Structure-Based Mechanism for Early PLP-Mediated Steps of Rabbit Cytosolic Serine Hydroxymethyltransferase Reaction

Serine hydroxymethyltransferase catalyzes the reversible interconversion of L-serine and glycine with transfer of one-carbon groups to and from tetrahydrofolate. Active site residue Thr254 is known to be involved in the transaldimination reaction, a crucial step in the catalytic mechanism of all pyridoxal 5′-phosphate- (PLP-) dependent enzymes, which determines binding of substrates and release of products. In order to better understand the role of Thr254, we have expressed, characterized, and determined the crystal structures of rabbit cytosolic serine hydroxymethyltransferase T254A and T254C mutant forms, in the absence and presence of substrates. These mutants accumulate a kinetically stable gem-diamine intermediate, and their crystal structures show differences in the active site with respect to wild type. The kinetic and crystallographic data acquired with mutant enzymes permit us to infer that conversion of gem-diamine to external aldimine is significantly slowed because intermediates are trapped into an anomalous position by a misorientation of the PLP ring, and a new energy barrier hampers the transaldimination reaction. This barrier likely arises from the loss of the stabilizing hydrogen bond between the hydroxymethyl group of Thr254 and the ε-amino group of active site Lys257, which stabilizes the external aldimine intermediate in wild type SHMTs

Molecular Defects of Vitamin B6 Metabolism Associated with Neonatal Epileptic Encephalopathy

Neonatal epileptic encephalopathy (NEE) is a seizure disorder that occurs within hours from birth and arises from central nervous system (CNS) dysfunctions of various origins, including metabolic or inflammatory conditions, abnormalities of brain structure and cerebrovascular diseases. In some rare circumstances, NEE is refractory to conventional antiepileptic drugs (AEDs) but responds very well to treatment with vitamin B6 in the form of either pyridoxine (PN) or pyridoxal 5’-phosphate (PLP). Vitamin B6-dependent NEE derives either from a deficiency of PLP, from inborn errors in enzymes, such as pyridoxine 5’-phosphate oxidase (PNPOx) and pyridoxal kinase (PL kinase) involved in the PLP salvage pathway or from inherited mutations of enzymes, such as -aminoadipic semialdehyde dehydrogenase (also known as antiquitin) involved in other metabolic pathways, which lead to the accumulation of intermediates that react with PLP, reducing its availability. Clinical phenotypes observed in vitamin B6-dependent NEE patients may include fetal distress, hypoglycemia, acidosis, anemia, and asphyxia. The health state of untreated patients may undergo progressive deterioration, which can lead to death within weeks. Surviving children are usually mentally retarded and are dependent on vitamin B6 to control the disease. Several known cases of B-dependent NEE, however do not or only mildly manifest some of the above clinical features, and are characterized by mild to moderate developmental delay. This chapter will review the molecular mechanism of how in-born errors in PNPOx or antiquitin affect PLP levels in the cell and lead to NEE. We will also review important clinical and general features associated with PLP dependent NEE, and provide some directions for clinicians to diagnose and treat or manage the diseas

Crystal Structures of Human Pyridoxal Kinase in Complex with the Neurotoxins, Ginkgotoxin and Theophylline: Insights into Pyridoxal Kinase Inhibition

Several drugs and natural compounds are known to be highly neurotoxic, triggering epileptic convulsions or seizures, and causing headaches, agitations, as well as other neuronal symptoms. The neurotoxic effects of some of these compounds, including theophylline and ginkgotoxin, have been traced to their inhibitory activity against human pyridoxal kinase (hPL kinase), resulting in deficiency of the active cofactor form of vitamin B6, pyridoxal 5′-phosphate (PLP). Pyridoxal (PL), an inactive form of vitamin B6 is converted to PLP by PL kinase. PLP is the B6 vitamer required as a cofactor for over 160 enzymatic activities essential in primary and secondary metabolism. We have performed structural and kinetic studies on hPL kinase with several potential inhibitors, including ginkgotoxin and theophylline. The structural studies show ginkgotoxin and theophylline bound at the substrate site, and are involved in similar protein interactions as the natural substrate, PL. Interestingly, the phosphorylated product of ginkgotoxin is also observed bound at the active site. This work provides insights into the molecular basis of hPL kinase inhibition and may provide a working hypothesis to quickly screen or identify neurotoxic drugs as potential hPL kinase inhibitors. Such adverse effects may be prevented by administration of an appropriate form of vitamin B6, or provide clues of how to modify these drugs to help reduce their hPL kinase inhibitory effects

Globally Distributed Drug Discovery of New Antibiotics: Design and Combinatorial Synthesis of Amino Acid Derivatives in the Organic Chemistry Laboratory

An experiment for the synthesis of N-acyl derivatives of natural amino acids has been developed as part of the Distributed Drug Discovery (D3) program. Students use solid-phase synthesis techniques to complete a three-step, combinatorial synthesis of six products, which are analyzed using LC–MS and NMR spectroscopy. This protocol is suitable for introductory organic laboratory students and has been successfully implemented at multiple academic sites internationally. Accompanying prelab activities introduce students to SciFinder and to medicinal chemistry design principles. Pairing of these activities with the laboratory work provides students an authentic and cohesive research project experience

Intergroup struggles over victimhood in violent conflict: The victim-perpetrator paradigm

Most groups in violent, intergroup conflict perceive themselves to be the primary or sole victims of that conflict. This often results in contention over who may claim victim status and complicates a central aim of post-conflict processes, which is to acknowledge and address harms experienced by the victims. Drawing from victimology scholarship and intergroup relations theory, this article proposes the victim-perpetrator paradigm as a framework to analyse how, why and to what end groups in conflict construct and maintain their claims to the moral status of victim. This interdisciplinary paradigm builds on the knowledge that groups utilise the ‘ideal victim’ construction to exemplify their own innocence and blamelessness in contrast to the wickedness of the perpetrator, setting the two categories as separate and mutually exclusive even where experiences of violence have been complex. Additionally, this construction provides for a core intergroup need to achieve positive social identity, which groups may enhance by demonstrating a maximum differentiation between the in-group as victims and those out-groups identified as perpetrators. The paradigm contributes greater knowledge on the social roots of victim contention in conflict, as well as how groups legitimise their violence against out-groups during and after conflict

Nuclear localised more sulphur accumulation1 epigenetically regulates sulphur homeostasis in Arabidopsis thaliana

Sulphur (S) is an essential element for all living organisms. The uptake, assimilation and metabolism of S in plants are well studied. However, the regulation of S homeostasis remains largely unknown. Here, we report on the identification and characterisation of the more sulphur accumulation1 (msa1-1) mutant. The MSA1 protein is localized to the nucleus and is required for both S adenosylmethionine (SAM) production and DNA methylation. Loss of function of the nuclear localised MSA1 leads to a reduction in SAM in roots and a strong S-deficiency response even at ample S supply, causing an over- accumulation of sulphate, sulphite, cysteine and glutathione. Supplementation with SAM suppresses this high S phenotype. Furthermore, mutation of MSA1 affects genome-wide DNA methylation, including the methylation of S-deficiency responsive genes. Elevated S accumulation in msa1-1 requires the increased expression of the sulphate transporter genes SULTR1;1 and SULTR1;2 which are also differentially methylated in msa1-1. Our results suggest a novel function for MSA1 in the nucleus in regulating SAM biosynthesis and maintaining S homeostasis epigenetically via DNA methylation

Multi-Institution Research and Education Collaboration Identifies New Antimicrobial Compounds

New antibiotics are urgently needed to address increasing rates of multidrug resistant infections. Seventy-six diversely functionalized compounds, comprising five structural scaffolds, were synthesized and tested for their ability to inhibit microbial growth. Twenty-six compounds showed activity in the primary phenotypic screen at the Community for Open Antimicrobial Drug Discovery (CO-ADD). Follow-up testing of active molecules confirmed that two unnatural dipeptides inhibit the growth of Cryptococcus neoformans with a minimum inhibitory concentration (MIC) ≤ 8 μg/mL. Syntheses were carried out by undergraduate students at five schools implementing Distributed Drug Discovery (D3) programs. This report showcases that a collaborative research and educational process is a powerful approach to discover new molecules inhibiting microbial growth. Educational gains for students engaged in this project are highlighted in parallel to the research advances. Aspects of D3 that contribute to its success, including an emphasis on reproducibility of procedures, are discussed to underscore the power of this approach to solve important research problems and to inform other coupled chemical biology research and teaching endeavors