René Jørgensen

Structural and mechanistic basis for a new mode of glycosyltransferase inhibition.

Glycosyltransferases are carbohydrate-active enzymes with essential roles in numerous important biological processes. We have developed a novel donor analogue for galactosyltransferases which locks a representative target enzyme in a catalytically inactive conformation, thus almost completely abolishing sugar transfer. Results with other galactosyltransferases suggest that this novel and unique mode of glycosyltransferase inhibition is, very likely, generally applicable to other members of this very important enzyme family also.

Weak A phenotypes associated with novel ABO alleles carrying the A2‐related 1061C deletion and various missense substitutions

BACKGROUND: The 1061delC single‐nucleotide polymorphism (SNP) has been reported mostly in the context of the common A2[A201] allele and typically produces an A2 phenotype. This study evaluated new Aweak alleles, each containing 1061delC.

Yeast as a tool for characterizing mono‐ADP‐ribosyltransferase toxins

The emergence of bacterial antibiotic resistance poses a significant challenge in the pursuit of novel therapeutics, making new strategies for drug discovery imperative. We have developed a yeast growth-defect phenotypic screen to help solve this current dilemma. This approach facilitates the identification and characterization of a new diphtheria toxin (DT) group, ADP-ribosyltransferase toxins from pathogenic bacteria. In addition, this assay utilizes Saccharomyces cerevisiae, a reliable model for bacterial toxin expression, to streamline the identification and characterization of new inhibitors against this group of bacterial toxins that may be useful for antimicrobial therapies. We show that a mutant of the elongation factor 2 target protein in yeast, G701R, confers resistance to all DT group toxins and recovers the growth-defect phenotype in yeast. We also demonstrate the ability of a potent small-molecule toxin inhibitor, 1,8-naphthalimide (NAP), to alleviate the growth defect caused by toxin expression in yeast. Moreover, we determined the crystal structure of the NAP inhibitor-toxin complex at near-atomic resolution to provide insight into the inhibitory mechanism. Finally, the NAP inhibitor shows therapeutic protective effects against toxin invasion of mammalian cells, including human lung cells.

Newly discovered and characterized antivirulence compounds inhibit bacterial mono-ADP-ribosyltransferase toxins.

ABSTRACT The mono-ADP-ribosyltransferase toxins are bacterial virulence factors that contribute to many disease states in plants, animals, and humans. These toxins function as enzymes that target various host proteins and covalently attach an ADP-ribose moiety that alters target protein function. We tested compounds from a virtual screen of commercially available compounds combined with a directed poly(ADP-ribose) polymerase (PARP) inhibitor library and found several compounds that bind tightly and inhibit toxins from Pseudomonas aeruginosa and Vibrio cholerae. The most efficacious compounds completely protected human lung epithelial cells against the cytotoxicity of these bacterial virulence factors. Moreover, we determined high-resolution crystal structures of the best inhibitors in complex with cholix toxin to reveal important criteria for inhibitor binding and mechanism of action. These results provide new insight into development of antivirulence compounds for treating many bacterial diseases.

Stepwise emergence of azole, echinocandin and amphotericin B multidrug resistance in vivo in Candida albicans orchestrated by multiple genetic alterations

OBJECTIVESnThe objective of this study was to characterize the underlying molecular mechanisms in consecutive clinical Candida albicans isolates from a single patient displaying stepwise-acquired multidrug resistance.nnnMETHODSnNine clinical isolates (P-1 to P-9) were susceptibility tested by EUCAST EDef 7.2 and Etest. P-4, P-5, P-7, P-8 and P-9 were available for further studies. Relatedness was evaluated by MLST. Additional genes were analysed by sequencing (including FKS1, ERG11, ERG2 and TAC1) and gene expression by quantitative PCR (CDR1, CDR2 and ERG11). UV-spectrophotometry and GC-MS were used for sterol analyses. In vivo virulence was determined in the insect model Galleria mellonella and evaluated by log-rank Mantel-Cox tests.nnnRESULTSnP-1u200a+u200aP-2 were susceptible, P-3u200a+u200aP-4 fluconazole resistant, P-5 pan-azole resistant, P-6u200a+u200aP-7 pan-azole and echinocandin resistant and P-8u200a+u200aP-9 MDR. MLST supported genetic relatedness among clinical isolates. P-4 harboured four changes in Erg11 (E266D, G307S, G450E and V488I), increased expression of ERG11 and CDR2 and a change in Tac1 (R688Q). P-5, P-7, P-8 and P-9 had an additional change in Erg11 (A61E), increased expression of CDR1, CDR2 and ERG11 (except for P-7) and a different amino acid change in Tac1 (R673L). Echinocandin-resistant isolates harboured the Fks1 S645P alteration. Polyene-resistant P-8u200a+u200aP-9 lacked ergosterol and harboured a frameshift mutation in ERG2 (F105SfsX23). Virulence was attenuated (but equivalent) in the clinical isolates, but higher than in the azole- and echinocandin-resistant unrelated control strain.nnnCONCLUSIONSnC. albicans demonstrates a diverse capacity to adapt to antifungal exposure. Potentially novel resistance-inducing mutations in TAC1, ERG11 and ERG2 require independent validation.

Base-modified Donor Analogues Reveal Novel Dynamic Features of a Glycosyltransferase.

Background: Modified UDP-Gal donor substrates with 5-formylthienyl and 5-phenyl substituents on the uracil base exhibit differential inhibition patterns for glycosyltransferases. Results: Structural studies reveal a new enzyme loop folding mode for the 5-formylthienyl analogue. Conclusion: Differential inhibition is attributed to alternate enzyme conformational changes and interactions with the respective inhibitors. Significance: The conformational plasticity of glycosyltransferases can be exploited in designing novel inhibitors. Glycosyltransferases (GTs) are enzymes that are involved, as Natures “glycosylation reagents,” in many fundamental biological processes including cell adhesion and blood group biosynthesis. Although of similar importance to that of other large enzyme families such as protein kinases and proteases, the undisputed potential of GTs for chemical biology and drug discovery has remained largely unrealized to date. This is due, at least in part, to a relative lack of GT inhibitors and tool compounds for structural, mechanistic, and cellular studies. In this study, we have used a novel class of GT donor analogues to obtain new structural and enzymological information for a representative blood group GT. These analogues interfere with the folding of an internal loop and the C terminus, which are essential for catalysis. Our experiments have led to the discovery of an entirely new active site folding mode for this enzyme family, which can be targeted in inhibitor development, similar to the DFG motif in protein kinases. Taken together, our results provide new insights into substrate binding, dynamics, and utilization in this important enzyme family, which can very likely be harnessed for the rational development of new GT inhibitors and probes.

Identification of catalytically important amino acid residues for enzymatic reduction of glyoxylate in plants

NADPH-dependent glyoxylate reductases from Arabidopsis thaliana (AtGLYR) convert both glyoxylate and succinic semialdehyde into their corresponding hydroxyacid equivalents. The primary sequence of cytosolic AtGLYR1 reveals several sequence elements that are consistent with the β-HAD (β-hydroxyacid dehydrogenase) protein family, whose members include 3-hydroxyisobutyrate dehydrogenase, tartronate semialdehyde reductase and 6-phosphogluconate dehydrogenase. Here, site-directed mutagenesis was utilized to identify catalytically important amino acid residues for glyoxylate reduction in AtGLYR1. Kinetic studies and binding assays established that Lys170 is essential for catalysis, Phe231, Asp239, Ser121 and Thr95 are more important in substrate binding than in catalysis, and Asn174 is more important in catalysis. The low activity of the mutant enzymes precluded kinetic studies with succinic semialdehyde. The crystal structure of AtGLYR1 in the absence of substrate was solved to 2.1Å by molecular replacement using a previously unrecognized member of the β-HAD family, cytokine-like nuclear factor, thereby enabling the 3-D structure of the protein to be modeled with substrate and co-factor. Structural alignment of AtGLYR1 with β-HAD family members provided support for the essentiality of Lys170, Phe173, Asp239, Ser121, Asn174 and Thr95 in the active site and preliminary support for an acid/base catalytic mechanism involving Lys170 as the general acid and a conserved active-site water molecule. This information established that AtGLYR1 is a member of the β-HAD protein family. Sequence and activity comparisons indicated that AtGLYR1 and the plastidial AtGLYR2 possess structural features that are absent in Arabidopsis hydroxypyruvate reductases and probably account for their stronger preference for glyoxylate over hydroxypyruvate.

Novel UDP-GalNAc Derivative Structures Provide Insight into the Donor Specificity of Human Blood Group Glycosyltransferase.

Two closely related glycosyltransferases are responsible for the final step of the biosynthesis of ABO(H) human blood group A and B antigens. The two enzymes differ by only four amino acid residues, which determine whether the enzymes transfer GalNAc from UDP-GalNAc or Gal from UDP-Gal to the H-antigen acceptor. The enzymes belong to the class of GT-A folded enzymes, grouped as GT6 in the CAZy database, and are characterized by a single domain with a metal dependent retaining reaction mechanism. However, the exact role of the four amino acid residues in the specificity of the enzymes is still unresolved. In this study, we report the first structural information of a dual specificity cis-AB blood group glycosyltransferase in complex with a synthetic UDP-GalNAc derivative. Interestingly, the GalNAc moiety adopts an unusual yet catalytically productive conformation in the binding pocket, which is different from the “tucked under” conformation previously observed for the UDP-Gal donor. In addition, we show that this UDP-GalNAc derivative in complex with the H-antigen acceptor provokes the same unusual binding pocket closure as seen for the corresponding UDP-Gal derivative. Despite this, the two derivatives show vastly different kinetic properties. Our results provide a important structural insight into the donor substrate specificity and utilization in blood group biosynthesis, which can very likely be exploited for the development of new glycosyltransferase inhibitors and probes.

Protein expression, characterization, crystallization and preliminary X-ray crystallographic analysis of a Fic protein from Clostridium difficile.

Fic domains in proteins are found in abundance in nature from the simplest prokaryotes to animals. Interestingly, Fic domains found in two virulence factors of Gram-negative bacteria have recently been demonstrated to catalyse the transfer of the AMP moiety from ATP to small host GTPases. This post-translational modification has attracted considerable interest and a role for adenylylation in pathology and physiology is emerging. This work was aimed at the structural characterization of a newly identified Fic protein of the Gram-positive bacterium Clostridium difficile. A constitutively active inhibitory helix mutant of C. difficile Fic was overexpressed in Escherichia coli, purified and crystallized by the vapour-diffusion technique. Preliminary X-ray crystallographic analysis shows that the crystals diffract to at least 1.68u2005Å resolution at a synchrotron X-ray source. The crystals belonged to the orthorhombic space group P2₁2₁2₁, with unit-cell parameters a=45.6, b=80.8, c=144.7u2005Å, α=β=γ=90°. Two molecules per asymmetric unit corresponds to a Matthews coefficient of 2.37u2005Å3u2005Da(-1) and a solvent content of 48%.

Biophysical comparison of diphtheria and tetanus toxins with the formaldehyde-detoxified toxoids, the main components of diphtheria and tetanus vaccines

Diphtheria and tetanus vaccines are produced by chemical detoxification of the native toxins through formaldehyde treatment. Diphtheria toxin (DT)# is secreted from the pathogenic Corynebacterium d...