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. (This research is partially supported by a grant from the National Institutes of Health, GM42588.)

Selected Recent Publications:

"Differential Effects of Bromination on Substrates and Inhibitors of Kynureninase from Pseudomonas fluorescens", Heiss, C., Anderson, J. and Phillips, R. S., Org. Biomol. Chem., 1, 288-295 (2003).

"Indole can act as an Extracellular Signal to Regulate Biofilm Formation in Escherichia coli and in other Indole-producing Bacteria" , Di Martino, P., Fursy, R., Bret, L. Sundararaju, B. and Phillips, R. S., Can. J. Microbiol., 43, 443-449 (2003).

"Detection of Open and Closed Conformations of Tryptophan Synthase by ¹⁵N-HSQC NMR of Bound 1-[¹⁵N]-L-Tryptophan" , Osborne, A., Teng , Q., Miles, E. W., and Phillips, R. S., J. Biol. Chem., 278, 44083-44090 (2003).

"The Three Dimensional Structure of Kynureninase from Pseudomonas fluorescens" , Momany, C., Levdikov, V., Blagova, L., and Phillips, R. S., Biochemistry, 43, 1193-1203 (2004).

"Reaction of beta-Benzoyl-L-alanine with Kynureninase from Pseudomonas fluorescens: Detection of a New Reaction Intermediate and a Change in Rate-determining Step" , Gawandi, V., Liskey, D., Lima, S., and Phillips, R. S., Biochemistry, 43, 3230-7 (2004).

“The Reaction of Indole with the Aminoacrylate Intermediate of Salmonella typhimurium Tryptophan Synthase:  Observation of a Primary Kinetic Isotope Effect with 3-[²H]-Indole”, Cash, M. T., Miles, E. W.  and Phillips, R. S., Arch. Biochem. Biophys., 432, 233-243 (2004).

“Synthetic Applications of Tryptophan Synthase”, Phillips, R. S., Invited paper for special issue, “Integrating Biocatalysis into Organic Synthesis”, Tetrahedron: Asymmetry, 15, 2787-2792 (2004).

“The Role of Lys-256 in Citrobacter freundii Tyrosine Phenol-lyase in Monovalent Cation Activation”, Phillips, R. S., Chen, H. Y., Shim, D., Lima, S., Tavakoli, K. and Sundararaju, B., Biochemistry, 43, 14412-14419 (2004).

“Hydrostatic Pressure Affects the Conformational Equilibrium of Salmonella typhimurium Tryptophan Synthase”, Phillips, R. S., Miles, E. W., Holtermann G., and Goody, R. S., Biochemistry., 44, 7921-7928 (2005).

“Excited State Tautomerization of Azaindole”, Cash, M. T., Schreiner, P. R. and Phillips, R. S., Org. Biomol. Chem., 3, 3701-3706 (2005).

“Differential Effects of Temperature and Hydrostatic Pressure on the Formation of Quinonoid Intermediates from L-Trp and L-Met by H463F Mutant Escherichia coli Tryptophan Indole-lyase", Phillips, R. S. and Holtermann, G., Biochemistry, 44, 14289-97 (2005).

“Aminoacrylate Intermediates in the Reaction of Citrobacter freundii Tyrosine Phenol-lyase” Phillips, R. S., Faleev, N. G. and Chen, H. Y., Biochemistry, 45, 9575-9583 (2006).

"Asymmetric Reduction and Oxidation of Aromatic Ketones and Alcohols Using W110A Secondary Alcohol Dehydrogenase from Thermoanaerobacter ethanolicus", Musa, M. M., Ziegelmann-Fjeld, K. I., Vieille, C., Zeikus, J. G. and Phillips R. S., J. Org. Chem., 72, 30-34 (2007).

“Xerogel Encapsulated W110A Secondary Alcohol Dehydrogenase from Thermoanaerobacter ethanolicus Performs Asymmetric Reduction of Hydrophobic Ketones in Organic Solvents”, Musa, M. M., Ziegelmann-Fjeld, K. I., Vieille, C., Zeikus, J. G. and Phillips, R. S., Angew. Chem., Intl. Ed. Eng., 46, 3091-3094 (2007).

"Crystal Structure of Homo Sapiens Kynureninase", Lima, S., Khristoforov, R., Momany, C. and Phillips, R. S., Biochemistry, 46, 2735-2744 (2007).

“Quantitative effects of allosteric ligands and mutations on conformational equilibria in Salmonella typhimurium tryptophan synthase”, Phillips, R. S., McPhie P., Miles, E. W., Marchal, S., Lange, R., Arch. Biochem. Biophys., 470, 8-19 (2008).

“Activity and selectivity of W110A secondary alcohol dehydrogenase from Thermoanaerobacter ethanolicus in organic solvents and ionic liquids: mono- and biphasic media”, Musa, M. M., Ziegelmann-Fjeld, K. I., Vieille, C., and Phillips, R.S., Org. Biomol. Chem., 6, 887-892 (2008).

Pressure and temperature jump relaxation kinetics of the conformational change in Salmonella typhimurium tryptophan synthase L-serine complex: large activation compressibility and heat capacity changes demonstrate the contribution of solvation. Phillips, R. S., Miles, E. W., McPhie, P., Marchal, S., Georges, C., Dupont, Y., and Lange, R., J. Am. Chem. Soc., 130, 13580-13588 (2008).

“Regioselective nitration of N(alpha),N(1)-bis(trifluoroacetyl)-L-tryptophan methyl ester: Efficient synthesis of 2-nitro and 6-nitro-N-trifluoroacetyl-L-tryptophan methyl ester”, Osborne, A. S., Som, P., Metcalf, J. L., and Phillips, R. S. Bioorg. Med. Chem. Letts. (2008)

Members of the Phillips Group:

Robert S. Phillips , Professor of Chemistry and of Biochemistry and Molecular Biology

Austin Harris, Graduate Student, Chemistry

Sunil Kumar, Graduate Student, Chemistry

Nathan Lott, Graduate Student, Chemistry

Chandan Maitrani, Graduate student, Chemistry

Musa Musa, Graduate Student, Chemistry

Phanneth Som, Graduate Student, Chemistry

Quang (Johnny) Do, Graduate Student,Chemistry

Last updated: October 16, 2008

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