Summary: The gene context in microorganism genomes is of considerable help for identifying potential substrates. The C785_RS13685 gene in Herbaspirillum huttiense IAM 15032 is a member of the d‐altronate dehydratase protein family, and which functions as a d‐arabinonate dehydratase in vitro, is clustered with genes related to putative pentose metabolism. In the present study, further biochemical characterization and gene expression analyses revealed that l‐xylonate is a physiological substrate that is ultimately converted to α‐ketoglutarate via so‐called Route II of a non‐phosphorylative pathway. Several hexonates, including d‐altronate, d‐idonate and l‐gluconate, which are also substrates of C785_RS13685, also significantly up‐regulated the gene cluster containing C785_RS13685, suggesting a possibility that pyruvate and d‐ or l‐glycerate were ultimately produced (novel Route III). On the contrary, ACAV_RS08155 of Acidovorax avenae ATCC 19860, a homologous gene to C785_RS13685, functioned as a d‐altronate dehydratase in a novel l‐galactose pathway, through which l‐galactonate was epimerized at the C5 position by the sequential activity of two dehydrogenases, resulting in d‐altronate. Furthermore, this pathway completely overlapped with Route III of the non‐phosphorylative l‐fucose pathway. The 'substrate promiscuity' of d‐altronate dehydratase protein(s) is significantly expanded to 'metabolic promiscuity' in the d‐arabinose, sugar acid, l‐fucose and l‐galactose pathways. [ABSTRACT FROM AUTHOR]