Program

Stephen W. Ragsdale, Don M. Becker, Eisuke Murakami, Mihaela Simianu, Sharmin Allen. 

Department of Biochemistry, Beadle Center, University of Nebraska, Lincoln, NE 68588-0664. 

Methyl-CoM reductase (MCR) (Reaction 1) and heterodisulfide reductase (HDR) (Reaction 2) 

MCR: CH3-SCOM + CoB-SH→→→ CH4 + CoB-SS-COM (1) 

HDR: COB-SS-COM + 2 e→ CoB-SH + CoM-SH (2) 

catalyze key reactions in methane synthesis. CoM-SH and CoB-SH are cofactors that are unique to methanogens. MCR contains 2 mole of a nickel tetrahydrocorphinoid, Factor F430. Mechanistic studies of MCR have been thwarted by the unavailability of stabile, active enzyme. We recently developed a protocol to activate MCR that involves controlling the Ni ligation and redox state. Ni(I) was shown to be the active state of the enzyme. 

The MCR reaction produces CoB-SS-COM, which must be reduced by HDR to participate in another cycle of methanogenesis. HDR also is involved in vectorial proton translocation that is coupled to ATP synthesis. We recently characterized HDR from Methanosarcina thermophila and showed that it contains two b-type hemes and two Fe4S4 clusters. Our recent results have led to a proposed mechanism that can account for proton translocation and electron transfer through the four metal centers leading to reduction of the heterodisulfide. 

Adam K. Whiting, Masami Ito and Lawrence Que, Jr. 

Department of Chemistry and the Center for Metals in Biocatalysis University of Minnesota, Minneapolis, MN 55455 

p-Nitrocatechol (pNC) is a strong competitive inhibitor of the Mn(II)-dependent extradiol cleaving catechol dioxygenase (MndD). The distinctive EPR features of the pNC-MndD complex suggest direct coordination of pNC to the active site Mn(II) in a mode similar to native substrate binding. In this poster we report that the pNC-MndD complex also gives rise to a strong electronic absorbance (^max = 526 nm, 13,600 M-1cm-1) that is highly red-shifted relative to free pNC. 

Furthermore, under aerobic conditions this chromophore disappears in a very slow first-order process (kobs = 0.0005 min-1) concomitant with the appearance of an extradiol cleavage product confirming that pNC is in fact a slow substrate of MndD. These spectroscopic and functional properties of pNC-MndD differ greatly from those of the PNC complex of the analogous Fe(II)- dependent enzyme which exhibits a λmax at 427 nm and a 104-fold faster extradiol cleavage rate. 

Resonance Raman spectra of pNC-MndD as well as two synthetic pNC-Mn(II) model complexes leads us to conclude that pNC coordinates the active site Mn(II) as a dianion. The inability of dianionic pNC-MndD complex to turn over efficiently further supports a mechanism for extradiol cleavage in which monoanionic substrate-metal coordination is essential for activity.

Lydia Tabernero, Daniel A. Bochar, Victor W. Rodwell and Cynthia V. Stauffacher. 

Departments of Life Sciences and Biochemistry. Purdue University, West Lafayette, Indiana 47907. USA. 

HMG-CoA (3-hydroxy-3-methylglutaryl coenzyme A) reductase catalyzes the interconversion of HMG-CoA and mevalonate, a reaction at the root of the pathway leading to synthesis of isoprenoid lipids, including cholesterol and its derivatives. In mammals this reaction is the first committed step in cholesterol biosynthesis, therefore HMG-CoA reductase is a primary target enzyme for chemotherapy of hypercholesterolemias. 

The crystal structure of the HMG-CoA reductase from Pseudomonas mevalonii, [C.M. Lawrence, V.W. Rodwell, and C.V. Stauffacher. (1995) Science 268, 1758- 1762.] a catalytic model for this class of enzymes, appears to be a tightly bound dimer that brings together the conserved residues implicated in binding and catalysis at the subunit interfaces. Each monomer can be divided into two major domains. The large domain, which contains both the N and C termini, has an unusual conformation, showing three roughly triangular walls around a central 24-residue a-helix. The small domain, which binds the NAD(H) cofactor, has a unique binding fold built on a four-strand anti-parallel b-sheet. 

Non-productive ternary complexes of HMG-CoA and NAD, and mevalonate and NADH were produced by successive soaking of the apoenzyme crystals. The crystallographic structures show the closing of the flexible flap that forms half of the active site and that was missing in the initial structure of the apoenzyme. Furthermore we were able to locate the catalytic histidine in the flexible flap and to identify a new residue, Lys-267, involved in catalysis. Based on the analysis of the three-dimensional structures and mutagenesis data we propose a new mechanism for the interconversion of HMG-CoA and mevalonate catalyzed by HMG-CoA reductase.

Ronald E. Viola”, Maithri Jayasekera”, Jennifer Dunbar, Greg Farber#, Andrea Hadfield and Greg Petsko 

*University of Akron, Akron, Ohio, "Penn State University, State College, PA, *Brandeis University, Waltham, MA. 

One of the primary goals of mechanistic enzymology is to learn how enzymes accelerate chemical reactions. Two enzyme examples will be presented to show what site-directed mutagenesis studies and X-ray structural studies can tell us about the details of catalysis. L-Aspartase catalyzes the reversible deamination of aspartic acid to fumarate and ammonia. The structure of this tetrameric enzyme has been determined to 2.8Å resolution, and reveals a central core consisting of a 20-helix cluster. Site-directed mutagenesis studies have indicated the location of the active site, and have led to tentative assignments of both binding and catalytic residues in this enzyme. Aspartic acid is also the precursor for the biosynthesis of four amino acids in both plants and in microorganisms. After phosphorylation of the B-carboxyl group of aspartic acid, the enzyme aspartate-ß-semialdehyde dehydrogenase reduces this position to an aldehyde. This metabolic intermediate is located at a critical branch point in the biosynthetic pathway. Mutagenesis studies have previously identified the active site nucleophile in this enzyme, and some of the groups that are involved in substrate and coenzyme binding. The high-resolution structure of this dimeric enzyme has now been determined. An examination of the structure of the free enzyme and an enzyme-inactivator complex has disclosed some additional amino acids that appear to be of functional significance. This work was partially supported by a grant from the NIH (DK 47838).

A.F. Arendsen Y.B.M. Bulsink3, W.A.M. van den Berg, S.J. Marritt, W.R. Hagen' 

Department of Biochemistry, Wageningen, Dreijenlaan 3, NL-6703 HA Wageningen, the Netherlands 

M.C. Feiters 

b Department of Organic Chemistry, Catholic University Nijmegen, NL-6526 ED Nijmegen, the Netherlands 

J. Charnock, D. Collison, C.D. Garner 

c Department of Chemistry, Manchester University, Oxford Rd, Manchester M13 9PL, UK 

M. Kröckel, A.X. Trautweind 

d Institute of Physics, Ratzeburger Allee 160, D-23538 Lübeck, Germany 

J.M. hadden, G.L. Card, A.S. McAlpine, S. Bailey, V.N. Zaitsev, P.F. Lindley 

* CCLRC Daresbury Laboratory, Warrington, Cheshire, WA4 4AD, UK 

The three-dimensional structure of the native 'putative prismane' protein from Desulfovibrio vulgaris (Hildenborough) has been solved by the multiple isomorphous replacement technique to a resolution of 1.72 Å. The molecule does not contain a [6Fe-6S] cluster, but rather two 4Fe clusters some 12 Å apart and situated close to the interfaces formed by the three domains of the protein. Cluster 1 is a conventional [4Fe-4S] cubane, however, bound near the N-terminus by an unusual, sequencial sequence of four cysteine residues (Cys 3, 6, 15, 21). Cluster 2 is a novel structure with two μ2-sulphido bridges, two μ2-oxo bridges, and a partially occupied, unidentified μ2 bridge X. The protein ligands of cluster 2 are widely scattered through the second half of the sequence and include three cysteine residues (Cys 312, 434, 459), one persulphido- cysteine (Cys 406), two glutamates (Glu 268, 494), and one histidine (His 244). Its assymmetric, open structure suggests it to be the site of a catalytic activity. 

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V. Jo Davisson 

Department of Medicinal Chemistry and Molecular Pharmacology, 1333 Robert Heine Pharmacy Building, Purdue University, West Lafayette, IN 47907-1333 

One of the unifying principles in modern biology is that all organisms show marked similarities in their central metabolic pathways. This generalization is based upon a common evolutionary relationship among the constituent metabolic enzymes that control the chemistry associated with each of these pathways. De novo purine biosynthesis is a pathway common to all life forms (with the exception of some protoza) which represents a highly integrated process involving nitrogen, amino acid, carbohydrate, and one carbon metabolism. Because of the complexity and intensive energy commitments of purine metabolism, the expectation that biochemical similarities across kingdoms has prevailed. However, there are distinctions emerging from the detailed comparative biochemical studies of the enzymes in the pathway. Our recent studies of the ADE2 protein have identified a divergence in the step for conversion of 5'-aminoimidazole (AIR) ribonucleotide to 4-carboxy-5-aminoimidazole ribonucleotide (CAIR). This reaction represents a unique example of biological carboxylation chemistry since the substrate is an aromatic ring system and no cofactor is required. The enzyme systems from bacteria, fungi, vertebrates, and archaebacteria which catalyze this reaction have been isolated and the details of the true substrate requirements have been established. In addition, a series of inhibitors, and alternate substrates have been used to probe the divergence of the AIR carboxylases and to establish possibile significance of this enzyme as a target for antifungal agents. The results implicate a general relationship among CO2 requirements for different life forms and the molecular evolution of AIR carboxylases. 

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Barbara S. Gibbs, ‡ Todd J. Zahn,† YongQi Mu,† Judith S. Sebolt-Leopold,* and Richard A. Gibbs*,* 

*Department of Cell Biology, Parke-Davis Pharmaceutical Research, 2800 Plymouth Road, Ann Arbor, MI 48105, and †Department of Pharmaceutical Sciences, College of Pharmacy and Allied Health Professions, Wayne State University, 528 Shapero Hall, Detroit, MI 48202 

Inhibition of protein farnesyltransferase (FTase), the enzyme responsible for protein farnesylation, has become a key target for the rational design of cancer chemotherapeutic agents. We have found that certain novel farnesyl diphosphate analogs are potent inhibitors of mammalian FTase. Furthermore, the alcohol precursors of two of these compounds, namely 3-allylfarnesol and 3-vinylfarnesol, are able to efficiently block the growth of ras transformed cells. While the former compound is an inhibitor of protein farnesylation, the latter instead acts as an alternative substrate and leads to the abnormal prenylation of proteins with the 3-vinylfarnesyl group. This finding strongly implies that the specific structure of the farnesyl group is key to the biological activity of certain farnesylated proteins. 

Dehua Pei, P. T. Ravi Rajagopalan, Yaoming Wei, & Michael K. Chan 

Department of Chemistry and Ohio State Biochemistry Program, The Ohio State University, 100 W. 18th Avenue, Columbus, OH 43210. 

Peptide deformylase is involved in the maturation of bacterial proteins by removing the N-formyl group from newly synthesized polypeptides. It is an essential activity for bacterial survival but absent in eukaryotes, providing an ideal target for antibacterial chemotherapy. Despite of its discovery over thirty years ago, the enzyme has resisted any purification or characterization due to its extreme lability. We have overexpressed the E. coli deformylase and purified it to homogeneity. A variety of enzymological studies are being carried out on the enzyme. Its tertiary structure has been solved by X-ray crystallography. The deformylase contains one iron atom per polypeptide and represents a novel type of amide hydrolyase. The mechanism for its rapid inactivation has been determined. Potent inhibitors have been developed which eliminate deformylase activity in vitro and arrest bacterial cell growth in vivo.

K. K. Wong, M. S. Anderson, S. S. Eveland, S. Reddy, D. Kuo, R. Chabin, C. Fournier & D. L. Pompliano

Department of Biochemistry, Merck Research Laboratories, P. O. Box 2000, Rahway, NJ 07065. 

The peptidoglycan, a polymeric molecule that surrounds and shapes the bacterial cell, is composed of an array of glycan strands connected by short, unusual peptide crosslinks. Its biosynthesis starts in the cytoplasm with the condensation of phosphoenolpyruvate and UDP-N-acetylglucosamine catalyzed by MurA, and finishes across the plasma membrane in the periplasm with the transglycosylation and transpeptidation of the disaccharide pentapeptide monomers by the penicillin-binding proteins. The focus of the cytoplasmic steps is construction of the pentapeptide sidechain by stepwise addition of amino acids to lactyl carboxylate of UDP-N-acetylmuramic acid. These ADP-generating peptide-bond-forming reactions are catalyzed by MurC, D, E and F. Genetic studies have shown that these enzymes are essential for cell viability and, as such, are attractive targets for antibacterial chemotherapy. We have expressed all of the enzymes involved in the construction of the disaccharide pentapeptide monomer and reconstituted the pathway of biosynthesis in vitro. The kinetic behavior of the pathway has been modeled and the experimental conditions of its operation optimized to detect inhibitors of the component enzymes. Certain evolutionary relationships amongst the Mur enzymes suggest that a single inhibitor may inhibit more than one enyzme. The strategy and progress toward developing such inhibitors will be discussed.