Crystal Structures of β-1,4-Galactosyltransferase 7 Enzyme Reveal Conformational Changes and Substrate Binding

The β-1,4-galactosyltransferase 7 (β4GalT7) enzyme is involved in proteoglycan synthesis. In the presence of a manganese ion, it transfers galactose from UDP-galactose to xylose on a proteoglycan acceptor substrate. We present here the crystal structures of human β4GalT7 in open and closed conformations. A comparison of these crystal structures shows that, upon manganese and UDP or UDP-Gal binding, the enzyme undergoes conformational changes involving a small and a long loop. We also present the crystal structures of Drosophila wild-type β4GalT7 and D211N β4GalT7 mutant enzymes in the closed conformation in the presence of the acceptor substrate xylobiose and the donor substrate UDP-Gal, respectively. To understand the catalytic mechanism, we have crystallized the ternary complex of D211N β4GalT7 mutant enzyme in the presence of manganese with the donor and the acceptor substrates together in the same crystal structure. The galactose moiety of the bound UDP-Gal molecule forms seven hydrogen bonds with the protein molecule. The nonreducing end of the xylose moiety of xylobiose binds to the hydrophobic acceptor sugar binding pocket created by the conformational changes, whereas its extended xylose moiety forms hydrophobic interactions with a Tyr residue. In the ternary complex crystal structure, the nucleophile O4 oxygen atom of the xylose molecule is found in close proximity to the C1 and O5 atoms of the galactose moiety. This is the first time that a Michaelis complex of a glycosyltransferase has been described, and it clearly suggests an S_N2 type catalytic mechanism for the β4GalT7 enzyme.     

Intermediates in the Guanine Nucleotide Exchange Reaction of Rab8 Protein Catalyzed by Guanine Nucleotide Exchange Factors Rabin8 and GRAB

Small G-proteins of the Ras superfamily control the temporal and spatial coordination of intracellular signaling networks by acting as molecular on/off switches. Guanine nucleotide exchange factors (GEFs) regulate the activation of these G-proteins through catalytic replacement of GDP by GTP. During nucleotide exchange, three distinct substrate·enzyme complexes occur: a ternary complex with GDP at the start of the reaction (G-protein·GEF·GDP), an intermediary nucleotide-free binary complex (G-protein·GEF), and a ternary GTP complex after productive G-protein activation (G-protein·GEF·GTP). Here, we show structural snapshots of the full nucleotide exchange reaction sequence together with the G-protein substrates and products using Rabin8/GRAB (GEF) and Rab8 (G-protein) as a model system. Together with a thorough enzymatic characterization, our data provide a detailed view into the mechanism of Rabin8/GRAB-mediated nucleotide exchange.     

Role of Glycoside Phosphorylases in Mannose Foraging by Human Gut Bacteria

To metabolize both dietary fiber constituent carbohydrates and host glycans lining the intestinal epithelium, gut bacteria produce a wide range of carbohydrate-active enzymes, of which glycoside hydrolases are the main components. In this study, we describe the ability of phosphorylases to participate in the breakdown of human N-glycans, from an analysis of the substrate specificity of UhgbMP, a mannoside phosphorylase of the GH130 protein family discovered by functional metagenomics. UhgbMP is found to phosphorolyze β-d-Manp-1,4-β-d-GlcpNAc-1,4-d-GlcpNAc and is also a highly efficient enzyme to catalyze the synthesis of this precious N-glycan core oligosaccharide by reverse phosphorolysis. Analysis of sequence conservation within family GH130, mapped on a three-dimensional model of UhgbMP and supported by site-directed mutagenesis results, revealed two GH130 subfamilies and allowed the identification of key residues responsible for catalysis and substrate specificity. The analysis of the genomic context of 65 known GH130 sequences belonging to human gut bacteria indicates that the enzymes of the GH130_1 subfamily would be involved in mannan catabolism, whereas the enzymes belonging to the GH130_2 subfamily would rather work in synergy with glycoside hydrolases of the GH92 and GH18 families in the breakdown of N-glycans. The use of GH130 inhibitors as therapeutic agents or functional foods could thus be considered as an innovative strategy to inhibit N-glycan degradation, with the ultimate goal of protecting, or restoring, the epithelial barrier.     

Broad Salinity Tolerance as a Refuge from Predation in the Harmful Raphidophyte Alga Heterosigma akashiwo (Raphidophyceae)

The ability of harmful algal species to form dense, nearly monospecific blooms remains an ecological and evolutionary puzzle. We hypothesized that predation interacts with estuarine salinity gradients to promote blooms of Heterosigma akashiwo (Y. Hada) Y. Hada ex Y. Hara et M. Chihara, a cosmopolitan toxic raphidophyte. Specifically, H. akashiwo's broad salinity tolerance appears to provide a refuge from predation that enhances the net growth of H. akashiwo populations through several mechanisms. (1) Contrasting salinity tolerance of predators and prey. Estuarine H. akashiwo isolates from the west coast of North America grew rapidly at salinities as low as six, and distributed throughout experimental salinity gradients to salinities as low as three. In contrast, survival of most protistan predator species was restricted to salinities >15. (2) H. akashiwo physiological and behavioral plasticity. Acclimation to low salinity enhanced H. akashiwo's ability to accumulate and grow in low salinity waters. In addition, the presence of a ciliate predator altered H. akashiwo swimming behavior, promoting accumulation in low‐salinity surface layers inhospitable to the ciliate. (3) Negative effects of low salinity on predation processes. Ciliate predation rates decreased sharply at salinities <25 and, for one species, H. akashiwo toxicity increased at low salinities. Taken together, these behaviors and responses imply that blooms can readily initiate in low salinity waters where H. akashiwo would experience decreased predation pressure while maintaining near‐maximal growth rates. The salinity structure of a typical estuary would provide this HAB species a unique refuge from predation. Broad salinity tolerance in raphidophytes may have evolved in part as a response to selective pressures associated with predation.     

A Novel p53 Mutant Found in Iatrogenic Urothelial Cancers Is Dysfunctional and Can Be Rescued by a Second-site Global Suppressor Mutation

Exposure to herbal remedies containing the carcinogen aristolochic acid (AA) has been widespread in some regions of the world. Rare A→T TP53 mutations were recently discovered in AA-associated urothelial cancers. The near absence of these mutations among all other sequenced human tumors suggests that they could be biologically silent. There are no cell banks with established lines derived from human tumors with which to explore the influence of the novel mutants on p53 function and cellular behavior. To investigate their impact, we generated isogenic mutant clones by integrase-mediated cassette exchange at the p53 locus of platform (null) murine embryonic fibroblasts and kidney epithelial cells. Common tumor mutants (R248W, R273C) were compared with the AA-associated mutants N131Y, R249W, and Q104L. Assays of cell proliferation, migration, growth in soft agar, apoptosis, senescence, and gene expression revealed contrasting outcomes on cellular behavior following introduction of N131Y or Q104L. The N131Y mutant demonstrated a phenotype akin to common tumor mutants, whereas Q104L clone behavior resembled that of cells with wild-type p53. Wild-type p53 responses were restored in double-mutant cells harboring N131Y and N239Y, a second-site rescue mutation, suggesting that pharmaceutical reactivation of p53 function in tumors expressing N131Y could have therapeutic benefit. N131Y is likely to contribute directly to tumor phenotype and is a promising candidate biomarker of AA exposure and disease. Rare mutations thus do not necessarily point to sites where amino acid exchanges are phenotypically neutral. Encounter with mutagenic insults targeting cryptic sites can reveal specific signature hotspots.     

AlkB Dioxygenase Preferentially Repairs Protonated Substrates: SPECIFICITY AGAINST EXOCYCLIC ADDUCTS AND MOLECULAR MECHANISM OF ACTION*

Efficient repair by Escherichia coli AlkB dioxygenase of exocyclic DNA adducts 3,N⁴-ethenocytosine, 1,N⁶-ethenoadenine, 3,N⁴-α-hydroxyethanocytosine, and reported here for the first time 3,N⁴-α-hydroxypropanocytosine requires higher Fe(II) concentration than the reference 3-methylcytosine. The pH optimum for the repair follows the order of pK_a values for protonation of the adduct, suggesting that positively charged substrates favorably interact with the negatively charged carboxylic group of Asp-135 side chain in the enzyme active center. This interaction is supported by molecular modeling, indicating that 1,N⁶-ethenoadenine and 3,N⁴-ethenocytosine are bound to AlkB more favorably in their protonated cationic forms. An analysis of the pattern of intermolecular interactions that stabilize the location of the ligand points to a role of Asp-135 in recognition of the adduct in its protonated form. Moreover, ab initio calculations also underline the role of substrate protonation in lowering the free energy barrier of the transition state of epoxidation of the etheno adducts studied. The observed time courses of repair of mixtures of stereoisomers of 3,N⁴-α-hydroxyethanocytosine or 3,N⁴-α-hydroxypropanocytosine are unequivocally two-exponential curves, indicating that the respective isomers are repaired by AlkB with different efficiencies. Molecular modeling of these adducts bound by AlkB allowed evaluation of the participation of their possible conformational states in the enzymatic reaction.     

Roles of Long-range Electrostatic Domain Interactions and K+ in Phosphoenzyme Transition of Ca2+-ATPase

Sarcoplasmic reticulum Ca²⁺-ATPase couples the motions and rearrangements of three cytoplasmic domains (A, P, and N) with Ca²⁺ transport. We explored the role of electrostatic force in the domain dynamics in a rate-limiting phosphoenzyme (EP) transition by a systematic approach combining electrostatic screening with salts, computer analysis of electric fields in crystal structures, and mutations. Low KCl concentration activated and increasing salt above 0.1 m inhibited the EP transition. A plot of the logarithm of the transition rate versus the square of the mean activity coefficient of the protein gave a linear relationship allowing division of the activation energy into an electrostatic component and a non-electrostatic component in which the screenable electrostatic forces are shielded by salt. Results show that the structural change in the transition is sterically restricted, but that strong electrostatic forces, when K⁺ is specifically bound at the P domain, come into play to accelerate the reaction. Electric field analysis revealed long-range electrostatic interactions between the N and P domains around their hinge. Mutations of the residues directly involved and other charged residues at the hinge disrupted in parallel the electric field and the structural transition. Favorable electrostatics evidently provides a low energy path for the critical N domain motion toward the P domain, overcoming steric restriction. The systematic approach employed here is, in general, a powerful tool for understanding the structural mechanisms of enzymes.     

A New Way to Degrade Heme: THE MYCOBACTERIUM TUBERCULOSIS ENZYME MhuD CATALYZES HEME DEGRADATION WITHOUT GENERATING CO*?

MhuD is an oxygen-dependent heme-degrading enzyme from Mycobacterium tuberculosis with high sequence similarity (∼45%) to Staphylococcus aureus IsdG and IsdI. Spectroscopic and mutagenesis studies indicate that the catalytically active 1:1 heme-MhuD complex has an active site structure similar to those of IsdG and IsdI, including the nonplanarity (ruffling) of the heme group bound to the enzyme. Distinct from the canonical heme degradation, we have found that the MhuD catalysis does not generate CO. Product analyses by electrospray ionization-MS and NMR show that MhuD cleaves heme at the α-meso position but retains the meso-carbon atom at the cleavage site, which is removed by canonical heme oxygenases. The novel tetrapyrrole product of MhuD, termed “mycobilin,” has an aldehyde group at the cleavage site and a carbonyl group at either the β-meso or the δ-meso position. Consequently, MhuD catalysis does not involve verdoheme, the key intermediate of ring cleavage by canonical heme oxygenase enzymes. Ruffled heme is apparently responsible for the heme degradation mechanism unique to MhuD. In addition, MhuD heme degradation without CO liberation is biologically significant as one of the signals of M. tuberculosis transition to dormancy is mediated by the production of host CO.     

GENETIC ANALYSIS OF MATE DISCRIMINATION IN DROSOPHILA SIMULANS

Courtship is an elaborate behavior that conveys information about the identity of animal species and suitability of individual males as mates. In Drosophila, there is extensive evidence that females are capable of evaluating and comparing male courtships, and accepting or rejecting males as mates. These relatively simple responses minimize random sexual encounters involving subpar conspecific males and heterospecific males, and over generations can potentially select novel physical and behavioral traits. Despite its evolutionary and behavioral significance, little is still known about the genes involved in mating choice and how choices for novel males and females arise during evolution. Drosophila simulans and Drosophila sechellia are two recently diverged species of Drosophila in which females have a preference for conspecific males. Here we analyzed a total of 1748 F2 hybrid females between these two species and found a small number of dominant genes controlling the preference for D. simulans males. We also mapped two redundant X‐linked loci of mating choice, Macho‐XA and Macho‐XB, and show that neither one is required for female attractiveness. Together, our results reveal part of the genetic architecture that allows D. simulans females to recognize, mate, and successfully generate progenies with D. simulans males.     

Therapeutic and pharmacokinetic characterizations of an anti-amyloidogenic bis-styrylbenzene derivative for Alzheimer’s disease treatment

Alzheimer’s disease drug discovery regarding exploration into the molecules and processes has focused on the intrinsic causes of the brain disorder correlated with the accumulation of amyloid-β. An anti-amyloidogenic bis-styrylbenzene derivative, KMS80013, showed excellent oral bioavailability (F = 46.2%), facilitated brain penetration (26%, iv) in mouse and target specific in vivo efficacy in acute AD mouse model attenuating the cognitive deficiency in Y-maze test. Acute toxicity (LD₅₀ >2000 mg/kg) and hERG channel inhibition (14% at 10 μM) results indicated safety of KMS80013.