Biology subjects

Hull, K. G.

Publications and source records attributed to Hull, K. G..

3 recordsLinked to original sources

bioRxiv2022

Roles of the second-shell amino acid R266 in other members of the MLE subgroup of the enolase superfamily

Catalytic promiscuity is the coincidental ability to catalyze non-biological reactions in the same active site as the native biological reaction. Several lines of evidence show that catalytic promiscuity plays a role in the evolution of new enzyme functions. Thus, studying catalytic promiscuity can help identify structural features that predispose an enzyme to evolve new functions. This study identifies such a pre-adaptive residue in an N-succinylamino acid racemase/o-succinylbenzoate synthase (NSAR/OSBS) enzymes from the NSAR/OSBS subfamily. Previously, we identified a point mutation, R266Q, in the catalytically promiscuous Amycolatopsis sp. T-1-60 NSAR/OSBS that has a deleterious effect on NSAR activity with a lesser effect on OSBS activity (Truong et al., in preparation). We demonstrated that R266 was a pre-adaptive feature that enabled the emergence and evolution of NSAR activity in AmyNSAR/OSBS. We examined the role of the residue R266 in the evolution of NSAR activity by examining the effects of the single substitution R266Q in other members of the NSAR/OSBS subfamily including Enterococcus faecalis NSAR/OSBS, Roseiflexus castenholzii NSAR/OSBS, Lysinibacillus varians NSAR/OSBS, and Listeria innocua NSAR/OSBS, which have been previously characterized to carry out both OSBS and NSAR activities efficiently. RcNSAR/OSBS, LvNSAR/OSBS, EfNSAR/OSBS, and LiNSAR/OSBS are 49, 48, 32, and 28% identical, respectively, to AmyNSAR/OSBS. We found that while the R266Q mutation decreases NSAR activity more than OSBS activity, as expected, in most NSAR/OSBS members, the differential effects of the R266Q substitution on NSAR and OSBS activities are not as striking as observed in AmyNSAR/OSBS. In some homologs, the R266Q mutation has very deleterious effects on both OSBS and NSAR activities. Furthermore, the mutation unexpectedly decreases OSBS activity more than NSAR activity in LiNSAR/OSBS. Thus, the effects of R266Q on NSAR and OSBS activities depend on differences in sequence context between members of the NSAR/OSBS subfamily, demonstrating the complex role of epistasis in the evolution of NSAR activity in the NSAR/OSBS subfamily.

biochemistry↗

bioRxiv2020

Necator americanus Ancylostoma secreted protein-2 (Na-ASP-2) selectively binds an ascaroside (ascr#3)

During their infective stages, hookworms release excretory-secretory (E-S) products, including small molecules and proteins, to help evade and suppress the hosts immune system. Small molecules found in E-S products of mammalian hookworms include nematode derived metabolites like ascarosides, which are composed of the sugar ascarylose linked to a fatty acid side chain. Ascarosides play vital roles in signaling, development, reproduction, and survival. The most abundant proteins found in hookworm E-S products are members of the protein family known as Ancylostoma secreted protein (ASP). ASP belongs to the SCP/TAPS (sperm-coating protein / Tpx / antigen 5 / pathogenesis related-1 / Sc7) superfamily of proteins, members of which have previously been shown to bind to eicosanoids and fatty acids. These molecules are structurally similar to the fatty acid moieties of ascarosides. The objective of this study was to determine if the hookworm ASP; N. americanus Ancylostoma secreted protein 2 (Na-ASP-2) binds to the ascarosides or their fatty acid moieties. We describe investigations of our hypothesis that there is a functional relationship between the major secreted proteins and signaling small molecules found in hookworm E-S products. To accomplish this, several ascarosides and their fatty acid moieties were synthesized and tested for in vitro binding to Na-ASP-2 using a ligand competition assay and microscale thermophoresis. Our results reveal that the fatty acid moieties of the ascarosides, bind specifically to the palmitic acid binding cavity of Na-ASP-2. Additionally, ascr#3, an ascaroside that is present in mammalian hookworm E-S products binds to the palmitic acid binding cavity of Na-ASP-2, whereas oscr#10 which is not found in hookworm E-S products does not bind. Future studies are required to determine the structural basis of ascaroside binding by Na-ASP-2 and to understand the physiological significance of these observations.

biochemistry↗

bioRxiv2018

Occidiofungin, an actin binding antifungal with in vivo efficacy in a vulvovaginal candidiasis infection

Current antifungal treatment options are plagued with rapidly increasing occurrence of resistance, high degree of toxicity and a limited spectrum of activity. The need to develop a novel antifungal with a unique target, wider spectrum of activity, and reduced toxicity to the host, is urgent. We have identified and characterized one such compound named occidiofungin that is produced by the soil bacterium Burkholderia contaminans MS14. This study identifies the primary cellular target of the antifungal, which was determined to be actin. Actin binding metabolites are generally characterized by their ability to inhibit polymerization or depolymerization of actin filaments, which presumably accounts for their severe toxicity. Occidiofungin, instead, has a subtler effect on actin dynamics that triggers apoptotic cell death. We were able to demonstrate the effectiveness of the antifungal in treating a vulvovaginal yeast infection in a murine model. This discovery puts occidiofungin in a unique class of actin-binding antifungal compounds with minimal reported toxicity to the host. The results of this study are important for the development of a novel class of antifungals that could fill the existing gap in treatment options for fungal infections.\n\nAuthor summaryWidespread resistance to antifungal compounds currently in use has been alarming. Identification and development of a new class of antifungals with a novel cellular target is desperately needed. This study describes the assays carried out to determine the molecular target and evaluate efficacy of one such novel antifungal compound called occidiofungin. Occidiofungin modified with a functional alkyne group enabled affinity purification assays and localization studies in yeast. These studies led to the identification of the actin binding property of occidiofungin. Actin-binding by secondary metabolites often exhibit severe host toxicity, but this does not appear to be the case for occidiofungin. We have previously been able to administer occidiofungin to mice at concentrations in the range of 5 mg/kg without any serious complications. We were able to demonstrate the effectiveness of the antifungal in treating a vaginal fungal infection in a murine model. The results outlined in this manuscript establish that occidiofungin is an efficacious compound with a novel molecular target, putting it in a completely new class of antifungals.

microbiology↗