Christopher T. Walsh, Ph.D. - US grants

Four problems in flavoenzyme structure and catalytic mechanism are to be studied, reflecting both the diversity of electron transfer chemistry available to flavin coenzymes and the biological range of reactions utilizing vitamin B2 derivatives. 1) We shall continue studies on the mer A gene and its encoded enzyme, mercuric ion reductase, conferring resistance to inorganic mercuric salts to bacteria that carry this and associated genes of the mer operon. Site-directed mutagenesis of sulfhydryl groups will continue to be a focus. 2) Cyclohexanone Oxygenase enables soil bacteria to grow on cycloalkanones as carbon source and is of interest as the best characterized biological Baeyer- Villiger oxygenation catalyst. We have cloned and sequenced the Acinetobacter gene and will continue analysis of structure and catalytic mechanism for oxygen activation and transfer to cosubstrates. 3) Cyclobutane-containing intrachain thymine dimers are the major lesions in UV-damaged DNA, and in several species (prokaryotes and eukaryotes) these can be repaired in a visible-light driven photomonomerization reaction. The photoreactivation enzyme from streptomycetes, blue green alga Anacystis nidulans, and from methanobacteria appear to contain both FAD and the 8-hydroxy-5-deazaflavin factor F420. We will analyze how visible light is utilized in this two coenzyme sequence to photorepair DNA. 4) In kinetoplastid-containing parasites such as trypanosomes and leishmania there is very little glutathione; most glutathione is modified as the N1,8-bis spermidinyl derivative known as trypanothione. We have determined there is no detectable glutathione reductase in these parasites but rather a specific trypanothione reductase which we have purified to homogeneity and characterized as an analogous flavoenzyme. We are in the process of cloning and sequencing the gene from various trypanosomatids and will continue molecular biology and enzymology studies on this enzyme, likely target for antiparasitic agents.

The goals of this proposal are the study of enzymatic reaction mechanisms. In this project period the focus is on the reaction mechanisms of two kinds of enzyme systems involved in the biosynthesis of peptide antibiotics. The first is the enzymatic heterocyclization machinery involved in the conversion of the 69aa Microcin A protein, an antibiotic precursor, to Microcin B17, an antibiotic targetted against E.coli DNA gyrase, in which 14 residues (six gly, four ser, four cys) have been posttranslationally modified to four thiazole and four oxazole rings, essential for antibiotic activity. The second goal is analysis of the enzymatic strategies used by multimodular enzymes that make peptide bonds nonribosomally, e.g. in the biosynthesis of peptide antibiotics and iron-chelatina siderophores. The example to be studied is the four enzyme system, Ent B,D,E,F, responsible for formation of the E. coli iron chelator enterobactin. In particular the EntF enzyme has four domains (condensation, adenylation, peptidyl carrier protein and thioesterase) whose functions in assembly of the (dihydroxybenzoyl)-serine trilactone, enterobactin, will be analyzed in terms of covalent priming, initiation, elongation, and termination strategies.

histogenesis; cell transformation; genetic manipulation; retina;

The objectives of this research are to elucidate the role of 8-hydroxy-7-demethyl-5-deazaflavin coenzymes and nickel-dependent enzymes in the biogenesis of natural gas from C02 and H2 by methanogenic bacteria. The deazaflavins are structural and functional hybrids between nicotinamide and flavin coenzymes and are used as low potential redox currencies. We will elucidate redox stereochemistry, modes of deazaflavin biosynthesis, enzymic nucleotidylation reactions and other roles as coenzymes in the action of DNA photolyase, involved in photomonomerization of thymine dimer lesions in DNA. Over the past five years four novel nickel-containing enzymes have been detected and purified from methanognic bacteria. We are studying two nickel-containing hydrogenases, nickel-linked methyl-S-coenzyme M reductase, and a nickel-dependent CO dehydrogenase as nickel hydrogenation biocatalyst, nickel desulfurization biocatalyst, and nickel carbonylation biocatalyst, respectively. We shall continue efforts to analyze the structure of the nickel active sites to identify ligands nickel oxidation states and geometries to understand catalytic function.

Polyhydroxbuytyrate (PHB) can be produced by many bacteria by fermentation and can be easily obtained in quantity. It would be an excellent thermoplastic; it lacks toxicity and is biodegradable. However it is too crystalline and brittle which has hindered its commercial potential. It is, however, possible that these PHB biosynthetic enzymes could be used to make alternate polyester materials with improved properties. Thus it is planned to dissect the important and fundamental mechanism of how this class of biological molecules are assembled. It will be necessary to clone (or move) the three genes in the biosynthetic pathway. The genes will be sequenced, appropriate vectors will be constructed and the resulting three enzymes, thiolase, acetoacetyl CoA reductase and PHB synthase, will be purified to homogeneity. The regulation of the three genes will be studied and each purified enzyme characterized structurally and functionally. The ultimate objective is to shed light on the unexplored mechanisms of biological polyester formation of PHB analogues. This is an ideal team to conduct this investigations. Dr. Masamune, a physical organic chemist, Dr. Walsh, as skilled bioorganic chemist, and Dr. Sinsky an applied microbiologist. Support is recommended with a high priority.

This proposal requests funds to obtain a 300 MHz Nuclear Magnetic Resonance Spectrometer. The instrument will be used for research in the following areas of biology, biochemistry, and molecular biology. 1. enzyme mechanisms and molecular bases of enzyme inhibition 2. mechanism of DNA cleavage by neocarzinostatin and related antitumor proteins 3. mobility of lipid membrane phases and action of anesthetic molecules 4. conformation of peptides with hormonal activity 5. metabolism and mechanisms of interconversion of retinoids in visual pigment biology 6. two-dimensional NMR studies of the conformation of peptide fragments of parathyroid hormone and humoral hypercalcemia factor The nuclear magnetic resonance spectrometer will be a truly significant addition to the Department's instrumentation capabilities, enhancing both current and future research efforts.

The Principal Investigator and coinvestigators from the Department of Biological Chemistry and Molecular Pharmacology and Molecular Genetics at the Harvard Medical School and from the Department of Medicine at Massachusetts General Hospital request funds for a 500MHz NMR Spectrophotometer to conduct research into the structure and function of proteins and polypeptides. Specific research problems address proteins in mercury detoxification, (merR, A,B, gene products), microcins as antibacterial proteins, amylogenins, Ca++-sequestering proteins in bone enamel, the role of key active site residues in the ADO-Pribosylating toxins, diptheria toxin and P. aeruginosa exotoxin-A, the nature of the DNA binding domains conned transcriptional activation domains in the yeast transcriptional activating protein GCN-4, and structure-function studies in insulin folding. Strong expertise in site-directed and selective mutagenesis coupled with experience in overproduction or proteins or protein fragments in quantity by high field NMR methods.

This renewal proposal deals with the enzymes involved in providing and incorporating D-alanine (D-ala) and the dipeptide D-Ala-D-Ala into the peptoglycan layer of cell walls in bacteria. Three enzymes comprise the D-Alanine branch, alanine racemase, D-Ala-D-Ala ligase and the D-Ala-D-Ala adding enzyme. In the past grant period (s) all three enzymes from E.coli and Salmonella typhimurium were purified to homogeneity for the first time. In the new grant period will be devoted to the study of the mechanism, structure/function of each of the genes and the encolded enzymes. In addition, the first enzyme in the peptoglycan pathway, the UDP-Nacetylglucosamine enolpyruvyltransferase will be purified and characterized. The results will greatly enchance our understanding of hw bacteria grow and build their cell walls. They may also lead to new strategies for designing effective chemotherapeutic agents against bacterial infections.

This work seeks to identify genes responsible for the developmental specification of the many cell types in the vertebrate nervous system. Most vertebrate neural cell fate decisions do not seem to depend only upon the lineage of neural cells, but instead reflect the serial recruitment of multipotential neural progenitor cells in response to tissue signals. A description of these fundamental processes is important in understanding the pathogenesis not only of developmental disorders of the brain, but also of tumors, in which such developmental control genes are often affected. Genetic studies in Drosophila show that several genes involved in cell fate decisions contain "zinc finger" DNA binding motifs, which allow them to regulate transcription. Despite clues that similar genes may have important roles in vertebrates as well, the relative lack of vertebrate genetic systems makes analysis difficult. The advent of retroviral vector systems offers several strategies for elucidating the functional roles of these zinc finger genes. In this research, a family of zinc finger genes will first be identified from the mouse using the polymerae chain reaction (PCR), and then will be cloned and sequenced. Wild type, or mutated or truncated versions of these genes, will then be cloned into retroviral shuttle vectors. These vectors allow the alteration of gene expression in vitro, using specifically engineered cell lines or primary neural cultures, or in vivo in the intact retina or cerebral cortex. Cellular phenotypes of altered expression, such as transformation, alteration in proliferation rate, or persistent differentiation (or blockage of differentiation) into specific cell types, can then be determined. Once phenotypes have been observed in these experiments, the "downstream" genes which are in turn regulated by the zinc-finger genes will be sought by using a modification of PCR.

This proposal focuses on enzymes in bacterial cell wall assembly of the peptidoglycan (PG) component, a structure unique to bacteria and known to be the target of several clinically useful antibiotics. Experiments are proposed in two areas: (1) the first committed step in the PG biosynthesis, an unusual enolpyruvyl transfer from PEP to UDP-Nacetyl gluco-samine to produce UDPenolpyruvyl G1cNAc, the scaffolding element for peptide assembly and (2) the D-ala-D-ala termini of PG that form the high affinity site for the antibiotic vancomycin. We have recently cloned, sequenced and purified to homogeneity MurZ, the enolpyruvyl transferase, and propose to study its catalytic mechanism and the mechanism of time-dependent inactivation of this enzyme by the antibiotic fosfomycin, an epoxypropane phosphonate in clinical use in Europe. No molecular information is known about the specificity of fosfomycin for MurZ and structure/function studies on catalytic mechanism could lead to improved antibiotic design against this target. Vancomycin resistance arises in life-threatening gram positive bacterial infections (e.g., endocarditis) when Van resistance genes encode five new proteins, VanS, R, H, A, X. We have recently overproduced, purified and characterized VanH, a D-specific a-ketoacid reductase and VanA, a D-Ala- D-X ligase and shown that they act in concert to make D-Ala-D-Lactate (a depsipeptide) and allow replacement of the normal D-Ala-D-Ala PG terminus by D-Ala-D-Lactate and that no longer recognizes vancomycin. We propose to further study the molecular mechanism of vancomycin resistance by comparison of VanA with the chromosomal D-Ala-D-Ala ligases as well as to purify and characterize VanS and VanR, a proposed two component regulatory system for control of VanH, A, X transcription. Knowledge of the Van resistance proteins may permit design of drugs, such as phos- phinate dipeptidomimetics, to revert vancomycin resistant bacteria to sensitivity.

The objectives of this proposal are to perform molecular and mechanistic analyses of the Vitamin K-dependent carboxylase, a liver endoplasmic reticular enzyme that carries out post-translational carboxylation of the most amino proximal 10-12 glutamyl residues in proteins such as prothrombin and Factor IX involved in blood coagulation. The resultant gamma-carboxyglutamyl (Gla) residues are high affinity calcium binding sites crucial for platelet membrane binding and for initiation of coagulation cascades. Now that this unusual O2 and dihydro vitamin K- utilizing enzyme has been purified, cloned, sequenced, and recombinant enzyme expressed (usefully for purification in baculovirus), we plan to perform analyses on molecular recognition of specific proteins and on catalytic mechanism. Specific aims include: recognition of substrates at both the upstream propeptide recognition site (gamma-carboxylation recognition site) and the active site, directionality and processivity of glutamyl residue carboxylation and quantitation of binding of enzyme to peptide substrates containing 1 to 10 glutamates (28mers to 59mers) by surface plasmon resonance and capillary zone electrophoresis techniques. Analysis of mechanism will include characterization of the enzyme as a hydroquinone epoxidase, and study of the timing of gamma-CH cleavage and carboxylation relative to KH2 and O2 reaction. Mechanistic insight may permit rational design of carboxylase inhibitors that complement such oral anticoagulants as the coumarins which inhibit the subsequent enzyme that recycles vitamin K epoxide.

Iron chelating siderophores and several peptide antibiotics are assembled by nonribosomal peptide synthase complexes via a thioltemplate mechanism. The siderophore Yersiniabactin, a key virulence factor in Yersinia infections, has both an aryl-N-Cap initiating biosynthesis and five membered sulfur heterocycles (thiazolines, thiazolidines resulting from cyclization of cysteines) that coordinate iron. The research proposed here involves determination of the genes responsible for yersiniabactin biosynthesis and characterization of the first enzymatic steps that involve activation of salicylic acid, amide bond formation to cysteine residues and cyclization to the thiazoline. We propose to purify YbtE and domains for the 200KD high molecular weight protein 2 of Y. pestis and analyze the anticipated enzymatic activity, including posttranslational phosphopantetheinylation (Ppant), salicyl-S-Ppant enzyme loading, salicyl-cys-S-enzyme formation, and cyclization to a salicyl-thiazoline-S-enzyme intermediate. These studies should decipher the logic of enzymatic assembly not only of virulence-conferring siderophores yersiniabactin and anguibactin but also for peptide antibiotics such as actinomycin, pristinamycin, and the antitumor agent bleomycin which use aryl N-cap imitation and/or thiazoline, oxazline- forming steps. The research will utilize the enzymology expertise in the PI s group and the yersiniabacteria genetic, microbiology and pathogenesis expertise in the co-investigator s group to decipher the molecular mechanisms for virulence determining siderophore biogenesis.

D. Gibson is a professor at the Hebrew University. He became experienced with mass spectrometry during his MS degree program, and has used instrumental techniques, particularly NMR, throughout his career. During his current sabbatical at the Resource, he will update his earlier experience by carrying out projects that require MALDI and ESI, both in areas related to his own research interests, the interactions of anticancer agents with oligonucleotides, and in collaborations with Resource and external personnel. Upon his return to Israel, his institution is planning to purchase both MALDI and ESI mass spectrometers, and Prof. Gibson will assume responsibility for oversight of these new instruments. N. Kelleher received his PhD at Cornell University under the direction of F. W. McLafferty and T. Begley. His thesis project centered on determinations of properties of proteins using electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry. He is presently a postdoctoral fellow in the research group of C. T. Walsh at Harvard Medical School. In order to broaden his experience with a variety of mass spectral techniques, Dr. Kelleher is now undertaking experiments that involve electrospray ionization triple quadrupole tandem mass spectrometry and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, under the guidance of Resource staff.

DESCRIPTION (Applicant's abstract): The glycopeptide antibiotics of the vancomycin family are potent antibacterial agents that can cure life-threatening bacterial infections by complexation of the D-Ala-D-Ala termini of peptidoglycan intermediates. The dramatic rise in resistance to vancomycin by grain positive pathogens (e.g., Vancomycin Resistant Enterococci) impels investigation into routes to more effective vancomycin analogs. Given the low likelihood of practical total syntheses, understanding of the enzymatic assembly of these nonribosomal peptide antibiotics offers prospect for subsequent reprogramming for combinatorial biosynthesis. This proposal deals with three phases of the enzymatic biosynthesis of vancomycin: (1) the beginning stages of the Nonribosomal Peptide Synthetase (NRPS) assembly line that makes the initial acyclic heptapeptide aglycone of vancomycin family members; (2) the post NRPS enzymatic tailoring of the heptapeptide, including oxidative crosslinking at the aryl side chains of residues 2,4,6,5 and 7, chlorination at 2 and 6, and glycosylation at 4 and 6; (3) the biogenesis of the 4-OH-phenylglycine incorporated at residues 4 and 5 of the core and 3,5-dihydroxyphenylglycine incorporated at residue 7. These aromatic amino acids are key sites for the crosslinking that produces the rigid cup-shaped architecture of the crosslinked core that enables recognition of the peptidoglycan termini and antibiotic action.

EXCEED THE SPACE PROVIDED. Iron chelators are synthesized, exported and retrieved by bacteria in iron-deficient microenvironments aid thus chelatars, also known as siderophores, serve as virulence factors for infections in vertebrates by pathogens such as Yersinia pestis in plague, Vibrio cholerae in cholera epidemics, and Pseudomonas aeruginosa in lung infection in CF patients. The respective siderophores, yersiniabactin (Ybt), vibriobactin(Vib), and pyochelin(Pch),are produced by non-ribosomal peptide synthetases(NRPS),activated by iron depletion. Yersiniabactin is assembled as a hybrid of a nonribosornal peptide and a polyketide (PK) by a mixed NRPS/PKS assembly line. The NRPS/PKS and PKS/NRPS switchpointsof this assembly line will be examined with purified multimodular subunits of Ybt synthetase. Ybt also has a tandem bithiazoline ring system and unusual C-methylations from Cyclization(Cy) and C-methylation(MT) domains embedded in the 17 domain Ybt assembly line. The mechanisms of the Cy and MT catalytic domains will be examined. The Pch assembly line involves four proteins and a cascade of acyl-S-enzyme intermediates that will be analyzed for timing and locus of specific chain elongation steps, including heterocyclic thiazoline ring reduction to thiazolidine and then subsequent N-methylationby the PchG subunit and the N-MT domain of the PchF subunit respectively. Because of the similarity of the Ybt and PCh siderophores protein-protein recognitionof domains and modules within and between the Pch and Ybt assembly lines will be examined to begin to establish rules for combinatorial biosynthesis of such NRP molecules. The third pathogenic siderophore system, the Vib synthetase assembles a branched siderophore and produces an oxazoline ring (from threonine) rather than the thiazoline riongs (from cysteine) in Pch and Ybt. There are two Cy domains and three Condensation (C) domains in the Vib assembly line and the function of all five of thesechain-elongation catalytic domains will be examined by biochemical analysis and mutagenesis to decipher the molecular logic of these NR.PS assemblies. Understanding of mechanism, e.g. of the heterocyclizing Cy domains may allow inhibitor design to block siderophore production by these multimodular enzymes.

DESCRIPTION (provided by applicant): The Harvard-MIT MD-Ph.D. Program provides an integrated approach to educating physician-scientists who will become leaders in American medicine and biomedical research. In this program, students combine medical studies at Harvard Medical School with graduate studies at Harvard or MIT. The program offers students the largest collection of academic laboratories in the world for research training, complemented by teaching hospitals that are poised to rapidly translate basic discoveries into new clinical applications. Students choose between two medical education curricula: a hybrid learning approach that combines small-group teaching and problem-oriented learning with more traditional teaching methods (New Pathway), or a traditional curriculum with an emphasis on science and technology (Health Sciences and Technology). Both curricula include a set of rigorous clinical clerkships at the Harvard teaching hospitals. Students also choose from among the four graduate programs in the Division of Medical Sciences at Harvard Medical School, other graduate programs in the Harvard Graduate School of Arts and Sciences, and programs in the Graduate Schools of Science and Engineering at MIT. The medical and scientific training components are integrated throughout the program, beginning with a course in the Molecular Biology of Human Disease and a laboratory research rotation that are taken by all MSTP students during the summer before the first academic year. Although not all MD-Ph.D. students are awarded funding at the time of matriculation, the program is designed to include all students at Harvard Medical School who are simultaneously pursuing the MD and Ph.D. degrees. Unfunded students can enter the program at the time of enrollment in a Ph.D. program. The program provides academic and mentoring support to approximately 130 students, taking advantage of a large, committed faculty. Approximately 125 faculty members are directly involved with the program through service on program committees and/or participation as MD-Ph.D. student thesis advisors. Mentoring, advising, and all program activities are available both to students who are funded by MSTP and to students who are not. Other training grants, individual NIH investigator (R01) awards, individual student fellowships, departmental funds, hospital funds and unrestricted institutional funds are used to supplement MSTP student support.

DESCRIPTION (provided by applicant):Nature assembles thousands of polyketide (PK) and nonribosomal peptide (NRP) natural product antibiotics on multimodular enzymatic assembly lines. Growing chains are tethered as a series of elongating covalent acyl-S-protein intermediates, attached by phosphopantetheinyl chains to carrier protein domains, typically one per module. This proposal examines catalytic machinery and chemical logic for two sets of maturation steps in antibiotic natural products. Nascent scaffolds are subject to tailoring reactions by dedicated enzymes that often are crucial for maturation of target biological activities. In Specific Aim 1 we prose examination of scope and mechanism of two types of oxidative tailoring enzymes for NRP and NR-PK hybrids: halogenases and oxygenases. Tailoring halogenases use either FADH2 or mononuclear nonheme FeII, dependent on electronic demand of the carbon site in the antibiotic. We plan to use halogenase gene probes to clone biosynthetic gene clusters, e.g. for kutzneride, and examine scope of the halogenative tailoring. We shall also examine the activity and mechanism of tailoring oxygenases that act in antibiotic scaffold maturations. Specific Aim 2 addresses catalytic domains in assembly lines involved in novel chemistry during chain elongation and termination steps. These include ester backbone linkages in place of amides, the recruitment of transglutaminase homologs as condensation domains that act in trans, formation of C-terminal pyrrolidinedione rings, and reductive chain termination steps.

DESCRIPTION (provided by applicant): The objectives of this proposal are to understand key aspects of the multicomponent assembly line of enzymes that produce the important antibiotic vancomycin and its congeners. A cluster of almost 30 genes encodes enzymes that provide dedicated monomers to be incorporated, enzymes that constitute a 27 step assembly line for producing the D-D-L-D-D-L-L- acyclic heptapeptide scaffold and tailoring enzymes that crosslink and glycosylate the peptide backbone to create the active antibiotic. Specific aim 1 proposes continued study of the biogenesis of 3,,5-dihydroxyphenyiglycine (Dpg), a key residue for subsequent crosslinking of the scaffold, and also the 3-OH-meta-chlorotyrosines incorporated at residues 2 and 6 of the peptide. Specific aim 2 examines the third subunit CepC of the chloroeremomycin assembly line and congeneric assembly lines (vancomycin, teicoplanin) that contains the seventh (last) module. This chain termination modules activates and incorporates Dpg as the terminal residue of the heptapeptidyl chain and then releases this full length peptide from its covalent attachment to the last carrier protein domain, PCP7, by action of the thioesterase domain in CepC. Aim 3 of the proposal studies two types of post-assembly line tailoring enzymes. The first group are hemeprotein oxidases involved in making the three crosslinks in vancomycin (2-4, 4-6, 5-7) and the four crosslinks in teicoplanin (2-4, 4-6, 5-7, 1-3), creating the rigidified peptide skeleton required for recognition of bacterial cell wall peptidoglycan. The second group is the acyl transferases that act on the teicoplanin subgroup to convert the glycopeptides to lipoglycopeptides, modifications that provide regain of potency against some phenotypic forms of vancomycin resistant enterococci. The knowledge base gained in these three aims will provide insight into molecular logic to enable subsequent combinatorial biosynthetic antibiotic variants.

The overall goal of this project is to learn about the interaction of domains in non-ribosomal polypeptide synthetase modules, using the 142 kDa E. coli EntF. This protein consists of four domains, C, A, T and TE that act as chain elongation and termination module to make and release the enterobactin siderophore. We are interested in how the domains cooperate to synthesize enterobactin, and how the HS-pantetheinyl-P group bound to the TE domain affects the interaction of domains, and how this may relate to the assembly- line function of the protein. The multidomain protein is a model system for development of NMR for large proteins The specific aims are: Specific Aim 1.. Complete structure determination of the 36 kDa A-T didomain fragment of the 142kDa EntF Specific Aim 2 Studies of structures and interactions of the in cis C and A domains centering on the T domain scaffold and how these partner proteins dock with it SpecificAim 3. Studies of the structure of the A-T 70 kDa didomain of EntF Specific Aim 4. . Interaction of the HS-pantetheinyl-P prosthetic group (isotopically labeled or spin-labeled )on the two T domains in EntB and EntF with different domains in cis and in trans (C, A, TE) in unacylated and acylated forms

The first focus is on enterobacterial strains that turn on biosynthetic genes for the siderophore enterobactin in microenvironments where iron is limiting, including vertebrate hosts. Enterobactin binds ferric iron avidly and is subjected to specific uptake by the producing bacteria. Some pathogenic gram negatives further elaborate the enterobactin scaffold to make C-glucosylated forms, also known as salmochelins from their discovery in iron-scavenging salmonella typhi strains. Salmochelin-producing bacteria tailor the Ent scaffold under direction of the Iro gene cluster IroBCDEN. We will study the enzymatic mechanism for C- glucosylation by IroB and determine to what extent the glucosylation of the dihydroxybenzene rings of the Ent scaffold interfere with sequestration of the siderophore by the mammalian host protein siderocalin. Failure to sequester the modified enterobactins should correlate with increased pathogenicity of the bacteria. The second focus is on the productuiion of the siderophore acinteobactin by the gram negative respiratory pathogen Acinetobacter baumanii. Siderophore production correlates with increased virulence. Acinetobactin has all three known-iron chelating groups, catechol, thiazoline, and hydroxamate, built into its skeleon by a nonribosomal peptide synthetase assembly line. The six genes BasABCDEF encode the siderophore synthetase assembly line. We plan to overproduce each protein and evaluate the following unusual featiures predicted for assembly line ooperations: action of two free standing adenylation and thiiolation domains, cyclodehdration by tandem condensation domains, hydroxamate formation during chain termination in siderophore maturation. Characterization of the salrnochelin and acinetobactin biosynthesizing enzymes will be the foundation for subsequent evaluation of enzyme inhibitors that might block siderophore production.

Normal development of the human cerebral cortex is essential for cognitive function, and is disrupted in human neurological disorders such as mental retardation and epilepsy. Positional cloning efforts by our lab and others have identified several genes required for normal neuronal migration, including DCX, FLNA, ARFGEF2, Reelin, Dab1 and others. Since many of the mechanisms involved in axon outgrowth and neuronal migration are shared, some of these genes also have effects on axon outgrowth. Many of these genes encode cytoplasmic proteins that exert their effects via interactions with signaling pathways that are not yet well defined. The overall goal of this proposal is to analyze the role of the Reelin/Dab1 pathway, and of doublecortin (Dcx) and the doublecortin-like kinase (Dclk) in neuronal migration and axon outgrowth. These two pathways appear to converge on the control of microtubules and the regulation of process outgrowth in neurons. Specific aim 1 will analyze the role of the Reelin/Dab1 pathway in control of the leading process of migrating neurons. Specific aim 2 will analyze the role of Dclk in activity-related axon outgrowth and normal synaptic remodeling. Specific aim 3 will analyze the interacting roles of Dcx and Dclk in controlling the normal migration of neurons to the cerebral cortex, and the normal outgrowth and targeting of cortical axons: We hope that this work will not only improve out understanding of the normal role of these genes in cortical development but may also identify additional candidate genes for other human developmental disorders.

DESCRIPTION (provided by applicant): Most clinically used antibiotics are natural products from microbial sources and are built of peptide or polyketide frameworks or hybrids thereof. In the biogenesis of peptide antibiotics of both ribosomal and nonribosomal origin one of Nature's strategies is to morph the amino acid side chains and also the peptide backbones into scaffolds that are conformationally restricted by cyclization to populate biologically active conformers. A complementary strategy is to generate functional groups within restricted architectures that lead to potent inhibition of specific targets. In this application we propose examination of both aspects to decipher enzymatic strategies for side chain and backbone cyclizations during peptide antibiotic scaffold maturation as well as generation of unusual functional groups that underlie antibiotic structure and function. In specific aim 1 we propose to decipher the enzymatic strategies for making stable N-P bonds in the ribosomal peptide antibiotic Microcin C7 and the nonribosomal peptide phaseolotoxin. In specific aim 2 we will examine the enzymatic machinery for construction of "syrbactin" antibiotics with 12 membered enamide macrolactam rings as conditional electrophiles that irreversibly target the proteasome. In specific aim 3 we examine antibiotic and pheromone synthases that convert the indole side chain of tryptophan to a rigidified tricyclic system as well as looking at the construction of an eight member macrocycle involving the indole ring. We also examine the enzymes that mediate conversion of Phe and Tyr side chains into rigidified bicyclic and tricyclic frameworks in toxin and antibiotic generation. PUBLIC HEALTH RELEVANCE: There is a constant need for new antibiotics as multiply drug resistant bacterial pathogens emerge. Most antibiotics are built on natural product scaffolds. This application examines sets of enzymes that build in conformational constraints and unusual side chains into natural peptide-based frameworks during antibiotic construction. These biosynthetic enzymes involve morphing of the amino acid building blocks, both in side chains and in peptide bond connectivity, to create the rigidified scaffolds with conditionally reactive functional groups that lead to biologically active conformers of antibiotics.