Phylogenetic Investigation of the 100 kDa Hexokinase Enzyme Family with the Topiary Ancestral Sequence Reconstruction Pipeline

The 100 kDa hexokinase (HK) enzyme family represents an attractive model to investigate the molecular origins of allosteric regulation in multidomain enzymes. Extant HK homologs are subject to various allosteric phenomena, including activation and inhibition by both homotropic and heterotropic ligands. Here, we report the results of a phylogenetic investigation of this enzyme family using the recently developed Topiary ancestral sequence reconstruction pipeline. The results agree with prior studies that used a smaller number of sequences from individual HK domains and suggest that modern HK3 isozymes diverged first from a 100 kDa ancestor, followed by gene duplication and divergence of the HK2 isozymes. A subsequent gene duplication event led to divergence of HK1 and the hexokinase domain containing protein 1 (HKDC1). To probe the ability of Topiary to yield functional, allosterically regulated ancestral enzymes, we resurrected and biochemically characterized two HKs from early vertebrate evolution, Anc1 and Anc2. Both enzymes were functionally similar to extant HK1, and possessed a low activity, regulatory N-terminal domain that governs allosteric regulation of the C-terminal active site by two heterotropic effectors, glucose 6-phosphate and inorganic phosphate. Neither ancestor was subject to homotropic regulation by substrate glucose, a characteristic observed in several extant HK3 family members. Our phylogenetic analysis provides a foundation for investigating the evolution of allostery in this enzyme family. It also demonstrates the need to sequence and biochemically characterize additional full-length HKs, especially those from jawless vertebrates, to enable more robust inferences of ancestral regulatory traits.