Current Research

Enzymes are remarkable biocatalysts, not only for the dramatic rate accelerations (up to 1020 fold) that they provide, but also for the high degree of substrate specificity, regiospecificity and stereospecificity that these reactions exhibit. The work in my laboratory is focused on the chemical basis for how enzymes achieve such high rates and reaction specificity. Two groups of enzymes are currently under study in the laboratory: 1) Pyridoxal 5'-phosphate (PLP; vitamin B6) dependent enzymes, and, 2) Alcohol dehydrogenases. Tyrosine phenol-lyase and tryptophan indole-lyase are two PLP-dependent enzymes that catalyze the hydrolytic cleavage of tyrosine or tryptophan to phenol or indole, respectively, and ammonium pyruvate. Although the amino acid sequences and three dimensional structures of the two enzymes are very similar, these enzyme are specific for their physiological substrates. We are determining the chemical mechanisms of both enzymes by synthesis of substrate and transition state analogs, steady state and rapid-scanning stopped-flow kinetics, and by using site-directed mutagenesis. We are also altering the substrate specificity by mutagenesis to identify the amino acids which determine the reaction specificity. Another PLP-dependent enzyme being studied in my laboratory is kynureninase. We have cloned this enzyme from Pseudomonas fluorescens and Homo sapiens and we have studied the mechanism by steady state and pre-steady state kinetic methods. Recently, we have determined the crystal structures of bacterial and human kynureninases, and we are determining the structural basis for the differences in reaction specificity. We have also synthesized potent mechanism based inhibitors of kynureninase that could be useful as drugs. In other work, we are studying a thermostable secondary alcohol dehydrogenase (SADH) isolated from a thermophilic bacterium. We demonstrated a novel temperature dependent reversal of stereospecificity of SADH in the reaction of 2-butanol. We are currently investigating a mutant SADH with specificity for aromatic substrates. A new project uses hydrostatic pressure as a probe of conformational changes in enzymes and proteins.  (This research was partially supported by a grant from the National Institutes of Health and the National Science Foundation.)

Selected Publications

Kumar, S. ; Gawandi, V. B. ; Capito, N. ; Phillips, R. S. Substituent effects on the reaction of beta-benzoylalanines with Pseudomonas fluorescens kynureninase. Biochemistry 2010, 49, 7913-9.
Lima, S. ; Kumar, S. ; Gawandi, V. ; Momany, C. ; Phillips, R. S. Crystal structure of the Homo sapiens kynureninase-3-hydroxyhippuric acid inhibitor complex: insights into the molecular basis of kynureninase substrate specificity. Journal of medicinal chemistry 2009, 52, 389-96.
Lima, S. ; Sundararaju, B. ; Huang, C. ; Khristoforov, R. ; Momany, C. ; Phillips, R. S. The crystal structure of the Pseudomonas dacunhae aspartate-beta-decarboxylase dodecamer reveals an unknown oligomeric assembly for a pyridoxal-5'-phosphate-dependent enzyme. Journal of molecular biology 2009, 388, 98-108.
Marchal, S. ; Font, J. ; Ribó, M. ; Vilanova, M. ; Phillips, R. S. ; Lange, R. ; Torrent, J. Asymmetric kinetics of protein structural changes. Accounts of chemical research 2009, 42, 778-87.
Musa, M. M. ; Ziegelmann-Fjeld, K. I. ; Vieille, C. ; Zeikus, G. J. ; Phillips, R. S. Xerogel-encapsulated W110A secondary alcohol dehydrogenase from Thermoanaerobacter ethanolicus performs asymmetric reduction of hydrophobic ketones in organic solvents. Angewandte Chemie (International ed. in English) 2007, 46, 3091-4.