Characterisation and Inhibition of Carnitine Biosynthesis Oxygenases

Fatty acids are a major energy source for humans and most other life forms. In order for fatty acids to be metabolised they must be transported into intracellular compartments called mitochondria, where they are metabolised by the oxygen dependent process of respiration to generate chemical energy that is essential for absolutely everything we do. However, when we consume more fatty acids than we can metabolise they are stored, leading to weight gain and, ultimately, obesity and related diseases. The process of fatty acid transport into mitochondria requires their conjugation to a molecule called carnitine, so named because it is present at high levels in animal muscle, i.e. meat. We humans both make carnitine and obtain it from our diet. Carnitine is widely used as a dietery supplement because it promotes fat metabolism. The starting points for carnitine production in humans are proteins that are degraded to give an unusual amino acid called trimethyl-lysine or TML. TML is converted into carnitine by a series of enzyme-catalysed steps, two of which are catalysed by oxygenases, i.e. enzymes that use oxygen for catalysis. Mildronate, a clinically used compound, inhibits at least one of these oxygenases. Mildronate is given to patients after they have had a heart attack, in order to inhibit fatty acid biosynthesis. This is because fatty acid biosynthesis produces damaging reactive oxidising species as byproducts and these are thought to be particularly dangerous after heart attacks. We have recently made a breakthrough in work aimed at understanding how Mildronate works by solving crystal structures of its target. We have also found that Mildronate is not a simple enzyme inhibitor but is, in fact, a substrate undergoing an unprecedented reaction involving rearrangement of its molecular structure. This work has also placed us in an exceptionally good position to understand how Mildronate actually works in human cells and to unravel the molecular details of how we and other animals make carnitine. We are particularly interested in exploring links between carnitine biosynthesis and genetics because, when it is present in DNA binding proteins called histones, TML is also involved in regulating which genes are 'turned on and off' in cells. Importantly we have good evidence that we can develop improved versions of Mildronate, which we will make available to the large community of researchers interested in fatty acid metabolism. We will investigate the structure and mechanism of the oxygenases of carnitine biosynthesis and develop improved inhibitors that will be useful as functional probes for the physiological roles of the carnitine biosynthesis oxygenases. Building upon our discovery the unusual chemistry of the carnitine biosynthesis enzymes, we will also evaluate their potential for the production of chemicals that will be of use to the pharmaceutical industry.

Carnitine (CAR) is required for fatty acid transport into mitochondria and has roles in animals including transporting beta-oxidation products, toxin excretion, fat storage, and maintaining (acyl)CoA homeostasis. In humans/other mammals CAR is produced endogenously and obtained from the diet. CAR is used as a dietary supplement and is produced in racemic form by synthesis and in chiral form by fermentation. In animals CAR is produced in 4 steps from N-epsilon-trimethyllysine (TML) which in turn is produced (predominantly) by histone degradation. The first and last steps in carnitine biosynthesis are catalysed by 2-oxoglutarate oxygenases: TML hydroxylase (TMLH) and gamma-butyrobetaine hydroxylase (BBOX). BBOX is assigned as the target of Mildronate, which is given to patients after myocardial infarction with a view to limiting fatty acid biosynthesis and associated damage by reactive oxidising species. Work on BBOX and TMLH has been hampered by the lack of recombinant expression systems and a lack of structural information. We have developed procedures for producing recombinant human BBOX/TMLH and solved BBOX structures in complex with its gamma-butyrobetaine substrate and Mildronate and have pioneered an efficient and cost-effective assay for BBOX. We have found that Mildronate is a competitive substrate, undergoing an unprecedented rearrangement reaction. We propose to carry out structural and biochemical studies on TMLH/BBOX to develop a detailed understanding of their cosubstrate dependence, how their substrate selectivities are determined, to produce selective and potent inhibitors of BBOX/TMLH to be used in functional assignment work, to investigate factors regulating BBOX/TMLH activity in cells (including links to chromatin methylation status) and to evaluate their potential as biocatalysts. This work is enabled by the assays and reagents we have developed for many 2OG oxygenases and we hope will be of interest to a wide range of scientists.

We are committed to ensuring that our publicly-funded work achieves wide and substantial impact, in particular with respect to translating fundamental research to make impacts on the health of society. Carnitine is of central importance to animal metabolism, hence studies on its biosynthesis are of general interest and have the potential to become textbook studies. In addition to the basic (bio)chemistry communities the work has the potential to make a much wider impact because of its relevance to healthy and safe food, pharmaceutical development and industrial biotechnology. Carnitine is very widely used as food supplement thus studies on its biosynthesis and regulation will have direct relevance to the users of carnitine. Aside from its vital role in fatty acid metabolism, carnitine (biosynthesis) is also linked to a range of diseases, e.g. a defect in trimethyllysine biosynthesis is a common X-linked inborn error and may be a risk factor for autism (PNAS, 2012, 109, 7974-7981). Carnitine also has roles in nutritional redox homeostasis and regulation of longevity genes factors that are of very widespread (patho)physiological interest (see e.g. Am. J. Clin. Nutr. 2009, 89, 71-76; Life Sci. 2006, 78, 803-811). The enzymes of carnitine biosynthesis are the proposed targets for a clinically used compound that shows promise for the treatment of damage caused by heart attacks and validation of this mechanism, together with the development of improved inhibitors, will be of interest to the pharmaceutical industry and te many academic researchers working on cardiovascular disease. Further, enzymes from the same family as the carnitine biosynthesis oxygenases that are the focus of our current proposal (2-oxoglutarate oxygenases) are current medicinal chemistry targets, including the hypoxia inducible factor prolyl-hydroylases and the JmjC histone demethylases. To date the carnitine biosynthesis enzymes have not be available to use in counter-screens; our work will enable this to be achieved by the development of both in vitro and in cells assays, and thus will help to contribute to the development of pharmaceuticals aimed at treating anemia, ischemia, inflammatory related disorders and cancer. Finally, we aim to investigate the use of carnitine biosynthesis oxygenases as biocatalysts. We have found that one of the target enzymes catalyses an unprecedented rearrangement and can convert an achiral meso-compound into chiral compounds with 2 or 3 chiral centres in a single step. The development of this work has potential application in the field of Industrial Biotechnology (a BBSRC Highlight area), including for the production of pharmaceutical precursors. Overall, we believe the work will have impact well-beyond the community of enzymologists, as has been the case for our other work on human oxygenases (>10000 citations) which has helped to enable new pharmaceutical targets and has provided insights into a range of signalling pathways relevant to health and disease.