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Phosphoglucomutase 1 (PGM1) plays a central role in glucose homeostasis in human cells. Missense variants of this enzyme cause an inborn error of metabolism, which is categorized as a congenital disorder of glycosylation. Here, two disease-related variants of PGM1, T337M and G391V, which are both located in domain 3 of the four-domain protein, were characterized via X-ray crystallography and biochemical assays. The studies show multiple impacts resulting from these dysfunctional variants, including both short- and long-range structural perturbations. In the T337M variant these are limited to a small shift in an active-site loop, consistent with reduced enzyme activity. In contrast, the G391V variant produces a cascade of structural perturbations, including displacement of both the catalytic phosphoserine and metal-binding loops. This work reinforces several themes that were found in prior studies of dysfunctional PGM1 variants, including increased structural flexibility and the outsized impacts of mutations affecting interdomain interfaces. The molecular mechanisms of PGM1 variants have implications for newly described inherited disorders of related enzymes.

Once experimentally prohibitive, structural studies of individual missense variants in proteins are increasingly feasible, and can provide a new level of insight into human genetic disease. One example of this is the recently identified inborn error of metabolism known as phosphoglucomutase-1 (PGM1) deficiency. Just as different variants of a protein can produce different patient phenotypes, they may also produce distinct biochemical phenotypes, affecting properties such as catalytic activity, protein stability, or 3D structure/dynamics. Experimental studies of missense variants, and particularly structural characterization, can reveal details of the underlying biochemical pathomechanisms of missense variants. Here, we review four examples of enzyme dysfunction observed in disease-related variants of PGM1. These studies are based on 11 crystal structures of wild-type (WT) and mutant enzymes, and multiple biochemical assays. Lessons learned include the value of comparing mutant and WT structures, synergy between structural and biochemical studies, and the rich understanding of molecular pathomechanism provided by experimental characterization relative to the use of predictive algorithms. We further note functional insights into the WT enzyme that can be gained from the study of pathogenic variants., Competing Interests: Declaration of competing interest The author declares no conflict of interest., (Copyright © 2020. Published by Elsevier B.V.)

Missense variants of human phosphoglucomutase 1 (PGM1) cause the inherited metabolic disease known as PGM1 deficiency. This condition is categorised as both a glycogen storage disease and a congenital disorder of glycosylation. Approximately 20 missense variants of PGM1 are linked to PGM1 deficiency, and biochemical studies have suggested that they fall into two general categories: those affecting the active site and catalytic efficiency, and those that appear to impair protein folding and/or stability. In this study, we characterise a novel variant of Arg422, a residue distal from the active site of PGM1 and the site of a previously identified disease-related variant (Arg422Trp). In prior studies, the R422W variant was found to produce insoluble protein in a recombinant expression system, precluding further in vitro characterisation. Here we investigate an alternative variant of this residue, Arg422Gln, which is amenable to experimental characterisation presumably due to its more conservative physicochemical substitution. Biochemical, crystallographic, and computational studies of R422Q establish that this variant causes only minor changes in catalytic efficiency and 3D structure, but is nonetheless dramatically reduced in stability. Unexpectedly, binding of a substrate analog is found to further destabilise the protein, in contrast to its stabilising effect on wild-type PGM1 and several other missense variants. This work establishes Arg422 as a lynchpin residue for the stability of PGM1 and supports the impairment of protein stability as a pathomechanism for variants that cause PGM1 deficiency. SYNOPSIS: Biochemical and structural studies of a missense variant far from the active site of human PGM1 identify a residue with a key role in enzyme stability., (© 2020 SSIEM.)

The enzyme phosphoglucomutase 1 (PGM1) plays a central role in glucose homeostasis. Clinical studies have identified mutations in human PGM1 as the cause of PGM1 deficiency, an inherited metabolic disease. One residue, Asp263, has two known variants associated with disease: D263G and D263Y. Biochemical studies have shown that these mutants are soluble and well folded, but have significant catalytic impairment. To better understand this catalytic defect, we determined crystal structures of these two missense variants, both of which reveal a similar and indirect structural change due to the loss of a conserved salt bridge between Asp263 and Arg293. The arginine reorients into the active site, making interactions with residues responsible for substrate binding. Biochemical studies also show that the catalytic phosphoserine of the missense variants is more stable to hydrolysis relative to wild-type enzyme. The structural perturbation resulting from mutation of this single amino acid reveals the molecular mechanism underlying PGM1 deficiency in these missense variants., Database: Structural data are available in the PDB under the accession numbers 5JN5 and 5TR2., (© 2017 Federation of European Biochemical Societies.)

Objective: To define phenotypic groups and identify predictors of disease severity in patients with phosphoglucomutase-1 deficiency (PGM1-CDG)., Study Design: We evaluated 27 patients with PGM1-CDG who were divided into 3 phenotypic groups, and group assignment was validated by a scoring system, the Tulane PGM1-CDG Rating Scale (TPCRS). This scale evaluates measurable clinical features of PGM1-CDG. We examined the relationship between genotype, enzyme activity, and TPCRS score by using regression analysis. Associations between the most common clinical features and disease severity were evaluated by principal component analysis., Results: We found a statistically significant stratification of the TPCRS scores among the phenotypic groups (P < .001). Regression analysis showed that there is no significant correlation between genotype, enzyme activity, and TPCRS score. Principal component analysis identified 5 variables that contributed to 54% variance in the cohort and are predictive of disease severity: congenital malformation, cardiac involvement, endocrine deficiency, myopathy, and growth., Conclusions: We established a scoring algorithm to reliably evaluate disease severity in patients with PGM1-CDG on the basis of their clinical history and presentation. We also identified 5 clinical features that are predictors of disease severity; 2 of these features can be evaluated by physical examination, without the need for specific diagnostic testing and thus allow for rapid assessment and initiation of therapy., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Human phosphoglucomutase 1 (PGM1) plays a central role in cellular glucose homeostasis, mediating the switch between glycolysis and gluconeogenesis through the conversion of glucose 1-phosphate and glucose 6-phosphate. Recent clinical studies have identified mutations in this enzyme as the cause of PGM1 deficiency, an inborn error of metabolism classified as both a glycogen storage disease and a congenital disorder of glycosylation. Reported here are the first crystal structures of two disease-related missense variants of PGM1, along with the structure of the wild-type enzyme. Two independent glycine-to-arginine substitutions (G121R and G291R), both affecting key active site loops of PGM1, are found to induce regions of structural disorder, as evidenced by a nearly complete loss of electron density for as many as 23 aa. The disordered regions are not contiguous in sequence to the site of mutation, and even cross domain boundaries. Other structural rearrangements include changes in the conformations of loops and side chains, some of which occur nearly 20 Å away from the site of mutation. The induced structural disorder is correlated with increased sensitivity to proteolysis and lower-resolution diffraction, particularly for the G291R variant. Examination of the multi-domain effects of these G➔R mutations establishes a correlation between interdomain interfaces of the enzyme and missense variants of PGM1 associated with disease. These crystal structures provide the first insights into the structural basis of enzyme dysfunction in PGM1 deficiency and highlight a growing role for biophysical characterization of proteins in the field of precision medicine., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Recent studies have identified phosphoglucomutase 1 (PGM1) deficiency as an inherited metabolic disorder in humans. PGM1 deficiency is classified as both a muscle glycogenosis (type XIV) and a congenital disorder of glycosylation of types I and II. Affected patients show multiple disease phenotypes, reflecting the central role of the enzyme in glucose homeostasis, where it catalyzes the interconversion of glucose 1-phosphate and glucose 6-phosphate. The influence of PGM1 deficiency on protein glycosylation patterns is also widespread, affecting both biosynthesis and processing of glycans and their precursors. To date, 21 different mutations involved in PGM1 deficiency have been identified, including 13 missense mutations resulting in single amino acid changes. Growing clinical interest in PGM1 deficiency prompts a review of the molecular context of these mutations in the three-dimensional structure of the protein. Here the known crystal structure of PGM from rabbit (97 % sequence identity to human) is used to analyze the mutations associated with disease and find that many map to regions with clear significance to enzyme function. In particular, amino acids in and around the active site cleft are frequently involved, including regions responsible for catalysis, binding of the metal ion required for activity, and interactions with the phosphosugar substrate. Several of the known mutations, however, are distant from the active site and appear to manifest their effects indirectly. An understanding of how the different mutations that cause PGM1 deficiency affect enzyme structure and function is foundational to providing clinical prognosis and the development of effective treatment strategies.

Recent studies have identified phosphoglucomutase 1 (PGM1) deficiency as an inherited metabolic disorder in humans. Affected patients show multiple disease phenotypes, including dilated cardiomyopathy, exercise intolerance, and hepatopathy, reflecting the central role of the enzyme in glucose metabolism. We present here the first in vitro biochemical characterization of 13 missense mutations involved in PGM1 deficiency. The biochemical phenotypes of the PGM1 mutants cluster into two groups: those with compromised catalysis and those with possible folding defects. Relative to the recombinant wild-type enzyme, certain missense mutants show greatly decreased expression of soluble protein and/or increased aggregation. In contrast, other missense variants are well behaved in solution, but show dramatic reductions in enzyme activity, with kcat/Km often <1.5% of wild-type. Modest changes in protein conformation and flexibility are also apparent in some of the catalytically impaired variants. In the case of the G291R mutant, severely compromised activity is linked to the inability of a key active site serine to be phosphorylated, a prerequisite for catalysis. Our results complement previous in vivo studies, which suggest that both protein misfolding and catalytic impairment may play a role in PGM1 deficiency., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)

Summary Human phosphoglucomutase 1 (PGM1) plays a central role in cellular glucose homeostasis, catalyzing the conversion of glucose 1-phosphate and glucose 6-phosphate. Recently, missense variants of this enzyme were identified as causing an inborn error of metabolism, PGM1 deficiency, with features of a glycogen storage disease and a congenital disorder of glycosylation. Previous studies of selected PGM1 variants have revealed various mechanisms for enzyme dysfunction, including regions of structural disorder and side-chain rearrangements within the active site. Here, we examine variants within a substrate-binding loop in domain 4 (D4) of PGM1 that cause extreme impairment of activity. Biochemical, structural, and computational studies demonstrate multiple detrimental impacts resulting from these variants, including loss of conserved ligand-binding interactions and reduced mobility of the D4 loop, due to perturbation of its conformational ensemble. These potentially synergistic effects make this conserved ligand-binding loop a hotspot for disease-related variants in PGM1 and related enzymes. Graphical Abstract Highlights • Crystal structures of PGM1 missense variants in an active site loop are presented • Mutations cause direct effects on ligand binding and indirect effects on loop mobility • The conformational ensemble of the wild-type enzyme is altered in the variants • The loop is a hotspot for disease-related mutations in PGM1 Disease-related variants phosphoglucomutase 1 (PGM1) cause an inherited metabolic deficiency in humans. Stiers et al. show that mutations in a key active site region of the enzyme produce multiple detrimental impacts, including reduced flexibility of a loop required for substrate binding. [ABSTRACT FROM AUTHOR]