Tandem metalloenzymes gate plant cell entry by pathogenic fungi.

Global food security is endangered by fungal phytopathogens causing devastating crop production losses. Many of these pathogens use specialized appressoria cells to puncture plant cuticles. Here, we unveil a pair of alcohol oxidase-peroxidase enzymes to be essential for pathogenicity.

Determination of biocatalytic parameters of a copper radical oxidase using real-time reaction progress monitoring.

An Auxiliary Activity Family 5 (AA5) copper-radical alcohol oxidase (AlcOx) with promiscuous activity towards simple alkyl and aromatic alcohols was evaluated using real-time reaction progress monitoring. Reaction kinetics using variable time normalization analysis (VTNA) were determined from reaction progress curves. By this approach, a detailed view of the entire reaction time course under various conditions was obtained and used to identify parameters that will inform further process optimization development.

On the Catalytic Activity of a GT1 Family Glycosyltransferase from ISP5230.

GT1 family glycosyltansferase, Sv0189, from ISP5230 (ATCC 10721) was characterized. The recombinantly produced protein Sv0189 possessed UDP-glycosyltransferase activity. Screening, using an assay employing unnatural nitrophenyl glycosides as activated donors, resulted in the discovery of a broad substrate scope with respect to both acceptor molecules and donor sugars.

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-NH group of the Orn side chain results in collapse of the oxazolone ring and formation of a stable six-membered lactam.

Correction: Jadomycins, put a bigger ring in it: isolation of seven- to ten-membered ring analogues.

Correction for 'Jadomycins, put a bigger ring in it: isolation of seven- to ten-membered ring analogues' by Camilo F. Martinez-Farina et al., Chem.

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.

Furan and Lactam Jadomycin Biosynthetic Congeners Isolated from Streptomyces venezuelae ISP5230 Cultured with N-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 N-trifluoroacetyl-l-lysine (TFAL) congener. The compounds were isolated from Streptomyces venezuelae ISP5230 cultures grown with TFAL.

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.

MgF and AlF transition state analogue complexes of yeast phosphoglycerate kinase.

The phospho-transfer mechanism of yeast phosphoglycerate kinase (PGK) has been probed through formation of trifluoromagnesate (MgF) and tetrafluoroaluminate (AlF) transition state analogue complexes and analyzed using F, H waterLOGSY and H chemical shift perturbation NMR spectroscopy. We observed the first F 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.

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.

Polyphosphate-containing bisubstrate analogues as inhibitors of a bacterial cell wall thymidylyltransferase.

A series of polyphosphate containing sugar nucleotide analogues were synthesized and evaluated as bisubstrate inhibitors of α-D-glucose 1-phosphate thymidylyltransferase Cps2L, the first enzyme in Streptococcus pneumoniael-rhamnose biosynthesis, and a novel antibacterial target. WaterLOGSY NMR spectroscopy demonstrated binding of bisubstrate analogues to Cps2L and a spectrophotometric coupled assay was used to determine apparent Ki values.

Mechanistic evaluation of a nucleoside tetraphosphate with a thymidylyltransferase.

Pyrimidine polyphosphates were first detected in cells 5 decades ago; however, their biological significance remains only partially resolved. Such nucleoside polyphosphates are believed to be produced nonspecifically by promiscuous enzymes. Herein, synthetically prepared deoxythymidine 5'-tetraphosphate (p4dT) was evaluated with a thymidylyltransferase, Cps2L.

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

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).