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Research is presented demonstrating the proof of principle that non-natural secondary metabolite glycosylation patterns can be constructed through a selection of heterologous gene combinations.

A novel strategy for the practical synthesis of Gram negative cell wall antigens is presented. Compared to existing synthetic routes to 3-omicron-alpha-L-rhamnopyranosyl-D-galactopyranosides, this novel approach provides a 3-omicron-alpha-L-rhamnopyranosyl-D-galactal that can be easily derivatized from the reducing end. It has been used to afford a simplified chromophoric substrate which allows for a new quantitative ManT(super beta4) assay.

Unnatural amino acid mutagenesis was employed to substitute the active site tyrosine residue in human glutathione transferase A1-1 with fluorinated tyrosine analogues with 5.3-10 pKa site specifically. Tyrosine residue was replaced by 2-fluorotyrosine, 3-fluorotyrosine, 3,5-difluorotyrosine and 2,3,5,6-tetrafluorotyrosine. Results show that the slight proton location shift does not alter glutathione transferase behavior unless 2,3,5,6-tetrafluorotyrosine is introduced.

Mutational analysis on backbone hydrogen bonds in staphylococcal nuclease was conducted to demonstrate main chain-main chain hydrogen bonding interactions. Unnatural amino acid mutagenesis was then used to replace two varying chain hydrogen-bonded pairs of amide groups present in antiparallel beta-sheet. Results show a decrease of about 1.5-2.5 kcal/mol in protein stability when a weaker hydrogen bond acceptor substituted the backbone of amide groups in a beta-sheet secondary structure.

An analysis of hydrogen bonding strengths in proteins using unnatural amino acids show that the intramolecular hydrogen bonds in folded proteins make a net favorable contribution of 1-2 kcal per mol to protein stability relative to the corresponding hydrogen bonds to the solvent in the denatured state. Hydrogen bonds play an important role in finding the secondary and tertiary structure of proteins. The values of hydrogen bond stabilization are determined from mutational studies in which one member of a hydrogen bonded pair is deleted.

Kinetic studies enable the determination of free energies of denaturation for various Staphylococcal nuclease mutants having tyrosine-glutanate hydrogen bonds of different strengths. Fluorine ring substitutions modify the acidity of the tyrosine hydroxyl group and thereby modulates the strength of the hydrogen bond. Each fluorine substitution increases the stabilization energy by an average of 0.56 kcal mol. Linear free energy relation between the pKa of the tyrosine hydroxyl group and free energy change of the mutant proteins indicates the higher stability of intramolecular hydrogen bonds in folded proteins compared to the hydrogen bonds in the unfolded state.

CalE6 from Micromonospora echinospora is a (pyridoxal 5' phosphate) PLP-dependent methionine ?-lyase involved in the biosynthesis of calicheamicins. We report the crystal structure of a CalE6 2-(N-morpholino)ethanesulfonic acid complex showing ligand-induced rotation of Tyr100, which stacks with PLP, resembling the corresponding tyrosine rotation of true catalytic intermediates of CalE6 homologs. Elastic network modeling and crystallographic ensemble refinement reveal mobility of the N-terminal loop, which involves both tetrameric assembly and PLP binding. Modeling and comparative structuralanalysis of PLP-dependent enzymes involved in Cys/Met metabolism shine light on the functional implications of the intrinsic dynamic properties of CalE6 in catalysis and holoenzyme maturation. [ABSTRACT FROM AUTHOR]

Structure analysis and ensemble refinement of the apo-structure of thymidine diphosphate (TDP)-rhamnose 3'-O-methyltransferase reveal a gate for substrate entry and product release. TDP-rhamnose 3'-O-methyltransferase (CalS11) catalyses a 3'-O-methylation of TDP-rhamnose, an intermediate in the biosynthesis of enediyne antitumor antibiotic calicheamicin. CalS11 operates at the sugar nucleotide stage prior to glycosylation step. Here, we present the crystal structure of the apo form of CalS 11 at 1.89 Å resolution. We propose that the L2 loop functions as a gate facilitating and/or providing specificity for substrate entry or promoting product release. Ensemble refinement analysis slightly improves the crystallographic refinement statistics and furthermore provides a compelling way to visualize the dynamic model of loop L2, supporting the understanding of its proposed role in catalysis. [ABSTRACT FROM AUTHOR]

EPR studies on the pyridoxamine 5'-phosphate (PMP) induced catalysis of the PMP-dependent enzyme, CDP-6-deoxy-L-threo-D-glycero-4-hexulose-3-dehydrase (E1), reveals that PMP mediates anion induced dehydration and is an E3-electron receptor in the redox reaction. The presence of the E1 (2Fe-2S) center and the radical intermediate proves that the PMP-induced enzyme redox reactions have a radical mechanism. PMP is the amine form of the B6 coenzyme.

The gene coding for E1(ascC) from a Yersinia pseudotuberculosis lambda gt11 subgenomic library was cloned through hybridization with a degenerate oligonucleotide probe built from the N-terminal sequence of the purified wild-type E. This was done to investigate the biosynthesis of 3, 6-dideoxyhexoses, and represents the characterization of the first pyridoxamine 5'-phosphate (PMP)-dependent iron-sulfur containing enzyme needed for this biosynthesis.

Biosynthesis of deoxy sugars such as 2,6 and 4,76-dideoxyhexoses prevalent in a wide range of bioactive compounds and 3,6-dideoxyhexoses prevalent in the lipopolysaccharides of Yersinia follows a pathway similar to that of the biosynthesis of ascarylose. The pathway of the biosynthesis is characterized by an initial CDP-dextrorotary(D)-glucose 4,6-dehydratase (Eod)-catalyzed conversion of CDP-D-glucose to 6-deoxy-D-glycero-L-thero-4-hexulose. This is succeeded by a C-3 deoxygenation involving 2 successive reactions catalyzed by CDP-6-deoxy-laerorotatory(L)-threo-D-glycero-4-hexulose-3-dehydrase (EI), a PMP-dependent iron-sulfur containing enzyme and CDP-6-deoxy-delta-glucoseen reductase (E3), a (2Fe-2S)-containing flavoprotein. The biosynthesis terminates by a stereospecific C-4 reduction, concurrent with a probable C-5 epimerization, resulting in a field of the 3,6-dideoxyhexoses.

Researchers cloned and characterized the gene cluster encoding the proteins of the major biosynthetic passage related to CDP-D-glucose-Ascarylose biosynthetic genes, which is the first total characterization-correlation of gene products with their catalytic activities for a whole 3,6-dideoxyhexose pathway. The study offers specific molecular proof supporting the proposed chromology in the biosynthetis of ascarylose and other 3,6-dideoxyhexoses.