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The use of pharmacological chaperones (PCs) to stabilize specific enzymes and impart a therapeutic benefit is an emerging strategy in drug discovery. However, designing molecules that can bind optimally to their targets at physiological pH remains a major challenge. Our previous study found that dibasic polyhydroxylated pyrrolidine 5exhibited superior pH-selective inhibitory activity and chaperoning activity for human α-galactosidase A (α-Gal A) compared with its monobasic parent molecule, 4. To further investigate the role of different C-2 moieties on the pH-selectivity and protecting effects of these compounds, we designed and synthesized a library of monobasic and dibasic iminosugars, screened them for α-Gal A-stabilizing activity using thermal shift and heat-induced denaturation assays, and characterized the mechanistic basis for this stabilization using X-ray crystallography and binding assays. We noted that the dibasic iminosugars 5and 20protect α-Gal A from denaturation and inactivation at lower concentrations than monobasic or other N-substituted derivatives; a finding attributed to the nitrogen on the C-2 methylene of 5and 20, which forms the bifurcated salt bridges (BSBs) with two carboxyl residues, E203 and D231. Additionally, the formation of BSBs at pH 7.0 and the electrostatic repulsion between the vicinal ammonium cations of dibasic iminosugars at pH 4.5 are responsible for their pH-selective binding to α-Gal A. Moreover, compounds 5and 20demonstrated promising results in improving enzyme replacement therapy and exhibited significant chaperoning effects in Fabry cells. These findings suggest amino-iminosugars 5and 20as useful models to demonstrate how an additional exocyclic amino group can improve their pH-selectivity and protecting effects, providing new insights for the design of pH-selective PCs.

The catalog of enzymes known to catalyze the nucleophile-assisted formation of C–C bonds is extremely small, and there is presently no definitive example of a biological Rauhut-Currier reaction. Biosynthesis of the polyketide insecticide spinosyn A in Saccharopolyspora spinosainvolves a [4 + 2]-cycloaddition and a subsequent intramolecular C–C bond formation catalyzed by SpnF and SpnL, respectively. Isotope tracer experiments and kinetic isotope effects, however, imply that the SpnL-catalyzed reaction proceeds without initial deprotonation of the substrate. The crystal structure of SpnL exhibits high similarity to SAM-dependent methyltransferases as well as SpnF. The residue Cys60 is also shown to reside in the SpnL active site, and the Cys60Ala SpnL mutant is found to be devoid of activity. Moreover, SpnL is covalently modified at Cys60 and irreversibly inactivated when it is coincubated with a fluorinated substrate analogue designed as a suicide inactivator of nucleophile-assisted C–C bond formation. These results suggest that SpnL catalyzes a biological Rauhut–Currier reaction.

Recent research on the structure and mechanism of DNA polymerases has continued to generate fundamentally important features, including a noncanonical pathway involving “prebinding” of metal-bound dNTP (MdNTP) in the absence of DNA. While this noncanonical mechanism was shown to be a possible subset for African swine fever DNA polymerase X (Pol X) and human Pol λ, it remains unknown whether it could be the primary pathway for a DNA polymerase. Pol μ is a unique member of the X-family with multiple functions and with unusual Mn2+preference. Here we report that Pol μ not only prebinds MdNTP in a catalytically active conformation but also exerts a Mn2+over Mg2+preference at this early stage of catalysis, for various functions: incorporation of dNTP into a single nucleotide gapped DNA, incorporation of rNTP in the nonhomologous end joining (NHEJ) repair, incorporation of dNTP to an ssDNA, and incorporation of an 8-oxo-dGTP opposite template dA (mismatched) or dC (matched). The structural basis of this noncanonical mechanism and Mn2+over Mg2+preference in these functions was analyzed by solving 19 structures of prebinding binary complexes, precatalytic ternary complexes, and product complexes. The results suggest that the noncanonical pathway is functionally relevant for the multiple functions of Pol μ. Overall, this work provides the structural and mechanistic basis for the long-standing puzzle in the Mn2+preference of Pol μ and expands the landscape of the possible mechanisms of DNA polymerases to include both mechanistic pathways.

While cryo-EM is revolutionizing structural biology, its impact on enzymology is yet to be fully demonstrated. The ketol-acid reductoisomerase (KARI) catalyzes conversion of (2S)-acetolactate or (2S)-aceto-2-hydroxybutyrate to 2,3-dihydroxy-3-alkylbutyrate. We found that KARI from archaea Sulfolobus solfataricus(Sso-KARI) is unusual in being a dodecamer, bispecific to NADH and NADPH, and losing activity above pH 7.8. While crystals were obtainable only at pH 8.5, cryo-EM structures were solved at pH 7.5 and 8.5 for Sso-KARI:2Mg2+. The results showed that the distances of the two catalytic Mg2+ions are lengthened in both structures at pH 8.5. We next solved cryo-EM structures of two Sso-KARI complexes, with NADH+inhibitor and NADPH+inhibitor at pH 7.5, which indicate that the bispecificity can be attributed to a unique asparagine at the cofactor binding loop. Unexpectedly, Sso-KARI also differs from other KARI enzymes in lacking “induced-fit”, reflecting structural rigidity. Thus, cryo-EM is powerful for structural and mechanistic enzymology.

Trypanosomatid parasites are the causative agents of many neglected tropical diseases, including the leishmaniases, Chagas disease, and human African trypanosomiasis. They exploit unusual vacuolar soluble pyrophosphatases (VSPs), absent in humans, for cell growth and virulence and, as such, are drug targets. Here, we report the crystal structures of VSP1s from Trypanosoma cruziand T. brucei, together with that of the T. cruziprotein bound to a bisphosphonate inhibitor. Both VSP1s form a hybrid structure containing an (N-terminal) EF-hand domain fused to a (C-terminal) pyrophosphatase domain. The two domains are connected via an extended loop of about 17 residues. Crystallographic analysis and size exclusion chromatography indicate that the VSP1s form tetramers containing head-to-tail dimers. Phosphate and diphosphate ligands bind in the PPase substrate-binding pocket and interact with several conserved residues, and a bisphosphonate inhibitor (BPH-1260) binds to the same site. On the basis of Cytoscape and other bioinformatics analyses, it is apparent that similar folds will be found in most if not all trypanosomatid VSP1s, including those found in insects (Angomonas deanei, Strigomonas culicis), plant pathogens (Phytomonasspp.), and Leishmaniaspp. Overall, the results are of general interest since they open the way to structure-based drug design for many of the neglected tropical diseases.

Fassförmig: Das Enzym MoeO5 katalysiert den Transfer der C15‐Einheit von Farnesylpyrophosphat an die 2‐Hydroxygruppe von 3‐Phosphoglycerat unter Bildung von 2‐(Z,E)‐Farnesyl‐3‐phosphoglycerat (FPG; Ligand im Zentrum der gezeigten Struktur). Röntgenkristallstrukturen zeigen, dass MoeO5 die Fass‐Struktur einer Triosephosphatisomerase hat, und das FPG aufgrund des langen λ3‐Loops (magenta im Bild) in einer gebogenen Tasche bindet.

Barrel‐shaped: The enzyme MoeO5 catalyzes the transfer of the C15moiety of farnesyl pyrophosphate to the 2‐hydroxy group of 3‐phosphoglycerate to give 2‐(Z,E)‐farnesyl‐3‐phosphoglycerate (FPG; ligand in the center of the shown structure). X‐ray crystallographic structures showed that MoeO5 forms a triose‐phosphate‐isomerase barrel structure and binds FPG in a curved pocket, mainly as a result of its long λ3 loop (magenta in picture).

d-Psicose 3-epimerase (DPEase) is demonstrated to be useful in the bioproduction of d-psicose, a rare hexose sugar, from d-fructose, found plenty in nature. Clostridium cellulolyticumH10 has recently been identified as a DPEase that can epimerize d-fructose to yield d-psicose with a much higher conversion rate when compared with the conventionally used DTEase. In this study, the crystal structure of the C. cellulolyticumDPEase was determined. The enzyme assembles into a tetramer and each subunit shows a (β/α)8TIM barrel fold with a Mn2+metal ion in the active site. Additional crystal structures of the enzyme in complex with substrates/products (d-psicose, d-fructose, d-tagatose and d-sorbose) were also determined. From the complex structures of C. cellulolyticumDPEase with d-psicose and d-fructose, the enzyme has much more interactions with d-psicose than d-fructose by forming more hydrogen bonds between the substrate and the active site residues. Accordingly, based on these ketohexose-bound complex structures, a C3-O3 proton-exchange mechanism for the conversion between d-psicose and d-fructose is proposed here. These results provide a clear idea for the deprotonation/protonation roles of E150 and E244 in catalysis.

AbstractPlatinum(II) complexes have been demonstrated to form covalent bonds with sulfur-donating ligands (in glutathione, metallothionein and other sulfur-containing biomolecules) or coordination bonds with nitrogen-donating ligands (such as histidine and guanine). To investigate how these compounds interact with cysteine proteases, we chose terpyridine platinum(II) (TP-Pt(II)) complexes as a model system. By using X-ray crystallography, we demonstrated that TP-Pt(II) formed a covalent bond with the catalytic cysteine residue in pyroglutamyl peptidase I. Moreover, by using MALDI (matrix-assisted laser desorption/ionization) and TOF-TOF (time of flight) mass spectrometry, we elucidated that the TP-Pt(II) complex formed a covalent bond with the active-site cysteine residue in two other types of cysteine protease. Taken together, the results unequivocally showed that TP-Pt(II) complexes can selectively bind to the active site of most cysteine proteases. Our findings here can be useful in the design of new anti-cancer, anti-parasite or anti-virus platinum(II) compounds.

Zwei Schleifen, die sich nach innen zum aktiven Zentrum von α‐Fucosidase bewegen, führen zu einer geschlossenen Konformation des Komplexes mit Inhibitoren mit Ki‐Werten vom Mikro‐ bis zum Nanomolbereich. Bei Inhibitoren mit subnanomolaren Ki‐Werten treten zwar keine weiteren Konformationsänderungen in den beiden Schleifen auf, aber die Schleifen werden durch Wasserstoffbrücken und hydrophobe Wechselwirkungen weiter stabilisiert.

Platelet glycoprotein Ib (GPIb)-binding proteins (GPIb-BPs) from snake venoms are usually C-type lectins, which target specific sites of GPIbα and elicit distinct effects on platelets. In the present paper, we report a tetrameric platelet-agglutinating factor (molecular mass 121.1 kDa), termed mucrocetin, purified from the venom of Taiwan habu (Trimeresurus mucrosquamatus). Mucrocetin is a GPIbα agonist with a binding site distinct from that of flavocetin-A (a snake venom GPIbα antagonist) on GPIbα, in spite of the high sequence identity (94.6%) between the two venom lectins. The crystal structure of mucrocetin was solved and refined to 2.8 Å (1 Å=0.1 nm) resolution, which shows an interesting crystal packing of six-layer cylinders of doughnut-shaped molecules. The four αβ heterodimers are arranged in an unusual square-shaped ring stabilized by four interdimer ‘head-to-tail’ disulphide bridges. Detailed structural comparison between mucrocetin and flavocetin-A suggests that their disparate platelet effects are probably attributable to different charge distributions on the putative concave binding surface. A unique positively charged patch on the binding surface of mucrocetin, formed by Lys102, Lys108, Lys109 and Arg123 in the α-subunit coupled with Lys22, Lys102, Lys116 and Arg117 in the β-subunit, appears to be the primary determinant of its platelet-agglutinating activity. Conceivably, this interesting venom factor may provide a useful tool to study platelet agglutination by binding to the GPIb–IX–V complex.

RD1 is a 7-kDa globular protein from the Antarctic eel pout Lycodichthys dearborni. It belongs to type III of the four types of antifreeze proteins (AFPs) found in marine fishes living at subzero temperatures. For type III AFP, a potential ice-binding flat surface has been identified and is imbedded with side chains capable of making hydrogen bonds with a specific lattice plane on ice. So far, all crystallographic studies on type III AFPs were carried out using the Atlantic ocean pout Macrozoarces americanus as the source organism. Here we present the crystal structure of a type III AFP from a different zoarcid fish, and at an ultra-high resolution of 0.62Å. The protein fold of RD1 comprises a compact globular domain with two internal tandem motifs arranged about a pseudo-dyad symmetry. Each motif of the “pretzel fold” includes four short β-strands and a 310 helix. There is a novel internal cavity of 45Å3 surrounded by eight conserved nonpolar residues. The model contains several residues with alternate conformations, and a number of split water molecules, probably caused by alternate interactions with the protein molecule. After extensive refinement that includes hydrogen atoms, significant residual electron densities associated with the electrons of peptides and many other bonds could be visualized.

The Escherichia coliundecaprayl-pyrophosphate synthase (UPPs) structure has been solved using the single wavelength anomalous diffraction method. The putative substrate-binding site is located near the end of the βA-strand with Asp-26 playing a critical catalytic role. In both subunits, an elongated hydrophobic tunnel is found, surrounded by four β-strands (βA-βB-βD-βC) and two helices (α2 and α3) and lined at the bottom with large residues Ile-62, Leu-137, Val-105, and His-103. The product distributions formed by the use of the I62A, V105A, and H103A mutants are similar to those observed for wild-type UPPs. Catalysis by the L137A UPPs, on the other hand, results in predominantly the formation of the C70polymer rather than the C55polymer. Ala-69 and Ala-143 are located near the top of the tunnel. In contrast to the A143V reaction, the C30intermediate is formed to a greater extent and is longer lived in the process catalyzed by the A69L mutant. These findings suggest that the small side chain of Ala-69 is required for rapid elongation to the C55product, whereas the large hydrophobic side chain of Leu-137 is required to limit the elongation to the C55product. The roles of residues located on a flexible loop were investigated. The S71A, N74A, or R77A mutants displayed 25–200-fold decrease in kcatvalues. W75A showed an 8-fold increase of the FPP Kmvalue, and 22–33-fold increases in the IPP Kmvalues were observed for E81A and S71A. The loop may function to bridge the interaction of IPP with FPP, needed to initiate the condensation reaction and serve as a hinge to control the substrate binding and product release.

The growth mechanisms and physical properties of the orthorhombic crystal form of beef liver catalase were investigated using in situatomic force microscopy (AFM). It was observed that the crystals grow in the 〈001〉 direction by an unusual progression of sequential two‐dimensional nuclei of half unit‐cell layers corresponding to the `bottoms' and `tops' of unit cells. These were easily discriminated by their alternating asymmetric shapes and their strong growth‐rate anisotropy. This pattern has not previously been observed with other macromolecular crystals. Orthorhombic beef liver catalase crystals exhibit an extremely high defect density and incorporate great numbers of misoriented microcrystals, revealed intact by etching experiments, which may explain their marginal diffraction properties. To facilitate interpretation of AFM results in terms of intermolecular interactions, the structure of the orthorhombic crystals, having an entire tetramer of the enzyme as the asymmetric unit, was solved by molecular replacement using a model derived from a trigonal crystal form. It was subsequently refined by conventional techniques. Although the packing of molecules in the two unit cells was substantially different, with very few exceptions no significant differences in the molecular structures were observed. In addition, no statistically significant deviation from ideal 222 molecular symmetry appeared within the tetramer. The packing of molecules in the crystal revealed by X‐ray analysis explained in a satisfying way the process of crystal growth revealed by AFM.

Tyrosine aminotransferase catalyzes transamination for both dicarboxylic and aromatic amino‐acid substrates. The substrate‐free Escherichia colityrosine aminotransferase (eTAT) bound with the cofactor pyridoxal 5′‐phosphate (PLP) was crystallized in the trigonal space group P32. A low‐resolution crystal structure of eTAT was determined by molecular‐replacement methods. The overall folding of eTAT resembles that of the aspartate aminotransferases, with the two identical subunits forming a dimer in which each monomer binds a PLP molecule viaa covalent bond linked to the ɛ‐NH2group of Lys258. Comparison of the structure of eTAT with those of the open, half‐open or closed form of chicken or E. coliaspartate aminotransferases shows the eTAT structure to be in the open conformation.

Kato: Mast Therapeutics: Consultancy; Bayer: Research Funding.

Sphingosine 1-phosphate (S1P) is a bioactive signaling lipid highly enriched in mature erythrocytes. Previous study has revealed that levels of S1P are significantly elevated in patients and mice with Sickle Cell Disease (SCD), a devastating and highly prevalent genetic hemolytic disorder that causes life-threatening hemolysis, tissue damage, and organ dysfunction with very limited treatment. Moreover, the activity of S1P generating enzyme-Sphingosine Kinase 1 (SphK1) is increased in human and mouse SCD erythrocytes, and inhibition of SphK1 decreased erythrocyte sickling. However, the structural and functional basis for the pathogenic nature of S1P in SCD remains obscure.Here, we report that increased erythrocyte S1P promotes pathogenic metabolic reprogramming coupled to increased channeling of glucose to glycolysis rather than through the pentose phosphate pathway (PPP). Suppressed PPP causes compromised glutathione homeostasis and increased oxidative stress, while enhanced glycolysis induces production of 2,3-bisphosphoglycerate (2,3-BPG) and thus increasing deoxygenated sickle Hb (deoxyHbS), deoxyHbS polymerization, sickling, hemolysis and disease progression. S1P functioning intracellularly binds to deoxyHbS, facilitates deoxyHbS anchoring to the membrane, induces release of membrane-bound glycolytic enzymes and in turn switches glucose flux towards glycolysis relative to the PPP. Extending from SCD, we unexpectedly found that S1P and 2,3-BPG work synergistically to decrease both HbA and HbS oxygen binding affinity. The crystal structure of HbA complexed with S1P alone or in combination with 2,3-BPG at 1.9 Å resolution revealed the overall architecture and unique features of S1P-2,3-BPG-deoxyHbA complex. In the presence of 2,3-BPG, S1P binds to the surface of 2,3-BPG-deoxyHbA and causes additional conformation changes to the T-state Hb. Phosphate moiety of the surface bound S1P engages in a highly positive region close to a1-heme while its aliphatic chain snakes along a shallow cavity making hydrophobic interactions in the "switch region", as well as with b2-heme like a molecular "sticky tape" with the last 3-4 carbon atoms sticking out into bulk solvent. Altogether, our findings provide functional and structural bases underlying pathogenic consequences of elevated S1P in SCD and its potential role in normal erythrocyte physiology.

Neue Antibiotikawerden gebraucht, weil Multiresistenzen, z. B. die von Staphylococcus aureus(MRSA), immer häufiger werden. Der erste Schritt der Biosynthese des vielversprechenden Wirkstoffs Moenomycin wird von der Prenyltransferase MoeO5 katalysiert, die eine Triosephosphatisomerase‐Fassstruktur hat, wie E. Oldfield, R.‐T. Guo et al. in der Zuschrift auf S. 4233 ff.zeigen. Die erste Doppelbindung der übertragenen Farnesylgruppe geht eine trans‐cis‐Isomerisierung ein.

New antibioticsare needed because of the increasing occurrence of multidrug resistance, as seen in Staphylococcus aureus(MRSA). The initiating step of the biosynthesis of the next‐generation drug moenomycin is catalyzed by MoeO5, which is a prenyltransferase with triose‐phosphate‐isomerase barrel structure as was shown by E. Oldfield, R.‐T. Guo, and co‐workers in their Communication on page 4157 ff.The transferred farnesyl group undergoes a trans‐to‐cisconversion in its first double bond.

Helicobacter pylori colonizes the human gastric epithelium and causes diseases such as gastritis, peptic ulcers, and stomach cancer. Undecaprenyl pyrophosphate synthase (UPPS), which catalyzes consecutive condensation reactions of farnesyl pyrophosphate with eight isopentenyl pyrophosphate to form lipid carrier for bacterial peptidoglycan biosynthesis, represents a potential target for developing new antibiotics. In this study, we solved the crystal structure of H. pylori UPPS and performed virtual screening of inhibitors from a library of 58,635 compounds. Two hits were found to exhibit differential activities against Helicobacter pylori and Escherichia coli UPPS, giving the possibility of developing antibiotics specially targeting pathogenic H. pylori without killing the intestinal E. coli.