36 research outputs found
Biosynthesis and Incorporation of Nonproteinogenic Amino Acids into Non-ribosomal Peptide Natural Products
Thesis advisor: Steven D. BrunerComplex and unique enzymology is often behind the biosynthesis of natural products. This thesis is focused on how non-proteinogenic amino acids are biosynthesized and then incorporated into natural products. Chapters two, three and four deal with a unique dioxygenase found in vancomycin biosynthesis. Chapter five elaborates on the biochemical characterization along with efforts toward structural characterization of a terminal non-ribosomal peptide synthetase module. The vancomycin biosynthetic enzyme DpgC belongs to a small class of oxygenation enzymes that are not dependent on an accessory cofactor or metal ion. The detailed mechanism of cofactor-independent oxygenases has not been established. We have solved the first structure of an enzyme of this oxygenase class complexed with a bound substrate mimic. The use of a designed, synthetic substrate analog allows unique insights into the chemistry of oxygen activation. The structure confirms the absence of cofactors, and electron density consistent with molecular oxygen is present adjacent to the site of oxidation on the substrate. Molecular oxygen is bound in a small hydrophobic pocket and the substrate provides the reducing power to activate oxygen for downstream chemical steps. Our results resolve the unique and complex chemistry of DpgC, a key enzyme in the biosynthetic pathway of an important class of antibiotics. Mechanistic parallels exist between DpgC and cofactor-dependent flavoenzymes, providing information regarding the general mechanism of enzymatic oxygen activation.Thesis (PhD) — Boston College, 2008.Submitted to: Boston College. Graduate School of Arts and Sciences.Discipline: Chemistry
A ternary complex of hydroxycinnamoyl-CoA hydratase–lyase (HCHL) with acetyl-CoA and vanillin gives insights into substrate specificity and mechanism
Fine tuning of the catalytic effect of a metal-free porphyrin on the homogeneous oxygen reduction
Structure elucidation and biosynthesis of fuscachelins, peptide siderophores from the moderate thermophile Thermobifida fusca
Bacteria belonging to the order Actinomycetales have proven to be an important source of biologically active and often therapeutically useful natural products. The characterization of orphan biosynthetic gene clusters is an emerging and valuable approach to the discovery of novel small molecules. Analysis of the recently sequenced genome of the thermophilic actinomycete Thermobifida fusca revealed an orphan nonribosomal peptide biosynthetic gene cluster coding for an unknown siderophore natural product. T. fusca is a model organism for the study of thermostable cellulases and is a major degrader of plant cell walls. Here, we report the isolation and structure elucidation of the fuscachelins, siderophore natural products produced by T. fusca. In addition, we report the purification and biochemical characterization of the termination module of the nonribosomal peptide synthetase. Biochemical analysis of adenylation domain specificity supports the assignment of this gene cluster as the producer of the fuscachelin siderophores. The proposed nonribosomal peptide biosynthetic pathway exhibits several atypical features, including a macrocyclizing thioesterase that produces a 10-membered cyclic depsipeptide and a nonlinear assembly line, resulting in the unique heterodimeric architecture of the siderophore natural product
Substituent effects on the phenol coupling reaction catalyzed by the vancomycin biosynthetic P450 enzyme OxyB
Oxidative phenol coupling reactions are required to establish the cross-linked heptapeptide backbone of vancomycin. The first cross-linking reaction, catalyzed by the P450 enzyme OxyB, is dramatically slower when a chlorine substituent is present in the hexapeptide-S-PCP substrate and is abolished when chlorine is introduced into a potential heptapeptide-S-PCP substrate
Studien zur Aktivität des P450-Enzymes OxyB in der Biosynthese von Vancomycin: Einfluss der Chlor- und Hydroxy-Substituenten
In Vivo Investigation of the Roles of FdmM and FdmM1 in Fredericamycin Biosynthesis Unveiling a New Family of Oxygenases*
Fredericamycin (FDM) A, a highly oxidized aromatic pentadecaketide natural product, exhibits potent cytotoxicity and has been studied as a new anticancer drug lead. The FDM biosynthetic gene cluster has been previously cloned from Streptomyces griseus ATCC 49344 and successfully expressed in the heterologous host Streptomyces albus J1074. The fdmM and fdmM1 genes code for two proteins with high sequence homology to each other but unknown function. In-frame deletion of each of the genes from the fdm cluster was accomplished in the S. albus host. Each mutant failed to produce FDM A and the key biosynthetic intermediate FDM E but produced various new metabolites, the titers of which were dramatically increased via overexpression of an fdm pathway-specific activator fdmR1. The ΔfdmM mutant strain accumulated three new compounds FDM M-1, FDM M-2, and FDM M-3, whereas the ΔfdmM1 mutant strain produced one new compound FDM M1-1. Isolation and structural characterization of these compounds enable us to propose that FdmM and FdmM1 catalyze the C-6 and C-8 hydroxylations for FDM biosynthesis, respectively. Homologs of FdmM and FdmM1 can be found in biosynthetic gene clusters of many other aromatic polyketides, ranging from dodecaketides to pentadecaketides, but to date all of them were annotated as proteins of unknown function. Based on the findings reported here for FdmM and FdmM1, we now propose similar functions for those proteins, and FdmM and FdmM1 therefore represent an emerging family of novel oxygenases responsible for hydroxylation of aromatic polyketide natural products