David L. Jakeman

Culture conditions improving the production of jadomycin B.

The jadomycins are a unique family of benzoxazolophenanthridine antibiotics produced by Streptomyces venezuelae ISP5230 following heat or ethanol shock or phage infection. We have modified the culture conditions by altering the carbon source, buffer, inoculum size, and timing of ethanol shock, thereby reducing growing times and improving jadomycin B production. Our optimized conditions use glucose as the carbon source, MOPS as buffer, low concentrations of phosphate, a defined inoculum concentration and an immediate ethanol shock to induce jadomycin B production; results that contrast previous studies. The altered media will facilitate the isolation of related jadomycin B congeners.

Antimicrobial activities of jadomycin B and structurally related analogues.

ABSTRACT Natural products are leads for new antibiotics as a result of their structural complexity and diversity. We have isolated a series of structurally related polyketide-derived natural products from Streptomyces venezuelae ISP5230. The most active of these jadomycin analogues showed good activity against a variety of staphylococci, including methicillin-resistant Staphylococcus aureus.

Stereochemical Integrity of Oxazolone Ring-Containing Jadomycins

The jadomycins are a series of natural products produced by Streptomyces venzuelae ISP5230 in response to ethanol shock. A unique structural feature of these angucyclines is the oxazolone ring, the formation of which is catalyzed by condensation of a biosynthetic aldehyde intermediate and an amino acid. The feeding of enantiomeric forms of α‐amino acids indicates that the amino acid is incorporated by S. venezuelae ISP5230 without isomerization at the α‐carbon. The characterization of the first two six‐membered E‐ring‐containing jadomycins is reported. These precursor‐directed biosynthesis studies indicate flexibility in the acceptor substrate specificity of the glycosyltransferase, JadS. Analysis of cytotoxicity data against two human breast cancer cell lines indicates that the nature of the substitution at the α‐carbon, rather than the stereochemistry, influences biological activity.

Structure-Activity Analysis of Quorum-Sensing Signaling Peptides from Streptococcus mutans

Streptococcus mutans secretes and utilizes a 21-amino-acid signaling peptide pheromone to initiate quorum sensing for genetic competence, biofilm formation, stress responses, and bacteriocin production. In this study, we designed and synthesized a series of truncated peptides and peptides with amino acid substitutions to investigate their structure-activity relationships based on the three-dimensional structures of S. mutans wild-type signaling peptide UA159sp and C-terminally truncated peptide TPC3 from mutant JH1005 defective in genetic competence. By analyzing these peptides, we demonstrated that the signaling peptide of S. mutans has at least two functional domains. The C-terminal structural motif consisting of a sequence of polar hydrophobic charged residues is crucial for activation of the signal transduction pathway, while the core alpha-helical structure extending from residue 5 to the end of the peptide is required for receptor binding. Peptides in which three or more residues were deleted from the C terminus did not induce genetic competence but competitively inhibited quorum sensing activated by UA159sp. Disruption of the amphipathic alpha-helix by replacing the Phe-7, Phe-11, or Phe-15 residue with a hydrophilic residue resulted in a significant reduction in or complete loss of the activity of the peptide. In contrast to the C-terminally truncated peptides, these peptides with amino acid substitutions did not compete with UA159sp to activate quorum sensing, suggesting that disruption of the hydrophobic face of the alpha-helical structure results in a peptide that is not able to bind to the receptor. This study is the first study to recognize the importance of the signaling peptide C-terminal residues in streptococcal quorum sensing.

Myristoylation, a Protruding Loop, and Structural Plasticity Are Essential Features of a Nonenveloped Virus Fusion Peptide Motif

Members of the fusion-associated small transmembrane (FAST) protein family are a distinct class of membrane fusion proteins encoded by nonenveloped fusogenic reoviruses. The 125-residue p14 FAST protein of reptilian reovirus has an ∼38-residue myristoylated N-terminal ectodomain containing a moderately apolar N-proximal region, termed the hydrophobic patch. Mutagenic analysis indicated sequence-specific elements in the N-proximal portion of the p14 hydrophobic patch affected cell-cell fusion activity, independent of overall effects on the relative hydrophobicity of the motif. Circular dichroism (CD) of a myristoylated peptide representing the majority of the p14 ectodomain suggested this region is mostly disordered in solution but assumes increased structure in an apolar environment. From NMR spectroscopic data and simulated annealing, the soluble nonmyristoylated p14 ectodomain peptide consists of an N-proximal extended loop flanked by two proline hinges. The remaining two-thirds of the ectodomain peptide structure is disordered, consistent with predictions based on CD spectra of the myristoylated peptide. The myristoylated p14 ectodomain peptide, but not a nonmyristoylated version of the same peptide nor a myristoylated scrambled peptide, mediated extensive lipid mixing in a liposome fusion assay. Based on the lipid mixing activity, structural plasticity, environmentally induced conformational changes, and kinked structures predicted for the p14 ectodomain and hydrophobic patch (all features associated with fusion peptides), we propose that the majority of the p14 ectodomain is composed of a fusion peptide motif, the first such motif dependent on myristoylation for membrane fusion activity.

Chemoenzymatic Synthesis, Inhibition Studies, and X-ray Crystallographic Analysis of the Phosphono Analog of UDP-Galp as an Inhibitor and Mechanistic Probe for UDP-Galactopyranose Mutase

UDP (uridine diphosphate) galactopyranose mutase (UGM) is involved in the cell wall biosynthesis of many pathogenic microorganisms. UGM catalyzes the reversible conversion of UDP-α-D-galactopyranose into UDP-α-D-galactofuranose, with the latter being the precursor of galactofuranose (Galf) residues in cell walls. Glycoconjugates of Galf are essential components in the cell wall of various pathogenic bacteria, including Mycobacterium tuberculosis, the causative agent of tuberculosis. The absence of Galf in humans and its bacterial requirement make UGM a potential target for developing novel antibacterial agents. In this article, we report the synthesis, inhibitory activity, and X-ray crystallographic studies of UDP-phosphono-galactopyranose, a nonhydrolyzable C-glycosidic phosphonate. This is the first report on the synthesis of a phosphonate analog of UDP-α-D-galactopyranose by a chemoenzymatic phosphoryl coupling method. The phosphonate was evaluated against three bacterial UGMs and showed only moderate inhibition. We determined the crystal structure of the phosphonate analog bound to Deinococcus radiodurans UGM at 2.6 Å resolution. The phosphonate analog is bound in a novel conformation not observed in UGM-substrate complex structures or in other enzyme-sugar nucleotide phosphonate complexes. This complex structure provides a structural basis for the observed micromolar inhibition towards UGM. Steric clashes, loss of electrostatic stabilization between an active-site arginine (Arg305) and the phosphonate analog, and a 180° flip of the hexose moiety account for the differences in the binding orientations of the isosteric phosphonate analog and the physiological substrate. This provides new insight into the ability of a sugar-nucleotide-binding enzyme to orient a substrate analog in an unexpected geometry and should be taken into consideration in designing such enzyme inhibitors.

Diverse DNA-cleaving capacities of the jadomycins through precursor-directed biosynthesis.

Gel mobility assays were used to establish that some members of the jadomycin family of natural products act as DNA cleaving agents. Moreover, it was found that subtle structural changes generated through the use of precursor-directed biosynthesis lead to marked effects on the DNA-damaging properties of these glycosylated polyketide-derived natural products.

α-Fluorophosphonates reveal how a phosphomutase conserves transition state conformation over hexose recognition in its two-step reaction.

Significance Enzymes that use the same active site to catalyze two native, sequential reactions are extraordinary. Structural studies of phosphohexose mutases are particularly informative, permitting direct comparison of the organization of catalysis of phosphoryl transfer involving two different substrates. The present study of β-phosphoglucomutase (βPGM) deploys chemical synthesis of substrate analogs to enable detailed NMR and X-ray structural analysis of both steps of its catalytic activity. It reveals how βPGM conserves fidelity of transition state organization while maintaining substrate recognition for its two steps by prioritizing positioning of both phosphates over direct hexose recognition for the second step. It identifies the structural basis for the strong discrimination by βPGM between two, diastereoisomeric α-fluoromethylenephosphonate analogs of β-d-glucose 1-phosphate. β-Phosphoglucomutase (βPGM) catalyzes isomerization of β-d-glucose 1-phosphate (βG1P) into d-glucose 6-phosphate (G6P) via sequential phosphoryl transfer steps using a β-d-glucose 1,6-bisphosphate (βG16BP) intermediate. Synthetic fluoromethylenephosphonate and methylenephosphonate analogs of βG1P deliver novel step 1 transition state analog (TSA) complexes for βPGM, incorporating trifluoromagnesate and tetrafluoroaluminate surrogates of the phosphoryl group. Within an invariant protein conformation, the β-d-glucopyranose ring in the βG1P TSA complexes (step 1) is flipped over and shifted relative to the G6P TSA complexes (step 2). Its equatorial hydroxyl groups are hydrogen-bonded directly to the enzyme rather than indirectly via water molecules as in step 2. The (C)O–P bond orientation for binding the phosphate in the inert phosphate site differs by ∼30° between steps 1 and 2. By contrast, the orientations for the axial O–Mg–O alignment for the TSA of the phosphoryl group in the catalytic site differ by only ∼5°, and the atoms representing the five phosphorus-bonded oxygens in the two transition states (TSs) are virtually superimposable. The conformation of βG16BP in step 1 does not fit into the same invariant active site for step 2 by simple positional interchange of the phosphates: the TS alignment is achieved by conformational change of the hexose rather than the protein.

Eight-membered ring-containing jadomycins: implications for non-enzymatic natural products biosynthesis.

Jadomycin Oct (1) was isolated from Streptomyces venezuelae ISP5230 and characterized as a structurally unique eight-membered l-ornithine ring-containing jadomycin. The structure was elucidated through the semisynthetic derivatization of starting material via chemoselective acylation of the l-ornithine α-amino group using activated succinimidyl esters. Incorporation of 5-aminovaleric acid led to jadomycin AVA, a second eight-membered ring-containing jadomycin. These natural products illustrate the structural diversity permissible from a non-enzymatic step within a biosynthetic pathway and exemplifies the potential for discovery of novel scaffolds.

Engineering Ribonucleoside Triphosphate Specificity in a Thymidylyltransferase

Natures glycosylation catalysts, glycosyltransferases, indirectly manipulate and control many important biological processes by transferring sugar nucleotide donors onto acceptors. Challenging chemical synthesis impedes synthetic access to sugar nucleotides and limits the study of many glycosyltransferases. Enzymatic access to sugar nucleotides is a rapidly expanding avenue of research, limited only by the substrate specificity of the enzyme. We have explored the promiscuous thymidylyltransferase from Streptococcus pneumoniae, Cps2L, and enhanced its uridylyltransferase and guanidyltransferase activities by active site engineering. Mutagenesis at position Q24 resulted in a variant with 10-, 3-, and 2-fold enhancement of UDP-glucosamine, UDP-mannose, and UDP- N-acetylglucosamine production, respectively. New catalytic activities were observed for the Cps2L variant over the wild-type enzyme, including the formation of GDP-mannose. The variant was evaluated as a catalyst for the formation of a series of dTDP- and UDP-furanoses and notably produced dTDP-Gal f in 90% yield and UDP-Ara f in 30% yield after 12 h. A series of 3- O-alkylglucose 1-phosphates were also evaluated as substrates, and notable conversions to UDP-3- O-methylglucose and UDP-3- O-dodecylglucose were achieved with the variant but not the wild-type enzyme. The Q24S variant also enhanced essentially all thymidylyltransferase activities relative to the wild-type enzyme. Comparison of active sites of uridylyltransferases and thymidylyltransferases with products bound indicate the Q24S variant to be a new approach in broadening nucleotidylyltransferase activity.