Conversion of waste poly(vinyl chloride) to branched polyethylene mediated by silylium ions.

Full dechlorination of poly(vinyl chloride) (PVC) in a controlled manner to yield useful polymeric and chlorinated products is of great interest for the processing of PVC waste. Forming polyethylene (PE) without corrosive by-products would allow for a pre-treatment of PE wastes that are often contaminated with PVC. Herein, full dechlorination of PVC has been achieved via generation of silylium ions in situ, to furnish PE products. Complete dechlorination of PVC can be achieved in 2 hours, yielding organic polymer that has similar spectroscopic and thermal signatures of branched PE, with no observable chlorine. The degree of branching can be tuned between 31 and 57 branches per 1000 carbons, with melting temperatures ranging from 51 to 93 °C. This method is applicable to not only pure PVC, but also commercial PVC products. Depending on if the PVC products are separated from plasticizers, different melting points of the resulting PE are observed. PVC dechlorination in the presence of PE waste is also shown. This is the first report of being able to cleanly convert PVC waste to PE in high yields and tune the thermal properties of the PE product, highlighting the remarkable control that silylium ion mediated transformations enables compared to past chemical methods.

Choline Halide-Based Deep Eutectic Solvents as Biocompatible Catalysts for the Alternating Copolymerization of Epoxides and Cyclic Anhydrides.

Aliphatic polyesters have received considerable attention in recent years due to their biodegradability and biocompatible, mechanical, and thermal properties that can make them a suitable alternative to today's commercialized polymers. The ring-opening copolymerization (ROCOP) of epoxides and cyclic anhydrides is a route to synthesize a diverse array of polyesters that could be useful in many applications. However, the catalysts used rarely consider biocompatible catalysts in the case that any are left in the polymer. To the best of our knowledge, we report the first example of using deep eutectic solvents (DESs) as biocompatible catalysts for this target ROCOP with polymerization activity for at least six diverse monomer pairs. Choline halide salts are active for this polymerization, with dried salts showing polymerization slower than that of those conducted in air. Hydrogen bonding with water is hypothesized to enhance the rate-determining step of epoxide ring opening. While the presence of water improves the rate of polymerization, it also acts as a chain transfer agent, leading to smaller molar mass polymers than intended. Combining the choline halide salts with urea or ethylene glycol hydrogen bond donors in air led to DES catalysts that reacted similarly to the salts exposed to air. However, when generating these DESs in air-free conditions, they showed similar rates of polymerization without a drop in polymer molar mass. The hydrogen bonding provided by urea and ethylene glycol seems to promote the rate increase without serving as a chain transfer agent. Results reported herein display the promising potential of biocompatible catalyst systems for this ROCOP process as well as introducing the use of hydrogen bonding to enhance polymerization rates.

Structural basis for Lewis antigen synthesis by the α1,3-fucosyltransferase FUT9.

Mammalian cell surface and secreted glycoproteins exhibit remarkable glycan structural diversity that contributes to numerous physiological and pathogenic interactions. Terminal glycan structures include Lewis antigens synthesized by a collection of α1,3/4-fucosyltransferases (CAZy GT10 family). At present, the only available crystallographic structure of a GT10 member is that of the Helicobacter pylori α1,3-fucosyltransferase, but mammalian GT10 fucosyltransferases are distinct in sequence and substrate specificity compared with the bacterial enzyme. Here, we determined crystal structures of human FUT9, an α1,3-fucosyltransferase that generates Lewisx and Lewisy antigens, in complex with GDP, acceptor glycans, and as a FUT9-donor analog-acceptor Michaelis complex. The structures reveal substrate specificity determinants and allow prediction of a catalytic model supported by kinetic analyses of numerous active site mutants. Comparisons with other GT10 fucosyltransferases and GT-B fold glycosyltransferases provide evidence for modular evolution of donor- and acceptor-binding sites and specificity for Lewis antigen synthesis among mammalian GT10 fucosyltransferases.

Simple yttrium salts as highly active and controlled catalysts for the atom-efficient synthesis of high molecular weight polyesters.

The ring-opening copolymerization (ROCOP) of epoxides and cyclic anhydrides is a promising route to sustainable aliphatic polyesters with diverse mechanical and thermal properties. Here, simple yttrium chloride salts (YCl3THF3.5 and YCl3·6H2O), in combination with a bis(triphenylphosphoranylidene)ammonium chloride [PPN]Cl cocatalyst, are used as efficient and controlled catalysts for ten epoxide and anhydride combinations. In comparison to past literature, this simple salt system exhibits competitive turn-over frequencies (TOFs) for most monomer pairs. Despite no supporting ligand framework, these salts provide excellent control of dispersity, with suppression of side reactions. Using these catalysts, the highest molecular weight reported to date (302.2 kDa) has been obtained with a monosubstituted epoxide and tricyclic anhydride. These data indicate that excellent molecular weight control and suppression of side reactions for ROCOP of epoxides and cyclic anhydrides can coincide with high activity using a simple catalytic system, warranting further research in working towards industrial viability.

Catalytic methods for chemical recycling or upcycling of commercial polymers.

Polymers (plastics) have transformed our lives by providing access to inexpensive and versatile materials with a variety of useful properties. While polymers have improved our lives in many ways, their longevity has created some unintended consequences. The extreme stability and durability of most commercial polymers, combined with the lack of equivalent degradable alternatives and ineffective collection and recycling policies, have led to an accumulation of polymers in landfills and oceans. This problem is reaching a critical threat to the environment, creating a demand for immediate action. Chemical recycling and upcycling involve the conversion of polymer materials into their original monomers, fuels or chemical precursors for value-added products. These approaches are the most promising for value-recovery of post-consumer polymer products; however, they are often cost-prohibitive in comparison to current recycling and disposal methods. Catalysts can be used to accelerate and improve product selectivity for chemical recycling and upcycling of polymers. This review aims to not only highlight and describe the tremendous efforts towards the development of improved catalysts for well-known chemical recycling processes, but also identify new promising methods for catalytic recycling or upcycling of the most abundant commercial polymers.

Tuning π-Acceptor/σ-Donor Ratio of the 2-Isocyanoazulene Ligand: Non-Fluorinated Rival of Pentafluorophenyl Isocyanide and Trifluorovinyl Isocyanide Discovered.

Isocyanoazulenes (CNAz) constitute a relatively new class of isocyanoarenes that offers rich structural and electronic diversification of the organic isocyanide ligand platform. This article considers a series of 2-isocyano-1,3-X2-azulene ligands (X = H, Me, CO2Et, Br, and CN) and the corresponding zero-valent complexes thereof, [(OC)5Cr(2-isocyano-1,3-X2-azulene)]. Air- and thermally stable, X-ray structurally characterized 2-isocyano-1,3-dimethylazulene may be viewed as a non-benzenoid aromatic congener of 2,6-dimethyphenyl isocyanide (2,6-xylyl isocyanide), a longtime "workhorse" aryl isocyanide ligand in coordination chemistry. Single crystal X-ray crystallographic {Cr-CNAz bond distances}, cyclic voltametric {E1/2(Cr0/1+)}, 13C NMR {δ(13CN), δ(13CO)}, UV-vis {dπ(Cr) → pπ*(CNAz) Metal-to-Ligand Charge Transfer}, and FTIR {νN≡C, νC≡O, kC≡O} analyses of the [(OC)5Cr(2-isocyano-1,3-X2-azulene)] complexes provided a multifaceted, quantitative assessment of the π-acceptor/σ-donor characteristics of the above five 2-isocyanoazulenes. In particular, the following inverse linear relationships were documented: δ(13COtrans) vs. δ(13CN), δ(13COcis) vs. δ(13CN), and δ(13COtrans) vs. kC≡O,trans force constant. Remarkably, the net electron withdrawing capability of the 2-isocyano-1,3-dicyanoazulene ligand rivals those of perfluorinated isocyanides CNC6F5 and CNC2F3.

Comparison of human poly-N-acetyl-lactosamine synthase structure with GT-A fold glycosyltransferases supports a modular assembly of catalytic subsites.

Poly-N-acetyl-lactosamine (poly-LacNAc) structures are composed of repeating [-Galß(1,4)-GlcNAcß(1,3)-]n glycan extensions. They are found on both N- and O-glycoproteins and glycolipids and play an important role in development, immune function, and human disease. The majority of mammalian poly-LacNAc is synthesized by the alternating iterative action of ß1,3-N-acetylglucosaminyltransferase 2 (B3GNT2) and ß1,4-galactosyltransferases. B3GNT2 is in the largest mammalian glycosyltransferase family, GT31, but little is known about the structure, substrate recognition, or catalysis by family members. Here we report the structures of human B3GNT2 in complex with UDP:Mg2+ and in complex with both UDP:Mg2+ and a glycan acceptor, lacto-N-neotetraose. The B3GNT2 structure conserves the GT-A fold and the DxD motif that coordinates a Mg2+ ion for binding the UDP-GlcNAc sugar donor. The acceptor complex shows interactions with only the terminal Galß(1,4)-GlcNAcß(1,3)- disaccharide unit, which likely explains the specificity for both N- and O-glycan acceptors. Modeling of the UDP-GlcNAc donor supports a direct displacement inverting catalytic mechanism. Comparative structural analysis indicates that nucleotide sugar donors for GT-A fold glycosyltransferases bind in similar positions and conformations without conserving interacting residues, even for enzymes that use the same donor substrate. In contrast, the B3GNT2 acceptor binding site is consistent with prior models suggesting that the evolution of acceptor specificity involves loops inserted into the stable GT-A fold. These observations support the hypothesis that GT-A fold glycosyltransferases employ coevolving donor, acceptor, and catalytic subsite modules as templates to achieve the complex diversity of glycan linkages in biological systems.

Characterizing human α-1,6-fucosyltransferase (FUT8) substrate specificity and structural similarities with related fucosyltransferases.

Mammalian Asn-linked glycans are extensively processed as they transit the secretory pathway to generate diverse glycans on cell surface and secreted glycoproteins. Additional modification of the glycan core by α-1,6-fucose addition to the innermost GlcNAc residue (core fucosylation) is catalyzed by an α-1,6-fucosyltransferase (FUT8). The importance of core fucosylation can be seen in the complex pathological phenotypes of FUT8 null mice, which display defects in cellular signaling, development, and subsequent neonatal lethality. Elevated core fucosylation has also been identified in several human cancers. However, the structural basis for FUT8 substrate specificity remains unknown.Here, using various crystal structures of FUT8 in complex with a donor substrate analog, and with four distinct glycan acceptors, we identify the molecular basis for FUT8 specificity and activity. The ordering of three active site loops corresponds to an increased occupancy for bound GDP, suggesting an induced-fit folding of the donor-binding subsite. Structures of the various acceptor complexes were compared with kinetic data on FUT8 active site mutants and with specificity data from a library of glycan acceptors to reveal how binding site complementarity and steric hindrance can tune substrate affinity. The FUT8 structure was also compared with other known fucosyltransferases to identify conserved and divergent structural features for donor and acceptor recognition and catalysis. These data provide insights into the evolution of modular templates for donor and acceptor recognition among GT-B fold glycosyltransferases in the synthesis of diverse glycan structures in biological systems.

A redox-active switch in fructosamine-3-kinases expands the regulatory repertoire of the protein kinase superfamily.

Aberrant regulation of metabolic kinases by altered redox homeostasis substantially contributes to aging and various diseases, such as diabetes. We found that the catalytic activity of a conserved family of fructosamine-3-kinases (FN3Ks), which are evolutionarily related to eukaryotic protein kinases, is regulated by redox-sensitive cysteine residues in the kinase domain. The crystal structure of the FN3K homolog from Arabidopsis thaliana revealed that it forms an unexpected strand-exchange dimer in which the ATP-binding P-loop and adjoining ß strands are swapped between two chains in the dimer. This dimeric configuration is characterized by strained interchain disulfide bonds that stabilize the P-loop in an extended conformation. Mutational analysis and solution studies confirmed that the strained disulfides function as redox "switches" to reversibly regulate the activity and dimerization of FN3K. Human FN3K, which contains an equivalent P-loop Cys, was also redox sensitive, whereas ancestral bacterial FN3K homologs, which lack a P-loop Cys, were not. Furthermore, CRISPR-mediated knockout of FN3K in human liver cancer cells altered the abundance of redox metabolites, including an increase in glutathione. We propose that redox regulation evolved in FN3K homologs in response to changing cellular redox conditions. Our findings provide insights into the origin and evolution of redox regulation in the protein kinase superfamily and may open new avenues for targeting human FN3K in diabetic complications.

A terminal α3-galactose modification regulates an E3 ubiquitin ligase subunit in Toxoplasma gondii.

Skp1, a subunit of E3 Skp1/Cullin-1/F-box protein ubiquitin ligases, is modified by a prolyl hydroxylase that mediates O2 regulation of the social amoeba Dictyostelium and the parasite Toxoplasma gondii The full effect of hydroxylation requires modification of the hydroxyproline by a pentasaccharide that, in Dictyostelium, influences Skp1 structure to favor assembly of Skp1/F-box protein subcomplexes. In Toxoplasma, the presence of a contrasting penultimate sugar assembled by a different glycosyltransferase enables testing of the conformational control model. To define the final sugar and its linkage, here we identified the glycosyltransferase that completes the glycan and found that it is closely related to glycogenin, an enzyme that may prime glycogen synthesis in yeast and animals. However, the Toxoplasma enzyme catalyzes formation of a Galα1,3Glcα linkage rather than the Glcα1,4Glcα linkage formed by glycogenin. Kinetic and crystallographic experiments showed that the glycosyltransferase Gat1 is specific for Skp1 in Toxoplasma and also in another protist, the crop pathogen Pythium ultimum The fifth sugar is important for glycan function as indicated by the slow-growth phenotype of gat1Δ parasites. Computational analyses indicated that, despite the sequence difference, the Toxoplasma glycan still assumes an ordered conformation that controls Skp1 structure and revealed the importance of nonpolar packing interactions of the fifth sugar. The substitution of glycosyltransferases in Toxoplasma and Pythium by an unrelated bifunctional enzyme that assembles a distinct but structurally compatible glycan in Dictyostelium is a remarkable case of convergent evolution, which emphasizes the importance of the terminal α-galactose and establishes the phylogenetic breadth of Skp1 glycoregulation.

Conservation of Atypical Allostery in C. elegans UDP-Glucose Dehydrogenase.

Human UDP-glucose dehydrogenase (hUGDH) oxidizes uridine diphosphate (UDP)-glucose to UDP-glucuronic acid, an essential substrate in the phase II metabolism of drugs. The activity of hUGDH is controlled by an atypical allosteric mechanism in which the feedback inhibitor UDP-xylose competes with the substrate for the active site and triggers a buried allosteric switch to produce an inactive complex (EΩ). Previous comparisons with a nonallosteric UGDH identified six large-to-small substitutions that produce packing defects in the protein core and provide the conformational flexibility necessary for the allosteric transition. Here, we test the hypothesis that these large-to-small substitutions form a motif that can be used to identify allosteric UGDHs. Caenorhabditis elegans UGDH (cUGDH) conserves this motif with the exception of an Ala-to-Pro substitution in position 109. The crystal structures of unliganded and UDP-xylose bound cUGDH show that the A109P substitution is accommodated by an Asn-to-Ser substitution at position 290. Steady-state analysis and sedimentation velocity studies show that the allosteric transition is conserved in cUGDH. The enzyme also exhibits hysteresis in progress curves and negative cooperativity with respect to NAD+ binding. Both of these phenomena are conserved in the human enzyme, which is strong evidence that these represent fundamental features of atypical allostery in UGDH. A phylogenetic analysis of UGDH shows that the atypical allostery motif is ancient and identifies a potential transition point in the evolution of the UGDH family.

The structural and biochemical impacts of monomerizing human acetylcholinesterase.

Serving a critical role in neurotransmission, human acetylcholinesterase (hAChE) is the target of organophosphate nerve agents. Hence, there is an active interest in studying the mechanism of inhibition and recovery of enzymatic activity, which could lead to better countermeasures against nerve agents. As hAChE is found in different oligomeric assemblies, certain approaches to studying it have been problematic. Herein, we examine the biochemical and structural impact of monomerizing hAChE by using two mutations: L380R/F535K. The activities of monomeric hAChE L380R/F535K and dimeric hAChE were determined to be comparable utilizing a modified Ellman's assay. To investigate the influence of subunit-subunit interactions on the structure of hAChE, a 2.1 Å X-ray crystallographic structure was determined. Apart from minor shifts along the dimer interface, the overall structure of the hAChE L380R/F535K mutant is similar to that of dimeric hAChE. To probe whether the plasticity of the active site was overtly impacted by monomerizing hAChE, the kinetic constants of (PR/S ) - VX (ethyl({2-[bis(propan-2-yl)amino]ethyl}sulfanyl)(methyl)phosphinate) inhibition and subsequent rescue of hAChE L380R/F535K activity with HI-6 (1-(2'-hydroxyiminomethyl-1'-pyridinium)-3-(4'-carbamoyl-1-pyridinium)) were determined and found to be comparable to those of dimeric hAChE. Thus, hAChE L380R/F535K could be used as a substitute for dimeric hAChE when experimentally probing the ability of the hAChE active site to accommodate future nerve agent threats or judge the ability of new therapeutics to access the active site.

Hysteresis and Allostery in Human UDP-Glucose Dehydrogenase Require a Flexible Protein Core.

Human UDP-glucose dehydrogenase (hUGDH) oxidizes UDP-glucose to UDP-glucuronic acid, an essential substrate in the phase II metabolism of drugs. The activity of hUGDH is regulated by the conformation of a buried allosteric switch (T131 loop/α6 helix). Substrate binding induces the allosteric switch to slowly isomerize from an inactive E* conformation to the active E state, which can be observed as enzyme hysteresis. When the feedback inhibitor UDP-xylose binds, the allosteric switch and surrounding residues in the protein core repack, converting the hexamer into an inactive, horseshoe-shaped complex (EΩ). This allosteric transition is facilitated by large cavities and declivities in the protein core that provide the space required to accommodate the alternate packing arrangements. Here, we have used the A104L substitution to fill a cavity in the E state and sterically prevent repacking of the core into the EΩ state. Steady state analysis shows that hUGDHA104L binds UDP-xylose with lower affinity and that the inhibition is no longer cooperative. This means that the allosteric transition to the high-UDP-xylose affinity EΩ state is blocked by the substitution. The crystal structures of hUGDHA104L show that the allosteric switch still adopts the E and E* states, albeit with a more rigid protein core. However, the progress curves of hUGDHA104L do not show hysteresis, which suggests that the E* and E states are now in rapid equilibrium. Our data suggest that hysteresis in native hUGDH originates from the conformational entropy of the E* state protein core.

The entropic force generated by intrinsically disordered segments tunes protein function.

Protein structures are dynamic and can explore a large conformational landscape1,2. Only some of these structural substates are important for protein function (such as ligand binding, catalysis and regulation)3-5. How evolution shapes the structural ensemble to optimize a specific function is poorly understood3,4. One of the constraints on the evolution of proteins is the stability of the folded 'native' state. Despite this, 44% of the human proteome contains intrinsically disordered peptide segments greater than 30 residues in length6, the majority of which have no known function7-9. Here we show that the entropic force produced by an intrinsically disordered carboxy terminus (ID-tail) shifts the conformational ensemble of human UDP-α-D-glucose-6-dehydrogenase (UGDH) towards a substate with a high affinity for an allosteric inhibitor. The function of the ID-tail does not depend on its sequence or chemical composition. Instead, the affinity enhancement can be accurately predicted based on the length of the intrinsically disordered segment, and is consistent with the entropic force generated by an unstructured peptide attached to the protein surface10-13. Our data show that the unfolded state of the ID-tail rectifies the dynamics and structure of UGDH to favour inhibitor binding. Because this entropic rectifier does not have any sequence or structural constraints, it is an easily acquired adaptation. This model implies that evolution selects for disordered segments to tune the energy landscape of proteins, which may explain the persistence of intrinsic disorder in the proteome.

The crystal structure of Proteus vulgaris tryptophan indole-lyase complexed with oxindolyl-L-alanine: implications for the reaction mechanism.

Tryptophan indole-lyase (TIL) is a bacterial enzyme which catalyzes the reversible formation of indole and ammonium pyruvate from L-tryptophan. Oxindolyl-L-alanine (OIA) is an inhibitor of TIL, with a Ki value of about 5 µM. The crystal structure of the complex of Proteus vulgaris TIL with OIA has now been determined at 2.1 Šresolution. The ligand forms a closed quinonoid complex with the pyridoxal 5'-phosphate (PLP) cofactor. The small domain rotates about 10° to close the active site, bringing His458 into position to donate a hydrogen bond to Asp133, which also accepts a hydrogen bond from the heterocyclic NH of the inhibitor. This brings Phe37 and Phe459 into van der Waals contact with the aromatic ring of OIA. Mutation of the homologous Phe464 in Escherichia coli TIL to Ala results in a 500-fold decrease in kcat/Km for L-tryptophan, with less effect on the reaction of other nonphysiological ß-elimination substrates. Stopped-flow kinetic experiments of F464A TIL show that the mutation has no effect on the formation of quinonoid intermediates. An aminoacrylate intermediate is observed in the reaction of F464A TIL with S-ethyl-L-cysteine and benzimidazole. A model of the L-tryptophan quinonoid complex with PLP in the active site of P. vulgaris TIL shows that there would be a severe clash of Phe459 (∼1.5 Šapart) and Phe37 (∼2 Šapart) with the benzene ring of the substrate. It is proposed that this creates distortion of the substrate aromatic ring out of plane and moves the substrate upwards on the reaction coordinate towards the transition state, thus reducing the activation energy and accelerating the enzymatic reaction.

Human N-acetylglucosaminyltransferase II substrate recognition uses a modular architecture that includes a convergent exosite.

Asn-linked oligosaccharides are extensively modified during transit through the secretory pathway, first by trimming of the nascent glycan chains and subsequently by initiating and extending multiple oligosaccharide branches from the trimannosyl glycan core. Trimming and branching pathway steps are highly ordered and hierarchal based on the precise substrate specificities of the individual biosynthetic enzymes. A key committed step in the synthesis of complex-type glycans is catalyzed by N-acetylglucosaminyltransferase II (MGAT2), an enzyme that generates the second GlcNAcß1,2- branch from the trimannosyl glycan core using UDP-GlcNAc as the sugar donor. We determined the structure of human MGAT2 as a Mn2+-UDP donor analog complex and as a GlcNAcMan3GlcNAc2-Asn acceptor complex to reveal the structural basis for substrate recognition and catalysis. The enzyme exhibits a GT-A Rossmann-like fold that employs conserved divalent cation-dependent substrate interactions with the UDP-GlcNAc donor. MGAT2 interactions with the extended glycan acceptor are distinct from other related glycosyltransferases. These interactions are composed of a catalytic subsite that binds the Man-α1,6- monosaccharide acceptor and a distal exosite pocket that binds the GlcNAc-ß1,2Man-α1,3Manß- substrate "recognition arm." Recognition arm interactions are similar to the enzyme-substrate interactions for Golgi α-mannosidase II, a glycoside hydrolase that acts just before MGAT2 in the Asn-linked glycan biosynthetic pathway. These data suggest that substrate binding by MGAT2 employs both conserved and convergent catalytic subsite modules to provide substrate selectivity and catalysis. More broadly, the MGAT2 active-site architecture demonstrates how glycosyltransferases create complementary modular templates for regiospecific extension of glycan structures in mammalian cells.

Allostery and Hysteresis Are Coupled in Human UDP-Glucose Dehydrogenase.

Human UDP-glucose dehydrogenase (hUGDH) is regulated by an atypical allosteric mechanism in which the feedback inhibitor UDP-xylose (UDP-Xyl) competes with the substrate for the active site. Binding of UDP-Xyl triggers the T131-loop/α6 allosteric switch, which converts the hexameric structure of hUGDH into an inactive, horseshoe-shaped complex (EΩ). This allosteric transition buries residue A136 in the protein core to produce a subunit interface that favors the EΩ structure. Here we use a methionine substitution to prevent the burial of A136 and trap the T131-loop/α6 switch in the active conformation. We show that hUGDHA136M does not exhibit substrate cooperativity, which is strong evidence that the methionine substitution prevents the formation of the low-UDP-Glc-affinity EΩ state. In addition, the inhibitor affinity of hUGDHA136M is reduced 14-fold, which most likely represents the Ki for competitive inhibition in the absence of the allosteric transition to the higher-affinity EΩ state. hUGDH also displays a lag in progress curves, which is caused by a slow, substrate-induced isomerization that activates the enzyme. Stopped-flow analysis shows that hUGDHA136M does not exhibit hysteresis, which suggests that the T131-loop/α6 switch is the source of the slow isomerization. This interpretation is supported by the 2.05 Å resolution crystal structure of hUGDHA136M, which shows that the A136M substitution has stabilized the active conformation of the T131-loop/α6 allosteric switch. This work shows that the T131-loop/α6 allosteric switch couples allostery and hysteresis in hUGDH.

Evidence of Kinetic Cooperativity in Dimeric Ketopantoate Reductase from Staphylococcus aureus.

Ketopantoate reductase (KPR) catalyzes the NADPH-dependent production of pantoate, an essential precursor in the biosynthesis of coenzyme A. Previous structural studies have been limited to Escherichia coli KPR, a monomeric enzyme that follows a sequential ordered mechanism. Here we report the crystal structure of the Staphylococcus aureus enzyme at 1.8 Å resolution, the first description of a dimeric KPR. Using sedimentation velocity analysis, we show that the S. aureus KPR dimer is stable in solution. In fact, our structural analysis shows that the dimeric assembly we identify is present in the majority of KPR crystal structures. Steady state analysis of S. aureus KPR reveals strong positive cooperativity with respect to NADPH (Hill coefficient of 2.5). In contrast, high concentrations of the substrate ketopantoate (KP) inhibit the activity of the enzyme. These observations are consistent with a random addition mechanism in which the initial binding of NADPH is the kinetically preferred path. In fact, Förster resonance energy transfer studies of the equilibrium binding of NADPH show only a small degree of cooperativity between subunits (Hill coefficient of 1.3). Thus, the apparently strong cooperativity observed in substrate saturation curves is due to a kinetic process that favors NADPH binding first. This interpretation is consistent with our analysis of the A181L substitution, which increases the Km of ketopantoate 844-fold, without affecting kcat. The crystal structure of KPRA181L shows that the substitution displaces Ser239, which is known to be important for the binding affinity of KP. The decrease in KP affinity would enhance the already kinetically preferred NADPH binding path, making the random mechanism appear to be sequentially ordered and reducing the kinetic cooperativity. Consistent with this interpretation, the NADPH saturation curve for KPRA181L is hyperbolic.

Man o' war mutation in UDP-α-D-xylose synthase favors the abortive catalytic cycle and uncovers a latent potential for hexamer formation.

The man o' war (mow) phenotype in zebrafish is characterized by severe craniofacial defects due to a missense mutation in UDP-α-d-xylose synthase (UXS), an essential enzyme in proteoglycan biosynthesis. The mow mutation is located in the UXS dimer interface ∼16 Å away from the active site, suggesting an indirect effect on the enzyme mechanism. We have examined the structural and catalytic consequences of the mow mutation (R236H) in the soluble fragment of human UXS (hUXS), which shares 93% sequence identity with the zebrafish enzyme. In solution, hUXS dimers undergo a concentration-dependent association to form a tetramer. Sedimentation velocity studies show that the R236H substitution induces the formation of a new hexameric species. Using two new crystal structures of the hexamer, we show that R236H and R236A substitutions cause a local unfolding of the active site that allows for a rotation of the dimer interface necessary to form the hexamer. The disordered active sites in the R236H and R236A mutant constructs displace Y231, the essential acid/base catalyst in the UXS reaction mechanism. The loss of Y231 favors an abortive catalytic cycle in which the reaction intermediate, UDP-α-d-4-keto-xylose, is not reduced to the final product, UDP-α-d-xylose. Surprisingly, the mow-induced hexamer is almost identical to the hexamers formed by the deeply divergent UXS homologues from Staphylococcus aureus and Helicobacter pylori (21% and 16% sequence identity, respectively). The persistence of a latent hexamer-building interface in the human enzyme suggests that the ancestral UXS may have been a hexamer.

Hysteresis in human UDP-glucose dehydrogenase is due to a restrained hexameric structure that favors feedback inhibition.

Human UDP-α-d-glucose-6-dehydrogenase (hUGDH) displays hysteresis because of a slow isomerization from an inactive state (E*) to an active state (E). Here we show that the structure of E* constrains hUGDH in a conformation that favors feedback inhibition at physiological pH. The feedback inhibitor UDP-α-d-xylose (UDP-Xyl) competes with the substrate UDP-α-d-glucose for the active site. Upon binding, UDP-Xyl triggers an allosteric switch that changes the structure and affinity of the intersubunit interface to form a stable but inactive horseshoe-shaped hexamer. Using sedimentation velocity studies and a new crystal structure, we show that E* represents a stable conformational intermediate between the active and feedback-inhibited conformations. Because the allosteric switch occludes the cofactor and substrate binding sites in the inactive hexamer, the intermediate conformation observed in the crystal structure is consistent with the E* transient observed in relaxation studies. Steady-state analysis shows that the E* conformation enhances the affinity of hUGDH for the allosteric inhibitor UDP-Xyl by 8.6-fold (Ki = 810 nM). We present a model in which the constrained quaternary structure permits a small effector molecule to leverage a disproportionately large allosteric response.