Kinetic and Structural Characterization of Trypanosoma cruzi Hypoxanthine-Guanine-Xanthine Phosphoribosyltransferases and Repurposing of Transition-State Analogue Inhibitors.

Over 70 million people are currently at risk of developing Chagas Disease (CD) infection, with more than 8 million people already infected worldwide. Current treatments are limited and innovative therapies are required. Trypanosoma cruzi, the etiological agent of CD, is a purine auxotroph that relies on phosphoribosyltransferases to salvage purine bases from their hosts for the formation of purine nucleoside monophosphates. Hypoxanthine-guanine-xanthine phosphoribosyltransferases (HGXPRTs) catalyze the salvage of 6-oxopurines and are promising targets for the treatment of CD. HGXPRTs catalyze the formation of inosine, guanosine, and xanthosine monophosphates from 5-phospho-d-ribose 1-pyrophosphate and the nucleobases hypoxanthine, guanine, and xanthine, respectively. T. cruzi possesses four HG(X)PRT isoforms. We previously reported the kinetic characterization and inhibition of two isoforms, TcHGPRTs, demonstrating their catalytic equivalence. Here, we characterize the two remaining isoforms, revealing nearly identical HGXPRT activities in vitro and identifying for the first time T. cruzi enzymes with XPRT activity, clarifying their previous annotation. TcHGXPRT follows an ordered kinetic mechanism with a postchemistry event as the rate-limiting step(s) of catalysis. Its crystallographic structures reveal implications for catalysis and substrate specificity. A set of transition-state analogue inhibitors (TSAIs) initially developed to target the malarial orthologue were re-evaluated, with the most potent compound binding to TcHGXPRT with nanomolar affinity, validating the repurposing of TSAIs to expedite the discovery of lead compounds against orthologous enzymes. We identified mechanistic and structural features that can be exploited in the optimization of inhibitors effective against TcHGPRT and TcHGXPRT concomitantly, which is an important feature when targeting essential enzymes with overlapping activities.

Elucidation of tropane alkaloid biosynthesis in Erythroxylum coca using a microbial pathway discovery platform.

Tropane alkaloids (TAs) are heterocyclic nitrogenous metabolites found across seven orders of angiosperms, including Malpighiales (Erythroxylaceae) and Solanales (Solanaceae). Despite the well-established euphorigenic properties of Erythroxylaceae TAs like cocaine, their biosynthetic pathway remains incomplete. Using yeast as a screening platform, we identified and characterized the missing steps of TA biosynthesis in Erythroxylum coca. We first characterize putative E. coca polyamine synthase- and amine oxidase-like enzymes in vitro, in yeast, and in planta to show that the first tropane ring closure in Erythroxylaceae occurs via bifunctional spermidine synthase/N-methyltransferases and both flavin- and copper-dependent amine oxidases. We next identify a SABATH family methyltransferase responsible for the 2-carbomethoxy moiety characteristic of Erythroxylaceae TAs and demonstrate that its coexpression with methylecgonone reductase in yeast engineered to express the Solanaceae TA pathway enables the production of a hybrid TA with structural features of both lineages. Finally, we use clustering analysis of Erythroxylum transcriptome datasets to discover a cytochrome P450 of the CYP81A family responsible for the second tropane ring closure in Erythroxylaceae, and demonstrate the function of the core coca TA pathway in vivo via reconstruction and de novo biosynthesis of methylecgonine in yeast. Collectively, our results provide strong evidence that TA biosynthesis in Erythroxylaceae and Solanaceae is polyphyletic and that independent recruitment of unique biosynthetic mechanisms and enzyme classes occurred at nearly every step in the evolution of this pathway.

United States tea: A synopsis of ongoing tea research and solutions to United States tea production issues.

Tea is a steeped beverage made from the leaves of Camellia sinensis. Globally, this healthy, caffeine-containing drink is one of the most widely consumed beverages. At least 50 countries produce tea and most of the production information and tea research is derived from international sources. Here, we discuss information related to tea production, genetics, and chemistry as well as production issues that affect or are likely to affect emerging tea production and research in the United States. With this review, we relay current knowledge on tea production, threats to tea production, and solutions to production problems to inform this emerging market in the United States.

Kinetic Characterization and Inhibition of Trypanosoma cruzi Hypoxanthine-Guanine Phosphoribosyltransferases.

Chagas disease, caused by the parasitic protozoan Trypanosoma cruzi, affects over 8 million people worldwide. Current antiparasitic treatments for Chagas disease are ineffective in treating advanced, chronic stages of the disease, and are noted for their toxicity. Like most parasitic protozoa, T. cruzi is unable to synthesize purines de novo, and relies on the salvage of preformed purines from the host. Hypoxanthine-guanine phosphoribosyltransferases (HGPRTs) are enzymes that are critical for the salvage of preformed purines, catalyzing the formation of inosine monophosphate (IMP) and guanosine monophosphate (GMP) from the nucleobases hypoxanthine and guanine, respectively. Due to the central role of HGPRTs in purine salvage, these enzymes are promising targets for the development of new treatment methods for Chagas disease. In this study, we characterized two gene products in the T. cruzi CL Brener strain that encodes enzymes with functionally identical HGPRT activities in vitro: TcA (TcCLB.509693.70) and TcC (TcCLB.506457.30). The TcC isozyme was kinetically characterized to reveal mechanistic details on catalysis, including identification of the rate-limiting step(s) of catalysis. Furthermore, we identified and characterized inhibitors of T. cruzi HGPRTs originally developed as transition-state analogue inhibitors (TSAIs) of Plasmodium falciparum hypoxanthine-guanine-xanthine phosphoribosyltransferase (PfHGXPRT), where the most potent compound bound to T. cruzi HGPRT with low nanomolar affinity. Our results validated the repurposing of TSAIs to serve as selective inhibitors for orthologous molecular targets, where primary and secondary structures as well as putatively common chemical mechanisms are conserved.

The design of protozoan phosphoribosyltransferase inhibitors containing non-charged phosphate mimic residues.

Phosphate groups play essential roles in biological processes, including retention inside biological membranes. Phosphodiesters link nucleic acids, and the reversible transfer of phosphate groups is essential in energy metabolism and cell-signalling processes. Phosphorylated metabolic intermediates are known targets for metabolic and disease-related disorders, and the enzymes involved in these pathways recognize phosphate groups in their catalytic sites. Therapeutics that target these enzymes can require charged (ionic) entities to capture the binding energy of ionic substrates. Such compounds are not cell-permeable and require pro-drug strategies for efficacy as therapeutics. Protozoan parasites such as Plasmodium and Trypanosoma spp. are unable to synthesise purines de novo and rely on the salvage of purines from the host cell to synthesise free purine bases. Purine phosphoribosyltransfereases (PPRTases) play a crucial role for purine salvage and are potential target for drug development. Here we present attempts to design inhibitors of PPRTases that are non-ionic and show affinity for the nucleotide 5'-phosphate binding site. Inhibitor design was based on known potent ionic inhibitors, reported phosphate mimics and computational modelling studies.

Characterization of adenine phosphoribosyltransferase (APRT) activity in Trypanosoma brucei brucei: Only one of the two isoforms is kinetically active.

Human African Trypanosomiasis (HAT), also known as sleeping sickness, is a Neglected Tropical Disease endemic to 36 African countries, with approximately 70 million people currently at risk for infection. Current therapeutics are suboptimal due to toxicity, adverse side effects, and emerging resistance. Thus, both effective and affordable treatments are urgently needed. The causative agent of HAT is the protozoan Trypanosoma brucei ssp. Annotation of its genome confirms previous observations that T. brucei is a purine auxotroph. Incapable of de novo purine synthesis, these protozoan parasites rely on purine phosphoribosyltransferases to salvage purines from their hosts for the synthesis of purine monophosphates. Complete and accurate genome annotations in combination with the identification and characterization of the catalytic activity of purine salvage enzymes enables the development of target-specific therapies in addition to providing a deeper understanding of purine metabolism in T. brucei. In trypanosomes, purine phosphoribosyltransferases represent promising drug targets due to their essential and central role in purine salvage. Enzymes involved in adenine and adenosine salvage, such as adenine phosphoribosyltransferases (APRTs, EC 2.4.2.7), are of particular interest for their potential role in the activation of adenine and adenosine-based pro-drugs. Analysis of the T. brucei genome shows two putative aprt genes: APRT1 (Tb927.7.1780) and APRT2 (Tb927.7.1790). Here we report studies of the catalytic activity of each putative APRT, revealing that of the two T. brucei putative APRTs, only APRT1 is kinetically active, thereby signifying a genomic misannotation of Tb927.7.1790 (putative APRT2). Reliable genome annotation is necessary to establish potential drug targets and identify enzymes involved in adenine and adenosine-based pro-drug activation.