Research in our group concerns the enzymology of multi-protein allosteric complexes, 3-oxo carboxylic acid reductases, tRNA modification, and human histidine kinases. We employ a breadth of strategies and techniques (e.g. molecular biology, protein expression and purification, steady-state and pre-steady-state kinetics, isotopic labelling of substrates and enzymes, kinetic and binding isotope effects, density-functional theory calculations, and protein crystallography) to elucidate all aspects of the mechanism of an enzymatic reaction. We harness that information to design specific enzyme inhibitors and to modify the substrate specificities of enzymes.
The biosynthesis of histidine involves some unusual enzymatic reactions, and provides an excellent opportunity to apply kinetic isotope effects and computational chemistry to paint an experimentally constrained picture of the reactions’ transition-state structures. Furthermore, isotopic labelling of the enzymes permits the contribution of femtosecond dynamics to the overall probability of transition-state barrier crossing to be gauged. Efforts are focused on the first enzyme of the pathway, the hetero-octameric ATP phosphoribosyltransferase (ATPPRT). ATPPRT is allosterically inhibited by histidine via the HisZ subunit, while the active site is found in the HisG subunit. We are investigating the mechanism of reaction of the ATPPRT from Psychrobacter arcticus, a cold-adapted bacterium from the Siberian permafrost. The long-term goal is to understand the principles that underpin enzyme catalysis and allostery in multi-protein complexes.
5-aminolevulinic acid biosynthesis in Staphylococcus aureus
5-Aminolevulic acid (5-ALA) is the common precursor in the biosynthesis of tetrapyrroles such as heme and chlorophylls. In bacteria, 5-ALA biosynthesis is catalysed by two enzymes, glutamyl-tRNA reductase (GluTR) and glutamate-1-semialdehyde 2,1-aminomutase. These enzymes are absent in humans, who generate 5-ALA via the Shemin pathway. Both GluTR and GSAAM have been shown to be essential in Staphylococcus aureus, the leading cause of hospital-associated infections worldwide. S. aureus isolates have emerged that are resistant to most antibiotics in clinical use. They are collectively referred to as methicillin-resistant Staphylococcus aureus (MRSA). Therefore, there is an urgent need to develop new drugs to treat MRSA infections. 5-ALA biosynthesis is a potential target for antibiotic development. We are investigating the reaction mechanism of the first enzyme in the pathway, GluTR, This enzyme catalyses the formation of glutamate-1-semialdehyde from glutamyl-tRNA and NADPH. Our goal is the design of GluTR inhibitors that can be further developed into antibiotics.
Protein histidine phosphorylation in H. sapiens
Protein histidine phosphorylation has recently had its importance recognized in mammalian biochemical signalling. The enzyme nucleoside diphosphate kinase B (NDPK-B) has emerged as the sole bona fide protein histidine kinase identified hitherto in humans, catalysing the phosphorylation of a histidine residue in the C-terminus of the Ca2+-activated potassium channel 3.1 (KCa3.1), an essential step for activation of the channel and subsequent activation and proliferation of TH1 and TH2 lymphocytes. As activity of KCa3.1 has been linked to autoimmune conditions such as Crohn’s disease and ulcerative colitis, inhibition of NDPK-B is a potential strategy to treat these illnesses. Our goal is therefore to elucidate the mechanism of NDPK-B-catalysed protein histidine kinase reaction and design specific inhibitors of the enzyme. Besides serving as a starting point for new immunomodulator development, such inhibitors can be used as chemical tools to probe the role of NDPK-B in additional histidine phosphorylation-mediated signalling pathways.