Coenzymology: biochemistry of vitamin biogenesis and cofactor-containing enzymes 767 Biophysical tools to monitor enzyme–ligand interactions of enzymes involved in vitamin biosynthesis A. Ciulli 1 and C. Abell University Chemical Laboratory, Lensfield Road, Cambridge CB2 1EW, U.K. Abstract Knowledge of biomolecular interactions is of importance to our understanding of biological processes such as enzyme catalysis and inhibition. Biophysical techniques enable sensitive detection and accurate charac- terization of binding and are therefore powerful tools in enzymology and rational drug design. The applications of NMR spectroscopy and isothermal titration calorimetry to study enzyme–ligand interactions will be discussed. Recent work on ketopantoate reductase, which catalyses an important step on the biosynthetic pathway to vitamin B5, is used to illustrate the potential of this approach. Introduction Many enzymes bind non-protein groups known as cofactors and require them to carry out catalysis. Most of the organic cofactors, which are called coenzymes, are vitamins or their derivatives. Vitamins are synthesized in vivo by bacteria, fungi and plants and are essential for their survival. Conse- quently, enzymes involved in the biosynthetic pathways to vitamins are potential targets of novel antibiotics and herbi- cides. And because these enzymes are absent from humans, such drugs are more likely to be selective and to have minimal side effects [1]. Enzyme inhibitors are useful chemical tools to study biological systems and are commonly identified by random screening or designed from a knowledge of the enzyme struc- ture and of the reaction mechanism. In the last few years, there has been an emergence of new approaches to drug dis- covery based on the identification of molecular fragments binding weakly to enzymes and their subsequent rational development into more potent inhibitors driven by structural information [2]. Biophysical methods that directly monitor protein–ligand interactions are used to study binding of small ligands such as coenzymes, substrates and inhibitors to enzymes and are often more suitable than conventional bio- assays to screen and detect fragments [3]. The applications of X-ray crystallography [4] and NMR spectroscopy [5] are now well established in fragment-based drug discovery to provide structural information on potential protein-binding sites for small molecules. Other biophysical techniques such as MS, ITC (isothermal titration calorimetry) and surface plasmon resonance are also becoming increasingly popular to characterize biomolecular interactions in proteomics [6] and drug discovery [7]. Although these methods can be Key words: calorimetry, drug design, enzyme–ligand interaction, ketopantoate reductase, NMR, vitamin biosynthesis. Abbreviations used: CPMG, Carr–Purcell–Meiboom–Gill; ITC, isothermal titration calorimetry; KPR, ketopantoate reductase; WaterLOGSY, water–ligand observed via gradient spectroscopy. 1 To whom correspondence should be addressed (email ac313@cam.ac.uk). slow, material-intensive and therefore are not generally suit- able for high-throughput ligand screening, they enable the direct detection of binding in solution, which minimizes any interference from non-specific associations, and allow access to weak affinities and detailed characterization of the thermodynamics and kinetics of the interaction. Biophysical methods are therefore powerful tools in enzymology and are now being developed as alternative lead discovery tools. Model system studies: KPR (ketopantoate reductase) Pantothenate (vitamin B5) is the precursor to CoA and the phosphopantetheine moiety of acyl-carrier proteins. It is an essential nutrient in animals and the biosynthetic pathway is limited to bacteria and plants [8]. Four enzymes are respon- sible for pantothenate biosynthesis – KPHMT (ketopantoate hydroxymethyltransferase; EC 2.1.2.11) encoded by the panB gene, KPR (EC 1.1.1.169) encoded by the panE gene, ADC (aspartate decarboxylase; EC 4.1.1.11) encoded by the panD gene and finally PS (pantothenate synthetase; EC 6.3.2.1) encoded by the panC gene. The three-dimensional structures of all Escherichia coli enzymes have been recently solved by X-ray crystallography [9]. We are currently developing novel approaches to inhibitor design using a range of biophysical methods and the panto- thenate enzymes as targets. Three enzymes in the pathway – KPHMT, KPR and PS – require coenzymes to catalyse their reactions – Me-THF (methylene tetrahydrofolate), NADPH and ATP respectively. Coenzyme-utilizing enzymes and, more generally, multisubstrate systems are particularly suit- able model systems for biophysical methods and to apply fragment-based approaches. Coenzyme and substrate bind- ing occur at separate pockets of the enzyme active sites to form the intermolecular complexes and the interactions can be studied in the absence of catalytic turnover under equilibrium conditions. Drugs can be designed to bind either the more C  2005 Biochemical Society