Program

James D. Patrone, Jiangwei Yao, and GARRY D. DOTSON Department of Medicinal Chemistry, College of Pharmacy, University of Michigan, Ann Arbor, MI 48109 

Coenzyme A (CoA) biosynthesis from (R)-pantothenate has been shown to be conserved and essential for microorganism growth and survival. The critical nature of CoA to the integrity and viability of cells makes the biosynthetic pathway leading to its production an intriguing target for antimicrobial development. Phosphopantothenoylcysteine synthetase (PPCS) is responsible for installing the biologically reactive cystamine moiety contained within the phosphopantetheine prosthetic group of CoA. This enzyme catalyzes the nucleotide triphosphate (NTP)-dependent amide bond formation between the carboxyl group of (R)- phosphopantothenate and the amine moiety of L-cysteine. Herein, detailed kinetic and chemical studies show that the bacterial enzymes are very specific for CTP, while the human enzyme can use both ATP and CTP with similar affinity. Utilizing initial velocity, product inhibition, and isotopic labeling studies, we describe the kinetic and chemical mechanism of the monofunctional PPCSS from Enterococcus faecalis and human. Both enzymes proceed through a phosphopantothenoyl nucleotide, mixed anhydride intermediate, and utilize a Bi Uni Uni Bi Ping Pong kinetic mechanism. Both structural and kinetic characterization studies on PPCS have shown differences in the nucleobase binding site between the bacterial and human enzyme. We have designed and synthesized mimics of the phosphopantothenoyl cytidylate intermediate formed during PPCS catalysis. These compounds were evaluated in vitro against PPCS from human and several species of bacteria and show marked potency and selectivity towards the bacterial enzymes.

Zhuojun Guo, Krishanthi S. Karunatilaka and DAVID RUEDA 

Department of Chemistry, Wayne State University, Detroit, MI 48236 

Spliceosomes catalyze the maturation of precursor mRNAs from yeast to humans. Their catalytic core comprises three small nuclear RNAs (U2, U5 and U6) involved in substrate positioning and catalysis. It has been postulated, but never shown experimentally, that the U2/U6 complex adopts at least two conformations that reflect different activation states. We have used single-molecule fluorescence to probe the structural dynamics of a protein-free RNA complex modeling U2/U6 from yeast and mutants of highly conserved regions. Our data show the presence of at least three distinct conformations in equilibrium. The minimal folding pathway consists of a two-step process with an obligatory intermediate. The first step is strongly magnesium dependent and we provide evidence suggesting the second corresponds to the formation of the genetically conserved helix IB. Site-specific mutations in the highly conserved AGC triad and the U80 base in U6 suggest that the observed conformational dynamics correlate with residues that play an important role in splicing. a Phase 1 clinical trial.

EMILY SCOTT, Linda Blake, The Department of Medicinal Chemistry, The University of Kansas, 1251 Wescoe Hall Dr., Lawrence, KS 66045 

Many human cytochrome P450 enzymes metabolize xenobiotic compounds, including drugs, so that they can be subsequently cleared from the body. Although evidence of cytochrome P450 inhibition often halts development of new drug candidates, cytochrome P450 enzymes can also be intentional therapeutic targets. Cytochrome P450 2A13 (CYP2A13) is a human respiratory tract enzyme responsible for metabolism of the nicotine- derived molecule 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), one of the two most prevalent and carcinogenic compounds in tobacco. CYP2A13 activates NNK to generate diazonium metabolites that in turn form DNA adducts and can cause lung cancer. Thus, CYP2A13 inhibition is expected to reduce DNA damage and lung cancer in nicotine- exposed individuals. Since CYP2A13 is 93.5% identical to the human liver CYP2A6 enzyme, selective inhibition poses a substantial challenge. Simple, selective inhibitors of CYP2A13 were first identified from high throughput screening. A small library of benzylmorpholine analogs has been generated and evaluated for differential ligand binding and inhibition of human cytochrome P450 2A enzymes. Structure-activity relationships are currently being defined to optimize both the selectivity and inhibition of the lung CYP2A13 enzyme. Identification of these selective inhibitors of CYP2A13 constitutes a novel approach to lung cancer chemoprevention by targeting reduction of the in vivo formation of nicotine-derived carcinogens without requiring changes in tobacco use, a primary source of mortality accounting for 1.4 million deaths annually. 

MARTIN NEWCOMB, John H. Horner, Xin Sheng, Rui Zhang, Xinting Yuan, and Qin Wang, Department of Chemistry, University of Illinois at Chicago, 845 W. Taylor St., Chicago, IL 60607 

The oxidant in cytochrome P450 enzymes has long been assumed to be an iron(IV)-oxo porphyrin radical cation termed Compound I, but only fleeting evidence for detection of P450 Compounds I from rapid mixing studies of P450 enzymes with chemical oxidants exists. As an alternative entry to Compounds I, our group developed a method in which irradiation of iron(IV)-oxo neutral porphyrin species (Compounds II) produces Compounds I by photo-ejection. Compounds II, in turn, can be prepared by reaction of some resting P450 enzymes with peroxynitrite (PN). The sequence of PN treatment followed by photolysis has given P450 Compounds I that have been studied at ambient temperature via stopped-flow mixing and laser flash photolysis. At temperatures as low as -50 °C, P450 Compounds I can be prepared quantitatively for spectroscopic and kinetic studies in 50% glycerol solutions by PN treatment followed by photolysis with a powerful pulsed lamp. Kinetic studies of P450 Compounds I reactions include epoxidations of alkenes and styrene and hydroxylations of high reactivity (benzylic), modest reactivity (adjacent to heteroatoms), and low reactivity C- H positions (lauric acid). The presentation will discuss the methods for production of P450 Compounds I and physical and kinetic studies of these intermediates.

DENNIS J. STUEHR, Jesus Tejero, Ashis Biswas, Zhi-Qiang Wang, Richard C. Page, Mohammed Mahfuzul Haque, Craig Hemann, Jay L. Zweier, and Saurav Misra

Department of Pathobiology and Department of Molecular Cardiology, Cleveland Clinic, Cleveland, OH, Department of Chemistry, Kent State University Tuscarawas, New Philadelphia, OH, Davis Heart & Lung Research Institute, Ohio State University, Columbus, OH. 

Nitric oxide synthases (NOS) are heme-thiolate enzymes that generate nitric oxide from L- Arginine. The mechanisms by which NOS generate and control reactivity of their heme-oxy intermediates is of current interest. NOS enzymes have a proximal tryptophan that hydrogen bonds with the heme-thiolate. To assess its importance we replaced Trp with His in the inducible NOS (INOSoxy). The W188H mutation had small effects on L-Arg binding and on enzyme heme-CO and heme-NO absorbance spectra, but increased the heme midpoint potential by 88 mV relative to wild-type iNOSoxy. The crystal structure showed that the His 188 imidazole was in position to form a stronger hydrogen bond with the heme thiolate. Analysis of a single turnover L-Arg hydroxylation reaction revealed that a new heme species built up during the reaction. Its UV-visible spectra suggests it is a Compound 1-like species (FelV=O porph+). Formation of this intermediate required tetrahydrobiopterin (HB) be bound within iNOSoxy, and its buildup was kinetically coupled to the formation of the bound H4B radical. Moreover, the disappearance of the intermediate correlated kinetically with L- Arg hydroxylation. These data establish that the intermediate is kinetically and chemically competent. Our results suggest that the W188H mutation stabilizes the Compound 1 intermediate in NOS, whose reactivity then becomes rate-limiting for L-Arg hydroxylation. We propose that Trp188 facilitates NO synthesis by tuning the heme-thiolate midpoint potential, which in turn may control reactivity of Compound Itoward NOS substrates. Additional research that tests this and related hypotheses will be discussed.

SYLVIE GARNEAU-TSODIKOVA Department of Medicinal Chemistry and Life Sciences Institute, University of Michigan, 210 Washtenaw Ave, Ann Arbor, MI 48109, U.S.A. 

Aminoglycosides are broad-spectrum antibiotics commonly used for the treatment of serious. bacterial infections. Over six decades of intensive clinical use of aminoglycosides has led to the emergence of bacterial resistance to this family of drugs. A highly prevalent mode of bacterial resistance to aminoglycosides evolved through the acquisition of enzymes that modify the antibiotics. These enzymes perform three chemical alterations of the drug: N- acetylation by acetyltransferases (AACs), adenylation by adenyltransferases (ANTs), or phosphorylation by phosphotransferases (APHs). We took advantage of the substrate and cosubstrate promiscuity of AACs (AAC(3)-IV and AAC(6”)-APH(2”)) to generate novel N- acylated aminoglycoside antibiotics.

AYANO SAKAI,' Heidi J. Imker,2 Chakrapani Kalyanaraman,3 Alexander A. Fedorov,* Elena V. Fedorov, Margaret E. Glasner, Patricia C. Babbitt,36 Steven C. Almo, Matthew P. Jacobson,3 and John A. Gerlt

' 'Departments of Biochemistry and Chemistry, University of Illinois at Urbana-Champaign, 600 South Mathews Ave, Urbana, IL 61801, Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, 25 Shattuck St, Boston, MA 02115, 'Department of Pharmaceutical Chemistry, University of California, 600 16th St, San Francisco, CA 94158, "Department of Biochemistry, Albert Einstein College of Medicine, Bronx, NY 10461, USA Department of Biochemistry & Biophysics, Texas A&M University, 2128 TAMU, College Station, TX 77843, "Department of Biopharmaceutical Sciences, University of California, 1700 4th St, San Francisco, CA 94158. 

This study introduces a new strategy for assigning correct functions to members of the enolase superfamily. Using the mechanistically diverse muconate lactonizing enzyme (MLE) subgroup of the enolase superfamily, in silico docking of a ligand library to a homology-modeled structure was used to predict substrate specificity and to facilitate experimental screening of N-acylamino acid and dipeptide libraries. Although the potential libraries of possible amino acid derivatives can be enormous, our modeling and docking approach for targeting subsets of substrates is practical when no clear genomic context is available for function assignment. An uncharacterized member in the MLE subgroup from Enterococcus faecalis with low sequence identity to other characterized members in the dipeptide epimerase family was selected for application and development of our new approach. The closest homologue with characterized structure and function is the L-Ala- D/L-Glu epimerase (AEE) from Bacillus subtilis (31% sequence identity), whose structures are deposited at the PDB. The study resulted in close agreement between computational prediction and experimental result of substrate preference which led to function assignment of E. faecalis dipeptide epimerase. A successful example of the computational approach proved that a homology-model based protein structure was comparable to the native protein. structures. Additionally, this study indicates water molecules playing a significant role in protein-ligand binding calculation, which advances our computational approach for further understanding of the function/structure relationship in the enolase superfamily.

THOMAS M. MAKRIS', Mrinmoy Chakrabarti2, Eckard Münck2, and John D. Lipscomb' 

'Dept. of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Minneapolis, MN, 55455 

2Dept. of Chemistry, Carnegie Mellon University, Pittsburgh, PA 15213 

Natural product assembly lines often involve the use of tailoring enzymes which are critical for the resulting structure and bioactivity of the non-ribosomal peptide product. These include several types of oxygenases, which are involved in the biosynthesis of pharmaceutically important antimicrobial and cytostatic compounds. Notably, non-heme diiron monooxygenases are not known to catalyze these reactions. Analysis of the chloramphenicol biosynthesis gene cluster, chosen as a model system because of its structural simplicity, has now revealed the presence of two diiron monooxygenases, which we have recently recombinantly expressed and characterized. The first, a beta-hydroxylase, represents a novel diiron oxygenase motif which also appears to be utilized in the biosynthetic pathways of a number of pharmaceutical mainstays. We present optical, EPR, and Mössbauer spectra to establish the nature of the diiron cofactor. Also, analytical methods are used in concert with stopped flow kinetics to determine the substrate specificity, mechanism, and regulation. The second enzyme, an N-oxygenase, belongs to a group that has been the subject of considerable debate regarding metal ion specificity and mechanism. We present the spectroscopic characterization of the enzyme, and utilizing metal titration and activity studies, unambiguously assign the diiron cofactor. Its role in chloramphenicol biosynthesis will be discussed. 

YUANYUAN CHEN', Michael L. Gleghorn2, Elena K. Davydova3, Lucia B. Rothman- Denes3, Katsuhiko S. Murakami2 and Paul R. Carey' 

'Department of Biochemistry, Case Western Reserve University 

'Department of Biochemistry and Molecular Biology, The Pennsylvania State University 'Department of Molecular Genetics and Cell Biology, University of Chicago, Chicago 

The de novo transcription, an unique phosphoryl transfer reaction forming a 2-mer RNA from two nucleotide triphosphates, is observed and characterized in single crystals of N4 phage virion-encapsulated RNA polymerase (RNAP) using the complementary techniques of X-ray crystallography and Raman spectroscopy. The X-ray studies provide exquisite structure details of four species on the reaction pathway: the binary complex of the enzyme and a 36 nt DNA template, two pre-catalytic intermediate complexes and a post-catalytic complex with the 2-mer RNA product in the active site, and these structural snapshots reveal conformational changes of the enzyme, DNA and nucleotides associated during the catalysis. Raman spectroscopy is applied to monitor the formation of the pre-catalytic and post- catalytic complexes from the binary complex in real-time. By soaking isotopically labeled substrates and metals into RNAP-DNA crystals, different events including substrate-DNA base pairing, active site O-helix conformational change, formation of metal binding sites, decrease of substrates and increase of products, are observed as a function of time. The Raman results provide important temporal information linking the X-ray snapshots to provide a more complete account of de novo transcription. 

ERIC M. KOEHN', Todd Fleischmann', John A. Conrad2, Bruce A. Palfey2, Scott A. Lesley3, Irimpan I. Mathews & Amnon Kohen1 

'Department of Chemistry, University of Iowa, Iowa City, Iowa 52242. 2Department of Biological Chemistry, University of Michigan Medical School, Ann Arbor, Michigan 48109. 3The Joint Center for Structural Genomics at the Genomics Institute of Novartis Research. Foundation, San Diego, California 92121. 4Stanford Synchrotron Radiation Laboratory, Stanford University, Menlo Park, California 94025. 

Biosynthesis of the DNA base thymine depends on activity of the enzyme thymidylate synthase to catalyze the methylation of the uracil moiety of 2'-deoxyuridine-5'- monophosphate (dUMP). All known thymidylate synthases rely on an active site residue of the enzyme to activate the substrate, dUMP. This functionality has been demonstrated for classical thymidylate synthases, including human thymidylate synthase, and is instrumental in mechanism-based inhibition of these enzymes. Here we report an example of thymidylate biosynthesis that occurs without an enzymatic nucleophile [Koehn, E. M. et al. Nature, 458, 919-923 (2009)]. This unusual biosynthetic pathway occurs in organisms containing the thyX gene, which codes for a flavin-dependent thymidylate synthase (FDTS), and is present in several human pathogens. Our findings suggested that the putative active site nucleophile is not required for FDTS catalysis, and no alternative nucleophilic residues capable of serving this function can be identified. Instead, our findings suggest that a hydride equivalent is transferred from the reduced flavin cofactor directly to the uracil ring, followed by an isomerization of the intermediate to form the product, 2'-deoxythymidine-5'-monophosphate (dTMP). These observations indicate a very different chemical cascade than that of classical thymidylate synthases or any other known biological methylation. The findings and chemical mechanism proposed here, together with available structural data, suggest that selective inhibition of FDTSs, with little effect on human thymine biosynthesis, should be feasible. Because several human pathogens depend on FDTS for DNA biosynthesis, its unique mechanism makes it an attractive target for antibiotic drugs.