Enzyme regulation is vital for metabolic adaptability in living systems. Fine control of enzyme activity is often delivered through post-translational mechanisms, such as allostery or allokairy. β-phosphoglucomutase (βPGM) from Lactococcus lactis is a phosphoryl transfer enzyme required for complete catabolism of trehalose and maltose, through the isomerisation of β-glucose 1-phosphate to glucose 6-phosphate via β-glucose 1,6-bisphosphate. Surprisingly for a gatekeeper of glycolysis, no fine control mechanism of βPGM has yet been reported. Herein, we describe allomorphy, a post-translational control mechanism of enzyme activity. In βPGM, isomerisation of the K145-P146 peptide bond results in the population of two conformers that have different activities owing to repositioning of the K145 sidechain. In vivo phosphorylating agents, such as fructose 1,6-bisphosphate, generate phosphorylated forms of both conformers, leading to a lag phase in activity until the more active phosphorylated conformer dominates. In contrast, the reaction intermediate β-glucose 1,6-bisphosphate, whose concentration depends on the β-glucose 1-phosphate concentration, couples the conformational switch and the phosphorylation step, resulting in the rapid generation of the more active phosphorylated conformer. In enabling different behaviours for different allomorphic activators, allomorphy allows an organism to maximise its responsiveness to environmental changes while minimising the diversion of valuable metabolites.

Green synthesis strategy generating a valuable metabolite through manipulation of the catalytic magnesium coordination of an enzyme.

β-Phosphoglucomutase (βPGM) is a magnesium-dependent phosphoryl transfer enzyme that catalyses the reversible isomerisation of β-glucose 1-phosphate and glucose 6-phosphate, via two phosphoryl transfer steps and a β-glucose 1,6-bisphosphate intermediate. Substrate-free βPGM is an essential component of the catalytic cycle and an understanding of its dynamics would present significant insights into βPGM functionality, and enzyme catalysed phosphoryl transfer in general. Previously, 30 residues around the active site of substrate-free βPGM WT were identified as undergoing extensive millisecond dynamics and were unassignable. Here we report 1 H, 15 N and 13 C backbone resonance assignments of the P146A variant (βPGM P146A ) in its substrate-free form, where the K145–A146 peptide bond adopts a trans conformation in contrast to all crystal structures of βPGM WT , where the K145–P146 peptide bond is cis. In βPGM P146A millisecond dynamics are suppressed for all but 17 residues, allowing 92% of backbone resonances to be assigned. Secondary structure predictions using TALOS-N reflect βPGM crystal structures, and a chemical shift comparison between substrate-free βPGM P146A and βPGM WT confirms that the solution conformations are very similar, except for the D137–A147 loop. Hence, the isomerisation state of the 145–146 peptide bond has little effect on structure but the cis conformation triggers millisecond dynamics in the hinge (V12–T16), the nucleophile (D8) and residues that coordinate the transferring phosphate group (D8 and S114–S116), and the D137–A147 loop (V141–A142 and K145). These millisecond dynamics occur in addition to those for residues involved in coordinating the catalytic Mg II ion and the L44–L53 loop responsible for substrate discrimination.

Understanding the factors that underpin the enormous catalytic proficiencies of enzymes is fundamental to catalysis and enzyme design. Enzymes are, in part, able to achieve high catalytic proficiencies by utilizing the binding energy derived from nonreacting portions of the substrate. In particular, enzymes with substrates containing a nonreacting phosphodianion group coordinated in a distal site have been suggested to exploit this binding energy primarily to facilitate a conformational change from an open inactive form to a closed active form, rather than to either induce ground state destabilization or stabilize the transition state. However, detailed structural evidence for the model is limited. Here, we use β-phosphoglucomutase (βPGM) to investigate the relationship between binding a phosphodianion group in a distal site, the adoption of a closed enzyme form, and catalytic proficiency. βPGM catalyzes the isomerization of β-glucose 1-phosphate to glucose 6-phosphate via phosphoryl transfer reactions in the proximal site, while coordinating a phosphodianion group of the substrate(s) in a distal site. βPGM has one of the largest catalytic proficiencies measured and undergoes significant domain closure during its catalytic cycle. We find that side chain substitution at the distal site results in decreased substrate binding that destabilizes the closed active form but is not sufficient to preclude the adoption of a fully closed, near-transition state conformation. Furthermore, we reveal that binding of a phosphodianion group in the distal site stimulates domain closure even in the absence of a transferring phosphoryl group in the proximal site, explaining the previously reported β-glucose 1-phosphate inhibition. Finally, our results support a trend whereby enzymes with high catalytic proficiencies involving phosphorylated substrates exhibit a greater requirement to stabilize the closed active form.

<p>Manipulation of enzyme behaviour represents a sustainable technology that can be harnessed to enhance the production of valuable metabolites and chemical precursors. b-glucose 1,6-bisphosphate (bG16BP) is a native reaction intermediate in the catalytic cycle of b-phosphoglucomutase (bPGM) that has been proposed as a treatment for human congenital disorder of glycosylation involving phosphomannomutase 2 (PMM2). Studies of both bPGM and PMM2 could benefit from a green and high-yielding method for bG16BP production. Three strategies have been reported previously for the synthesis of bG16BP; however, each of these methods either delivers low yields or uses chemicals and procedures with significant environmental impacts. Herein, through combined use of NMR spectroscopy, kinetic assays and site-directed mutagenesis, we report the efficient enzymatic synthesis of anomer-specific bG16BP using a variant of bPGM. Further purification, employing a simple environmentally considerate precipitation procedure requiring only a standard biochemical toolset, results in a product with high purity and yield. Moreover, this synthesis strategy illustrates how manipulation of the catalytic magnesium coordination of an enzyme can be utilised to generate large quantities of a valuable metabolite.</p>