The investigators are studying the mechanism of pyridoxal-5'-phosphate-dependent enzymes that break or form carbon-carbon bonds. These enzymes include tryptophan indole-lyase, tyrosine phenol-lyase, tryptophan synthase, bacterial and human kynureninases, and aspartate beta-decarboxylase. They are evaluating the reaction mechanism of tyrosine phenol-lyase and tryptophan indole-lyase. The investigators are determining the roles of specific amino acids in catalysis and reaction specificity by both site-directed mutagenesis and by synthesis of a C-terminal peptide containing unnatural amino acids. The investigators are probing the mechanism of the reaction of these enzymes by various kinetic techniques, including rapid scanning stopped flow spectrophotometry and rapid chemical quench. They will design, synthesize, and evaluate novel inhibitors of tryptophan indole-lyase that may be useful in the treatment of Haemophilus influenzae meningitis. The potent competitive inhibitors of kynureninase that they are designing and synthesizing, based on their studies of the bacterial enzyme, have been patented and are undergoing studies to evaluate their potential in the treatment of neurological disorders such as stroke. The investigators will determine and compare the structure and reaction mechanism of bacterial and human kynureninases as well as aspartate beta-decarboxylase. More information on the structure and mechanism of kynureninases mat lead to the design of other more selective and potent inhibitors. The central role of pyridoxal-5'phosphate-dependent enzymes in the metabolism of amino acids and amines and in the biosynthesis of important bioactive metabolites, makes them attractive targets for the design of mechanism-based inhibitors which may be useful for drugs. Thus the results of this research are likely to make a significant contribution to the goal of improving health care for Americans in the future.
|Phillips, Robert S (2015) Chemistry and diversity of pyridoxal-5'-phosphate dependent enzymes. Biochim Biophys Acta 1854:1167-74|
|Phillips, Robert S; Demidkina, Tatyana V; Faleev, Nicolai G (2014) The role of substrate strain in the mechanism of the carbon-carbon lyases. Bioorg Chem 57:198-205|
|Phillips, Robert S (2014) Structure and mechanism of kynureninase. Arch Biochem Biophys 544:69-74|
|Phillips, Robert S (2011) Structure, mechanism, and substrate specificity of kynureninase. Biochim Biophys Acta 1814:1481-8|
|Lima, Santiago; Khristoforov, Roman; Momany, Cory et al. (2007) Crystal structure of Homo sapiens kynureninase. Biochemistry 46:2735-44|