UDP-Galactopyranose mutase (UGM) is a flavin-containing enzyme that catalyses the reversible conversion of UDP-Galactopyranose (UDP-Galp) to UDP-Galactofuranose (UDPGalf) and plays a key role in the biosynthesis of the mycobacterial cell wall galactofuran. A soluble, active form of UGM from Mycobacterium tuberculosis (MtUGM) was obtained from a dual His 6 -MBP tagged MtUGM construct. We present the first complex structures of MtUGM with bound substrate UDP-Galp (both oxidized flavin and reduced flavin). In addition, we have determined the complex structures of MtUGM with inhibitors (UDP and the dideoxytetrafluorinated analogs of both UDP-Galp (UDP-F 4 -Galp) and UDP-Galf (UDP-F 4 -Galf)), which represent the first complex structures of UGM with an analogue in the furanose form, as well as the first structures of dideoxy-tetrafluorinated sugar analogs bound to a protein. These structures provide detailed insight into ligand recognition by MtUGM and show a similar overall binding mode as reported for other prokaryotic UGMs. The binding of the ligand induces conformational changes in the enzyme, allowing ligand binding and active site closure. In addition, the complex structure of MtUGM with UDP-F 4 -Galf reveals the first detailed insight into how the furanose moiety binds to UGM. In particular, this study confirmed that the furanoside adopts a high energy conformation ( 4 E) within the catalytic pocket. Moreover, these investigations provide structural insights to the enhanced binding of the dideoxy-tetrafluorinated sugars compared to unmodified analogs. These results will help in the design of carbohydrate mimetics and drug development, and show the enormous possibilities on the use of polyfluorination in the design of carbohydrate mimetics.
UDP-galactopyranose mutase (UGM), a key enzyme in the biosynthesis of mycobacterial cell walls, is a potential target for the treatment of tuberculosis. In this work, we investigate binding models of a non-substrate-like inhibitor, MS-208, with M. tuberculosis UGM. Initial saturation transfer difference (STD) NMR experiments indicated a lack of direct competition between MS-208 and the enzyme substrate, and subsequent kinetic assays showed mixed inhibition. We thus hypothesized that MS-208 binds at an allosteric binding site (A-site) instead of the enzyme active site (S-site). A candidate A-site was identified in a subsequent computational study, and the overall hypothesis was supported by ensuing mutagenesis studies of the A-site. Further molecular dynamics studies led us to propose that MS-208 inhibition occurs by preventing complete closure of an active site mobile loop that is necessary for productive substrate binding. The results suggest the presence of an A-site with potential druggability, opening up new opportunities for the development of novel drug candidates against tuberculosis.
The present research develops an innovative nutrient recovery strategy for capturing of un-utilised nutrients from agri-food byproducts using a combination of solid state fermentation (SSF) and insect rearing. SSF of borage and flaxseed meals were performed using GRAS organisms to release indigenous nutrients and to produce additional nutrients. Proximate analysis of the SSF-meals showed increases in both lipid and protein contents. Black soldier fly larvae (BSFL) were then grown on these SSF-meals for 12 day. The meals fermented singularly with Lactobacillus plantarum or Aspergillus niger displayed up to 30% enhancement in BSFL biomass, and dual fermentation with L. plantarum and Aspergillus oryzae resulted in an additional 10% enhancement. These examinations showed SFL use over-90% of proteins and lipids in SSF-meals. The results indicate that fermentation of these low-value meals can boost the efficacy of larval growth and the recovery of nutrients from agricultural byproducts as larval biomass.Nutrient recovery using SSF and insect rearing J.R.A. Kuttiyatveetil et al.
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