Key structural role of a conserved cis-proline revealed by the P285S variant of soybean serine hydroxymethyltransferase 8.

The enzyme serine hydroxymethyltransferase (SHMT) plays a key role in folate metabolism and is conserved in all kingdoms of life. SHMT is a pyridoxal 5'-phosphate (PLP) - dependent enzyme that catalyzes the conversion of L-serine and (6S)-tetrahydrofolate to glycine and 5,10-methylene tetrahydrofolate. Crystal structures of multiple members of the SHMT family have shown that the enzyme has a single conserved cis proline, which is located near the active site.

Structural insights into binding of polyglutamylated tetrahydrofolate by serine hydroxymethyltransferase 8 from soybean.

Tetrahydrofolate and its derivatives participate in one-carbon transfer reactions in all organisms. The cellular form of tetrahydrofolate (THF) is modified by multiple glutamate residues and polyglutamylation plays a key role in organellar and cellular folate homeostasis. In addition, polyglutamylation of THF is known to increase the binding affinity to enzymes in the folate cycle, many of which can utilize polyglutamylated THF as a substrate.

Structural and functional analysis of two SHMT8 variants associated with soybean cyst nematode resistance.

Two amino acid variants in soybean serine hydroxymethyltransferase 8 (SHMT8) are associated with resistance to the soybean cyst nematode (SCN), a devastating agricultural pathogen with worldwide economic impacts on soybean production. SHMT8 is a cytoplasmic enzyme that catalyzes the pyridoxal 5-phosphate-dependent conversion of serine and tetrahydrofolate (THF) to glycine and 5,10-methylenetetrahydrofolate. A previous study of the P130R/N358Y double variant of SHMT8, identified in the SCN-resistant soybean cultivar (cv.

Tracer metabolomics reveals the role of aldose reductase in glycosylation.

Abnormal polyol metabolism is predominantly associated with diabetes, where excess glucose is converted to sorbitol by aldose reductase (AR). Recently, abnormal polyol metabolism has been implicated in phosphomannomutase 2 congenital disorder of glycosylation (PMM2-CDG) and an AR inhibitor, epalrestat, proposed as a potential therapy. Considering that the PMM2 enzyme is not directly involved in polyol metabolism, the increased polyol production and epalrestat's therapeutic mechanism in PMM2-CDG remained elusive.

Effects of the T337M and G391V disease-related variants on human phosphoglucomutase 1: structural disruptions large and small.

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.

Enzyme dysfunction at atomic resolution: Disease-associated variants of human phosphoglucomutase-1.

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.

Development of a Homogeneous Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET) Assay for the Inhibition of Keap1-Nrf2 Protein-Protein Interaction.

The transcription factor, nuclear factor erythroid 2-related factor 2 (Nrf2), plays a major role in regulating the antioxidant defense system through the Kelch-like ECH-associated protein 1-Nrf2-antioxidant response element (Keap1-Nrf2-ARE) pathway. Small-molecule inhibitors targeting Keap1-Nrf2 protein-protein interaction (PPI) decrease the rate of Nrf2 degradation by the 26S proteasome and thus increase the intracellular level of Nrf2, which translocates into the nucleus, leading to upregulated expression of cytoprotective and antioxidant enzymes. Such inhibitors can be developed into potential preventive and therapeutic agents of diseases caused by oxidative damage.

A missense variant remote from the active site impairs stability of human phosphoglucomutase 1.

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.

Impaired folate binding of serine hydroxymethyltransferase 8 from soybean underlies resistance to the soybean cyst nematode.

Management of the agricultural pathogen soybean cyst nematode (SCN) relies on the use of SCN-resistant soybean cultivars, a strategy that has been failing in recent years. An underutilized source of resistance in the soybean genotype Peking is linked to two polymorphisms in serine hydroxy-methyltransferase 8 (SHMT8). SHMT is a pyridoxal 5'-phosphate-dependent enzyme that converts l-serine and (6)-tetrahydrofolate to glycine and 5,10-methylenetetrahydrofolate.

Inhibitory Evaluation of αPMM/PGM from : Chemical Synthesis, Enzyme Kinetics, and Protein Crystallographic Study.

α-Phosphomannomutase/phosphoglucomutase (αPMM/PGM) from is involved in bacterial cell wall assembly and is implicated in virulence, yet few studies have addressed αPMM/PGM inhibition from this important Gram-negative bacterial human pathogen. Four structurally different α-d-glucopyranose 1-phosphate (αG1P) derivatives including 1--fluoromethylated analogues (-), 1,2-cyclic phosph(on)ate analogues (-), isosteric methylene phosphono analogues ( and ), and 6-fluoro-αG1P (), were synthesized and assessed as potential time-dependent or reversible αPMM/PGM inhibitors. The resulting kinetic data were consistent with the crystallographic structures of the highly homologous αPGM with inhibitors and - binding to the enzyme active site (1.

Synthesis, Derivatization, and Structural Analysis of Phosphorylated Mono-, Di-, and Trifluorinated d-Gluco-heptuloses by Glucokinase: Tunable Phosphoglucomutase Inhibition.

Glucokinase phosphorylated a series of C-1 fluorinated α-d-gluco-heptuloses. These phosphorylated products were discovered to be inhibitors of α-phosphomannomutase/phosphoglucomutase (αPMM/PGM) and β-phosphoglucomutase (βPGM). Inhibition potency with both mutases inversely correlated to the degree of fluorination.

Structural and dynamical description of the enzymatic reaction of a phosphohexomutase.

Enzymes are known to adopt various conformations at different points along their catalytic cycles. Here, we present a comprehensive analysis of 15 isomorphous, high resolution crystal structures of the enzyme phosphoglucomutase from the bacterium . The protein was captured in distinct states critical to function, including enzyme-substrate, enzyme-product, and enzyme-intermediate complexes.

Assessment and Impacts of Phosphorylation on Protein Flexibility of the α-d-Phosphohexomutases.

Enzymes in the α-d-phosphohexomutase (PHM) superfamily catalyze a multistep reaction, entailing two successive phosphoryl transfers. Key to this reaction is a conserved phosphoserine in the active site, which serves alternately as a phosphoryl donor and acceptor during the catalytic cycle. In addition to its role in the enzyme mechanism, the phosphorylation state of the catalytic phosphoserine has recently been found to have widespread effects on the structural flexibility of enzymes in this superfamily.

A Hotspot for Disease-Associated Variants of Human PGM1 Is Associated with Impaired Ligand Binding and Loop Dynamics.

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.

Phosphorylation-Dependent Effects on the Structural Flexibility of Phosphoglucosamine Mutase from .

Phosphoglucosamine mutase (PNGM) is an evolutionarily conserved bacterial enzyme in the peptidoglycan biosynthetic pathway, catalyzing the reversible conversion between glucosamine 1- and 6-phosphate. Previous structural studies of PNGM from the pathogen revealed its dimeric assembly and highlighted the rotational mobility of its C-terminal domain. Recent studies of two other enzymes in the same superfamily have demonstrated the long-range effects on the conformational flexibility associated with phosphorylation of the conserved, active site phosphoserine involved in phosphoryl transfer.

Sequence-structure relationships, expression profiles, and disease-associated mutations in the paralogs of phosphoglucomutase 1.

The key metabolic enzyme phosphoglucomutase 1 (PGM1) controls glucose homeostasis in most human cells. Four proteins related to PGM1, known as PGM2, PGM2L1, PGM3 and PGM5, and referred to herein as paralogs, are encoded in the human genome. Although all members of the same enzyme superfamily, these proteins have distinct substrate preferences and different functional roles.

Multiple Ligand-Bound States of a Phosphohexomutase Revealed by Principal Component Analysis of NMR Peak Shifts.

Enzymes sample multiple conformations during their catalytic cycles. Chemical shifts from Nuclear Magnetic Resonance (NMR) are hypersensitive to conformational changes and ensembles in solution. Phosphomannomutase/phosphoglucomutase (PMM/PGM) is a ubiquitous four-domain enzyme that catalyzes phosphoryl transfer across phosphohexose substrates.

Biology, Mechanism, and Structure of Enzymes in the α-d-Phosphohexomutase Superfamily.

Enzymes in the α-d-phosphohexomutases superfamily catalyze the reversible conversion of phosphosugars, such as glucose 1-phosphate and glucose 6-phosphate. These reactions are fundamental to primary metabolism across the kingdoms of life and are required for a myriad of cellular processes, ranging from exopolysaccharide production to protein glycosylation. The subject of extensive mechanistic characterization during the latter half of the 20th century, these enzymes have recently benefitted from biophysical characterization, including X-ray crystallography, NMR, and hydrogen-deuterium exchange studies.

Structure and characterization of a class 3B proline utilization A: Ligand-induced dimerization and importance of the C-terminal domain for catalysis.

The bifunctional flavoenzyme proline utilization A (PutA) catalyzes the two-step oxidation of proline to glutamate using separate proline dehydrogenase (PRODH) and l-glutamate-γ-semialdehyde dehydrogenase active sites. Because PutAs catalyze sequential reactions, they are good systems for studying how metabolic enzymes communicate via substrate channeling. Although mechanistically similar, PutAs vary widely in domain architecture, oligomeric state, and quaternary structure, and these variations represent different structural solutions to the problem of sequestering a reactive metabolite.

Asp263 missense variants perturb the active site of human phosphoglucomutase 1.

Unlabelled: 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.

Data on the phosphorylation state of the catalytic serine of enzymes in the α-D-phosphohexomutase superfamily.

Most enzymes in the α-D-phosphohexomutase superfamily catalyze the reversible conversion of 1- to 6-phosphosugars. They play important roles in carbohydrate and sugar nucleotide metabolism, and participate in the biosynthesis of polysaccharides, glycolipids, and other exoproducts. Mutations in genes encoding these enzymes are associated with inherited metabolic diseases in humans, including glycogen storage disease and congenital disorders of glycosylation.

Synchrotron-based macromolecular crystallography module for an undergraduate biochemistry laboratory course.

This paper describes the introduction of synchrotron-based macromolecular crystallography (MX) into an undergraduate laboratory class. An introductory 2 week experimental module on MX, consisting of four laboratory sessions and two classroom lectures, was incorporated into a senior-level biochemistry class focused on a survey of biochemical techniques, including the experimental characterization of proteins. Students purified recombinant protein samples, set up crystallization plates and flash-cooled crystals for shipping to a synchrotron.

Induced Structural Disorder as a Molecular Mechanism for Enzyme Dysfunction in Phosphoglucomutase 1 Deficiency.

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.

Phosphorylation in the catalytic cleft stabilizes and attracts domains of a phosphohexomutase.

Phosphorylation can modulate the activities of enzymes. The phosphoryl donor in the catalytic cleft of α-D-phosphohexomutases is transiently dephosphorylated while the reaction intermediate completes a 180° reorientation within the cleft. The phosphorylated form of 52 kDa bacterial phosphomannomutase/phosphoglucomutase is less accessible to dye or protease, more stable to chemical denaturation, and widely stabilized against NMR-detected hydrogen exchange across the core of domain 3 to juxtaposed domain 4 (each by ≥ 1.