Chemistry Department

Our interests are generally in the relationship of protein three-dimensional structure to chemical function. To this end, research is focussed on the modification of the catalytic properties of a number of pharmaceutically or industrially important enzymes. The methods used are a combination of X-ray crystallography, design of transition-state analog inhibitors, and site-directed mutagenesis. The objectives are to learn how to re-engineer these catalysts to perform useful chemical reactions which may not occur efficiently with the naturally occurring enzyme, to dissect the individual steps in a mechanism and characterize them structurally, or to learn how to inhibit an enzyme specifically and selectively.

The proteins being studied currently include enzymes utilizing pyridoxal phosphate as cofactor, a GTP-binding protein, a DNA-binding protein, and several proteases. Different methods are being used to study these systems, including traditional kinetic and structural methods, and low-temperature and time-resolved x-ray structural methods. In addition, a new method for mapping of binding surfaces on proteins is being developed for the design of specific inhibitors.

Pyridoxal is capable of catalyzing several types of transformations. However, any one enzyme utilizing this cofactor does only one of them predominantly. The question therefore arises how the protein controls the chemical outcome of such transformations. We are studying a number of these enzymes structurally in order to begin to answer that question.

The expression of diphtheria toxin in toxigenic Corynebacterium diphtheria is controlled by a transition metal ion activated repressor DtxR. The repressor binds DNA after activation by the metal ion and thereby regulates expression of the toxin. The mechanism of activation is being studied structurally.

Serine proteases are important in cellular development, blood clotting, and a variety of defense mechanisms. Disorders involving these proteases are often linked to the absence or inefficiency of a specific inhibitor to control the activity of the enzyme. The design of such inhibitors requires detailed knowledge of the structure of the enzyme, of the enzyme complexed with inhibitors, and if possible, an understanding of the mechanisms of inhibition.

Selected Publications:

Gray JV, Petsko GA, Johnston GC, Ringe D, Singer RA, Werner-Washburne M. (2004) "Sleeping beauty": quiescence in Saccharomyces cerevisiae. Microbiol Mol Biol Rev. 68:187-206. [abstract]

Canet-Aviles RM, Wilson MA, Miller DW, Ahmad R, McLendon C, Bandyopadhyay S, Baptista MJ, Ringe D, Petsko GA, Cookson MR. (2004) The Parkinson's disease protein DJ-1 is neuroprotective due to cysteine-sulfinic acid-driven mitochondrial localization. Proc Natl Acad Sci U S A. 101:9103-8. [abstract]

Yang J, Wang Y, Woolridge EM, Arora V, Petsko GA, Kozarich JW, Ringe D. (2004) Crystal structure of 3-carboxy-cis,cis-muconate lactonizing enzyme from Pseudomonas putida, a fumarase class II type cycloisomerase: enzyme evolution in parallel pathways. Biochemistry. 43:10424-34. [abstract]

Vogan EM, Bellamacina C, He X, Liu HW, Ringe D, Petsko GA. (2004) Crystal structure at 1.8 A resolution of CDP-D-glucose 4,6-dehydratase from Yersinia pseudotuberculosis. Biochemistry. 43:3057-67. [abstract]

Pozharski E, Wilson MA, Hewagama A, Shanafelt AB, Petsko G, Ringe D. (2004) Anchoring a cationic ligand: the structure of the Fab fragment of the anti-morphine antibody 9B1 and its complex with morphine. J Mol Biol., 337:691-7. [abstract]

Ringe D, Wei Y, Boino KR, Ondrechen MJ. (2004) Protein structure to function: insights from computation. Cell Mol Life Sci. 61:387-92. [abstract]

Holyoak T, Kettner CA, Petsko GA, Fuller RS, Ringe D. (2004) Structural basis for differences in substrate selectivity in Kex2 and furin protein convertases. Biochemistry. 43:2412-21. [abstract]

Wilson MA, St Amour CV, Collins JL, Ringe D, Petsko GA. (2004) The 1.8-A resolution crystal structure of YDR533Cp from Saccharomyces cerevisiae: a member of the DJ-1/ThiJ/PfpI superfamily. Proc Natl Acad Sci U S A. 101:1531-6. [abstract]

Holyoak T, Fenn TD, Wilson MA, Moulin AG, Ringe D, Petsko GA. (2003) Malonate: a versatile cryoprotectant and stabilizing solution for salt-grown macromolecular crystals. Acta Crystallogr D Biol Crystallogr. 59(Pt 12):2356-8. [abstract]

Ringe D, Petsko GA. (2003) The 'glass transition' in protein dynamics: what it is, why it occurs, and how to exploit it. Biophys Chem. 105:667-80. [abstract]

Wilson MA, Collins JL, Hod Y, Ringe D, Petsko GA. (2003) The 1.1-A resolution crystal structure of DJ-1, the protein mutated in autosomal recessive early onset Parkinson's disease. Proc Natl Acad Sci U S A. 100:9256-61. [abstract]

D'Aquino JA, Ringe D. (2003) Determinants of the SRC homology domain 3-like fold. J Bacteriol. 185:4081-6. [abstract]

Holyoak T, Wilson MA, Fenn TD, Kettner CA, Petsko GA, Fuller RS, Ringe D. (2003) 2.4 A resolution crystal structure of the prototypical hormone-processing protease Kex2 in complex with an Ala-Lys-Arg boronic acid inhibitor. Biochemistry. 42:6709-18. [abstract]

Fenn TD, Stamper GF, Morollo AA, Ringe D. (2003) A side reaction of alanine racemase: transamination of cycloserine. Biochemistry. 42:5775-83. [abstract]

Beebe JA, Arabshahi A, Clifton JG, Ringe D, Petsko GA, Frey PA. (2003) Galactose mutarotase: pH dependence of enzymatic mutarotation. Biochemistry. 42:4414-20. [abstract]

Spiering MM, Ringe D, Murphy JR, Marletta MA. (2003) Metal stoichiometry and functional studies of the diphtheria toxin repressor. Proc Natl Acad Sci U S A. 100:3808-13. [abstract]

Kenyon GL, DeMarini DM, Fuchs E, Galas DJ, Kirsch JF, Leyh TS, Moos WH, Petsko GA, Ringe D, Rubin GM, Sheahan LC; National Research Council Steering Committee. (2002) Defining the mandate of proteomics in the post-genomics era: workshop report. Mol Cell Proteomics. 1:763-80. [abstract]

Desmarais, W., Bienvenue, D.L., Bzymek, K.P., Holz, R.C., Petsko, G.A., Ringe, D., (2002) "The 1.20 Å Resolution Crystal Structure of the Aminopeptidase from Aeromonas proteolytica Complexed with Tris: A Tale of Buffer Inhibition", Structure, 10:1063-72.

Taoka S., Lepore, B.W., Kabil, O., Ojha, S., Ringe, D., Banerjee, R. (2002), "Human Cystathionine beta-Synthase Is a Heme Sensor Protein. Evidence That the Redox Sensor is Heme and Not the Vicinal Cysteines in the CXXC Motif Seen in the Crystal Structure of the Truncated Enzyme", Biochemistry 41:10454-10461.

Bienvenue, D.L., Mathew, R.S., Ringe, D. Holz, R.C., (2002) "The Aminopeptidase from Aeromonas proteolytica can function as an esterase", J. Biol. Inorg. Chem., 7:129-135.

Vitkup, D., Ringe, D., Karplus, M., Petsko, G.A.(2002) "Why Protein R-factors are so large: A self-consistent analysis", Proteins, 46:345-354. [abstract]

Ringe, D.(2002), "Function by Serendipidy", Nature, 415:488-489.

Vogan, E.M., Bellamacina, C.R., He, X., Foxman, B.M., Ringe, D., Liu, H.W., Petsko, G.A. (2002). "Purification, crystallization and molecular symmetry of CDP-D glucose, 4, 6-dehydratase from Yersinia pseudotuberculosis", Acta Cryst. D. 58:370-373. [abstract]

Mattos, C. and Ringe, D. (2001) "Proteins in organic solvents", Current Opinion in Structural Biology, 11:761-764.

Ondrechen, M.J., Clifton, J. G., Ringe, D. (2001) "THEMATICS: A Simple Computational Predictor of Enzyme Function from Structure", Proc. Natl. Acad. Sci., USA, 98:12473-12478.

Hadfield, A., Shammas, C., Kryger, G., Ringe, D., Petsko, G.A., Ouyang, J., Viola, R. E. (2001) "Active Site Analysis of the Potential Antimicrobial Target Aspartate Semialdehyde Dehydrongenase", Biochemistry 40:14475-14483.

Amor, J.C., Horton, J., Zhu, X., Wang, Y., Sullards, C., Ringe, D., Cheng, X., Kahn, R.A. (2001) "Structures of yeast ARF2 and ARL1: distinct roless for the N-terminus in the structure and function of ARF family GTPases", J. Biol Chem. 276:42477-42484.

Pasternak A, White A, Jeffery CJ, Medina N, Cahoon M, Ringe D, Hedstrom L. (2001) The energetic cost of induced fit catalysis: Crystal structures of trypsinogen mutants with enhanced activity and inhibitor affinity. Protein Sci. 10:1331-42. [abstract]

Stamper C, Bennett B, Edwards T, Holz RC, Ringe D, Petsko G. (2001) Inhibition of the aminopeptidase from Aeromonas proteolytica by L-leucinephosphonic acid. Spectroscopic and crystallographic characterization of the transition state of peptide hydrolysis. Biochemistry. 40:7035-46. [abstract]

Chen CS, White A, Love J, Murphy JR, Ringe D. (2000) Methyl groups of thymine bases are important for nucleic acid recognition by DtxR. Biochemistry. 39:10397-407. [abstract]

Jeffery, C.J., Bahnson, B.J., Chien, W., Ringe, D., Petsko, G.A. (2000) Crystal Structure of Rabbit Phosphoglucose Isomerase, a Glycolytic Enzyme that Moonlights as Neuroleukin, Autocrine Motility Factor, and Differentiation Mediator, Biochemistry, 39:955-964.

Petsko, G. A. and Ringe, D. (2000) Observation of unstable species in enzyme-catalyzed transformations using protein crystallography, Current Opinion in Chemical Biology, 4:89-94. [abstract]

Vitkup, Dennis, Ringe, D., Petsko, G.A., and Karplus, Martin. (2000) Solvent Mobility and the Protein "Glass" Transition, Nature Structural Biology, 7:34-38. [abstract]

Schlichting, I., Berendzen, J., Chu, K., Stock, A.M., Maves, S.A., Benson, D.E., Sweet, R.M., Ringe, D., Petsko, G.A., Sligar, S.G. (2000) The Catalytic Pathway of Cytochrome P450 at Atomic Resolution, Science, 287:1615-1622.

McMillan, F.M., Cahoon, M., White, A., Hedstrom, L., Petsko, G.A., Ringe, D. (2000) 2.4 Å Crystal Structure of Borrelia burgdorferi IMP Dehydrogenase: Evidence of a Substrate Induced Hinged-Lid Motion by Loop 6, Biochemistry, 39:4533-4542. [abstract]

De Paola CC, Bennett B, Holz RC, Ringe D, Petsko GA. (1999) 1-Butaneboronic acid binding to Aeromonas proteolytica aminopeptidase: a case of arrested development, Biochemistry, 38:9048-53.

Hadfield A, Kryger G, Ouyang J, Petsko GA, Ringe D, Viola R. (1999) Structure of aspartate-beta-semialdehyde dehydrogenase from Escherichia coli, a key enzyme in the aspartate family of amino acid biosynthesis,. J Mol Biol, 289:991-1002.

Zhang Z, Komives EA, Sugio S, Blacklow SC, Narayana N, Xuong NH, Stock AM, Petsko GA, Ringe D. (1999) The role of water in the catalytic efficiency of triosephosphate isomerase. Biochemistry, 38:4389-97.

Morollo AA, Petsko GA, Ringe D. (1999) Structure of a Michaelis complex analogue: propionate binds in the substrate carboxylate site of alanine racemase. Biochemistry, 38:3293-301.

White A, Ding X, vanderSpek JC, Murphy JR, Ringe D. (1998) Structure of the metal-ion-activated diphtheria toxin repressor/tox operator complex. Nature 394:502-6.

Hasson MS, Muscate A, McLeish MJ, Polovnikova LS, Gerlt JA, Kenyon GL, Petsko GA, Ringe D (1998). The crystal structure of benzoylformate decarboxylase at 1.6 Å resolution: diversity of catalytic residues in thiamin diphosphate-dependent enzymes, Biochemistry, 37:9918-30.

Brenner C, Garrison P, Gilmour J, Peisach D, Ringe D, Petsko GA, Lowenstein JM. (1997) Crystal structures of HINT demonstrate that histidine triad proteins are GalT-related nucleotide-binding proteins. Nature Structural Biology, 4:231-8.