Enzymes containing pyridoxal-5'-phosphate (PLP), also known as vitamin B6, are ubiquitous in biology, performing essential reactions in metabolism of amino acids and amines. These enzymes catalyse a wide variety of reactions, including racemization, transamination, alpha- and beta- decarboxylation, retro-aldol cleavage, beta- and gamma-elimination and substitution. The central role of PLP-dependent enzymes in the metabolism of amino acids and amines, and the biosynthesis of important bioactive metabolites, makes them attractive targets for the design of mechanism- based inhibitors, which may be useful as drugs. The carbon-carbon lyases (tyrosine phenol-lyase and tryptophan indole-lyase) are bacterial enzymes which catalyse the hydrolytic beta-elimination of L-tyrosine or L- tryptophan to give, respectively, phenol or indole, and ammonium pyruvate. These enzymes are ideal models to study the relationship between structure and mechanisms in PLP enzymes. The structures and mechanisms of these enzymes will be studied by mutagenesis, X-ray crystallography, and pre- steady-state kinetics. Kynureninase catalyses the hydrolytic cleavage of L-kynurenine to give anthranilic acid and L-alanine. In eucaryotic organisms, the product of kynureninase, 3-hydroxyanthranilic acid, undergoes ring cleavage by a dioxygenase to give alpha-amino-beta- carboxymuconic semialdehyde, which can cyclize spontaneously to give quinolinic acid, a known neurotoxin. Inhibitors of kynureninase could thus be of value in the treatment of some serious neurological disorders, such as Huntington's chorea, ALS, stroke, epilepsy, and AIDS-related dementia. In order to aid in the design of new inhibitors, kynureninase will be cloned, sequenced, and the three dimensional structure determined. Last year, the product of nifS was found to be a PLP-dependent enzyme which catalyses the desulfurization of L-cysteine to give L-alanine and elemental sulfur. It has been proposed that this enzyme is required for the in vitro synthesis of iron-sulfur centers, which are necessary for electron transfer processes and are thus ubiquitous in biology. The mechanism of L-cysteine desulfurase will be studied by steady-state and pre-steady-state kinetic methods. The results of our continuing 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 |
