Structural and functional studies on a 3'-epimerase involved in the biosynthesis of dTDP-6-deoxy-D-allose.

Unusual deoxy sugars are often attached to natural products such as antibiotics, antifungals, and chemotherapeutic agents. One such sugar is mycinose, which has been found on the antibiotics chalcomycin and tylosin. An intermediate in the biosynthesis of mycinose is dTDP-6-deoxy-D-allose. Four enzymes are required for the production of dTDP-6-deoxy-D-allose in Streptomyces bikiniensis, a soil-dwelling microbe first isolated from the Bikini and Rongelap atolls. Here we describe a combined structural and functional study of the enzyme ChmJ, which reportedly catalyzes the third step in the pathway leading to dTDP-6-deoxy-D-allose formation. Specifically, it has been proposed that ChmJ is a 3'-epimerase that converts dTDP-4-keto-6-deoxyglucose to dTDP-4-keto-6-deoxyallose. This activity, however, has never been verified in vitro. As reported here, we demonstrate using (1)H nuclear magnetic resonance that ChmJ, indeed, functions as a 3'-epimerase. In addition, we determined the structure of ChmJ complexed with dTDP-quinovose to 2.0 Å resolution. The structure of ChmJ shows that it belongs to the well-characterized "cupin" superfamily. Two active site residues, His 60 and Tyr 130, were subsequently targeted for study via site-directed mutagenesis and kinetic analyses, and the three-dimensional architecture of the H60N/Y130F mutant protein was determined to 1.6 Å resolution. Finally, the structure of the apoenzyme was determined to 2.2 Å resolution. It has been previously suggested that the position of a conserved tyrosine, Tyr 130 in the case of ChmJ, determines whether an enzyme in this superfamily functions as a mono- or diepimerase. Our results indicate that the orientation of the tyrosine residue in ChmJ is a function of the ligand occupying the active site cleft.

Changes in tear protein pattern after photorefractive keratectomy.

PURPOSE: Changes in tear protein composition of patients who underwent photorefractive keratectomy (PRK) were analyzed. METHODS: Tear samples were obtained from 23 eyes of 23 patients immediately before PRK and on the fourth postoperative day with glass capillaries. Tear proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Digital image analysis and evaluation of the densitometric data of the electrophoretic separations were done with BioDoc-Analyze. RESULTS: Analysis of discriminance found a significant difference in the protein patterns (p < 0.001). This type of analysis of the electrophoretic densitographs uses all peak information simultaneously. A significant decrease (p < 0.005) in three of the main protein peaks--lactoferrin, immunoglobulin A heavy chain, and lysozyme--was also found after PRK. CONCLUSIONS: Excimer laser ablation of the cornea has an acute effect on lacrimal gland protein secretion. Changes in tear composition may lead to feelings of dryness and to a decrease in tear film stability postoperatively.