Stephanie M. Forget

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.

Synthesis and enzymatic evaluation of ketose phosphonates: the interplay between mutarotation, monofluorination and acidity

Ketose-phosphonates may adopt open chain, or α- or β-furanosyl, or α- or β-pyranosyl configurational isomers in aqueous solution. An HPLC and NMR analysis of a series of ketose-phosphonates with a thymidylyltransferase (dTDP-glucose pyrophosphorylase) implied a rapid dynamic equilibrium between the pyranosyl forms of gluco-ketose phosphonate leading to efficient production of unique sugar nucleotide analogues. The preparation of diastereomerically pure gluco-configured monofluoromethylenephosphonates enabled the determination of the thymidylyltransferase preference for CHF stereochemistry. The effects of acidity upon thymidylyltransferase substrate specificity were determined using a series of monofluoro- and difluoro- ketose-phosphonates. WaterLOGSY NMR spectroscopy demonstrated a switching of the ordered Bi-Bi mechanism with ketose-phosphonate substrates. Ketose-phosphonates are presented as a unique class of sugar 1-phosphate analogues with potential applications as glycosyltransferase probes.

Synthesis and evaluation of l-rhamnose 1C-phosphonates as nucleotidylyltransferase inhibitors.

We report the synthesis of a series of phosphonates and ketosephosphonates possessing an L-rhamnose scaffold with varying degrees of fluorination. These compounds were evaluated as potential inhibitors of α-D-glucose 1-phosphate thymidylyltransferase (Cps2L), the first enzyme in Streptococcus pneumoniae L-rhamnose biosynthesis, and a novel antibiotic target. Enzyme-substrate and enzyme-inhibitor binding experiments were performed using water-ligand observed binding via gradient spectroscopy (WaterLOGSY) NMR for known sugar nucleotide substrates and selected phosphonate analogues. IC50 values were measured and Ki values were calculated for inhibitors. New insights were gained into the binding promiscuity of enzymes within the prokaryotic L-rhamnose biosynthetic pathway (Cps2L, RmlB-D) and into the mechanism of inhibition for the most potent inhibitor in the series, L-rhamnose 1C-phosphonate.

Post Polyketide Synthase Carbon–Carbon Bond Formation in Type-II PKS-Derived Natural Products from Streptomyces venezuelae

Polyketide synthase (PKS) derived natural products are biosynthesized by head-to-tail addition of acetate and malonate extender units resulting in linear extended-polyketide chains. Despite the well-documented structural diversity associated with PKS-derived natural products, C-C chain branching deviating from the usual linear pattern is relatively rare. Herein, type-II PKS angucyclic natural products containing a hemiaminal functionality were identified and proposed as the parent of a series of C-C-branched analogues. These C-C linked acetate or pyruvate branching units were located at the α-positions on the extended polyketide chains of jadomycins incorporating 3- and 4-aminomethylbenzoic acids. Labeling studies utilizing [1-13C]-d-glucose provided mechanistic evidence that the C-C bond formation occurred as a result of a previously unidentified post-PKS processing, additional to the enzymes encoded within the biosynthetic gene cluster. Selected compounds were evaluated in cytotoxic or antimicrobial assays.

JadX is a Disparate Natural Product Binding Protein

We report that JadX, a protein of previously undetermined function coded for in the jadomycin biosynthetic gene cluster of Streptomyces venezuelae ISP5230, affects both chloramphenicol and jadomycin production levels in blocked mutants. Characterization of recombinant JadX through protein-ligand interactions by chemical shift perturbation and WaterLOGSY NMR spectroscopy resulted in the observation of binding between JadX and a series of jadomycins and between JadX and chloramphenicol, another natural product produced by S. venezuelae ISP5230. These results suggest JadX to be an unusual class of natural product binding protein involved in binding structurally disparate natural products. The ability for JadX to bind two different natural products in vitro and the ability to affect production of these secondary metabolites in vivo suggest a potential role in regulation or signaling. This is the first example of functional characterization of these JadX-like proteins, and provides insight into a previously unobserved regulatory process.

Furan and lactam jadomycin biosynthetic congeners Isolated from Streptomyces venezuelae ISP5230 cultured with Nε-Trifluoroacetyl-l-lysine

Angucycline antibiotics are composed of a classical four-ring angularly linked polyaromatic backbone. Differential cyclization chemistry of the A- and B-rings in jadomycin biosynthesis led to the discovery of two new furan analogues, while oxidation led to a ring-opened form of the jadomycin Nε-trifluoroacetyl-l-lysine (TFAL) congener. The compounds were isolated from Streptomyces venezuelae ISP5230 cultures grown with TFAL. Biosynthetic incorporation using d-[1-13C]-glucose in cultures enabled the unambiguous assignment of the aldehyde, alcohol, and amide functionalities present in these new congeners through NMR spectroscopy. Tandem mass spectrometry analysis of cultures grown with 15Nα- or 15Nε-lysine demonstrated the incorporation of Nα exclusively into the angucycline backbone, contrasting results with ornithine [J. Am. Chem. Soc. 2015, 137, 3271]. Compounds were evaluated against antimicrobial and cancer cell panels and found to possess good activity against Gram-positive bacteria.

Streptomyces venezuelae ISP5230 Maintains Excretion of Jadomycin upon Disruption of the MFS Transporter JadL Located within the Natural Product Biosynthetic Gene Cluster

JadL was identified as a Major Facilitator Superfamily (MFS) transporter (T.C. 2.A.1) through sequence homology. The protein is encoded by jadL, situated within the jadomycin biosynthetic gene cluster. JadL has, therefore, been assigned a putative role in host defense by exporting its probable substrates, the jadomycins, a family of secondary metabolites produced by Streptomyces venezuelae ISP5230. Herein, we evaluate this assumption through the construction and analysis of a jadL disrupted mutant, S. venezuelae VS678 (ΔjadL::aac(3)IV). Quantitative determination of jadomycin production with the jadL disrupted mutant did not show a significant decrease in production in comparison to the wildtype strain, as determined by HPLC and by tandem mass spectrometry. These results suggest that efflux of jadomycin occurs upon disruption of jadL, or that JadL is not involved in jadomycin efflux. Potentially, other transporters within S. venezuelae ISP5230 may adopt this role upon inactivation of JadL to export jadomycins.

Isolation of a jadomycin incorporating l -ornithine, analysis of antimicrobial activity and jadomycin reactive oxygen species (ROS) generation in MDA-MB-231 breast cancer cells

Herein, we report the characterization and antimicrobial activity of a previously unreported jadomycin (1) obtained from a culture of S. venezuelae ISP5230 with l-ornithine (Orn). 1 arises from the rearrangement of a putative five-membered ring containing jadomycin incorporating Orn, whereby intramolecular attack of the E-ring carbonyl from the δ-NH2 group of the Orn side chain results in collapse of the oxazolone ring and formation of a stable six-membered lactam. This rearrangement produces a jadomycin with a 3a hemiaminal position that is susceptible to solvolysis. A structure–activity relationship is discussed based on the antimicrobial activity of 1 compared to previously reported jadomycins, providing evidence that the presence of a 3a hemiaminal enhances activity against Gram-positive bacteria. Additionally, assays to quantify reactive oxygen species (ROS) generation and cell viability were performed using a series of nine jadomycins. Compound 1 was found to produce the highest ROS activity and to possess the greatest cytotoxicity against MDA-MB-231 breast cancer cells.

MgF3− and AlF4− transition state analogue complexes of yeast phosphoglycerate kinase

The phospho-transfer mechanism of yeast phosphoglycerate kinase (PGK) has been probed through formation of trifluoromagnesate (MgF3-) and tetrafluoroaluminate (AlF4-) transition state analogue complexes and analyzed using 19F, 1H waterLOGSY and 1H chemical shift perturbation NMR spectroscopy. We observed the first 19F NMR spectroscopic evidence for the formation of metal fluoride transition state analogues of yeast PGK and also observed significant changes to proton chemical shifts of PGK in the presence, but not in the absence, of fluoride upon titration of ligands, providing indirect evidence of the formation of a closed ternary transition state. WaterLOGSY NMR spectroscopy experiments using an uncompetitive model were used in an attempt to measure ligand binding affinities within the transition state analogue complexes.