Biology subjects

Miller, B. G.

Publications and source records attributed to Miller, B. G..

6 recordsLinked to original sources

bioRxiv2026

Conformational Expansion Underlies the Evolutionary Emergence of Redox Sensitivity in Vertebrate Glucokinases

Glucokinase (GCK) catalyzes the first step of glycolysis in pancreatic {beta}-cells, where it functions as the bodys primary glucose sensor. GCK is extremely sensitive to oxidative inactivation, both in vivo and in vitro. This characteristic provides a mechanism to regulate GCK activity via alterations in the cellular redox environment. To understand the molecular and evolutionary origins of redox regulation, we characterized the sensitivity to oxidative inactivation of four extant GCKs and five ancestral GCKs produced from a recent phylogenetic analysis of the vertebrate family. We find that two invertebrate GCKs are significantly less sensitive to oxidative inactivation compared to their vertebrate counterparts. We also demonstrate that an ancestral GCK from chordates (cGCK) is insensitive to oxidative inactivation, whereas an ancestral GCK from early vertebrates (vGCK) displays a degree of redox responsiveness comparable to the extant human enzyme. The redox insensitive cGCK ancestor lacks cysteine residues at two positions, Cys230 and Cys461, that are conserved in all redox sensitive ancestral and extant enzymes. We find that installation of cysteines at these positions is insufficient to install redox sensitivity into cGCK. Our data demonstrate that the appearance of redox responsiveness in GCKs coincides with an expansion in the conformational landscape of the protein that occurred during early vertebrate evolution. These observations support a model in which the emergence of redox sensitivity required the ability to sample a unique super-open conformation, an event that also facilitated the emergence of two orthogonal GCK regulatory strategies, allosteric regulation by substrate glucose and an inhibitory interaction with the glucokinase regulatory protein.

biochemistry↗

bioRxiv2026

Conformational Diversity and Substrate Specificity are Decoupled in Ancestral and Extant Glucokinases

Multi-functionality in extant enzymes, including the ability to transform multiple substrates, is thought to arise, in part, from conformational flexibility. The hexokinase protein family represents a classic model system for investigating the relationship between substrate specificity and conformational change. Within this family, human glucokinase (hGCK) displays notable degrees of conformational heterogeneity, including an intrinsically disordered loop. The extent to which these structural features contribute to the breadth of hGCKs substrate scope is unknown. Here, we investigate the substrate specificities of extant and ancestral glucokinases that span the evolutionary emergence of conformational heterogeneity in this family. We show that extant hGCK catalyzes the ATP-dependent phosphorylation of glucose, 2-deoxyglucose, mannose, glucosamine, fructose, allose and galactose with catalytic efficiencies ranging from 6.3 x 103 M-1 sec-1 to 0.33 M-1sec-1. A glucokinase ancestor from early vertebrate evolution (vGCK), which also displays conformational heterogeneity and disorder, phosphorylates these same seven substrates with similar kcat/Km values. An antecedent, chordate glucokinase (cGCK), which displays reduced conformational heterogeneity and lacks intrinsic disorder, also transforms these same substrates, but with higher overall catalytic efficiencies and markedly lower Km values. Notably, however, the ratios of kcat/Km values for individual substrate pairs, which define specificity, are unchanged for all three enzymes. Our results demonstrate that substrate specificity is not correlated with conformational diversity in GCKs and support a model in which the differences in catalytic efficiencies of various substrates arise from differences in the ability to form the ground state enzyme-carbohydrate binary complex.

biochemistry↗

bioRxiv2026

Structural Co-optation and Loss-of-function Underlie the Evolution of Regulatory Novelty in the Glucokinase Regulatory Protein

The glucokinase regulatory protein (GKRP) derives from an ancestral etherase. Despite existing as a single locus in the metazoans, GKRP evolved multiple novel functions unrelated to etherase activity. In jawed vertebrates, a protein-protein interaction (PPI) emerged that inhibits glucokinase (GCK) activity in the liver. This PPI is critical to maintaining glucose homeostasis. In mammals, GKRP is allosterically regulated by carbohydrates, with 6-phospharylated sugars promoting inhibition of GCK by GKRP, while 1-phosphorylated sugars relieve inhibition. Here, we use a vertical evolutionary approach to identify the genetic, biochemical, and biophysical mechanisms underlying the emergence of small-molecule allostery in GKRP. We pinpointed a single leucine to valine substitution in the N-terminus of GKRP from the ancestor of the euarchontoglires that, when introduced into the non-regulated placental mammal GKRP ancestor, installed sensitivity to sorbitol-6-phosphate (S6P). Interestingly, GKRPs inhibitory activity in the absence of S6P was reduced but unchanged in its presence. The mutation enabled co-optation of the ancestral etherase active site, which also existed as an ambiguous phosphorylated carbohydrate binding site in unregulated GKRPs. This substitution likely introduced an alternative conformation of the N-terminus causing apo-GKRP to sample a binding incompetent state prior to GCK binding. Our results suggest a simple model of the evolution of protein functional novelty where a single mutation can cause a large functional shift via co-optation of pre-existing structural features. Importantly, in contrast to many models of protein evolution, ours does not require the addition of new genetic material to realize a novel function such as small-molecule allosteric regulation.

biochemistry↗

bioRxiv2026

Evolution of Protein Regulation in the Vertebrate Glucose Sensor

Protein regulation is essential for cellular function and mis-regulation commonly causes disease. Despite this fact, we know little about how new regulatory strategies first emerge and how they evolve to act in concert to control complex physiological processes. Glucokinase (GCK), the bodys glucose sensor, lies at the heart of vertebrate glucose homeostasis and its activity is tightly controlled by multiple regulatory mechanisms. In the pancreas and liver, GCK is regulated by a unique form of monomeric allostery originating from the unliganded enzymes conformational dynamics. In the liver, GCK and GKRP form an inhibitory protein-protein interaction that sequesters GCK within the hepatocyte nucleus. Using a vertical, evolutionary approach, we resurrected extinct GCKs and GKRPs along correlated evolutionary trajectories. Using enzyme kinetics, limited proteolysis, hydrogen-deuterium exchange, high resolution NMR, and X-ray crystallography we determined the historical and molecular origins of protein regulation. Prior to the emergence of jawed vertebrates, a non-regulated GCK ancestor underwent a conformational expansion leading to monomeric allostery. This novel conformation includes an intrinsically disordered substrate binding loop. Paradoxically, the emergence of disorder did not require sequence change in the loop. The new GCK conformation also exposed a hydrophobic cleft. In the jawed vertebrate GKRP ancestor, a de novo loop insertion enabled exaptation of the pre-existing hydrophobic patch in GCK. Our results demonstrate how multiple, distinct regulatory strategies can arise at a central homeostatic control point through evolutionary addition of novel conformations. Additionally, our results provide a general mechanism for the emergence of heteromeric protein-protein interactions. Significance StatementGlucose homeostasis was a key innovation in vertebrate evolution. Here, we uncover the evolutionary basis of regulation in two key homeostatic proteins, glucokinase (GCK) and glucokinase regulatory protein (GKRP). We find that the unique cooperativity of vertebrate GCK resulted from an expansion of this enzymes conformational landscape. This expansion included sampling a new state and the emergence of intrinsic disorder, which did not require substitutions in the disordered region itself. We also discover that the GCK-GKRP interaction emerged when a pre-existing hydrophobic surface -- a structural spandrel resulting from prior conformational expansion -- was co-opted by loop insertion in GKRP, facilitating a new, inhibitory heteromeric interaction. Our results demonstrate how multiple, mechanistically distinct regulatory strategies arise from an ability to sample new protein conformations.

biochemistry↗

bioRxiv2026

Gene sharing during enzyme recruitment reveals distinct adaptive strategies including massive, sustained duplication without divergence

Organisms expand their metabolism by repurposing enzymes to perform new reactions. To be repurposed, an enzyme must balance its original and new functions if both contribute to fitness. If an enzyme cannot balance its functions, another candidate may take its place. Here, we used adaptive evolution on a glucokinase-deficient Escherichia coli containing four promiscuous surrogates to investigate enzyme recruitment when the preferred candidate, N-acetyl-D-mannosamine kinase (NanK), is under selective pressure to maintain its original function. We find that NanK is still recruited to restore glycolysis under conditions requiring both functions via two distinct mechanisms that leave native activity largely unaltered. In one mechanism, small-scale gene amplification precedes the appearance of two non-synonymous mutations in nanK that increase glucokinase activity but have little or no effect on N-acetyl-D-mannosamine kinase activity. In another mechanism, recruitment occurs via amplification of a [~]1000 base pair fragment that narrowly encompasses nanK and reaches copy numbers as high as 127. Despite maintenance of amplification for hundreds of generations, we observe no persistent mutations in any nanK duplicate at the level of resolution provided by 75X whole genome sequencing coverage. Our results demonstrate that gene sharing can alter the trajectory but not necessarily prevent the recruitment of a preferred promiscuous candidate during adaptive evolution when other, seemingly equal candidates are available. Our findings also reveal that evolution by Innovation-Amplification-Divergence may only be facilitated at moderate levels of gene amplification, and hindered by massive amplification, as increased gene copy number diminishes returns of individual adaptive point mutations. Classification: Evolution, Biochemistry SignificanceThe Innovation-Amplification-Divergence model posits that single multifunctional enzymes evolve into multiple monofunctional enzymes through two events: First, selective pressure on a multifunctional enzyme leads to a duplication of the gene encoding that enzyme in an organisms genome. Second, the duplicate gene copy can freely accumulate mutations that enhance one of the encoded enzymes functions while the original copy can accumulate mutations that enhance the enzymes other encoded function. Intriguingly, our results reveal sequence divergence only in cells that experience mild amplification, and no divergence in cells that experience massive amplification. This suggests that sequence divergence may be suppressed above a certain number of gene copies, providing a new perspective on a widely accepted theory of evolution.

biochemistry↗

bioRxiv2026

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.

biochemistry↗