The success of inactivated and live-attenuated vaccines has enhanced livestock productivity, promoted food security, and attenuated the morbidity and mortality of several human, animal, and zoonotic diseases. However, these traditional vaccine technologies are not without fault. The efficacy of inactivated vaccines can be suboptimal with particular pathogens and safety concerns arise with live-attenuated vaccines. Additionally, the rate of emerging infectious diseases continues to increase and with that the need to quickly deploy new vaccines. Unfortunately, first generation vaccines are not conducive to such urgencies. Within the last three decades, veterinary medicine has spearheaded the advancement in novel vaccine development to circumvent several of the flaws associated with classical vaccines. These third generation vaccines, including DNA, RNA and recombinant viral-vector vaccines, induce both humoral and cellular immune response, are economically manufactured, safe to use, and can be utilized to differentiate infected from vaccinated animals. The present article offers a review of commercially available novel vaccine technologies currently utilized in companion animal, food animal, and wildlife disease control.

Enzyme reactivity is often enhanced by changes in oxidation state, spin state, and metal–ligand covalency of associated metallocofactors. The development of spectroscopic methods for studying these processes coincidentally with structural rearrangements is essential for elucidating metalloenzyme mechanisms. Herein, we demonstrate the feasibility of collecting X-ray emission spectra of metalloenzyme crystals at a third-generation synchrotron source. In particular, we report the development of a von Hamos spectrometer for the collection of Fe Kβ emission optimized for analysis of dilute biological samples. We further showcase its application in crystals of the immunosuppressive heme-dependent enzyme indoleamine 2,3-dioxygenase. Spectra from protein crystals in different states were compared with relevant reference compounds. Complementary density functional calculations assessing covalency support our spectroscopic analysis and identify active site conformations that correlate to high- and low-spin states. These experiments validate the suitability of an X-ray emission approach for determining spin states of previously uncharacterized metalloenzyme reaction intermediates.

<div> <div> <div> <p>Keratinicyclins and keratinimicins are recently discovered glycopeptide antibiotics (GPAs). The latter are canonical GPAs with broad-spectrum activity against Gram-positive bacteria, while keratinicyclins form a new chemotype by virtue of an unusual oxazolidinone moiety and exhibit specific antibiosis against Clostridium difficile. Here, we investigated the three-dimensional structures and functional consequences for both molecules. Equilibrium binding studies showed tight binding by keratinimicin A, but not keratinicyclin B, to the peptidoglycan terminus. Using protein crystallography methods, we solved the X-ray crystal structures of both GPAs, which, in conjunction with DFT calculations, indicate that the inability of keratinicyclin B to bind the peptidoglycan is governed by steric factors. Keratinicyclin B, therefore, interferes with an alternative target to inhibit C. difficile growth, a conclusion confirmed by checkerboard analysis that revealed synergistic activity with vancomycin. Our results set the stage for identifying the molecular target of keratinicyclins and for exploring their therapeutic utility in combination with vancomycin. </p> </div> </div> </div>

Nitration reactions are crucial for many industrial syntheses; however, current protocols lack site specificity and employ hazardous chemicals. The noncanonical cytochrome P450 enzymes RufO and TxtE catalyze the only known direct aromatic nitration reactions in nature, making them attractive model systems for the development of analogous biocatalytic and/or biomimetic reactions that proceed under mild conditions. While the associated mechanism has been well-characterized in TxtE, much less is known about RufO. Herein we present the first structure of RufO alongside a series of computational and biochemical studies investigating its unusual reactivity. We demonstrate that free Ltyrosine is not readily accepted as a substrate despite previous reports to the contrary. Instead, we propose that RufO natively modifies L-tyrosine tethered to the peptidyl carrier protein of a nonribosomal peptide synthetase encoded by the same biosynthetic gene cluster and present both docking and molecular dynamics simulations consistent with this hypothesis. Our results expand the scope of direct enzymatic nitration reactions and provide the first evidence for such a modification of a peptide synthetase-bound substrate. Both of these insights may aid in the downstream development of biocatalytic approaches to synthesize rufomycin analogues and related drug candidates.