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Our immune system constantly samples peptides found inside the body as a means to detect foreign pathogens, infected cells, and tumorous cells. T cells, which carry out the critical task of distinguishing self from nonself peptides, can only survey peptides that are presented by the major histocompatibility complex protein. We investigated how the secondary structure of a peptide, namely, the polyproline II helix content, influences major histocompatibility complex binding. We synthesized 12 analogues of the wheat gluten derived α-I-gliadin peptide and tested their binding to the celiac disease associated HLA-DQ2 protein. Our analogue library represents a broad spectrum of polyproline II propensities, ranging from random coil structure to high polyproline II helix content. Overall, there was no noticeable correlation between the peptide polyproline II helix content and HLA-DQ2 binding. One analogue peptide, which has low polyproline II helix content, showed a 4.5-fold superior binding compared to native α-I-gliadin.

Postgenomic analysis revealed that many microorganisms carry numerous secondary metabolite biosynthetic genes on their genome. However, activities of those putative genes are not clearly reflected in the metabolic profile of the microorganisms, especially in fungi. A recent genome mining effort is promising in discovering new natural products. However, many fungi and other organisms are not amenable to molecular genetics manipulations, making the study difficult. Here we report successful engineering of Chaetomium globosum, a known producer of various valuable natural products, that allows its genetic manipulation via targeted homologous recombination. This strain permitted us to abolish transcriptional regulators associated with epigenetic silencing of secondary metabolite biosynthetic pathways, leading to the identification of the products generated by different gene clusters and isolation of novel secondary metabolites. We were able to identify six gene clusters that are responsible for the biosynthesis of 11 natural products previously known to be produced by C. globosum, including one cytochalasan and six azaphilone-type compounds. In addition, we isolated two new compounds, mollipilin A and B, that were only recently identified in a related Chaetomium species. Furthermore, our investigation into the mechanism of biosynthesis of those natural products in C. globosum also led to the discovery of a secondary metabolite, aureonitol, that acts like a transcriptional regulator for the biosynthesis of other secondary metabolites. Similar approaches should facilitate exploration of the untapped potential of fungal biosynthetic capability and identification of various unique biological functions that those secondary metabolites possess.

Redox enzymes play a central role in generating structural complexity during natural product biosynthesis. In the postassembly tailoring steps, redox cascades can transform nascent chemical scaffolds into structurally complex final products. Chaetoglobosin A (1) is biosynthesized by a hybrid polyketide synthase-nonribosomal peptide synthetase. It belongs to the chaetoglobosin family of natural products, comprising many analogs having different degrees of oxidation introduced during their biosynthesis. We report here the determination of the complete biosynthetic steps leading to the formation of 1 from prochaetoglobosin I (2). Each oxidation step was elucidated using Chaetomium globosum strains carrying various combinations of deletion of the three redox enzymes, one FAD-dependent monooxygenase, and two cytochrome P450 oxygenases, and in vivo biotransformation of intermediates by heterologous expression of the three genes in Saccharomyces cerevisiae. Five analogs were identified in this study as intermediates formed during oxidization of 2 to 1 by those redox enzymes. Furthermore, a stereochemical course of each oxidation step was clearly revealed with the absolute configurations of five intermediates determined from X-ray crystal structure. This approach allowed us to quickly determine the biosynthetic intermediates and the enzymes responsible for their formation. Moreover, by addressing the redox enzymes, we were able to discover that promiscuity of the redox enzymes allowed the formation of a network of pathways that results in a combinatorial formation of multiple intermediate compounds during the formation of 1 from 2. Our approach should expedite elucidation of pathways for other natural products biosynthesized by many uncharacterized enzymes of this fungus.

Our recent findings of the first epoxide hydrolase Lsd19, involved in lasalocid A biosynthesis, led us to investigate a long-standing controversial issue on the mechanism of enzymatic epoxide-opening cascades. The site-directed mutagenesis and domain dissection analysis to reveal the mechanism of the reaction catalyzed by Lsd19 is examined, especially in the role of acidic amino acid pair and catalytic domains.

The mechanisms by which solvents, antibodies, and albumins influence the rates of base-catalyzed reactions of benzisoxazoles have been explored theoretically. New experimental data on substituent effects and rates of reactions in several solvents, in an antibody, and in an albumin are reported. Quantum mechanical calculations were carried out for the reactions in water and acetonitrile, and docking of the transition state into a homology model of antibody 34E4 and an X-ray structure of human serum albumin was accomplished. A microenvironment made up of catalytic polar groups (glutamate in antibody 34E4 and lysine in human serum albumin) surrounded by relatively nonpolar groups is present in both catalytic proteins.

The siderophore petrobactin harbors unique 3,4-dihydroxybenzoyl iron-liganding groups. These moieties are known to be synthesized from shikimate pathway precursors, but no reports of the biosynthetic enzymes responsible for this conversion have been published. The gene encoding AsbF from Bacillus thuringiensis 97-27 was overexpressed in an Escherichia coli host. AsbF rapidly and efficiently transforms (-)-3-dehydroshikimate (DHS) into 3,4-dihydroxybenzoate (k(cat)(DHS) = 217 +/- 10 min(-1); K(m)(DHS) = 125 +/- 14 microM) at 37 degrees C and has an absolute requirement for divalent metal. Finally, the pH versus k(cat)(DHS) profile revealed two ionizable groups (pK(a1) = 7.9 +/- 0.1, and pK(a2) = 9.3 +/- 0.1).

Engineered biosynthetic pathways provide a powerful method for generating complex molecules. Precursor-directed biosynthesis, which combines chemical synthesis and enzymatic transformations, allows non-native starting materials to be incorporated into biosynthetic pathways. Using this approach, we achieved the production of the anticancer agent epothilone C in Escherichia coli. An E. coli strain was engineered to express the last three modules of the epothilone biosynthetic pathway (epoD-M6, epoE, and epoF) and the substrate required to complement the biosynthetic enzymes was obtained by chemical synthesis. Under high-density cell culture conditions, the E. coli strain processed exogenously fed synthetic substrate into epothilone C at levels comparable to the native host (1 mg/L) and at higher levels than other heterologous hosts. Importantly, this precursor-directed approach will allow chemical modifications to be introduced into the polyketide backbone and may ultimately provide access to epothilone analogues with improved pharmacological properties in quantities sufficient for clinical development.