysteresis, and strong positive cooperativity. These characteristics differentiate it from canonical, enterobacterial KOR decarboxylases. More broadly, our findings challenge the narrow substrate and functional scope traditionally assigned to the KOR-DC superfamily. We propose that many members of this large and diverse enzyme family catalyze distinct reactions and participate in previously unrecognized metabolic pathways, revealing a broader and more nuanced role for pyridoxal 5'-phosphate-dependent enzymes in microbial physiology and evolution.