Carol M. Taylor

Glycosides of hydroxyproline: some recent, unusual discoveries.

Glycosides of hydroxyproline (Hyp) in the plant cell wall matrix were discovered by Lamport and co-workers in the 1960s. Since then, much has been learned about these Hyp-rich glycoproteins. The intent of this review was to compare and contrast some less common structural motifs, in nontraditional roles, to uncover themes. Arabinosylation of short-peptide plant hormones is essential for growth, cell differentiation and defense. In a very recent development, prolyl hydroxylase and arabinosyltransferase activity has been shown to have a direct impact on the growth of root hairs in Arabidopsis thaliana. Pollen allergens of mugwort and ragweed contain proline-rich domains that are hydroxylated and glycosylated and play a structural role. In the case of mugwort, this domain also presents a significant immunogenic epitope. Major crops, including tobacco and maize, have been used to express and produce recombinant proteins of mammalian origin. The risks of plant-imposed glycosylation are discussed. In unicellular eukaryotes, Skp1 (a subunit of the E3(SCF) ubiquitin ligase complex) harbors a key Hyp residue that is modified by a linear pentasaccharide. These modifications may be involved in sensing oxygen levels. A few studies have probed the impact of glycosylation on the structure of Hyp-containing peptides. These have necessarily looked at small, synthetic molecules, since natural peptides and proteins are often isolable in only minuscule amounts and/or are heterogeneous in nature. The characterization of native structural motifs, together with the determination of glycopeptide conformation and properties, holds the key to rationalizing natures architectural design.

Requirements for Skp1 processing by cytosolic prolyl 4(trans)-hydroxylase and α-N-acetylglucosaminyltransferase enzymes involved in O2 signaling in dictyostelium

The social amoeba Dictyostelium expresses a hypoxia inducible factor-α (HIFα) type prolyl 4-hydroxylase (P4H1) and an α-N-acetylglucosaminyltransferase (Gnt1) that sequentially modify proline-143 of Skp1, a subunit of the SCF (Skp1/Cullin/F-box protein) class of E3 ubiquitin ligases. Prior genetic studies have implicated Skp1 and its modification by these enzymes in O(2) regulation of development, suggesting the existence of an ancient O(2)-sensing mechanism related to modification of the transcription factor HIFα by animal prolyl 4-hydroxylases (PHDs). To better understand the role of Skp1 in P4H1-dependent O(2) signaling, biochemical and biophysical studies were conducted to characterize the reaction product and the basis of Skp1 substrate selection by P4H1 and Gnt1. (1)H NMR demonstrated formation of 4(trans)-hydroxyproline as previously found for HIFα, and highly purified P4H1 was inhibited by Krebs cycle intermediates and other compounds that affect animal P4Hs. However, in contrast to hydroxylation of HIFα by PHDs, P4H1 depended on features of full-length Skp1, based on truncation, mutagenesis, and competitive inhibition studies. These features are conserved during animal evolution, as even mammalian Skp1, which lacks the target proline, became a good substrate upon its restoration. P4H1 recognition may depend on features conserved for SCF complex formation as heterodimerization with an F-box protein blocked Skp1 hydroxylation. The hydroxyproline-capping enzyme Gnt1 exhibited similar requirements for Skp1 as a substrate. These and other findings support a model in which the protist P4H1 conditionally hydroxylates Skp1 of E3(SCF)ubiquitin ligases to control half-lives of multiple targets, rather than the mechanism of animal PHDs where individual proteins are hydroxylated leading to ubiquitination by the evolutionarily related E3(VBC)ubiquitin ligases.

Peptides containing γ,δ,-dihydroxy-L-leucine

(+/-)-Dehydroleucine was prepared and resolved by porcine kidney acylase. Under the conditions of the Sharpless asymmetric dihydroxylation (SAD), employing AD-mix-alpha, N alpha-carbobenzyloxy-(2S)-4,5-dehydroleucine methyl ester (16) gave rise to a 6.5:1.0 mixture of gamma-lactones 17, favoring the 4R configuration. Such carbamate-protected alpha-amino-gamma-hydroxylactones are not recommended as intermediates for peptide synthesis, since model studies showed that lactone 13 was unreactive toward amines. Moreover, the lactone ring could not be opened hydrolytically without epimerization at C alpha. N alpha-carbobenzyloxy-(2S)-4,5-dehydroleucine (22) was condensed with valine ethyl ester (19) to give dipeptide 23. Treatment of 23 with AD-mix-beta, under the SAD conditions, converted the dehydroleucine residue to gamma,delta-dihydroxyleucine with 4S configuration, as occurs in alloviroidin (3), a natural product isolated from Amanita suballiacea.

Design, synthesis, and antibacterial properties of dual-ligand inhibitors of acetyl-CoA carboxylase.

There is an urgent demand for the development of new antibiotics due to the increase in drug-resistant pathogenic bacteria. A novel target is the multifunctional enzyme acetyl-CoA carboxylase (ACC), which catalyzes the first committed step in fatty acid synthesis and consists of two enzymes: biotin carboxylase and carboxyltransferase. Covalently attaching known inhibitors against these enzymes with saturated hydrocarbon linkers of different lengths generated dual-ligand inhibitors. Kinetic results revealed that the dual-ligands inhibited the ACC complex in the nanomolar range. Microbiology assays showed that the dual-ligand with a 15-carbon linker did not exhibit any antibacterial activity, while the dual-ligand with a 7-carbon linker displayed broad-spectrum antibacterial activity as well as a decreased susceptibility in the development of bacterial resistance. These results suggest that the properties of the linker are vital for antibacterial activity and show how inhibiting two different enzymes with the same compound increases the overall potency while also impeding the development of resistance.

Conformational changes associated with post-translational modifications of pro143 in Skp1 of dictyostelium -a dipeptide model system

Prolyl hydroxylation and subsequent glycosylation of the E3SCF ubiquitin ligase subunit Skp1 affects its conformation and its interaction with F-box proteins and, ultimately, O2-sensing in the organism. Taking a reductionist approach to understand the molecular basis for these effects, a series of end-capped Thr-Pro dipeptides was synthesized, tracking the sequential post-translational modifications that occur in the protein. The conformation of the pyrrolidine ring in each compound was gauged via coupling constants (3JHα,Hβ) and the electronegativity of the Cγ-substituents by chemical shifts (13C). The equilibrium between the cis–trans conformations about the central prolyl peptide bond was investigated by integration of signals corresponding to the two species in the 1H NMR spectra over a range of temperatures. These studies revealed an increasing preference for the trans-conformation in the order Pro < Hyp < [α-(1,4)GlcNAc]Hyp. Rates for the forward and reverse reactions, determined by magnetization transfer experiments, demonstrated a reduced rate for the trans-to-cis conversion and a significant increase in the cis-to-trans conversion upon hydroxylation of the proline residue in the dipeptide. NOE experiments suggest that the Thr side chain pushes the sugar away from the pyrrolidine ring. These effects, which depended on the presence of the N-terminal Thr residue, offer a mechanism to explain altered properties of the corresponding full-length proteins.

Crystal Structure of Carboxyltransferase from Staphylococcus aureus Bound to the Antibacterial Agent Moiramide B.

The dramatic increase in the prevalence of antibiotic-resistant bacteria has necessitated a search for new antibacterial agents against novel targets. Moiramide B is a natural product, broad-spectrum antibiotic that inhibits the carboxyltransferase component of acetyl-CoA carboxylase, which catalyzes the first committed step in fatty acid synthesis. Herein, we report the 2.6 Å resolution crystal structure of moiramide B bound to carboxyltransferase. An unanticipated but significant finding was that moiramide B bound as the enol/enolate. Crystallographic studies demonstrate that the (4S)-methyl succinimide moiety interacts with the oxyanion holes of the enzyme, supporting the notion that an anionic enolate is the active form of the antibacterial agent. Structure-activity studies demonstrate that the unsaturated fatty acid tail of moiramide B is needed only for entry into the bacterial cell. These results will allow the design of new antibacterial agents against the bacterial form of carboxyltransferase.

Influence of Sulfur on Acid-Mediated Enamide Formation

The acid-mediated condensation of acetamide with butanal dimethylacetal and EtSCH2CH(OMe)2, followed by dehydration, was investigated by electronic structure calculations that supported the prediction that the Z-geometry would be favored in the product. The reaction was investigated experimentally using suitably functionalized cysteine building blocks. Some side reactions and optimization of reaction conditions are reported, en route to identifying a mild, inexpensive Lewis acid that achieves a reasonable yield of (Z)-thioenamide 21 with high stereoselectivity.

Generating Isoform-Specific Antibodies : Lessons from Nucleocytoplasmic Glycoprotein Skp1

Antibodies that discriminate protein isoforms differing by modifications at specific amino acids have revolutionized studies of their functions. Skp1 is a novel nucleocytoplasmic glycoprotein that is hydroxylated at proline-143 and then O-glycosylated by a pentasaccharide attached via a GlcNAcα1, 4(trans)- hydroxyproline linkage. Skp1 isoform-specific antibodies were successfully obtained by immunizing mice or rabbits with KLH-coupled synthetic peptides bearing either unmodified Pro, 4(trans)-hydroxyproline, or D-GlcNAcα1,4 (trans)-hydroxyproline, and screening with corresponding BSA-conjugates or by Western blotting toward a panel of Skp1 isoforms. Antibodies specific for Skp1 or HO-Skp1 were not found in exhaustive murine trials, yet monospecific polyclonal antibodies were readily achieved in rabbits without crossadsorption. In all cases, antibodies were specific at the protein but not the peptide level, which suggests that conformation comprises part of the basis for recognition and which should be considered when developing screening strategies.